Victoria

Gascón
AVANCES EN LAS PROPIEDADES DE

Pérez
La presente Tesis Doctoral se ha centrado en el diseño y
optimización de materiales silíceos mesoestructurados híbridos BIOCATALIZADORES
organo-inorgánicos en los que se controla el tamaño de poro, la
morfología de partícula y la funcionalización de la superficie
para incorporar moléculas de enzima en su interior. Se han
DISEÑO Y OBTENCIÓN DE MATERIALES

Avances en las propiedades de biocatalizadores: Diseño y obtención de Materiales

obtenido biocatalizadores con elevadas cargas enzimáticas, altas
MESOPOROSOS ORDENADOS COMO

Mesoporosos Ordenados como soportes para la inmovilización no covalente de enzimas

actividades y eficiencias catalíticas. Además, se ha podido
localizar a las moléculas de enzima en el interior de los poros de
los soportes. El diámetro de poro diseñado a medida y la alta SOPORTES PARA LA INMOVILIZACIÓN
afinidad enzima-soporte producen alta retención de la enzima
mediante uniones no covalentes, y en algunos casos se consigue
NO COVALENTE DE ENZIMAS
evitar el lixiviado. La inmovilización y/o encapsulación de la
enzima protege de la inactivación frente a disolventes orgánicos y
se mejora la estabilidad térmica. Por todo ello, estos
biocatalizadores podrían cumplir potencialmente con los
requisitos para ser utilizados industrialmente.

TESIS DOCTORAL
2014

VICTORIA GASCÓN PÉREZ

 Victoria
Gascón
AVANCES EN LAS PROPIEDADES DE

Pérez
La presente Tesis Doctoral se ha centrado en el diseño y
optimización de materiales silíceos mesoestructurados híbridos BIOCATALIZADORES
organo-inorgánicos en los que se controla el tamaño de poro, la
morfología de partícula y la funcionalización de la superficie
para incorporar moléculas de enzima en su interior. Se han
DISEÑO Y OBTENCIÓN DE MATERIALES

Avances en las propiedades de biocatalizadores: Diseño y obtención de Materiales

obtenido biocatalizadores con elevadas cargas enzimáticas, altas
MESOPOROSOS ORDENADOS COMO

Mesoporosos Ordenados como soportes para la inmovilización no covalente de enzimas

actividades y eficiencias catalíticas. Además, se ha podido
localizar a las moléculas de enzima en el interior de los poros de
los soportes. El diámetro de poro diseñado a medida y la alta SOPORTES PARA LA INMOVILIZACIÓN
afinidad enzima-soporte producen alta retención de la enzima
mediante uniones no covalentes, y en algunos casos se consigue
NO COVALENTE DE ENZIMAS
evitar el lixiviado. La inmovilización y/o encapsulación de la
enzima protege de la inactivación frente a disolventes orgánicos y
se mejora la estabilidad térmica. Por todo ello, estos
biocatalizadores podrían cumplir potencialmente con los
requisitos para ser utilizados industrialmente.

TESIS DOCTORAL
2014

VICTORIA GASCÓN PÉREZ

 AVANCES EN LAS PROPIEDADES DE
BIOCATALIZADORES: DISEÑO Y OBTENCIÓN DE
MATERIALES MESOPOROSOS ORDENADOS
COMO SOPORTES PARA LA INMOVILIZACIÓN NO
COVALENTE DE ENZIMAS

MEMORIA

para optar al grado de Doctora en Química: Ciencia Interdisciplinar

por la Universidad Autónoma de Madrid presentada por:

VICTORIA GASCÓN PÉREZ

Directores:
Dra. Rosa María Blanco Martín y Dr. Carlos Márquez-Álvarez

Instituto de Catálisis y Petroleoquímica (ICP),

Consejo Superior de Investigaciones Científicas

Tutor:
Dr. Felix Pariente Alonso
Departamento de Química Analítica, Facultad de Ciencias
Universidad Autónoma de Madrid

MADRID, 2014
El presente trabajo de investigación titulado “Avances en las propiedades de
biocatalizadores: Diseño y obtención de Materiales Mesoporosos Ordenados
como soportes para la inmovilización no covalente de enzimas” constituye la
Memoria que presenta Dña. Victoria Gascón Pérez para optar al grado de doctora en
Química: Ciencia Interdisciplinar por la Facultad de Ciencias de la Universidad
Autónoma de Madrid. Ha sido realizado en los Laboratorios del Grupo de Tamices
Moleculares del Instituto de Catálisis y Petroleoquímica (ICP-CSIC) bajo la
dirección del Dr. Carlos Márquez Álvarez y la Dra. Rosa María Blanco Martín.

Y para que así conste, firmamos el presente certificado en Cantoblanco, a 5

de Septiembre de 2014.

Fdo: Dr. Carlos Márquez Álvarez Fdo: Dra. Rosa María Blanco Martín
AGRADECIMIENTOS

En primer lugar, agradecer al Ministerio de Economía y Competitividad

(MINECO) por concederme una ayuda predoctoral de Formación de Personal
Investigador (FPI-BES-2010-040839), que me ha permitido costearme mis gastos
durante el curso académico del Máster en Química Aplicada, y el primer año de
tesis doctoral. También al Ministerio de Educación (MECD) por la concesión de
una ayuda predoctoral de Formación Personal Universitario (FPU-AP-2010-
2145) con la que he podido dedicarme a la investigación los dos años restantes,
aunque debo criticar que sin darme la posibilidad de hacer una estancia
predoctoral en el extranjero.

También agradecer a mis directores, Rosa y Carlos, por todo lo que me

enseñan, por sus valiosos comentarios, consejos, por su constante ayuda,
disponibilidad y paciencia cada vez que surgen problemas en el laboratorio o fuera
de él. Por supuesto agradecer a Isabel Díaz, que desde la lejanía siempre ha estado
pendiente de los avances conseguidos. Espero seguir trabajando a vuestro lado.

Cómo no, agradecer a todos los integrantes del grupo de Tamices Moleculares,
en especial a Joaquín Pérez Pariente por darme la oportunidad de trabajar en su
grupo de investigación; a Enrique Sastre por estar siempre pendiente de las
gestiones del grupo; a Manuel Sánchez por tener siempre la puerta de su despacho
abierta para hablar de investigación o de MOF (otro gran campo de investigación
por descubrir, y en el que aún sólo hemos dado pequeñas pinceladas con las
enzimas); a Marisol, a Luis, y a “las chicas”: Isa, Teresa, Almudena, Ana, Irene,
Bea, Claudia, Pilar,... Igualmente quiero dar las gracias a Vicente Cortés que
siempre está dispuesto a ayudar, y a pasar un rato para charlar de ciencia,
noticias, o de la vida misma; y a Álvaro Mayoral por el manejo tan maravilloso de
los microscopios. También a todos los compañeros/as que han pasado por el
laboratorio 107 (Óscar, Elsa,…). Y en especial a mi amigo Manuel “El Mexicano”,
porque no hay día que no hablemos de México, o de comida rica, y a Nacho, el
historiador del grupo, y mi compañero de adicción al té. Así como a todo el
personal de apoyo a la investigación (Javi, Conchi, Rosa, Mercedes,…), de
mantenimiento (Eduardo, Jose, Armando,…), a Paloma de almacén, a Adrián y
Jesús de informática, así como a todo el personal de secretaría (Rosa, Nuria,…),
de limpieza (Pili, Mari Ángeles,…).

También tengo que darle las gracias a las amigas que he hecho en el instituto,
sobre todo a Cris por ser tan sutil y delicada (cuanto voy a echar de menos
nuestras profundas conversaciones sobre gatos); a Idoia “mi súper-bióloga” por el
buen equipo que hicimos durante los 6 meses que estuvo haciendo su proyecto; a
Andreina y a su “beba” Carolina (esta niña va a acabar siendo científica seguro,
sólo con oírnos durante los 9 meses de embarazo…); y a Marina por su positivismo
y alegría. Claramente, sin vosotras este trabajo habría sido mucho más duro, por
no decir imposible.

I would like to pass my gratitude also to my friends in Ethiopia who have been
here in our institute for visiting and scientific discussion. Specially, Negash by
naming his new daughter “Victoriya”, and for his reinforcement in the scientific
work and social life. Also Solomon, Mamo, Alemnew, Mark, Kiros, Taju and
Lijalem for their cooperation during their stay here in Spain and impressed me to
visit Ethiopia: “Bekirbu Ethiopiayanina inanten indemigobegn tesfa adergalew”.

En esta tesis no me puedo olvidar de mis antiguos directores de la Universidad

Rey Juan Carlos, Jesús Mª Arsuaga y Amaya Arencibia, gracias a vosotros entré
en este apasionante y arduo mundo de la investigación, y vosotros habéis sentado
las bases de lo que soy ahora, científica, y a lo que deseo dedicarme el resto de mi
vida.

Y a mis amigos/as de siempre que aunque nos veamos poco siempre estáis ahí.
En especial a Laura Torres y a Natalia por preocuparse por mí y escucharme (qué
sepáis que sois las protagonistas de mi novela literaria, sí o sí). A mi querido
Victor por sus conversaciones políticas (en menudos líos que me metes siempre); a
Darío que un día me enseño que “La vida no es estabilidad; es saber andar en
equilibrio”; a Manolo por coincidir conmigo en tantas cosas, y sobre todo por
introducirme en el mundo de los “deportes de alto riesgo”: si ahora toca parapente
motorizado, ¿qué será lo siguiente?; a Miguel por su arsenal de proteínas y
vitaminas, y sus conversaciones sobre la eterna juventud (¿en serio que no
duermes en formol?); a mis amigas de Toledo: Ruth (y a su familia) y Celia que
siempre se emociona con mis Microrrelatos; a mí “teacher” de equitación Curro
Bedoya (aunque nunca paso de la doma vaquera…); a mis queridos vecinos de
Alcorcón, Laura y Dani por cuidarme tanto; a Eli y a Álvaro por llamar a las
cosas por su nombre; a los amigos del partido, a los compis de Ágora...

Ante todo agradecer a mis padres, su inestimable ayuda, por apoyar mis
decisiones, enseñarme a no rendirme cuando las cosas se ponen difíciles, a levantar
la cabeza, coger aire y seguir adelante. Además, gracias a mi hermano Jorge,
súper Doctor en Informática Gráfica, Juegos y Realidad Virtual, mi ejemplo a
seguir, aunque últimamente es al contrario (aún nos queda pendiente bucear en
aguas tropicales…, no se me ha olvidado!). Y al resto de mi familia, en especial a
mi tía Mila y a mi tío Paco, por tener siempre la puerta de su casa abierta; a mi tía
favorita, Carmen, por sus sabios consejos. También a las que ya han dejado este
mundo, a mi tía Ele, y a mi tía Rosi, una parte de vosotras siempre permanecerá
en mi corazón.

Pero, sobre todo, tengo que darle las gracias a Fran, gracias por ser, por estar y
por existir, desde hoy entiendo que si cruzaste o estas en mi vida es porque eres
parte de mí. Todo este trabajo y esfuerzo te lo dedico a ti.

Agradezco todo lo que he recibido y todo lo que aún está por venir.

Victoria
A Fran
“Nunca te rindas, a veces la última llave es la que abre la puerta”
Anónimo
 Bajo el cielo, cuando algo se concibe como bello,
aparece lo feo.
Cuando todos reconocen algo como bueno,
surge lo malo.

Por lo tanto, ser y no-ser se engendran uno al otro,

Tener y no tener se originan juntos,
Difícil y fácil se producen mutuamente,
Largo y corto se contrastan uno al otro,
Alto y bajo se apoyan uno al otro,
Palabra y sentido se armonizan uno al otro,
Adelante y atrás se siguen uno al otro.
Esta es la ley de la naturaleza.

Por eso el sabio va de un lado al otro

sin hacer nada, enseñando sin hablar.
Las Diez Mil Cosas se elevan y caen sin cesar.
Pero él crea, sin buscar posesión;
Trabaja, sin esperar reconocimiento;
Hecho el trabajo, pronto lo olvida.
Porque no lo reclama,
Su mérito perdura para siempre.

Lao-Tse (Tao Te Ching)

ÍNDICE
 Índice

ÍNDICE DE CONTENIDOS

1. RESUMEN / ABSTRACT 1

2. ESTRUCTURA GENERAL DE LA TESIS 7

3. INTRODUCCIÓN 11

3.1. AVANCES EN LA INMOVILIZACIÓN DE ENZIMAS EN MATERIALES

MESOPOROSOS ORDENADOS…………………………………………………………... 13

3.2. ESTRATEGIAS PARA LA INMOVILIZACIÓN DE ENZIMAS SOBRE

MATERIALES MESOPOROSOS ORDENADOS.....……………..……………………… 18

3.3. MATERIALES SILICEOS MESOPOROSOS ORDENADOS......………..………… 20

3.3.1. Desarrollo de los materiales mesoporosos silíceos…………………..……………. 20

3.3.2. Expansores de micelas: SBA-15……………………………......…........……..….… 27

3.3.3. Funcionalización orgánica de los materiales silíceos mesoestructurados……...... 30

3.3.4. Materiales mesoestructurados híbridos organosilíceos (PMO)...….........…..…… 33

3.4. INMOVILIZACIÓN DE ENZIMAS………….........………………………..……….... 36

3.4.1. Factores que afectan a la inmovilización de enzimas…………..………………… 36

3.4.2. Enzimas objeto de estudio……………………………………….……………….… 40

3.4.3. Estrategias de inmovilización en materiales mesoporosos silíceos………………. 42

3.5. REFERENCIAS BIBLIOGRÁFICAS………….........……………………....……….... 48

4. OBJETIVOS 61

5. PUBLICACIONES CIENTÍFICAS 65

CAPÍTULO 1: Mesoporous silicas with tunable morphology for the

immobilization of laccase………………….……….…………………………. 67

ABSTRACT………..…………………………………………………………………..……... 68

1. INTRODUCTION…………………………………………………………………….…... 69

i
Índice

2. EXPERIMENTAL……………………………………………………………………...…. 70

2.1. Synthesis of the supports.……………………………………………..……………… 70

2.2. Characterization techniques........………………………………..………………...… 71

2.3. Protein determination and activity assay…………………………………………… 72

2.4. Immobilization of laccase on mesoporous silicates……………………………….… 73

2.5. Immobilized laccase leaching tests………………………………………..…………. 73

2.6. SDS-PAGE electrophoresis……………………………………………..…….……… 74

3. RESULTS AND DISCUSSION………………………………………………………...… 74

3.1. Characterization of supports.......…………..……………………………………...… 74

3.2. Immobilization of laccase and specific activity.…………………………………...... 78

3.3. Leaching test of immobilized laccase ……...….…………………………………….. 82

4. CONCLUSION……………………………………………………………………………. 84

5. SUPPLEMENTARY INFORMATION………………………………………….………. 86

6. REFERENCES……………………………………………………………………………. 87

CAPÍTULO 2: Efficient retention of laccase by non-covalent

immobilization on amino-functionalized ordered mesoporous silica…....… 91

ABSTRACT………..………………………………………………………………..………... 92

1. INTRODUCTION……………………………………………………………………....… 93

2. EXPERIMENTAL……………………………………………………………………....… 96

2.1. Materials and reagents...………………...…………………………………………… 96

2.2. Synthesis of supports........……………..………………………………………...…… 97

2.3. Characterization........………………..………………………………….……………. 98

2.4. Laccase activity assay………………………………………………………………… 99

2.5. Protein determination………………………………………………………….…...… 99

ii
 Índice

2.6. Immobilization of laccase on mesoporous silicates……...……………………...…... 100

2.7. Effect of pH on laccase activity…………………………………………………..…... 101

2.8. Leaching………………………………………...……………………………………... 102

2.9. Thermal stability test……….……………...………………………………….......….. 103

2.10. Evaluation of enzyme stability in the presence of ethanol…………………...…… 103

3. RESULTS AND DISCUSSION …………………...……………………...…………..….. 104

3.1. Characterization of the supports..........…………..……………………………….…. 104

3.2. Laccase immobilization…………………………………………………………….… 109

3.3. Effect of pH on the activity of laccase….………………...………………………...... 113

3.4. Enzyme leaching……………………...……...………………………………………... 115

3.5. Thermal stability…………………...….…………………………………….……..…. 117

3.6. Stability in ethanol-containing solution………………...………………………...…. 118

4. CONCLUSION……………………………………………………………………….....… 119

5. SUPPLEMENTARY INFORMATION………………………………………………...... 121

6. REFERENCES……………………………………………………………………….....… 123

CAPÍTULO 3: Hybrid periodic mesoporous organosilica designed to

improve the properties of immobilized enzymes……………...…...……...… 131

ABSTRACT………..…………………………………………………………...……...…..…. 132

1. INTRODUCTION………………………………………………………………….……... 133

2. EXPERIMENTAL…………………………………………………………………….…... 136

2.1. Materials and reagents....………………...………………………………………...… 136

2.2. Synthesis of hybrid periodic mesoporous materials……………………………...… 137

2.3. Aminodipropyl-bridged periodic mesoporous aminosilica (PMA)…………...…… 137

2.4. Functionalization of mesoporous materials by grafting…………….……………… 138

iii
Índice

2.5. Characterization of supports……………………….……………………………...… 138

2.6. Protein determination………..…………….…………….…………………………… 139

2.7. Enzymes immobilization……..…………………………….………………………… 140

2.8. Enzyme leaching study ……..………………………….…………………………..… 142

2.9. Enzyme activity assay………..………………………….………………………….… 142

2.10. Stability tests……….………..…………………………….…………………………. 143

3. RESULTS……………………………………………………………...………………...… 144

3.1. Characterization of the supports............………………………………………….…. 144

3.2. Lipase immobilization on PMO at different pH values……..……………………… 148

3.3. Laccase immobilization on PMA…………………………………………….………. 149

3.4. Leaching and electrophoresis…………………..…………………………………….. 151

3.5. Thermal stability………………………………..…………………………………...... 153

3.6. Stability in organic solvents………………………………………………..…….…... 153

4. DISCUSSION……………………………………………………...…………………...….. 157

5. CONCLUSION……………………………………………………………………..……... 160

6. SUPPLEMENTARY INFORMATION……………………………...…………...……… 161

7. REFERENCES……………………………………………………………………...…….. 164

CAPÍTULO 4: Designing functionalized mesoporous materials for

enzyme immobilization: Location enzymes by using advanced TEM
techniques……………………………………………………………………… 171

ABSTRACT………..……………………………………………………………………….… 172

1. INTRODUCTION……………………………………………………………………....… 173

2. EXPERIMENTAL SECTION…………………………………………...……..………… 174

2.1. Synthesis of pure silica SBA-15, expanded SBA-15, and functionalized OMM,
PMO and PMA………………………………………………………………..…………....... 174

iv
 Índice

2.2. Characterization of the OMM…………………………………………….…………. 174

2.3. Enzyme immobilization…….………………………………………….………..……. 175

2.4. Reproducibility…………………..……………………………………………………. 176

2.5. Leaching and stability…..…...……..………….…………………………………...…. 176

2.6. Laccases…………………..…...…………………..……………………………...…… 177

2.7. Lipases…………………..…...……………………..……..…………………………… 178

3. RESULTS AND DISCUSSION……………………...………………...…..…………...… 178

3.1. Immobilization of lipase……………..…..………………………………………...…. 178

3.2. Immobilization of laccase.……..…..………………………………….…………...…. 180

3.3. TEM studies.……...........……………………………………………………….……... 183

4. CONCLUSION……………………………………………………………….…………… 189

5. REFERENCES……………………………………………………………………………. 190

CAPÍTULO 5: Location of laccase in ordered mesoporous materials…….. 193

ABSTRACT………..…………………………………………………………………….....… 194

1. INTRODUCTION………………………………………………………………...……..... 195

2. METHODOLOGY AND RESULTS………………...………………….....………..…… 198

3. CONCLUSION……………………………………………………………………...…..… 203

4. REFERENCES……………………………………………………………………….…… 204

6. RESUMEN DE RESULTADOS 207

6.1. ANTECEDENTES….………………………………………………………………….... 209

6.2. DISCUSIÓN DE RESULTADOS…………………………....……….……………..…. 210

6.3 REFERENCIAS BIBLIOGRÁFICAS………………………………………………….. 247

v
Índice

7. CONCLUSIONES 251

8. ANEXOS 257

I. APORTACIONES CIENTÍFICAS….…………………..……………………………….. 259

II. INDICE DE FIGURAS…………..….…………………………..……………………….. 269

III. INDICE DE TABLAS…………..….……………………………..….………………….. 277

IV. LISTA DE ABREVIATURAS Y ACRÓNIMOS..………………….…………...…….. 279

vi
1. RESUMEN /
ABSTRACT
 Resumen

RESUMEN

En este trabajo se aborda de forma multidisciplinar la obtención de biocatalizadores

mediante el diseño y síntesis de materiales mesoporosos ordenados como soportes a
medida para la inmovilización de enzimas con distintas características.

Se han obtenido novedosos materiales silíceos e híbridos organosilíceos de tipo

MMO (Materiales Mesoporosos Ordenados) diseñados a partir de las características de
dos enzimas con distintas actividades y propiedades: Lacasa de Myceliophthora
thermophila, una oxidorreductasa multicobre que cataliza la oxidación de diferentes
compuestos fenólicos, aminas y ligninas, tiene un peso molecular de 110 KDa y un
punto isoeléctrico de 4,2, y lipasa de Candida antarctica B encargada de la hidrólisis de
triglicéridos a ácidos grasos libres y glicerol, su peso molecular es 32 KDa y su
superficie presenta un dominio hidrofóbico.

En la presente tesis doctoral se han sintetizado materiales silíceos mesoporosos

ordenados avanzados, con poros de tipo canal, para la inmovilización post-síntesis y
estabilización de lacasa. Estos materiales poseen una elevada superficie específica y
estrecha distribución de tamaño de poro, que resultan de un sistema ordenado de
mesoporos de simetría definida. Los materiales puramente silíceos se utilizaron como
soportes para estudiar las características morfológicas de las partículas y los canales de
los poros más adecuadas para inmovilizar lacasa. La funcionalización con grupos
orgánicos para aumentar la afinidad química hace necesaria la obtención de poros de
mayor diámetro para permitir una buena difusión del enzima que permita alcanzar altos
valores de carga enzimática. El tamaño de poro se ha adaptado a las dimensiones
moleculares de la enzima lacasa adecuando las condiciones de síntesis y mediante la
adición de moléculas orgánicas que actúan de expansores de las micelas de surfactante
que actúan de plantilla de la estructura porosa. Para la inmovilización de lacasa, la
superficie de estos materiales se ha modificado incorporando cadenas orgánicas que
contienen grupos funcionales de tipo amino, capaces de interaccionar
electrostáticamente con la enzima en unas condiciones de pH adecuadas. Esta

3
Resumen

funcionalización orgánica se ha realizado tanto por anclaje (posterior a la síntesis de la

estructura de sílice) como por cocondensación (incorporación directa durante la síntesis).

Además, se han sintetizado novedosas organosílices periódicas mesoporosas que

incorporan dentro de la propia pared del soporte tanto el grupo etileno para la
inmovilización de lipasa, como grupos amino para la inmovilización de lacasa, y con un
diámetro de poro suficientemente grande para inmovilizar a las enzimas. Se han
obtenido biocatalizadores con altas cargas enzimáticas y elevada actividad catalítica en
los que los tamaños de poro están ajustados a las dimensiones de la enzima
(confinamiento), y en los que la afinidad química enzima-soporte es alta (interacciones
hidrofóbicas con lipasa y electrostáticas con lacasa). La conjunción de estas dos
propiedades contribuye a la retención prácticamente irreversible de las enzimas en el
interior de los entramados porosos de los soportes, dificultando o incluso eliminando el
lixiviado de las enzimas, a pesar de la naturaleza no covalente de la unión al soporte. La
inmovilización mejora la estabilidad de las enzimas lacasa y lipasa en presencia de
etanol y metanol, respectivamente.

También, se ha desarrollado una estrategia para inmovilizar la enzima en el interior

del material mesoporoso durante la propia etapa de síntesis (inmovilización in-situ). Esto
permite encapsular la enzima lacasa en sistemas con poros de tipo caja-ventana, en los
que las cajas de grandes dimensiones que contienen la enzima, están comunicadas entre
sí por ventanas estrechas, de menor diámetro que las dimensiones de la enzima. La
enzima queda retenida en el interior de las cajas, evitando cambios estructurales de las
enzimas y protegiéndola del medio externo. Para la encapsulación in-situ de la enzima
dentro de los materiales silíceos mesoporosos se ha requerido un profundo estudio del
uso de diferentes surfactantes, aditivos y condiciones de síntesis para obtener la
encapsulación de lacasa sin que el medio de síntesis produzca la desactivación de la
misma. La adecuada selección de los surfactantes y aditivos ha sido un factor clave para
obtener micelas diferentes alrededor de las moléculas de enzimas que actúan como
plantillas del sistema, y que permiten obtener materiales silíceos mesoporosos que
contienen moléculas de enzima en su interior.

4
 Resumen

Finalmente, por primera vez se ha conseguido mediante estudios avanzados de

microscopía electrónica de transmisión (STEM-HAADF-EELS con aberración
corregida) localizar directamente a las enzimas en el interior de los poros y cavidades de
los soportes.

5
Abstract

ABSTRACT
The present multidisciplinar work focuses on obtaining heterogeneous biocatalysts by
means of the design and optimization of hybrid organic-inorganic siliceous
mesostructured materials in which the pore size, the particle morphology and the
functionalization of the surface are controlled to incorporate enzyme molecules inside
those materials.

New siliceous and hybrid organo-siliceous OMM (Ordered Mesoporous Materials)

have been designed and synthesized according to the characteristics of two enzymes
with different activities and properties: Laccase from Myceliophthora thermophila, a
multicopper oxidoreductase with a molecular weight of 110 KDa and an isoelectric point
of 4.2, which catalyzes the oxidation of different phenolic compounds, amines and
lignins; and lipase from Candida antarctica B, a hydrolase which is in charge of the
hydrolysis from triglycerides to free fatty acids and glycerol, its molecular weight is 32
KDa and its surface presents a hydrophobic domain.

Biocatalysts with high enzymatic loading, high activities and catalytic efficiencies
have been obtained. The tailor-made pore diameter and high enzyme-support affinity
result in high retention of the enzyme, which prevents leaching, despite the non-covalent
bonding. The enzyme immobilization and/or encapsulation protects it from inactivation
against organic solvents and improves thermal stability.

A strategy has been designed to encapsulate the enzyme inside the mesoporous
material during the support synthesis step (in-situ immobilization). To do this, a
thorough study has been made to analyze the use of different surfactants, additives and
synthesis conditions to obtain the laccase encapsulation without deactivation of the
enzyme by the synthesis medium.

Furthermore, for the first time it has been achieved to obtain direct evidence of the
location of enzyme molecules inside the pores and cavities of the supports by means of
advanced electron microscopy techniques (aberration corrected STEM-HAADF-EELS).

The synthesis approach used produced biocatalysts that potentially fulfill the
requirements to be used industrially.

6
2. ESTRUCTURA
GENERAL
 Estructura general

ESTRUCTURA GENERAL

La presente Tesis Doctoral consta de los siguientes apartados

1. Resumen

2. Estructura General

3. Introducción

4. Objetivos

5. Publicaciones científicas. Recoge los trabajos de investigación que han dado

lugar a esta Memoria de Tesis y se ordenan en cinco capítulos. El contenido
de estos artículos se ha mantenido en el idioma en que fueron publicados y
constan de su propia sección de Resumen, Materiales y Métodos, Resultados,
Discusión, Conclusiones, Material Suplementario y Referencias
Bibliográficas.

Capítulo 1: V. Gascón, I. Díaz, C. Márquez-Alvarez, R.M. Blanco, “Mesoporous

silica with tunable morphology for the immobilization of lacase”,
Molecules, 19 (2014) 7057-7071.

Capítulo 2: V. Gascón, C. Márquez-Álvarez, R.M. Blanco, “Efficient retention of

laccase by non-covalent immobilization on amino-functionalized ordered
mesoporous silica”, Appl. Catal. A-Gen., 482 (2014) 116-126.

Capítulo 3: V. Gascón, I. Díaz, R.M. Blanco, C. Márquez-Alvarez, “Hybrid periodic

mesoporous organosilica designed to improve properties of immobilized
enzymes”, RCS Adv., 4 (2014) 34356-34368.

Capítulo 4: A. Mayoral, R. Arenal, V. Gascon, C. Marquez-Alvarez, R.M. Blanco, I.

Diaz, “Designing Functionalized Mesoporous Materials for Enzyme
Immobilization: Locating Enzymes by Using Advanced TEM
Techniques”, ChemCatChem, 5 (2013) 903-909.

Capítulo 5: A. Mayoral, V. Gascón, R.M. Blanco, C. Márquez-Alvarez, I. Díaz,

“Location of laccase in ordered mesoporous materials”, APL Materials, 2
(2014) 113304-113311.

9
 Estructura general

Los siguientes capítulos aún no han sido enviados para su publicación, por lo que
no se incluyen en la presente memoria:

[6] V. Gascón, C. Márquez-Álvarez, R.M. Blanco, “In-Situ encapsulation of laccase

in mesoporous silicas”, Paper under preparation, (2014).

[7] V. Gascón, C. Márquez-Álvarez, R.M. Blanco, “Encapsulation of β-glucosidase

into mesoporous silica materials”, Paper under preparation, (2014).

[8] V. Gascón, I. Martín de Lucía, C. Márquez-Alvarez, R.M. Blanco, “Evaluation

of laccase biocatalysts supported on mesostructured silica for oxidation of phenols”,
Paper under preparation, (2014).

6. Resumen de resultados, que muestra los antecedentes de la línea de

investigación y un resumen global de todos los trabajos presentados. Así
como las líneas de trabajo a desarrollar a corto, medio y largo plazo.

7. Conclusiones globales de la tesis.

8. Anexos, que muestra las aportaciones científicas de la doctoranda, el índice

de figuras, índice de tablas y la lista de abreviaturas y acrónimos.

10
3. INTRODUCCION
 Introducción

3. INTRODUCCIÓN

3.1. AVANCES EN LA INMOVILIZACIÓN DE ENZIMAS EN

MATERIALES MESOPOROSOS ORDENADOS

Uno de los campos más apasionantes de la ciencia y de la tecnología actuales es el

dedicado a aprovechar las posibilidades del mundo de lo más pequeño (átomos y
moléculas), para crear y construir nuevos materiales, dispositivos o instrumentos con
potenciales y novedosas aplicaciones, tal es el caso de la nanociencia, que utilizando la
rama de ingeniería de materiales permite obtener materiales de nuevo diseño en la
nanoescala. Si además, unimos otro campo de investigación altamente novedoso como es
la biotecnología que emplea moléculas biológicas como las enzimas, tendremos en
nuestras manos dos líneas de investigación altamente innovadoras, que con sólidos
conocimientos de cada una de las materias por separado podrán converger en una única
línea de investigación interdisciplinar siendo esta última una potentísima herramienta que
integra las ventajas de cada una de ellas. En el año 2005, Linqiu Cao publicó un artículo
basado en la siguiente pregunta: “Inmovilización de enzimas: ¿Ciencia o arte?” [1], la
respuesta a esta pregunta se tratará de desvelar a lo largo de la siguiente memoria de Tesis
Doctoral. La Figura 3.1 representa cómo el diseño de una metodología adecuada para la
inmovilización de enzimas puede llegar a ser una mezcla compleja entre ciencia y arte, ya
que no existe un método general ni universal aplicable cuando se inmoviliza una enzima.

13
Introducción

· Elección del
material/funcionalización
· Diseño del tamaño y la
morfología de la partícula
Elección de la enzima
· Diseño para la adaptación
del tamaño y geometría de
poro

Interacción enzima-soporte

Preservar/mejorar la Reducir el lixiviado

actividad catalítica y
estabilidad

Figura 3.1. Esquema del diseño de la inmovilización de una enzima en un nanomaterial mediante
la estrategia post-síntesis.

El continuo desarrollo de la biotecnología y sus numerosas aplicaciones ofrecen

soluciones en diversos campos en los que se demanda una química verde y sostenible,
tales como: obtención de productos farmacéuticos, síntesis de química fina, desarrollo de
biosensores, análisis proteómicos y celdas de biocombustible, entre otros. La presencia de
enzimas es una constante en la mayoría de los procesos biotecnológicos. En comparación
con los procesos químicos tradicionales, en general, el uso de enzimas es más respetuoso
con el medioambiente y es especialmente ventajoso debido a la alta eficacia, alta quimio-,
regio-, y estéreo-selectividad y a rutas sintéticas más cortas [2]. Esto además abarata los
procesos debido al bajo coste energético que se requiere. Sin embargo, en muchos casos
la aplicación industrial de las enzimas en forma libre se ve obstaculizada por su baja
estabilidad operacional, y por las dificultades para su recuperación y reutilización [1, 3].

Los avances en biotecnología han promovido el uso de enzimas inmovilizadas para

una amplia variedad de aplicaciones [4-6]. La inmovilización de enzimas en materiales
sólidos proporciona un método para superar estos inconvenientes, es decir, permite
mejorar la estabilidad de las enzimas, facilita su separación y reutilización. También

14
 Introducción

pueden ser de aplicación en reactores que operan de forma discontinua o continua

mediante un adecuado diseño del biocatalizador y del reactor [7].

Por otra parte, la inmovilización sobre soportes sólidos permite el uso de enzimas en
diferentes procesos en cascada o multienzimáticos que debido a la especificidad de acción
de las enzimas, hace que no se produzcan reacciones laterales no deseadas; y la facilidad
para separar el biocatalizador de las otras especies presentes en la reacción hace que
mediante un diseño correcto se puedan evitar interacciones negativas entre enzimas
distintas y otros catalizadores químicos implicados [8].

Sin embargo, la inmovilización de enzimas también tiene desventajas que incluyen: la

pérdida de actividad durante la inmovilización que puede deberse a una alteración de la
conformación o estructura tridimensional de la enzima, o también puede deberse a la
inmovilización de agregados enzimáticos que suelen ser inactivos; las limitaciones
difusionales de sustratos y productos, asociados a la localización de la enzima en el
interior de un soporte sólido más o menos poroso, y los costes adicionales asociados con
el proceso de inmovilización o recuperación del soporte una vez que la enzima es
inactiva.

Hasta ahora, los soportes más empleados para preparar biocatalizadores a escala
industrial han sido; el gel de sílice, materiales de vidrio de poro controlado, polímeros
molecularmente impresos y perlas de polímero [3, 9, 10], así como resinas orgánicas,
como las que compone el Novozym 435® (Lipasa de Candida antarctica inmovilizada en
una resina acrílica de intercambio aniónico). Se han empleado diferentes enzimas
inmovilizadas covalentemente sobre soportes comerciales con grupos epoxi activados
como el Eupergit C® (esferas de poliacrilamida), o Sepabeads® (esferas de
polimetacrilato) [11-14].

Estudios previos sobre inmovilización de enzimas en diferentes soportes muestran que

el confinamiento de la enzima en un espacio suficientemente limitado puede aumentar su
estabilidad y reducir la desnaturalización al evitar en gran medida el desplegamiento de la
proteína [4]. Con el fin de aumentar la eficacia de las enzimas inmovilizadas, es altamente
conveniente desarrollar nuevos materiales que posean elevadas áreas superficiales y alta
porosidad que permitan alcanzar altas cargas enzimáticas, un tamaño de poro uniforme y
diseñado a medida, estructuras de poro ordenadas y una funcionalización química
adecuada para favorecer un microambiente apropiado a la enzima [15, 16].

15
Introducción

De acuerdo con la definición de la IUPAC, los materiales porosos se pueden dividir en

tres clases: microporosos (tamaño de poro inferior a 2 nm), mesoporosos (entre 2 y 50
nm) y macroporosos (superior a 50 nm). Durante los últimos años se ha producido la
expansión del campo de los materiales mesoporosos ordenados debido al elevado interés
tecnológico que despiertan sus potenciales aplicaciones como soportes de fases activas,
catalizadores heterogéneos o adsorbentes [17]. Los materiales mesoporosos son
excelentes candidatos para la inmovilización de enzimas cuando el tamaño de los
mesoporos coincide con el tamaño de la enzima [16].

De hecho, el desarrollo de materiales mesoporosos ordenados se inició hace poco más

de dos décadas con el deseo de desarrollar aplicaciones relacionadas con el tratamiento de
moléculas de gran volumen, para superar la limitación por el relativamente reducido
tamaño de poro de las zeolitas y materiales microporosos relacionados [7].

Sin embargo, durante mucho tiempo, no existía un método eficiente para controlar el
tamaño y la disposición ordenada de mesoporos. Los primeros materiales mesoporosos se
sintetizaron mediante el método sol-gel que permitía obtener materiales con grandes áreas
superficiales (~ 900 m2/g) y tamaño de poro entre 2 y 4 nm, pero con distribuciones de
tamaño de poro muy amplias. A estos materiales se los denominó FSM (Folded Sheet
Mesoporous Materials) [18].

En 1992, investigadores de la Mobil Research and Development Corporation

publicaron la síntesis de un grupo de materiales mesoestructurados silíceos sintetizados
con surfactantes (agregados de moléculas anfifílicas que actúan como agentes directores
de la estructura). Estos materiales con una matriz porosa regular uniforme constituyen la
familia M41S [19, 20]. Se caracterizan por poseer distribuciones de tamaño de poro
estrechas, entre 1,5 nm y 4 nm, y elevadas superficies específicas. Los principales grupos
de esta familia son el MCM-48, el MCM-41, y el MCM-50.

Desde entonces, se han realizado numerosas síntesis de materiales mesoporosos,

óxidos simples o mixtos cuya característica común es el empleo de surfactantes como
agentes directores de la estructura porosa [21-24]. De este modo, se han obtenido con
éxito estructuras bidimensionales y tridimensionales, es decir, formadas por canales
unidireccionales o bien por sistemas de cavidades interconectadas, dando lugar a
numerosos materiales mesoporosos diferentes. A diferencia de las zeolitas, los materiales
mesoporosos no presentan cristalinidad en las paredes de su estructura, sino que sus

16
 Introducción

paredes están formadas por sílice amorfa que presentan numerosos defectos estructurales,
debido a una condensación silanol-siloxano incompleta tras la hidrólisis, lo que provoca
la presencia de grupos silanol (Si–OH) terminales que pueden servir para funcionalizar su
superficie con grupos orgánicos. No obstante, estos sólidos sí presentan cierto
ordenamiento mesoscópico debido al ordenamiento de sus poros.

En la Tabla 3.1 se recogen algunos de los principales materiales mesoporosos silíceos

con estructuras con ordenamiento mesoscópico [23, 25-32].

Tabla 3.1. Características de diferentes estructuras mesoporosas.

Material Estructura Mecanismo* Tipo de poro

a partir de
FSM-16 hexagonal plana kanemita canales
MCM-41 hexagonal plana S+I- canales
MCM-48 cúbica bicontinua S+I- canales
HMS desordenada S0I0 canales
MSU desordenada N0I0 canales
KIT-1 3D desordenada S+I- canales
SBA-1 cúbica S+X - I+ 2 cavidades
SBA-2 hexagonal 3D S+I- geminal cavidades/canales
SBA-3 hexagonal plana S+X - I+ canales
SBA-6 hexagonal 3D S+I- 2 cavidades
SBA-8 rómbica S+I- geminal canales
SBA-11 cúbica N0H+X - I+ canales
SBA-12 hexagonal 3D N0H+X - I+ cavidades/canales
SBA-15 hexagonal plana N0H+X - I+ canales
SBA-16 cúbica 3D N0H+X - I+ cavidades/canales
FDU-1 cúbica 3D N0H+X - I+ cavidades/canales
FDU-2 cúbica 3D N0H+X - I+ cavidades/canales
FDU-12 cúbica 3D N0H+X - I+ cavidades/canales
FDU-5 cúbica bicontinua N0H+X - I+ cavidades/canales
MCF varias S-I+ varias cavidades
PMO 2D o 3D hexagonal varios canales

* Mecanismos de formación: iónicos (S+I-; S-I+; S+X- I+; S-X+ I-), neutros (S0I0; N0I0; SI),
electrostático débil (N0H+X - I+).

17
Introducción

En una primera etapa, los trabajos se centraron principalmente en el desarrollo de

adsorbentes de cationes metálicos o moléculas orgánicas, y catalizadores heterogéneos
basados en estos materiales [33].

Hasta 1996 no se intentó por primera vez la inmovilización de enzimas de pequeño

tamaño y bajo coste en materiales mesoporosos silíceos ordenados (MMSO). El primer
trabajo publicado por Díaz & Balkus trataba sobre la inmovilización de enzimas
globulares pequeñas, como las proteasas tripsina y papaína, así como la proteína
citocromo C que contiene el grupo hemo c, en MCM-41 con un diámetro de poro de 4 nm
[34]. Estos investigadores también probaron a inmovilizar peroxidasa pero el diámetro
molecular esférico de esta enzima es de 4,6 nm y no se inmovilizaba de forma
significativa en los mesoporos de la MCM-41 (sólo 0,4 mg/gMCM-41). Hasta que no se
desarrollaron materiales silíceos mesoporosos con mayores diámetros de poro como
SBA-15 y las espumas mesocelulares silíceas (MCFs, mesostructured cellular foams), no
se pudieron inmovilizar enzimas de mayor tamaño en este tipo de materiales.

Desde entonces, la evolución de la inmovilización de enzimas sobre MMSO ha ido en

paralelo a los avances en la síntesis de nuevos materiales mesoporosos ordenados, aunque
con un retardo en el tiempo de unos cuatro años [16]. Este desarrollo ha sido promovido
en parte por la introducción de nuevas técnicas de inmovilización, por la demanda de
nuevas aplicaciones y sistemas enzimáticos complejos, y por el descubrimiento y
optimización de técnicas de caracterización avanzadas.

3.2. ESTRATEGIAS PARA LA INMOVILIZACIÓN DE

ENZIMAS SOBRE MATERIALES MESOPOROSOS
ORDENADOS

Los soportes mesoporosos poseen gran área superficial, siendo este un requisito
importante para la adsorción de enzimas. También se pueden diseñar poros con un
tamaño y una disposición estructural adecuada, lo que limita las restricciones difusionales
de la enzima. Además, se deben considerar las interacciones que se establecen entre la
enzima y el soporte; en función de la naturaleza química de los grupos superficiales que
presenten las distintas enzimas, la superficie de los materiales mesoporosos ordenados
(MMO) se puede funcionalizar con grupos que potencien las interacciones y la afinidad

18
 Introducción

hacia la enzima. Por lo tanto, existe la posibilidad de diseñar las propiedades superficiales
de los MMO para que sean afines a la enzima específica que se quiera inmovilizar [35].

Los materiales mesoporosos ordenados se pueden preparar con diferentes

composiciones químicas que incluyen sílices, organosílices [36], óxidos metálicos
(óxidos de aluminio, titanio, hierro o circonio, entre otros) [37-39], sulfuros metálicos,
como por ejemplo de germanio [40], metales [41], carbones [42], y polímeros [3, 43], los
cuales se pueden diseñar con las propiedades superficiales específicas que se requieran
[44]. Este trabajo se ha desarrollado utilizando materiales de tipo silíceo (organosílices).

Además de seleccionar un soporte que cumpla todos los requisitos necesarios para la
enzima objetivo, se debe emplear la técnica de inmovilización más adecuada. Los
métodos de inmovilización utilizados con mayor frecuencia para la incorporación de
enzimas en materiales mesoporosos son [8, 9, 15, 45, 46]: 1) Adsorción física, a través de
fuerzas de van der Waals, interacciones hidrofóbicas, enlace de hidrógeno e interacciones
iónicas; 2) unión covalente; y 3) la encapsulación.

En general, cada una de estas técnicas tiene sus propias ventajas y desventajas que se
enumeran en la Tabla 3.2, no existiendo por tanto, un método genérico para la
inmovilización de enzimas en materiales mesoporosos [6, 45]. Se debe elegir
cuidadosamente el método y las condiciones que se requieren para la adecuada
inmovilización con el fin de preservar la actividad intrínseca y la selectividad de las
enzimas, y evitar la desnaturalización e inactivación de las mismas. Un protocolo de
inmovilización eficaz sólo puede ser desarrollado a partir de una serie de experimentos
dirigidos a establecer un equilibrio entre actividad, selectividad, estabilidad y coste.
Además, el proceso de inmovilización utilizado para cada enzima es único y depende de
cada enzima específica y del soporte empleado [7, 47]. La unión a soportes mediante
adsorción o enlace químico no covalente, y la encapsulación física son las estrategias de
inmovilización empleadas en la presente Tesis Doctoral.

19
Introducción

Tabla 3.2. Métodos aplicados para la inmovilización de enzimas en MMO.

Interacción Parámetros a
Métodos Propiedades
considerar
Fuerzas de
Van der
Hidrofobicidad o
Waals,
hidrofilidad,
interacción Simple,
punto
Adsorción física hidrofóbica, interacción
isoeléctrico,
enlace de débil
cargas
hidrógeno,
superficiales
interacción
iónica
Grupos
funcionales
superficiales,
Unión
Unión covalente Unión fuerte unión
química
irreversible,
cambios
conformacionales
Condiciones de
síntesis,
presencia de
Retención
Encapsulación Unión física aditivos,
física
temperatura, pH,
empleo de
surfactantes

3.3. MATERIALES SILICEOS MESOPOROSOS ORDENADOS

3.3.1. Desarrollo de los materiales mesoporosos silíceos

Los materiales mesoporosos silíceos ordenados (MMSO) son los materiales más
estudiados y utilizados de entre los materiales mesoporosos debido a sus aplicaciones
diversas. Los precursores silíceos son baratos, y debido a la flexibilidad del ángulo de
enlace Si-O-Si permite sintetizarlos de forma más sencilla y controlada en comparación
con otros óxidos metálicos como TiO2 o Al2O3, además los materiales silíceos presentan
una alta estabilidad química y mecánica debido a que las paredes de sílice son robustas.
Desde su descubrimiento se han desarrollado numerosos materiales distintos con
diferentes aplicaciones.

La obtención de estas estructuras porosas es posible gracias al empleo de surfactantes

o tensioactivos que actúan como agentes directores de la estructura para formar los
canales de poros que caracterizan a estos materiales. Los surfactantes son moléculas

20
 Introducción

anfifílicas que constan de al menos dos partes; uno grupo polar hidrófilo y un grupo
apolar hidrófobo [30]. En medio acuoso y a una temperatura fija los surfactantes se
comportan de tal forma que al aumentar su concentración en el medio y alcanzarse la
concentración micelar crítica (CMC), sus moléculas se ordenan formando agregados
moleculares llamados micelas (Figura 3.2). La CMC es diferente para cada tipo de
surfactante e incluso en un mismo surfactante, puede variar en función de las condiciones
fisicoquímicas, como el pH, la temperatura, la presencia de co-surfactantes o sales [48].
Otra forma de inducir la formación de micelas, de especial interés en copolímeros de tres
bloques de PEO-PPO-PEO, es aumentar la temperatura. La temperatura a la que
comienzan a formarse las micelas, ahora en una concentración fija de surfactante, se
conoce como la temperatura micelar crítica (CMT) [49].

Surfactante
Reorganización de micelas

Figura 3.2. Proceso de reorganización de micelas en forma cilíndrica.

La temperatura mínima a la que tiene lugar la formación de las micelas se conoce

como temperatura de Krafft. Por debajo de ella, no se dan los fenómenos de
autoagregación del surfactante, permaneciendo este en una fase cristalina segregada. En
medio acuoso las cabezas polares de las moléculas de surfactante agrupadas en micelas se
encuentran enfrentadas al medio exterior mientras que las colas apolares (cadenas
hidrocarbonadas) se orientan hacia el interior de la micela. El tipo y la forma de micelas
formadas dependen de la naturaleza del surfactante, de su estructura molecular y de su
concentración. Es muy común clasificar los surfactantes en función de la polaridad de las
cabezas del surfactante en: aniónicos, catiónicos, geminales, zwitterionicos, neutros y no
iónicos. En este trabajo se emplearán surfactantes de tipo no iónico, con nombre
comercial Pluronic que son copolímeros anfifílicos de tres bloques (poli(óxido de
etileno)x-poli(óxido de propileno)y-poli(óxido de etileno)x; (EO)x-(PO)y-(EO)x), que se
caracterizan por poseer una cadena de polióxido de etileno (EO)x que conforma la parte
hidrófila o cabeza del surfactante, y una región hidrófoba que se trata de un polióxido de
propileno (PO)y [50].

21
Introducción

La síntesis de los MMSO se produce mediante el autoensamblaje cooperativo de las

especies precursoras de la estructura inorgánica de sílice (alcóxidos metálicos) y las
micelas, de esta manera se forma la estructura ordenada del material con una simetría
definida, y mediante la posterior eliminación del agente director se consigue el soporte
con los poros vacíos [51-53]. La diferencia principal entre los distintos MMSO se origina
por la naturaleza del surfactante empleado que condiciona el mecanismo de formación y
las posibles estructuras ordenadas finales obtenidas. Basados en esta estrategia de síntesis,
se han obtenido una gran variedad de mesoestructuras en dos dimensiones (hexagonal,
p6mm) y en tres dimensiones (hexagonal, cúbica, cúbica bicontinua, etc.) con diferentes
tamaños de poro entre 2 y 50 nm.

Debido a las grandes dimensiones de las biomoléculas, de todos los posibles MMSO
sólo unos pocos han sido utilizados hasta la actualidad como soportes de proteínas o
enzimas. Los materiales preferentemente empleados para la inmovilización de enzimas
son FSM-16, MCM-41, SBA-15 y MCF, principalmente debido a que los protocolos de
síntesis son más simples y reproducibles. Otros MMSO que también se han empleado son
MCM-48, FSM-7, HMS, SBA-16, KIT-6, FDU-5, FDU-12, HMS, MSU y PMO [16, 54-
56]. Una comparación entre las dimensiones de las enzimas inmovilizadas y el tamaño de
poro de los MMSO se muestra en la Figura 3.3 tomada del trabajo [16].

22
 Introducción

Figura 3.3. Comparación entre el tamaño de poro de diferentes materiales silíceos mesoporosos y
el tamaño de las enzima inmovilizadas [16].

A continuación se comentarán brevemente las características más importantes de

algunos de los materiales empleados como soportes de enzimas:

FSM-16 y FSM-7 pertenecen a la familia de materiales FSM (Folded Sheet

Materials) que fueron desarrollados por Kuroda y colaboradores en 1990 [18] a partir de
un precursor de polisilicato laminar denominado kanemita, en presencia de surfactantes
catiónicos (como el catión hexadecil-trimetilamonio). En 1993, Inagaki y col. [57]
propusieron un mecanismo de formación de este material mesoporoso ordenado en dos
etapas, según el cual se produce un intercambio iónico de los cationes Na+ interlaminares
por los cationes orgánicos y el plegamiento y condensación de las láminas de silicato. El
diámetro de poro de estos materiales se puede ajustar entre 2 y 4 nm mediante la
variación de la longitud de cadena del alquilo del surfactante.

23
Introducción

En 1992, científicos de Mobil Oil Research and Development Corporation sintetizaron

de forma satisfactoria materiales mesoestructurados silíceos mediante un proceso sol-gel.
Estos materiales, a los que se denominó familia M41S, se caracterizan por poseer un
sistema poroso ordenado con una estrecha distribución de diámetros, acompañados de una
elevada área superficial (> 700 m2/g) y volumen de poro [19, 20, 23]. En la estrategia de
síntesis se emplearon agregados moleculares o surfactantes iónicos también de tipo
alquiltrimetilamonio que actúan como moldes o agentes directores de la estructura para
formar los canales porosos que caracterizan a estos materiales. Las tres mesoestructuras
más importantes de la familia M41S son la MCM-41, con una estructura porosa
hexagonal p6mm formada por poros unidireccionales no conectados entre sí (Figura 3.4
a), la MCM-48, que tiene una estructura porosa cúbica tridimensional Ia3d formada por
dos sistemas de canales tridimensionales (Figura 3.4 b), y la MCM-50, con estructura
laminar p2. La estructura del sistema de poros hexagonal del material MCM-41 hace que
sea el más destacado de la familia M41S debido a su mayor estabilidad y tamaño de poro
(~ 4 nm) que hace que sea muy atractivo para aplicaciones como soportes de
catalizadores, adsorbentes, detectores, etc. [23].

p6mm Ia3d

a) MCM-41 b) MCM-48

Figura 3.4. Representación esquemática de los materiales a) MCM-41 y b) MCM-48

correspondiente a la familia M41S [27, 58].

En lugar de los surfactantes iónicos, en 1995, Tanev y Pinnavaia [59] utilizaron aminas
primarias como plantillas (principalmente docecilamina) y precursores inorgánicos
neutros para preparar la síntesis del soporte mesoporoso HMS (Hexagonal Mesoporous
Silica) que presenta una estructura desordenada conocida como “agujeros de gusano”, con

24
 Introducción

canales mesoporosos pequeños de un tamaño de unos 2 nm. Ese mismo año, Bagshaw y
col. publicaron la síntesis de MSU-1 también con estructura desordenada [60].

Desde entonces se ha realizado una intensa investigación en la síntesis de MMSO con

mayores diámetros de poro en todo el mundo.

Dentro del campo de la sílice mesoestructurada, uno de los materiales más destacados
ha sido el SBA-15 (Santa Barbara Amorphous-15) desarrollado en 1998 por el grupo de
Zhao y colaboradores [61, 62]. Este material presenta un sistema de poros altamente
ordenados en empaquetamiento hexagonal bidimensional y los mayores tamaños de
poro encontrados en los materiales silíceos mesoestructurados. Además posee unas
propiedades texturales y estructurales que son posiblemente las más deseables en este
campo.

Este material se sintetiza mediante el mecanismo electrostático débil [59, 61, 62]
utilizando como director de la estructura un surfactante no iónico, del tipo copolímero
en bloques de polietileno y polipropileno, como por ejemplo el Pluronic P123
(EO20PO70EO20), donde EO se refiere a óxido de etileno y PO a óxido de propileno. Con
el empleo de este surfactante se obtiene una estructura porosa de simetría hexagonal
altamente ordenada y bidimensional, p6mm, un tamaño de poro muy uniforme que puede
alcanzar teóricamente 30 nm, y volúmenes de poro de hasta 2,5 cm3/g. En general, se
trata de materiales con paredes de óxido de silicio muy gruesas (de hasta 3 nm) lo que les
confiere mayor estabilidad térmica e hidrotérmica que la MCM-41 [62]. Otra ventaja muy
importante en nuestro caso se debe a que las interacciones que gobiernan la formación de
estos materiales son débiles, por lo que la eliminación del surfactante mediante extracción
a reflujo con etanol o calcinación es más sencilla [22], dando lugar a una estructura libre
de surfactante y con gran volumen de poro, útil para la inmovilización de enzimas
(Figura 3.5).

Las posibles fuentes de sílice que se emplean en la preparación del material SBA-15
son tetraetilortosilicato (TEOS), tetrametilortosilicato (TMOS) o tetrapropilortosilicato
(TPOS). Los canales hexagonales mesoporosos se forman en medio ácido (pH < 1) con
diferentes ácidos inorgánicos (HCl, HBr, HI, HNO3, H2SO4 o H3PO4) de manera que las
especies silíceas se encuentran protonadas. Las temperaturas de síntesis están
comprendidas generalmente entre 35 ºC y 80 ºC [61, 62]. En la Figura 3.5 se

25
Introducción

esquematiza el procedimiento de síntesis y eliminación del surfactante del material SBA-

15.

Surfactante Precursor silíceo SBA15

CH3CH2O OCH2CH3 HCl

+ Si
40 ºC
CH3CH2O OCH2CH3
2. Tratamiento hidrotérmal
Pluronic P123 TEOS 1. Hidrólisis y
condensación
3. Eliminación del surfactante

Figura 3.5. Esquema del mecanismo de síntesis para obtener el material silíceo de tipo SBA-15,
utilizando como surfactante P123 y TEOS (tetraetilortosilicato) como fuente de sílice en medio
ácido.

En la Figura 3.6 se representa la estructura porosa de este material. Como se observa,

además de los canales de mesoporos, el SBA-15 presenta microporos que, en función de
las condiciones de síntesis, pueden llegar a conectar los canales mesoporosos entre sí de
forma aleatoria [26].

La presencia de estos microporos se atribuye al carácter hidrofílico de las cadenas de

los bloques de poli(óxido de etileno) (EO), que penetran dentro de las paredes silíceas de
la SBA-15 durante las etapas de condensación de las especias de sílice y que, al eliminar
el surfactante, generan una microporosidad adicional. Como resultado de esta doble
porosidad, los canales no presentan una superficie uniforme, sino más bien una superficie
de textura irregular [63, 64].

Figura 3.6. Esquema de canales de mesoporos y microporos que conforman el material SBA-15.

26
 Introducción

SBA-16 es una sílice mesoporosa que tiene la particularidad de presentar un sistema de

poros del tipo caja y ventana dispuestas en una simetría tridimensional cúbica centrada en
el cuerpo Im m (Figura 3.7) [61]. Las cavidades, con un diámetro ~ 5-15 nm, están
conectadas entre sí por aberturas o ventanas de menor diámetro [65]. Al igual que la
SBA-15 se sintetiza en condiciones ácidas usando un surfactante no iónico de tipo
Pluronic. La mesofase se puede crear utilizando mezclas de Pluronic P123 y Pluronic
F127 (EO106PO70EO106) o en un sistema ternario compuesto por agua, butanol y Pluronic
F127. Según lo sugerido por estudios de cristalografía de rayos X [27, 51], cada uno de
los mesoporos de la SBA-16 se conecta a ocho mesoporos vecinos. De este modo el
tamaño de poro de una entrada o ventana de mesoporos a otra es, por lo general,
significativamente menor que el tamaño de mesoporos de la caja o cavidad, siendo por
tanto el tamaño de las ventanas el factor limitante para inmovilizar una enzima en el
interior de las cavidades. Por ello, parece indicado sintetizar este tipo de soportes en
presencia de la propia enzima (síntesis in-situ), y de hecho así se aborda en el presente
trabajo, ya que la estructura cúbica además de permitir una conectividad tridimensional,
reduce el lixiviado de la enzima debido al pequeño diámetro de las ventanas.

Im3m
SBA-16

Figura 3.7. Modelización de la estructura de SBA-16 utilizando como surfactante F127 [27, 58].

3.3.2. Expansores de micelas: SBA-15

Para intentar sintetizar materiales mesoporosos con estructura hexagonal con mayores
diámetros de poro y de forma controlada se han estudiado diferentes estrategias [52]: 1)
variación de la longitud de cadena del surfactante, principalmente aumentando el tamaño
de la parte hidrofóbica del copolímero de tres bloques del surfactante [19, 61, 62, 66]; 2)

27
Introducción

modificación de la temperatura y tiempo de síntesis (de la hidrólisis, condensación y

tratamiento hidrotermal) [65, 67-69]; 3) adición de cosolventes orgánicos, co-surfactantes
[70] y/o agentes expansores de micela; 4) adición de sales al medio de síntesis [50, 69]; o
bien, 5) mezclado o sustitución del precursor silíceo TEOS por metasilicato de sodio [71].

A continuación, se comentará cómo influye la adición de agentes expansores de micela

sobre el diámetro de poro final por su importancia en la presente memoria.

Agentes expansores de micelas

Un agente expansor es un reactivo orgánico no polar que se solubiliza en el núcleo

hidrofóbico de las micelas de surfactante y las expande, aumentando así el tamaño de
poro del material final [72]. Estos compuestos se añaden en el medio de síntesis a la
disolución micelar antes de la adición del precursor de sílice, como puede observarse
esquemáticamente en la Figura 3.8:

Cabeza hidrofílica
Agente expansor:
TIPB

Cola hidrofóbica
Surfactante
P123

Figura 3.8. Interacción del agente expansor TIPB (1,3,5-triisopropilbenceno) con las micelas de
surfactante P123.

Cuando Zhao y col., [62] publicaron su primer artículo sobre SBA-15 indicaron que
utilizando 1,3,5-trimetilbenceno (TMB) como agente expansor de micelas se podría
incrementar el diámetro de poro en una longitud comprendida entre 5 y 30 nm. Un año
después, en 1999, el grupo de Schmidt-Winkel y col. [73] adicionaron al medio de
síntesis de la SBA-15 cantidades suficientemente grandes de TMB, pero la adición del
agente orgánico de hinchamiento derivó en la formación de estructuras desordenadas
mesoporosas tridimensionales ultra-grandes formadas por grandes cavidades esféricas
interconectadas por ventanas. Mediante estudios se observó que el TMB inducía una
transformación de la fase altamente ordenada de la mesoestructura tipo SBA-15 a

28
 Introducción

espumas mesocelulares silíceas que se denominaron MCFs [74]. Además, se ha

demostrado que cuando la relación másica TMB/P123 es superior a ~ 0,2, es cuando las
micelas cambian su disposición de cilindros para dar la espuma mesocelular silícea [75].
El tamaño de las cavidades esféricas variaba de 22 a 42 nm y el diámetro de las ventanas
estaba en torno a ~ 10 nm [75]. El diámetro de cavidad y tamaño de la ventana de las
MCF se pueden controlar cambiando la cantidad del agente expansor y la temperatura de
síntesis.

Una alternativa, pero no tan eficiente, es la adición de polímeros de tipo óxido de

polipropileno (PO), idéntico al que forma parte del bloque hidrofóbico del surfactante. La
adición de este agente expansor puede aumentar el diámetro de poro desde 4 nm que se
obtendrían sin la adición del PO a unos 5-6 nm que se consiguen dependiendo del peso
molecular del polímero [76].

También se han utilizado alcanos (entre 8-20 carbonos) y benceno sustituido por
grupos metilo o isopropilo como agentes expansores. En el caso de los alcanos, se
obtienen estructuras de tipo MCF utilizando octano y nonano, pero con alcanos con 10 o
más átomos de carbono se pueden obtener estructuras con ordenamiento hexagonal; en el
caso concreto de decano el tamaño de poro de la SBA-15 se aumentó a 10-12 nm [77]. De
forma general, el tamaño de poro se incrementa a medida que disminuye la longitud de la
cadena del alcano empleado en una serie de C10 a C5, siendo debido este
comportamiento a la solubilidad de dichos compuestos en las micelas de Pluronic P123.
Al utilizar benceno sustituido, el tamaño de poro podría ser teóricamente diseñado entre
7-43 nm, aunque en la mayoría de los casos sólo se obtienen MCF.

Agentes expansores de micelas a baja temperatura y en presencia de sales

Alcanos de cadena más corta, entre 6 y 9 átomos de carbono, también se pueden

utilizar como agentes expansores si las primeras etapas de síntesis se realizan a bajas
temperaturas (20-30 ºC) antes del envejecimiento hidrotérmico, y se adiciona la sal
inorgánica, fluoruro de amonio (NH4F) [78]. Mediante la combinación de los efectos
anteriores (bajas temperaturas, presencia de sales y del alcano de cadena corta) se puede
sintetizar SBA-15 con poros que varían entre 13,4 nm (adicionando nonano) a 15,7 nm
(con hexano) y con diferentes morfologías de partícula [78, 79]. Además, el diámetro de
los mesoporos se puede incrementar gradualmente alargando el tiempo del tratamiento o

29
Introducción

envejecimiento hidrotérmico a una temperatura dada o fijando temperaturas más altas

durante dicho tratamiento; de esta forma ha sido posible ajustar el tamaño de los poros
entre 9,4 y 18,2 nm cuando se utilizó hexano como agente expansor [80].

Una alternativa a los alcanos es la utilización de 1,3,5-triisopropilbenceno (TIPB) en

una síntesis similar (a bajas temperaturas y añadiendo NH4F), pero la temperatura del gel
inicial se debe disminuir más aún (entre 12 – 20 ºC) [81-84]. Una vez más, la solubilidad
del agente expansor en el núcleo micelar es la clave para la formación de poros
suficientemente grandes. El grupo de Cao y col., obtuvo SBA-15 con una porosidad
ordenada utilizando temperaturas de síntesis inferiores a 14 ºC, estas condiciones dieron
lugar a poros de hasta 26 nm (el cálculo del diámetro se obtiene utilizando una ecuación
geométrica para materiales con estructura 2D hexagonal de poros cilíndricos que están
separadas por paredes microporosas [85, 86]), que es el mayor tamaño de poro de la
SBA-15 obtenido hasta la fecha [81, 84].

La disponibilidad del material SBA-15 con grandes diámetros de poro y con sus poros
cilíndricos bien definidos y ordenados mejora significativamente las oportunidades en el
uso de este importante material para confinar enzimas voluminosas en el mismo. Para ello
se deben seleccionar agentes expansores de micelas con una capacidad de hinchamiento
moderada para lograr una ampliación del diámetro de poro apreciable, pero evitando al
mismo tiempo la formación de una mesoestructura heterogénea y / o mal definida de tipo
MCF. En la presente Tesis se ha tratado de obtener sílices mesoporosas ordenadas con un
diámetro de poro adaptado a las dimensiones moleculares de las enzimas objeto de
estudio, utilizando en la síntesis expansores de micelas (TIPB) que interaccionan con el
surfactante, bajas temperaturas de síntesis y mediante la adición de sales, como NH4F.

3.3.3. Funcionalización orgánica de los materiales silíceos mesoestructurados

Un aspecto importante de los materiales mesoporosos ordenados es la posibilidad de

incorporar grupos funcionales orgánicos [87-91] en la superficie de los canales o dentro
de la estructura silícea con el objetivo de aumentar la afinidad química de la enzima. De
esta forma se consigue aunar confinamiento de la enzima y afinidad, para mejorar las
propiedades del biocatalizador, como se explicará con extensión en capítulos posteriores.

Para llevar a cabo la funcionalización orgánica de los materiales mesoporosos existen

dos métodos diferentes [90, 92, 93]: anclaje (posterior a la síntesis de la estructura de

30
 Introducción

sílice) y cocondensación (incorporación directa durante la síntesis). En ambos métodos

se producen uniones covalentes entre la estructura inorgánica de la sílice y los grupos
funcionales (Figura 3.9), pero en el caso del anclaje el organosilano reaccionan con los
grupos silanol superficiales, mientras que en la cocondensación el grupo funcional se
incorpora directamente en las paredes de los poros.

a b

Figura 3.9. Metodología de funcionalización en materiales mesoporosos: a) Anclaje, b)

cocondensación.

Método de Anclaje

En el método de anclaje se parte del material mesoestructurado silíceo del cual se ha

eliminado previamente el surfactante por calcinación o por extracción. El procedimiento
de anclaje está basado en la reacción de los grupos silanol superficiales (Si-OH) situados
en la pared del material mesoporoso, con los grupos metoxilo (CH3-O-) o etoxilo (CH3-
CH2-O-) de las moléculas precursoras de grupos funcionales orgánicos
(organoalcoxisilano que contiene el grupo funcional, por ejemplo, aminopropilo)
mediante reacciones de silanización [88, 94]. Tras la reacción de anclaje, la estructura
silícea se suele mantener sin alterar, salvo por una ligera disminución del volumen y del
diámetro de poro debida a la presencia de nuevas especies en el interior del mismo [95,
96]. Por lo tanto, se necesita un material de poro suficientemente grande para que, una
vez anclados los grupos funcionales, mantenga un diámetro adecuado para poder
inmovilizar las enzimas.

Si se desea un alto grado de funcionalización del material es necesario mantener una

elevada concentración superficial de grupos silanol tras la eliminación del surfactante. Si
esta se lleva a cabo mediante extracción con disolventes (alcoholes en el caso de utilizar

31
Introducción

surfactantes no iónicos), se minimiza la pérdida de grupos silanol superficiales. Pero

cuando se elimina el surfactante por calcinación se produce una condensación de grupos
silanol y tiene lugar una deshidroxilación de la estructura y, por tanto, se reduce el
número de puntos de anclaje. Esta situación es reversible mediante un tratamiento
posterior de hidratación del soporte por ebullición en agua, que debe continuar con un
tratamiento de deshidratación para eliminar el exceso de agua incompatible con la
funcionalización posterior. De esta manera se consigue suministrar un número adecuado
de grupos silanol para unir el grupo orgánico funcional [87, 97].

Este método se puede emplear para aumentar la hidrofobicidad de la superficie del

soporte (incorporando cadenas de grupos fenilo o alquilo), introducir grupos funcionales
reactivos (haluros, epóxidos, olefinas, etc.) que posteriormente reaccionen con otras
moléculas de interés o bien, para funcionalizar selectivamente las superficies internas y
externas. En la Figura 3.10 se muestra la funcionalización del material silíceo amorfo
MS-3030 en el que se han incorporado grupos octilo que aportan hidrofobicidad a los
soportes mediante el método de anclaje por reacción de silanización de los grupos
silanol superficiales con el organosilano, n-octiltrietoxisilano [98, 99].

-Si-O-H SiO
-Si-O-H SiO Si-(CH2)7-CH3
Reflujo en tolueno SiO
-Si-O-H
-Si-O-H SiO
-Si-O-H SiO Si-(CH2)7-CH3
-Si-O-H SiO

Sílice mesoporosa
MS-3030 n-octiltrietoxisilano MS-3030-octilo

Figura 3.10. Funcionalización del material silíceo amorfo comercial MS-3030 mediante anclaje.
El grupo orgánico incorporado es el n-octilo.

Método de cocondensación

Este método, también denominado síntesis directa o de un solo paso, se basa en la

condensación conjunta de las especies silíceas tipo tetraalcoxisilano (fuente precursora
inorgánica) y los correspondientes precursores tipo organoalcoxisilano (incorpora el
grupo orgánico) que contienen al menos un enlace Si-C, en presencia del surfactante [88,

32
 Introducción

92, 94]. El material mesoestructurado se obtiene ya funcionalizado tras la extracción del

surfactante (Fig. 3.11). Debido a la inestabilidad térmica de las moléculas orgánicas
incorporadas, no se puede eliminar el surfactante mediante calcinación.

La principal ventaja de este método frente al anclaje es que no se necesitan varias

etapas; además la distribución de grupos orgánicos incorporados es más homogénea a lo
largo de la superficie del material mesoporoso y se produce una menor disminución de la
porosidad. Desgraciadamente, no todos los tipos de funcionalidades orgánicas pueden ser
incorporadas de esta forma, ya que las condiciones de síntesis pueden no ser las
adecuadas, además, el diámetro de poro es difícilmente controlable y la mesoestructura
final no es tan ordenada, ya que el grado de ordenamiento mesoscópico disminuye al
aumentar la concentración del organosilano en la mezcla de síntesis [88].

TEOS

C2H5O OC2H5
Si
Micelas de C2H5O R Extracción del
surfactante surfactante
Organosilano

Figura 3.11. Funcionalización del material silíceo SBA-15 mediante cocondensación.

3.3.4. Materiales mesoestructurados híbridos organosilíceos (PMO)

En 1999 tres grupos de investigación descubrieron de forma simultánea e

independiente una nueva clase de materiales mesoestructurados silíceos que incorporan
especies orgánicas de diferente naturaleza dentro de las pareces silíceas denominados
PMOs (Periodic Mesoporous Organosilicas) [100-102].

La síntesis de estos materiales es similar a la de las sílices mesoporosas ordenadas

descritas en el apartado anterior, pero reemplazando el alcóxido de silicio por un
alcoxisilano del tipo (R’O)3Si-R-Si(OR’)3 (donde R es un grupo orgánico puente que está
entre dos átomos de silicio y R’ metilo o etilo), que polimeriza y condensa alrededor de

33
Introducción

las plantillas de surfactante. Los primeros PMO tenían diámetros de poro comprendidos
entre 2 y 3 nm.

Estos materiales combinan las características estructurales de la sílice mesoporosa

ordenada con la funcionalización química de los polímeros orgánicos [93, 103]. La
principal diferencia entre los PMO y los procedimientos de funcionalización anteriores es
que en los PMO el grupo orgánico está integrado en la estructura tridimensional de la
matriz de sílice a través de dos enlaces covalentes, y está distribuido de forma homogénea
en las paredes de los poros, siendo la carga incorporada de grupos orgánicos mayor,
además, se obtiene un sistema de poros totalmente desocupado y completamente
accesibles para inmovilizar enzimas. El procedimiento de síntesis resumido queda
esquematizado en la Figura 3.12.

El número de grupos orgánicos puente (R) empleados en el diseño de PMO ha

aumentado constantemente desde 1999 [103-106]. En las primeras investigaciones el
grupo R estaba limitado a puentes orgánicos relativamente simples, tales como alcanos y
alquenos de cadena corta (Figura 3.12).

Surfactante Alcóxido de
Pluronic P123 TEOS silicio
HCl
40 ºC

Agitación Extracción
rápida

Figura 3.12. Esquema del procedimiento de síntesis de PMO donde las unidades orgánicas R,
correspondientes al grupo funcional, se anclan dentro de las paredes del poro.

En principio puede parecer que el uso de los PMO hará posible incorporar cualquier
tipo de grupo orgánico en las paredes del material mesoporoso, en cualquier
concentración deseada, y ajustando las propiedades fisicoquímicas del material final. Sin
embargo, la realidad de la situación no es tan simple. Hay algunas limitaciones estrictas
en cuanto a los tipos de grupos orgánicos que se pueden utilizar que parecen estar

34
 Introducción

relacionados con su flexibilidad, y solubilidad. En cuanto a la flexibilidad, cuanto más

rígido sea el grupo orgánico puente, más probable es que el precursor sea capaz de auto-
ensamblar. Por ejemplo, cuando el grupo puente es o bien un grupo metileno o etileno el
auto-ensamblaje se produce y se obtienen materiales con estructuras ordenadas. Sin
embargo, si la cadena de alcanodiilo se alarga en un átomo de carbono a un grupo
propileno, el material resultante es un gel completamente amorfo. El fracaso del precursor
de propileno a auto-ensamblar se debe más a su flexibilidad que a su tamaño, ya que se
han publicado trabajos que utilizan precursores más grandes y más rígidos que pueden
auto-ensamblar. Por ejemplo, si se utiliza fenileno como grupo orgánico puente los
materiales resultantes son ordenados, a pesar de que el fenileno es más grande que el
grupo propileno [107].

Esto limita la síntesis de PMO ya que la mayoría de los precursores disponibles en el

mercado son bastante largos y flexibles, que los hace inadecuados para su uso en la
síntesis de PMO. Actualmente, se han desarrollado nuevos procedimientos sintéticos para
desarrollar precursores organosilanos que permiten la incorporación en la pared de los
PMO de una gran variedad de grupos orgánicos, que van desde compuestos aromáticos e
hidrocarburos a complejos metálicos y dendrímeros pequeños [108, 109], aunque los
procedimientos de síntesis son complejos y difíciles de reproducir. Aquí radica la
dificultad que se ha abordado con éxito en esta Tesis, y es la síntesis de un material
altamente ordenado de tipo PMA donde el grupo puente incorpora un grupo amino, y
además tiene un diámetro de poro suficientemente grande (10 nm) como para poder
retener en su interior a la enzima lacasa, como se muestra posteriormente en el Capítulo
3.

La primera inmovilización de proteínas en PMO fue en 2005 por Hudson y

colaboradores [54]. Por sus propiedades específicas, se considera que los PMO tienen alto
potencial de aplicación para la inmovilización de grandes moléculas tales como las
enzimas [110]. Sin embargo, hasta la fecha la mayoría de los PMO están limitados por su
pequeño tamaño de poro (2-6 nm) para la adsorción de moléculas grandes.

35
Introducción

3.4. INMOVILIZACIÓN DE ENZIMAS

3.4.1. Factores que afectan a la inmovilización de enzimas

 Dimensiones, forma

 Subunidades

 Estabilidad

Selección de la enzima  Punto isoeléctrico

 Aminoácidos superficiales

 Estructura cristalina

 Área superficial, tamaño de poro,

Selección del soporte poroso
volumen de poro, geometría del poro

 Grado de orden, espesor de pared

 Tamaño de partícula, morfología

 Grupos funcionales superficiales

 Cargas, punto isoeléctrico

 Estabilidad

Inmovilización de la enzima  Método de inmovilización

Adsorción  pH, Concentración del tampón

Unión covalente
 Temperatura
Encapsulación
 Velocidad de agitación

 Actividad, eficiencia catalítica

 Estabilidad
Biocatalizador
 Lixiviado

Figura 3.13. Factores a tener en cuenta a la hora de realizar una inmovilización de enzimas en
materiales porosos.

36
 Introducción

Para llevar a cabo una inmovilización de enzimas con éxito sobre soportes de sílice
porosa se deben tener en cuenta varios factores que influyen en gran medida en el
biocatalizador final. Estos incluyen las condiciones de experimentación tales como el pH,
la concentración del tampón y la temperatura de inmovilización; las propiedades
fisicoquímicas del material, su morfología, tamaño de partícula, tamaño de poro, y su
composición química; además de las propiedades de la propia enzima, como su tamaño,
la presencia de grupos polares (por ejemplo grupos amino de la lisina, arginina e histidina
y/o grupos ácidos de los residuos de aminoácidos glutámico y aspártico), así como la
presencia de regiones superficiales apolares, glicosilación proteica, etc. (Figura 3.13). A
continuación se va a explicar el efecto del tamaño de poro, el tamaño de partícula, y la
morfología en la inmovilización de enzimas:

Tamaño de poro

Existen numerosos estudios que indican que la carga y la actividad enzimática son
claramente dependientes de tamaño de poro del soporte silíceo [56, 111, 112]. Por lo
tanto, no todos los materiales de sílice porosos se pueden utilizar para la inmovilización
de enzimas. En los materiales con diámetros de poro demasiado pequeños para acomodar
moléculas de enzima éstas se adsorben sobre la superficie externa y, no van a estar
protegidas del medio externo [113]. Si por el contrario, los poros son mucho más grandes
que las moléculas de enzima, esta última puede no estar suficientemente protegida
(afectando a su actividad enzimática), ni retenida por el soporte pudiendo lixiviar de los
poros si está adsorbida físicamente [114]. En la Figura 3.14 se muestra un ejemplo del
efecto del diámetro de poro sobre la inmovilización de la enzima lacasa.

Por tanto, a la hora de diseñar un soporte se debe tener un compromiso entre el tamaño
de poro del material y el tamaño de la enzima para obtener una alta carga enzimática y
alta retención. De entre los estudios publicados sobre el tamaño de poro ideal, algunos
indican que un diámetro ligeramente superior al tamaño de la enzima es suficiente para
mejorar la estabilidad y retener a la enzima [16, 36, 112, 115-117]. Existen trabajos de los
años 90 que indicaban que en materiales silíceos amorfos, el diámetro óptimo de poro
debía ser al menos el doble que el de la enzima [118]. Sin embargo, estudios posteriores
han demostrado que esta relación óptima de tamaños con materiales ordenados es muy
inferior.

37
Introducción

Disminución del diámetro de poro

Lacasa

Figura 3.14. Efecto del diámetro de poro sobre la inmovilización de una enzima.

En bibliografía se pueden consultar algunos trabajos que estudian de forma destacada

el efecto del diámetro de poro en la inmovilización de enzimas. Por ejemplo, Ikemoto y
col. han inmovilizado peroxidasa del rábano (HRP), con unas dimensiones ~ 3,7 nm x 4,3
nm x 6,4 nm, en SBA-15 con un diámetro de poro de 7,6 nm, y han observado que tanto
la estabilidad térmica, cómo la estabilidad frente a determinados inhibidores de la enzima
(cloruro de guanidinio y urea), mejoraron significativamente en comparación con HRP
libre [119]. Recientemente, Sang y Coppens [120] utilizaron espectroscopia ATR-FTIR
con el fin de analizar la estructura secundaria de dos proteínas globulares en los poros
cilíndricos de SBA-15 con diferente tamaño, y demostraron una fuerte correlación entre
la conformación de la molécula y la geometría local de los mesoporos, de tal manera que
la proteína conserva su conformación nativa cuando se adsorbe en un poro con un tamaño
cercano a las dimensiones moleculares de la proteína.

Estructura, tamaño de partícula y morfología

Además del tamaño de los poros del soporte, la estructura de los MMSO juega un
papel importante con respecto a la carga y actividad de las enzimas inmovilizadas. Los
materiales como MCM-41, FSM-16 y SBA-15 (Figura 3.4) tienen una estructura de
poros de una dimensión, donde la difusión de la enzima hacia el interior de la partícula
puede estar limitada debido al bloqueo de los poros en la boca de los mismos.

38
 Introducción

Por el contrario, materiales como MCM-48, SBA-16 (Figura 3.7), FDU-12, y los
MCFs poseen una estructura tridimensional de poros interconectados que debería reducir
potencialmente las limitaciones difusionales, siempre y cuando la enzima sea capaz de
entrar a través de las ventanas. Por ejemplo, para el caso de una proteína pequeña,
citocromo c, Balkus y col., [34, 121] demostraron que la carga enzimática depende de la
estructura del soporte basándose en un estudio comparativo entre MCM-41, MCM-48 y
SBA-15, consiguiendo mayores cargas con el material MCM-48 (con una estructura
cúbica y tridimensional).

La morfología y el tamaño de partícula del material también tienen un efecto

importante en la inmovilización de enzimas, aunque estos parámetros a veces no se tienen
en cuenta cuando se comparan sílices diferentes. En 2003, Fan y col., [122, 123]
estudiaron la adsorción de lisozima en SBA-15 convencional y en SBA-15 con canales
más cortos y demostraron que el material con menor tamaño de partícula mejoraba la
capacidad de adsorción de la enzima, y se disminuía el tiempo para llegar al equilibrio de
adsorción. Además, en esos estudios demostraron que el número de entradas o aperturas
de los poros aumenta con la disminución del tamaño de partícula en las sílices
mesoporosas, lo que lleva a una evidente mejora de la adsorción de la enzima.

Recientemente, Gustafsson y col. [124] estudiaron la influencia del tamaño de

partícula en la inmovilización, para ello utilizaron tres tipos de soportes silíceos
mesoporosos (dos SBA-15 y una HMM (Hiroshima Mesoporous Materials)) con
diferentes tamaños de partículas (40 nm, 300 nm y 1000 nm, respectivamente), pero el
mismo diámetro de poro de 9 nm, para la inmovilización de dos lipasas de diferente
origen, observaron que aunque la carga enzimática fue similar para las muestras
utilizadas, las lipasas presentan más actividad específica cuando se inmovilizan sobre el
material con tamaños de partícula de 300 nm.

La morfología de una partícula tiene una fuerte influencia en su superficie externa y

por lo tanto en la longitud de la trayectoria de difusión. Las partículas con una forma
irregular y una superficie rugosa presentan un área superficial externa mayor que una
superficie lisa. Tanto el tamaño de partícula como la morfología son parámetros clave
para la velocidad de adsorción y la capacidad de carga de las proteínas en los materiales.
La presencia de moléculas de enzima en las zonas extremas del poro puede impedir el
acceso de nuevas moléculas, lo que produce un efecto de bloqueo. Esto se traduce en un

39
Introducción

peor aprovechamiento de la superficie interna que brindan los poros. Menores tamaños de
partícula y por tanto, menores longitudes de poro, pueden reducir este efecto de bloqueo,
debido a que se facilita la difusión. Una alta área superficial externa y tamaños de
partícula pequeños permiten a las proteínas acceder a través de diferentes entradas a los
poros pudiendo conseguir una adsorción rápida y altas cargas de proteína.

3.4.2. Enzimas objeto de estudio

El trabajo de investigación que aquí se presenta, se centra principalmente en la

inmovilización de lacasa, y también de lipasa, enzimas con diferentes propiedades y
actividades catalíticas. Esta variedad ha sido la base del desarrollo de distintas estrategias
de síntesis de materiales silíceos mesoporosos ordenados con propiedades químicas y
texturales adecuadas a cada una de ellas.

Lacasas

Las lacasas son oxidasas multicobre (EC 1.10.3.1; p-difenol:dioxígeno:óxido-

reductasa) que reducen el oxígeno molecular formando dos moléculas de agua y
simultáneamente oxidan un amplio rango de polifenoles y sustratos aromáticos mediante
la abstracción de cuatro electrones [125-128]. Las lacasas son producidas por una amplia
variedad de microorganismos, especialmente por plantas, hongos y algunas bacterias
[129], siendo las extraídas de hongos las más estudiadas [130].

Esta enzima ha sido empleada en forma inmovilizada para degradar compuestos

xenobióticos en residuos acuosos [131], en la detoxificación de compuestos fenólicos en
vinos, y en la obtención de hidrolizados de lignocelulosa antes de ser usados para la
fermentación alcohólica por Saccharomyces cerevisiae, encontrándose también la
participación de esta enzima en la decoloración de melazas y colorantes [128, 132-141].

La mayoría de las lacasas son monoméricas, con tres dominios globulares conectados
consecutivamente, contienen cuatro átomos de cobre que se distribuyen en diferentes
centros de la estructura, y tienen un importante papel en el mecanismo de la catálisis
enzimática. El centro de cobre mononuclear T1 es el sitio de oxidación de sustratos, desde
el cual los electrones son transferidos al centro trinuclear compuesto por un cobre tipo 2
(T2) y dos tipo 3 (T3), en donde tiene lugar la reducción del oxígeno molecular.

40
 Introducción

Las lacasas de hongo tienen un peso molecular de 60-110 kDa, un punto isoeléctrico
(pI) cercano a 4,0; en el caso de la lacasa de Myceliophthora thermophila (MtL) [142],
utilizada en este trabajo su peso molecular es de 110 kDa y su pI es de 4,2 [143, 144].

A partir de la secuencia de aminoácidos de una enzima y de su estructura cristalina se

puede obtener un modelo estructural completo, que mediante su análisis puede aportar
información adicional que conduce al conocimiento detallado de la estructura en sí, tales
como los aminoácidos que componen la superficie de la proteína. Es muy importante
conocer el mapeado electrostático e hidrofóbico de la superficie de la proteína para poder
dirigir las interacciones con el soporte. En el caso de MtL su estructura cristalina aún no
está resuelta, por lo que no está disponible dicha información en la base de datos de
proteínas PDB (Protein Data Bank), pero haciendo uso de las bases de datos se ha
encontrado una enzima homóloga, la lacasa de Melanocarpus albomyces (1GWO, chain
a) [145] con un 97,6 % de homología, y 73 % de identidad. En la Figura 3.15 se muestra
un modelo combinado que representa la superficie de la molécula biológica, junto con un
modelo de cintas, donde se han seleccionado en diferentes colores los aminoácidos
polares de carga negativa (aspártico y glutámico).

Figura 3.15. Estructura modelizada de lacasa de Melanocarpus albomyces. Los aminoácidos

aspártico y glutámico están resaltados en rojo y azul, respectivamente. Sus dimensiones
moleculares son 6,3 x 7,2 x 8,9 nm.

Lipasas

Las lipasas (EC. 3.1.1.3) son un tipo de enzimas hidrolíticas producidas por plantas,
animales y microorganismos. En condiciones fisiológicas, estas enzimas catalizan la
hidrólisis de triglicéridos de aceites y grasas para dar glicerol y ácidos grasos libres, mono
y diglicéridos, puesto que su función biológica es la hidrólisis de los lípidos.

41
Introducción

Las lipasas también catalizan esterificaciones (u otras reacciones de condensación

como transesterificaciones o interesterificaciones) en sistemas con reducido contenido en
agua. Esta diversidad, unido a su enantioselectividad hace que las lipasas sean muy
utilizadas como biocatalizadores no solo en la modificación de grasas y aceites sino
también en otras reacciones de síntesis química [14, 146, 147].

Las lipasas poseen un dominio hidrofóbico sobre su superficie constituido por una
serie de aminoácidos con cadenas laterales hidrofóbicas [148], que constituye una especie
de tapadera, de modo que cuando la enzima se encuentra en medio acuoso, estos grupos
interaccionan entre sí impidiendo el acceso de sustratos al centro activo. Sólo cuando en
el medio existe una interfase hidrofóbica, la tapadera se abre para permitir el acceso de
los triglicéridos al centro activo.

La lipasa que se utiliza en la presente Tesis es la procedente de Candida antárctica B

(CALB) [149]. Se trata de una variedad que carece de esta tapadera, y su centro activo es
más accesible al encontrarse más cerca de la superficie [98, 150, 151]. Aunque como
todas las lipasas posee un dominio hidrofóbico en su superficie lo que permite la
inmovilización sobre soportes con superficies hidrofóbicas [152-155]. Su peso molecular
es de unos 32 kDa, su punto isoeléctrico es de 6,0 y sus dimensiones son
aproximadamente de 3 x 4 x 5 nm (Fig. 3.16).

Figura 3.16. Estructura modelizada de la lipasa de Candida antartica B.

3.4.3. Estrategias de inmovilización en MMSO

Las estrategias de inmovilización utilizadas en la presente Tesis Doctoral han sido: 1)

incorporación de la enzima sobre un soporte ya existente (inmovilización post-síntesis), y
2) mediante la inmovilización in-situ o encapsulación en el interior de un material poroso

42
 Introducción

durante el propio proceso de síntesis. A continuación se va a mostrar las características

principales de estas estrategias.

Inmovilización post-síntesis

La presente Tesis se ha centrado en la inmovilización de enzimas mediante unión no

covalente (adsorción) (incluyendo en el término de adsorción a las interacciones iónicas,
hidrofóbicas, fuerzas de Van der Waals y puentes de hidrógeno) sobre un soporte
previamente sintetizado.

La adsorción es un proceso rápido, simple y de bajo coste para inmovilizar las enzimas
en diferentes soportes. Se realiza poniendo en contacto a la proteína y al soporte en unas
condiciones adecuadas. Por parte de la proteína las interacciones con el soporte se
producen a través de los grupos funcionales perteneciente a las cadenas laterales de los
aminoácidos superficiales. Con frecuencia también presentan mayor o menor grado de
glicosilación, por lo que poseen también carbohidratos en su superficie (sobre todo las
enzimas comerciales obtenidas por expresión en hongos o levaduras y fermentación). La
diferenciación sobre qué tipo de fuerzas de adsorción están actuando en la inmovilización
generalmente es muy difícil cuando la unión al soporte no es específica. Aun así, el
proceso es reversible, por lo que es posible eliminar a las enzimas una vez inactivadas, y
reutilizar el soporte puede ser costoso tanto en elaboración como en precio. Las fuerzas
de Van der Waals están presentes en todos los sistemas enzima-soporte, pero son
demasiado débiles para mantener fuertemente retenida la enzima. Los biocatalizadores
resultantes son propensos a la lixiviación y el efecto de estabilización es bajo. Las
interacciones hidrofóbicas se pueden establecer entre un soporte hidrófobo y la superficie
hidrófoba de determinadas enzimas (como la lipasa) y se atribuyen principalmente a un
efecto entrópico favorable asociado a las diferentes características que presentan las
moléculas de agua próximas a las superficies hidrofóbicas con respecto a aquellas que
están en el seno de la fase acuosa. La unión por enlace de hidrógeno puede llevarse a
cabo predominantemente con carbohidratos que están unidos de forma natural a la
superficie de la enzima o bien con aminoácidos polares y la superficie de determinados
soportes. Las interacciones iónicas se producen entre los residuos de aminoácidos
cargados y los grupos funcionales del soporte con carga opuesta (Figura 3.17). Además,
se deben tener en cuenta las interacciones electrostáticas que se establecen entre las
propias proteínas. De entre todas estas fuerzas las electrostáticas son las más fuertes

43
Introducción

seguidas por las interacciones hidrofóbicas, enlaces de hidrógeno y las fuerzas de Van der
Waals. Dado que en este sistema interaccionan muchos grupos funcionales, la adsorción
de la proteína no sólo ocurre a través de un solo punto de la misma, sino de varios, lo que
puede contribuir a su estabilidad.

Las condiciones suaves del proceso de adsorción son la principal ventaja de esta
técnica, así como la posibilidad de reutilización del soporte. La principal desventaja es el
carácter no permanente de las uniones, que permite el lixiviado de la proteína.

Sustrato Producto

 
 
 
 
 
Soporte de sílice

Soporte de sílice
 
 
 
 
 
 
 
 
 
Producto Sustrato

Figura 3.17. Representación de una enzima adsorbida mediante interacciones electrostáticas

sobre un soporte silíceo funcionalizado con un organosilano.

Encapsulación In-situ

La encapsulación de la enzima es un método atractivo entre las diferentes estrategias

de inmovilización que existen para mejorar la reutilización y la estabilidad de las
enzimas, ya que puede evitar cambios estructurales de las enzimas y se pueden separar o
proteger a las mismas de un entorno externo agresivo. Sin embargo, los métodos de
encapsulación actuales tienen limitaciones incluyendo la lixiviación de la enzima.

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 Introducción

El atrapamiento en sílice sol-gel es la técnica más utilizada para la encapsulación de la

enzima. Pero sin el empleo de surfactantes (o agentes directores de estructura) se forma
una red porosa en la que la enzima está atrapada al azar, existiendo limitaciones
difusionales de sustratos y productos, debido a la tortuosidad de la estructura [156, 157].

En apartados anteriores se ha destacado que mediante el empleo de surfactantes es

posible obtener materiales mesoporosos silíceos ordenados (MMSO) con alta área
superficial, diámetro de poro controlable, red porosa altamente ordenada y volumen de
poro grande. Para ello se requiere usar condiciones de pH muy ácidas, procesos de alta
temperatura y etapas de síntesis hidrotermal a altas presiones y temperatura, que
obviamente no son compatibles con la presencia “in-situ” de la mayoría de las enzimas,
ya que estas se desnaturalizan en condiciones drásticas. Por ello, el objetivo en la presente
Tesis ha sido encapsular las enzimas en MMSO en condiciones suaves y que además se
evite el lixiviado. Para ello se ha buscado obtener una estructura de los poros de tipo caja-
ventana, donde la enzima se encuentre alojada en espacios voluminosos (cajas)
conectadas entre sí por ventanas de menor diámetro que las dimensiones de la enzima. De
tal manera que la enzima esté atrapada en una estructura de "cáscara", siendo la cáscara
una capa de encapsulación de sílice robusta y estable para proteger a las enzimas, y que a
la vez sea porosa para permitir la difusión de sustratos y productos.

Actualmente existen pocos trabajos que han apostado por esta vía de inmovilización
in-situ o en un solo paso [158-161]. Los primeros estudios para alcanzar este objetivo se
realizaron en el grupo de investigación donde se ha desarrollado la presente Tesis [162].
Primeramente se partió de un material tipo SBA-16 (caja-ventana) y se realizó una
inmovilización post-síntesis con lipasa mediante interacciones electrostáticas. Como se
observa en la Figura 3.18 el tamaño de las cajas es lo suficientemente amplio para
albergar a la enzima, pero no así el diámetro de las ventanas que es muy similar a las
dimensiones de la lipasa. La carga enzimática que se podía alcanzar en un material
similar mediante inmovilización post-síntesis sólo era 5 mg/g de sílice. Esto significa que
probablemente sólo puede entrar en el interior, la enzima que está orientada de modo que
su dimensión más pequeña se enfrente a la ventana. Aunque la carga enzimática era baja,
es importante resaltar que sólo el 2 % de la enzima que había conseguido entrar lixivió
tras 2 horas, siendo un resultado muy prometedor.

45
Introducción

Lipasa de Candida antarctica B

Hidrolasa
4 nm

pI 6.0 5 nm
SBA-16

Diámetro de cavidad: 12.6 nm

Diámetro de ventana: 4.6 nm

Figura 3.18. Inmovilización de la enzima lipasa en SBA-16 mediante la estrategia post-síntesis.

Para solucionar este inconveniente y conseguir mayores cargas enzimáticas y que no

lixivie la enzima se requiere sintetizar el propio soporte caja-ventana alrededor de las
moléculas de enzima. Para lograr este objetivo se requirió de un profundo estudio sobre la
naturaleza del surfactante (de tipo Pluronic) y de las condiciones de síntesis (presencia de
sales, baja temperatura, pH moderadamente ácido). La naturaleza dual
hidrofílica/hidrofóbica de la lipasa, con marcada afinidad por las interfases hidrofóbicas,
indujo a pensar que en la fase micelar previa a la adición de la fuente de silicio se
incluiría la enzima en el interior de las micelas de surfactante, en íntimo contacto con su
núcleo hidrófobico. La posterior adición de un alcóxido de silicio daría lugar a un
material mesoporoso ordenado con un sistema de canales rellenos de surfactante, en el
interior del cual está alojada la lipasa. Finalmente, el surfactante se eliminó en
condiciones suaves para no afectar a las enzimas encapsuladas.

Después de estos brillantes resultados se trató de extrapolar el mismo protocolo para

encapsular diferentes enzimas: Peroxidasa de rábano (HRP) y tirosinasa (PPO, polifenol
oxidasa). En el caso de la HRP, la enzima quedó atrapada, pero la estructura resultante no
fue ordenada sino una espuma mesocelular (MCF). Para la PPO (polifenol oxidasa) se
obtuvo una estructura regular pero la enzima no estaba atrapada en ella [163].

46
 Introducción

Si se analizan estos resultados atendiendo a las diferentes características de las

enzimas: hidrofobicidad y punto isoeléctrico observamos que el punto isoeléctrico de la
lipasa es 6,0, por lo tanto, está cargado positivamente al pH de la síntesis (3,5). Pero sobre
todo, la hidrofobicidad de la superficie externa ayuda a las micelas a organizarse
alrededor de la molécula de enzima, colocándose la lipasa en el interior del núcleo
hidrofóbico mientras que las cabezas polares o hidrófilas quedarían orientadas hacia la
disolución acuosa externa. Como consecuencia las micelas se forman correctamente
alrededor de la enzima y el biocatalizador final es altamente ordenado con enzima
atrapada en su interior.

En el caso de la HRP su punto isoeléctrico es de 7,2, por lo que la enzima tiene

también una carga neta positiva a pH 3,5 que impulsa la formación de las micelas. Pero la
ausencia de una superficie hidrofóbica en esta enzima impide la correcta organización de
las micelas a diferencia de los que sucede con lipasa.

La enzima PPO tiene un punto isoeléctrico mucho menor, de 4,75, por lo que en el pH
de la síntesis de (3,5) la carga neta es cercana a cero. No hay ninguna fuerza que ayude a
la formación de micelas, y la enzima carece de una superficie hidrófoba. Por lo tanto, la
enzima y el agente surfactante funcionan por separado, y las micelas se forman
independientemente de la enzima. Así que en este caso se obtiene un material ordenado
que no contiene la enzima.

Uno de los retos planteados en esta tesis ha sido precisamente encapsular in situ la
lacasa (enzima de gran similitud con la polifenol oxidasa (PPO)). Para conseguirlo, se
propone una nueva estrategia, cómo se explica en el Capítulo 5.

La inmovilización de enzimas en materiales porosos diseñados a medida, tanto post-

síntesis como in-situ, es un campo relativamente joven en aplicaciones (menos de 15
años), por lo que su uso industrial a día de hoy aún no está explotado. Sin embargo, estos
materiales “a medida” ofrecen una posibilidad única para construir el soporte perfecto
para cada tipo de enzima y proceso catalítico.

47
 Introducción

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[142] R.M. Berka, I.V. Grigoriev, R. Otillar, A. Salamov, J. Grimwood, I. Reid, N. Ishmael, T.
John, C. Darmond, M.C. Moisan, B. Henrissat, P.M. Coutinho, V. Lombard, D.O. Natvig,
E. Lindquist, J. Schmutz, S. Lucas, P. Harris, J. Powlowski, A. Bellemare, D. Taylor, G.
Butler, R.P. de Vries, I.E. Allijn, J. van den Brink, S. Ushinsky, R. Storms, A.J. Powell,
I.T. Paulsen, L.D. Elbourne, S.E. Baker, J. Magnuson, S. Laboissiere, A.J. Clutterbuck,
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Rouvinen, Crystal structure of a laccase from Melanocarpus albomyces with an intact
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60
4. OBJETIVOS
Objetivos

4. OBJETIVOS

Este trabajo de investigación se enmarca en el ámbito del desarrollo de estrategias

adecuadas para optimizar la inmovilización y estabilización de enzimas de marcado
interés industrial en materiales silíceos mesoporosos. Concretamente, el objetivo
fundamental es el estudio de las características de distintos materiales sintetizados como
soportes para la inmovilización de enzimas, dado el interés en las posibilidades de
aplicación de estos biocatalizadores. El proyecto de Tesis se ha planteado desde el punto
de vista del diseño de materiales silíceos mesoporosos ordenados (MMO), y el estudio de
las interacciones de las enzimas con estos soportes de distintas estructuras y distinta
naturaleza química de sus superficies.

En esta Tesis nos planteamos la obtención de biocatalizadores mesoestructurados

basados principalmente en la enzima lacasa. No existe un método universal para obtener
catalizadores basados en enzimas inmovilizadas, ya que cada enzima posee determinadas
características químicas y estructurales, en las que se debe basar el diseño de los
materiales mesoestructurados a sintetizar para optimizar la inmovilización de la enzima.

Con esta idea, los objetivos concretos planteados han sido:

1. Síntesis de MMO con poros de tipo canal para la inmovilización post-síntesis de

lacasa.

Dadas las dimensiones de la enzima, se plantea la obtención de materiales que,

manteniendo la estructura ordenada de canales, presenten poros de gran diámetro para
permitir una buena difusión de la enzima. Este estudio debe incluir un estudio
comparativo con:

 Materiales “estándar”, es decir, en los que el diámetro de poro no se haya

aumentado.
 Materiales híbridos en los que se introducen grupos orgánicos (mediante
anclaje o cocondensación) para aumentar la afinidad química enzima-soporte

2. Síntesis de materiales híbridos PMO (organosílices mesoporosas periódicas), en

los que la función orgánica forma parte de la pared de los canales.

 Síntesis de materiales con grupos hidrofóbicos para inmovilizar lipasa

63
 Objetivos

 Síntesis de materiales con grupos amino para inmovilizar lacasa

Se podrán realizar estudios comparativos de ambos catalizadores entre sí, y de sus

propiedades específicas en comparación con los desarrollados en el punto 1.

3. Encapsulación in situ de lacasa.

Se desarrollará una estrategia de síntesis de los materiales mesoporosos en presencia

de la enzima para obtener en un solo paso el material silíceo conteniendo la enzima.
Para abordar este objetivo se propone un estudio de surfactantes y condiciones de
síntesis para obtener la encapsulación de lacasa manteniendo su actividad catalítica, y
posteriores estudios comparativos con el resto de biocatalizadores obtenidos.

4. La consecución de todos estos objetivos precisa del uso de diversas técnicas

físicoquímicas que serán aplicadas para caracterizar los biocatalizadores obtenidos de
manera completa:

Se emplearán diversas técnicas de caracterización de materiales con el objetivo de

optimizar el proceso de síntesis, para lo cual se realizará una detallada caracterización
fisicoquímica de los biocatalizadores obtenidos, principalmente mediante técnicas de
difracción de rayos X, adsorción-desorción de nitrógeno, microscopía electrónica de
barrido y de transmisión, análisis químico y análisis termogravimétrico. También
técnicas de bioquímica: como ensayos de actividad catalítica, cuantificación e
identificación de las moléculas proteicas (por Bradford y electroforesis,
respectivamente).

5. Evaluación de las propiedades catalíticas y estabilidad de los biocatalizadores.

Se ensayarán los biocatalizadores en reacciones modelo para evaluar su estabilidad

frente a disolventes orgánicos y temperatura. Además se evaluará la resistencia de las
enzimas al lixiviado, y el efecto del pH en la actividad enzimática.

64
5. PUBLICACIONES
CIENTÍFICAS
 Mesoporous silicas with tunable morphology for the immobilization of laccase

CAPÍTULO 1

Mesoporous silicas with tunable morphology for

the immobilization of laccase

Victoria Gascón, Isabel Díaz, Carlos Márquez-Álvarez and Rosa M. Blanco

Published in Molecules, 2014, vol. 19, pp. 7057‐7071.

DOI: 10.3390/molecules19067057

6.1 nm x 5.0 nm x 4.9 nm

Non-covalent immobilization of laccase

1µm 1µm

OMMs with different particle shape

67
Capítulo 1

ABSTRACT

Siliceous ordered mesoporous materials (OMM) are gaining interest as supports

for enzyme immobilization due to their uniform pore size, large surface area,
tunable pore network and the introduction of organic components to mesoporous
structure. We used SBA-15 type silica materials, which exhibit a regular 2D
hexagonal packing of cylindrical mesopores of uniform size, for non-covalent
immobilization of laccase. Synthesis conditions were adjusted in order to obtain
supports with different particle shape, where those with shorter channels had
higher loading capacity. Despite the similar isoelectric points of silica and laccase
and the close match between the size of laccase and the pore dimensions of these
SBA-15 materials, immobilization was achieved with very low leaching. Surface
modification of macro-/mesoporous amorphous silica by grafting of amine
moieties was proved to significantly increase the isoelectric point of this support
and improve the immobilization yield.

Keywords: amorphous silica; biocatalysts; enzyme immobilization;

functionalization; grafting; laccase; MS-3030; ordered mesoporous materials; SBA-
15; support.

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 Mesoporous silicas with tunable morphology for the immobilization of laccase

1. INTRODUCTION

Siliceous ordered mesoporous materials (OMMs) are gaining interest as supports

for enzyme immobilization because of their uniform and tunable pore size and large
surface area, which make them excellent hosts for the adsorption of bulky molecules
like enzymes [1-5]. OMMs are thermally, mechanically and chemically stable and
insoluble in the solution used for the immobilization process and their surface can be
grafted with different functionalities [6-10]. The immobilization of enzymes in
functionalized OMMs can increase the operational stability, durability and
importantly, the catalyst particles can be easily separated and reused without loss of
catalytic activity [11, 12].

To evaluate the suitability of a mesoporous material for enzyme immobilization

the following factors are to be considered: pore diameter, surface area, pore volume,
particle size, morphology, as well as surface modification. The effects of pore size
and enzyme-support material interactions have demonstrated to have significant roles
in the enzyme loading and activity. Surface chemical modification of the support
could further enhance interactions between the pore walls and the enzyme [12]. In
addition, the diffusion phenomena related to particle size are greatly affected by the
support morphology [4, 13].

Enzymes can be immobilized in mesoporous materials by different methods:

adsorption, covalent attachment or encapsulation [4]. Covalent attachment is not
advisable when pore size is close to enzyme dimension because of a “stopper” effect
which may result in low immobilization yield, and always requires chemical
modification of the enzyme which may result in catalytic activity decay. Adsorption
seems more suitable because both drawbacks are avoided. However the driving
forces of non-covalent immobilization are relatively weak interactions and, therefore,
a careful design of the mesoporous support is necessary.

Laccases (E.C. 1.10.3.2) are a group of multicopper enzymes of industrial interest

that catalyze the oxidation of phenolic compounds such as ortho- and para-diphenols
to their corresponding quinones with the concomitant reduction of oxygen to water
[14-18]. Their potential as biocatalysts for use in several biotechnological processes,
in nanobiotechnology applications, in food processing and in green chemistry [19-

69
Capítulo 1

26] is limited by their low operational stability, mostly due to their rapid inactivation
by different factors [12].

In this article we deal with the synthesis of SBA-15 type mesoporous silica
supports with different particle morphology offering improved diffusional properties.
SBA-15 with conventional rod type of particles, showing long channels (SBA-15-L)
is compared with hexagonal particles formed by short mesochannels (SBA-15-S).
Finally, we evaluate the incorporation of amine groups in large pore-size amorphous
silica via grafting method (MS-3030-N). The morphology of SBA-15 structure, the
effect of aminopropyl surface groups on the laccase immobilization behavior and
detailed characterization results will be discussed in order to shed some light on the
immobilization of enzymes. The laccase immobilization yields on pure silicas and
the functionalized amorphous silica, specific activity measurements as well as
enzyme leaching tests of immobilized laccase are also reported.

2. EXPERIMENTAL SECTION

2.1. Synthesis of the supports

Synthesis of SBA-15-L. Pure silica SBA-15 with elongated rod particles was
synthesized by using Pluronic P123 triblock copolymer according to the bibliography
[27]. First, Pluronic P123 (4 g) was dissolved at room temperature in 1.9 M aqueous
HCl solution (125 mL). The resulting solution was slowly stirred at 25 °C until the
solution became clear. TEOS (9.25 mL) was then added to the solution and the
resulting mixture was vigorously stirred at 40 °C for 20 h. Aging was then performed
at 110 °C under static conditions for 24 h in a closed Teflon container. Finally, the
white solid product was recovered by filtration, washed with water, air dried at room
temperature and calcined at 550 °C in air for 5 h in a furnace for removal of the
surfactant.

Synthesis of SBA-15-S. Pure silica SBA-15 with dish-shaped particles was

synthesized using Pluronic PE-10400 as structure directing agent and with
tetramethylorthosilicate (TMOS) as the silica source. This material has prepared
according to the method reported by Linton et al. [28, 29]. PE-10400 (2.5 g) was
dissolved in 1.6 M aqueous HCl solution (97.5 g). The mixture was stirred at room

70
 Mesoporous silicas with tunable morphology for the immobilization of laccase

temperature in a closed Teflon container until a homogeneous clear solution was

obtained. Then, the solution was heated at 55 °C for 1 h, after which TMOS (3.8 g) was
added and stirred vigorously at 55 °C for 24 h. Subsequently, the container was
transferred to an oven and kept at 80 °C for 24 h under static conditions. The
resultant product was filtered, washed thoroughly with dry ethanol and air-dried at
room temperature overnight. Finally, the surfactant template was removed by
calcination at 550 °C for 5 h in air.

Functionalization of MS-3030 by grafting with aminopropyl groups (MS-3030-

N). Commercial mesoporous amorphous silica material, MS-3030 manufactured by
PQ Corporation (Conshohocken, PA, USA), was functionalized with aminopropyl
groups by silanization reaction of surface silanol groups with
aminopropyltriethoxysilane (APTES) [8, 30]. Silica powder (5 g) was placed in a
round bottom flask and degassed at 80 °C under vacuum for 20 h to remove adsorbed
water [7]. This dried material was dispersed in dry toluene (100 mL), and APTES
(25 g) was added under constant stirring. The reaction mixture was refluxed under
stirring at atmospheric pressure in a nitrogen atmosphere for 24 h. After a slow
cooling of the reaction mixture, the resulting amino-functionalized material was
recovered by filtration, washed twice with dry toluene for removal of unreacted
reagent, followed by three times with acetone, and finally dried under vacuum for 24
h.

2.2. Characterization techniques

X-ray diffraction (XRD) patterns were acquired with a X’PERT diffractometer

(PANalytical, Almelo, The Netherlands) using the Cu-Kα (λ = 1.5406 Å) radiation.
The data were registered with two theta step size 0.02° and accumulation time 20 s.
Transmission electron micrographs (TEM) were obtained on a JEM-2100 electron
microscope (JEOL, Akishima, Japan) operated at 200 kV. A small amount of sample
powder was dispersed in ethanol and dropped on a holey carbon coated copper grid.
Scanning electron microscopy (SEM) images were collected with a Nova NanoSEM
230 FE-SEM microscope (FEI, Eindhoven, The Netherlands) with vCD detector. The
samples were prepared by placing material powder on double-sided graphite
adhesive tape mounted on the sample holder. Nitrogen adsorption-desorption
isotherms were measured at −196 °C on an ASAP 2420 device (Micromeritics,

71
Capítulo 1

Norcross, GA, USA). All calcined silicas before enzyme immobilization were
outgassed at 350 °C for 16 h under high vacuum prior to the measurements. The
specific surface area, SBET, was calculated from nitrogen adsorption data in the
relative pressure range from 0.04 to 0.2 using the BET (Brunauer-Emmett-Teller)
method. The total pore volume, Vp, was determined from the amount of nitrogen
adsorbed at a relative pressure of 0.97. Pore size distributions were determined from
the adsorption branch of the N2 isotherms using the BJH (Barrett-Joyner-Halenda)
model with cylindrical geometry of the pores. The BJH pore diameter, Dp BJH, is
defined as the position of the maximum of the pore size distribution. Quantitative
determination of the nitrogen content of organo-functionalized support was
performed using a CHNS-932 Elemental Analyser (LECO Corporation, St. Joseph,
MI, USA) with an AD-4 autobalance (Perkin Elmer, Shelton, CT, USA).
Thermogravimetric analyses were carried out using a TGA 7 instrument (Perkin
Elmer). Samples were heated in air atmosphere from 25 to 900 °C at a rate of 20
°C/min (see Supplementary Information SI 1).

2.3. Protein determination and activity assay

Protein content of commercial laccase extract was determined with the Bio-Rad
Protein Assay (Bio-Rad, München, Germany), based on the Bradford assay [31],
using bovine serum albumin (BSA) as protein standard. This extract was found to
contain a protein concentration of 1.3 mg/mL. Electrophoresis (lane 2 in Figure 9)
showed a unique band, so all protein was assigned to laccase.

Enzymatic activity of free and immobilized laccase was determined

spectrophotometrically by measuring the increase in absorbance at 530 nm caused by
the oxidation of syringaldazine in the presence of atmospheric oxygen to
tetramethoxyazobis(methylene) quinone [32, 33], using an 8453 UV-Vis
spectrophotometer (Agilent Technologies, Waldbronn, Germany) equipped with a
stirring device and temperature control. The reaction mixture consisted of 2200 µL
of potassium dihydrogen phosphate/disodium hydrogen phosphate buffer (pH 6.5,
100 mM), 60 µL of 1 mM syringaldazine solution dissolved in ethanol. 100 µL of
enzyme solution, suspension or supernatant, were added to the cuvette containing the
above substrate solution under stirring and the reaction was monitored continuously
for 5 min by measuring the absorption at 530 nm at 30 °C. One laccase activity unit

72
 Mesoporous silicas with tunable morphology for the immobilization of laccase

(U) is defined as the amount of enzyme required to oxidize 1 µmol of syringaldazine

per minute at 30 °C (ε530 nm = 65,000 M−1 cm−1). Each sample was measured three
times, and the average value was used.

2.4. Immobilization of laccase on mesoporous silicates

Immobilization of laccase on purely siliceous supports, namely SBA-15-L, SBA-

15-S and MS-3030, was performed by suspending the materials in enzyme solutions
at pH 3.5 (50 mM citric acid/trisodium citrate buffer). Amino-functionalized
material, MS-3030-N, was suspended in laccase solutions in 50 mM acetic
acid/sodium acetate buffer at pH 5.5.

To 10 mL enzyme solutions (containing 2.5 mg laccase), the support (50 mg) was
added and left in suspension under slow rotation at room temperature to allow
immobilization to occur by adsorption. Aliquots were withdrawn at given times, and
the enzymatic activity of the suspension and supernatant were analyzed by the
syringaldazine oxidation assay. The decrease in activity of the supernatant to a
minimum and constant value indicated the end point of the immobilization process.
At this equilibrium point, the solid was filtered off and washed with 50 mM buffer
(at the same pH as used for immobilization, pH 3.5 or 5.5). The solid samples were
first dried under vacuum and afterwards under dry nitrogen stream, and then stored at
4 °C in the refrigerator for later analysis. A mass balance was applied to calculate the
amount of enzyme immobilized on the supports.

To evaluate the enzymatic activity of laccase immobilized on mesoporous silicas,

10 mg of the respective dried biocatalysts were resuspended in 1 mL of 50 mM
buffer (pH 3.5 for biocatalysts prepared with purely siliceous supports or pH 5.5 for
those on amino-silica supports) and analyzed for remaining laccase activity by the
syringaldazine oxidation assay.

2.5. Immobilized laccase leaching tests

To measure the leaching of laccase from the supports, samples of the solid
containing the enzyme were suspended in 50 mM acetic acid/sodium acetate at pH
4.5 and the solid/liquid ratio was 1.25 mg/mL. At different times aliquots of the

73
Capítulo 1

suspensions were withdrawn and centrifuged, and the protein content of the
supernatants was measured according to Bradford assay [31].

2.6. SDS-PAGE electrophoresis

An attempt to verify the location of laccase inside the porous network of the
materials was made. Since solid samples are not suitable for electrophoresis, the
enzyme was forced to exit the pores. First, the laccase immobilized on different
materials was suspended in buffered solutions for 24 h at pH 4.5 as described above
to desorb all protein molecules adsorbed on the external surface of support particles.
The filtered solids were suspended in electrophoresis sample buffer (containing
sodium dodecyl sulfate, mercaptoethanol, bromophenol blue, Tris/HCl buffer pH 6.8
and glycerol) in the same amounts as in electrophoresis samples, and boiled for 5
min. In such denaturing conditions, the tertiary structure of the protein should be lost
and the lineal peptide chain should then be easily released from the pores. The
supernatants of these suspensions after centrifuging were withdrawn and analyzed by
SDS-PAGE electrophoresis [34].

3. RESULTS AND DISCUSSION

3.1. Characterization of supports

The SBA-15 materials were characterized by X-ray diffraction, scanning and

transmission electron microscopy to evaluate the morphology and internal structure
of the particles, and nitrogen adsorption, to obtain the textural properties of the
supports.

X-ray diffraction patterns of the SBA-15 materials synthesized with different

surfactants, conditions and silica sources corroborated the 2D p6mm mesostructure
with a unit cell parameter ao of 11.1 nm (Supplementary Information SI 2). The
desired effect of morphology modification was observed by scanning electron
microscopy. SEM images of SBA-15-L and SBA-15-S are shown in Figure 1. The
structure of SBA-15-L particles is fiber-like or elongated rod, formed by aggregates
of rods connected into ropelike macrostructures (Figure 1 A). The average particle
size is 0.75–1 µm in length and about 2 µm in width (Figure 1 B, C). SBA-15-S

74
 Mesoporous silicas with tunable morphology for the immobilization of laccase

particles exhibit hexagonal dish-shaped morphology or short rod (Figure 1 D–F).

The average particle size is 5 µm in diameter and 0.2 µm width. Moreover, the
alignment of channels with the axial direction of the rod-like particles on SBA-15-L
and SBA-15-S could be corroborated by transmission electron microscopy (TEM).

A B C

4 μm
3 μm 1 μm 500 nm

D E F

10 μm 3 μm 300 nm

Figure 1. SEM micrographs of mesoporous silicas: SBA-15-L (A–C), SBA-15-S (D–F).

Figure 2 displays representative TEM images of these materials showing the

projection in the direction perpendicular to the channels in the longitudinal axis of
sample SBA-15-L (Figure 2 A). The hexagonal symmetry of the pores, like a
honeycomb, characteristic of this kind of symmetry is also observed when the image
is taken in the direction parallel to the mesochannels and along the short axis of the
dish in sample SBA-15-S (Figure 2 B). In both cases, the images allow observing
the respective morphology, long rods in SBA-15-L and hexagonal dishes in SBA-15-
S.

XRD patterns of commercial amorphous silica, MS-3030, and amino-

functionalized sample, MS-3030-N, show disordered structure with no reflections at
low diffraction angles (data not shown). These materials were used to compare the
effect of the structure and pore diameter with OMMs. Figure 3 shows the SEM
images of MS-3030 (Figure 3 A) and MS-3030-N (Figure 3 B) revealing

75
Capítulo 1

fragmented spherical particles with diameter around 74 µm, indicating that there is
no alteration in silica morphology after amine-functionalization with
aminopropyltriethoxysilane (APTS).

A B

50 nm 50 nm

Figure 2. TEM images of calcined SBA-15-L in the direction perpendicular to

the pore axis (A) and SBA-15-S in the direction of the pore axis (B).

A B

30 μm 5 μm

Figure 3. SEM micrographs of amorphous silicas: MS-3030 (A) and MS-3030-N (B).

SBA-15-L and SBA-15-S exhibit type IV nitrogen adsorption-desorption

isotherms with hysteresis loops of H1 type associated with capillarity condensation at
high relative pressure (P/Po 0.7–0.8), which is characteristic of mesoporous materials
with hexagonal arrangement of cylindrical pore channels (Figure 4 A). Pore size
distribution curves calculated from the adsorption branch of the isotherms are shown
in the inset of Figure 4 A. The curves show very narrow pore size distribution with
7.8 and 6.4 nm mean pore size, respectively. SBA-15-L has a surface are of 609 m2/g
and pore volume of 1.0 cm3/g and SBA-15-S has 550 m2/g and 0.7 cm3/g,
respectively.

76
 Mesoporous silicas with tunable morphology for the immobilization of laccase

700
A

dV/dlog(Dp) (cm /g)

14

3
600 12
10
8

Vads (cm /g STP)

500 6
4
400 2
0
3
0 2 4 6 8 10 12 14
300 Dp (nm)

200

100 SBA-15-L
SBA-15-S
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
P/Po

B
1800 12
dV/dlog(Dp) (cm /g)

MS-3030
3

10
1600 MS-3030-N
8
1400
Vads (cm /g STP)

6
1200 4

1000 2
3

0
800 0 10 20 30 40 50 60 70 80

600 Dp (nm)
400
200
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
P/Po

Figure 4. N2 adsorption-desorption isotherms and pore size distributions: (A)

SBA-15-L and SBA-15-S; (B) MS-3030 and MS-3030-N.

The N2 isotherms of amorphous silica before and after functionalization (MS-3030

and MS-3030-N) are shown in Figure 4 B. Both materials exhibited type IV
isotherms, corresponding to mesoporous materials. The pore size distribution curves
of amorphous materials show a wider range, indicating heterogeneity in the porous
structure with average pore diameter of 30 nm. As compared to MS-3030, MS-3030-
N exhibited an expected slight decrease in BET surface area (from 296 to 236 m2/g)

77
Capítulo 1

and pore volume (from 2.5 to 2.1 cm3/g) due to grafting of aminopropyl organic
chains. The incorporation of the amine functional groups by grafting has been
confirmed by elemental analysis yielding a 1.81 mmol N per gram of SiO2 in MS-
3030-N material.

3.2. Immobilization of laccase and specific activity

The laccase used in this work was manufactured by pure culture fermentation of a
genetically modified strain of Aspergillus oryzae that contains the laccase gene
derived from Myceliophthora thermophila. Bioinformatics analysis was used to
determine the aminoacid sequence and 3D structure of this commercial laccase
because the crystal structure is not solved yet and its dimensions have not been
reported. An image of the laccase has been reconstructed and visualized using the
software package PyMOL [35]. With the use of PyMOL and repeated rotations of the
enzyme molecule, the average molecular size of laccase was found to be
approximately 6.1 nm × 5.0 nm × 4.9 nm (Figure 5 A).

A B
5 nm
6.1 nm

Figure 5. (A) Average dimensions of laccase measured in PyMOL (~6.1 nm ×

5.0 nm × 4.9 nm). (B) Surface distribution of amino acids in laccase constructed
in PyMOL (blue, basic amino acids; red, acidic amino acids; orange, polar
amino acids; white, nonpolar amino acids; pink, copper atoms).

The molecular weight estimates for the laccase obtained from SDS-PAGE gel
electrophoresis is around 80 kDa, consistent with other authors [36]. According to
the enzyme dimensions and to the modelling studies and the textural properties of the
supports, adsorption inside the pores of SBA-15 materials should be enabled. The
surface distribution of amino acids in laccase is displayed in Figure 5 B.

78
 Mesoporous silicas with tunable morphology for the immobilization of laccase

Electrostatic interactions between laccase molecules and the inner mesopore wall
were considered for enzyme immobilization. Immobilization of laccase was achieved
by suspending purely siliceous materials (SBA-15-L, SBA-15-S and MS-3030) in
laccase solution at pH 3.5. In these conditions, laccase molecules bear a positive net
charge (isoelectric point (pI) of laccase is 4.2). The silanol groups inside the
mesopores are deprotonated at pH 3.5 bearing a negatively charged surface, because
their isoelectric point is about 2.0.

Both ordered materials are similar in textural properties. However, despite the
moderately lower surface area and pore diameter of SBA-15-S, this support exhibited
moderately higher adsorption capacity compared to SBA-15-L (38.41 mg/g vs. 31.28
mg/g). The main difference comes from the different support morphology
highlighting the influence of this parameter on laccase immobilization. A schematic
illustration is proposed (Figure 6) showing the immobilization of laccase on the two
different supports. Laccase dimensions are of similar magnitude than channels pore
size, so the presence of an enzyme molecule inside may prevent the access of another
one, acting as a stopper or at least making enzyme diffusion more difficult.
Therefore, despite the smaller pore diameter of SBA-15-S, its shorter channels (of
about 170 nm) can accommodate more enzyme molecules per unit length than the
longer ones of SBA-15-L (1,000 nm length) where a part of the inner surface of the
channel is not accessible to laccase molecules. This also explains why this material
having a higher surface area does not result in a higher immobilization yield. This
behavior has also been observed by other authors. According to Fan and coworkers
[37], a SBA-15 sample with rod-like macromorphology displays faster loading
kinetics and a higher loading capacity for lysozyme than an SBA-15 sample with
fiber-like morphology. So, the morphology of OMM particles plays a significant role
in the efficiency of enzyme immobilization.

Regarding specific activity, the longer channels seem to challenge the diffusion of
substrates and products more than the shorter ones, as suggested by the lower value
in the long-channel SBA-15-L.

79
Capítulo 1

1000 nm

170 nm

Figure 6. Schematic illustration of the laccase immobilization on SBA-15-L

mesochannels (A) and SBA-15-S mesochannels (B).

The isoelectric points of the support and the enzyme are close, so the charge
densities at pH 3.5 are low. Grafting the support with amine groups shifts its pI to
higher value, so the interval becomes larger. Protonated terminal amine groups can
interact with deprotonated residues of aspartic acid and glutamic acid (pKa 4) at pH
5.5 which is an intermediate value between isoelectric points of enzyme and amino-
modified support, and it is far from these values. Therefore the respective densities of
charge should be higher than in the systems enzyme-siliceous supports at pH 3.5.
These high charge densities should drive stronger electrostatic interactions and favor
the immobilization of laccase. However, the close sizes of enzyme and pore diameter
does not enable to functionalize SBA-15 materials while leaving enough room for
enzyme diffusion, therefore a commercial siliceous material, MS-3030, with wider
pores was used for comparison. The 30 nm average pore size of this amorphous
material permits functionalization without compromising the access or diffusion of
enzyme molecules. Figure 7 and Table 1 displays the adsorption of laccase on
purely siliceous materials (SBA-15-L, SBA-15-S and MS-3030 at pH 3.5) and amine
grafted amorphous silica (MS-3030-N at pH 3.5 and pH 5.5) for initial amount of
enzyme solution of 50 mg/g support.

80
 Mesoporous silicas with tunable morphology for the immobilization of laccase

50
50

Adsorbed laccase (mg/g)

40
40

30
30

20
20

10
10

00
SBA-15-L SBA-15-S MS-3030 MS-3030-N MS-3030-N

Figure 7. Adsorption capacity of different supports. Solid bars: immobilization

at pH 3.5. Dashed bar: immobilization at pH 5.5.

Laccase was also adsorbed on purely siliceous MS-3030 at pH 3.5 with poor
results: 15.88 mg/g in the same immobilization time of 3 h. The pore tortuosity and
large particle size of this material may result in a slower diffusion of enzyme through
the pore network despite the wider average pore size of 30 nm. Immobilization on
MS-3030-N at pH 3.5 was negligible (2.32 mg/g, results not shown) because of
electrostatic repulsion between the positively charged enzyme and the protonated
amino groups of the support. However, immobilization on MS-3030-N performed at
pH 5.5 gave a 99 % yield despite the surface area is only 296 m2/g. At this pH,
enzyme and support surfaces have opposite charges and high charge densities, which
favors strong electrostatic interactions. This phenomenon indicates that amino groups
are appropriate for laccase immobilization at a suitable pH.

The activity of the biocatalysts was tested in the oxidation of syringaldazine.

Table 1 shows that the presence of the amine moieties in MS-3030-N also improves
the specific activity of the laccase. SBA-15-L and SBA-15-S display higher activity
than MS-3030, probably due to slower diffusion of substrate and product through the
tortuous porous network and the larger particle size of this latter material. In contrast,
SBA-15 materials offer an ordered structure with high pore connectivity and they are
assembled with smaller particles than amorphous silica, which should favor a shorter

81
Capítulo 1

diffusion course [38]. Other authors have also shown that materials with pore sizes
much larger than the enzyme size have an adverse effect on enzyme activity [39].

Table 1. Laccase immobilization characteristics and catalytic results for initial amount of
enzyme solution of 50 mg/g support.

Enzyme Specific
pH tc %
Support loading activity
immob.[a] (h)[b] Immob.[c]
(mg/g)[d] (U/mg)[e]

SBA-15-L 3.5 3 62.56 31.28 0.134

SBA-15-S 3.5 3 76.82 38.41 0.159

MS-3030 3.5 3 31.76 15.88 0.014

MS-3030-N 5.5 3 99.28 49.64 0.981

[a] pH of immobilization; [b] The time contact (tc) is the time required to
reach a constant activity of the supernatant towards the oxidation of
syringaldazine, indicative of maximum loading in the solid; [c] percent yield
of immobilization; [d] The enzyme loading is expressed in milligrams of
enzyme per gram of material; [e] The specific activity is expressed in units of
syringaldazine converted per milligram of enzyme inside the material.
Specific activity of soluble laccase: 5.68 U/mg.

3.3. Leaching test of immobilized laccase

The extent of leaching of laccase from the inner structure of supports in aqueous
media and the presence of laccase inside the porous network of the materials after the
leaching tests have been studied due to the non-covalent nature of the enzyme-
support binding through electrostatic interactions. Biocatalysts were incubated in
aqueous solution of low ionic strength buffer and high dilution at pH 4.5. In these
conditions, weak repulsion is established between negatively charged SBA-15-L or
SBA-15-S and laccase, and weak attraction is still maintained between the positively
charged MS-3030-N support and negatively charged laccase. In both cases low ionic
strength buffered solution is used just to preserve pH without forcing enzyme
leaching due to ionic competition anion-enzyme. These conditions are expected to
favor enzyme desorption from the siliceous materials more than from the amino-

82
 Mesoporous silicas with tunable morphology for the immobilization of laccase

modified one. Figure 8 summarizes these results, plotting the percentage of the
initial enzyme loading leached with time.

44
25
40
20
36
15
32
10
28
Leaching (%)
5
24 0
0 10 20 30 40 70 71 72
20
16
12
8
SBA-15-L
SBA-15-S
4
MS-3030-N
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

Figure 8. Leaching of enzyme from materials, expressed as percent of the initial

enzyme loading released as a function of time.

Two different kinds of patterns can be distinguished. Leaching profile in SBA-15-

L and SBA-15-S showed fast initial desorption values and a plateau suggesting that
the narrow pore size and confinement of the laccase minimize leaching of the
enzyme from the inner surfaces. The first initial slope is probably due to enzyme
molecules immobilized on external surfaces or close to the pore edges of the OMM
particles.

Despite the electrostatic attraction between the enzyme molecules and functional
groups on the surface remaining at pH 4.5, amine amorphous silica (MS-3030-N)
shows a continuous and fast release of enzyme, without reaching an equilibrium
point in the range of time studied (up to 72 h). This material with much larger pore
diameter offers no restriction to enzyme diffusion and is highly susceptible to
leaching during the operation. The confinement of the enzyme inside pores seems to
be more efficient to prevent leaching than the intensity of interaction with support.

From leaching tests, some laccase molecules seem to be adsorbed on the external
surface of SBA-15 particles. In order to check the presence of enzyme inside
particles, the biocatalysts previously incubated under the leaching conditions

83
Capítulo 1

described above were treated in denaturing conditions of SDS-PAGE and their

supernatants were assayed for electrophoresis. Linear polypeptide chain should
easily exit the pores after this treatment and be detected in the supernatants. Bands
were observed for all the samples which confirm that the enzyme is adsorbed inside
the pores of amorphous silica and both SBA-15 materials (Figure 9). Leaching
profile of MS-3030 is not displayed because laccase molecules were released from
the support during biocatalyst washing due to wide pores and weak attraction;
moreover the protein band obtained by electrophoresis is very thin (Figure 9, lane 5).

Figure 9. Electrophoresis in standard conditions. 1: Protein standard (high

range SDS-Page standard stained with coomassie G-250 stain; 2: Soluble laccase;
3: SBA-15-L; 4: SBA-15-S; 5: MS-3030; 6: MS-3030-N.

4. CONCLUSIONS

Two similar ordered mesoporous materials with well-defined morphologies and

highly uniform particle sizes synthesized using surfactant templates in acidic
solutions were tested as supports for laccase immobilization. The morphology of
SBA-15 demonstrated to play a significant role in the efficiency of enzyme
immobilization. Short SBA-15-S channels proved to be more efficient to carry higher
enzyme loading whereas restriction to enzyme diffusion in the longer SBA-15-L
channels resulted in lower enzyme loading and higher retention (or less leaching).
Also, some restriction to the diffusion of substrate or product may drive a lower
specific activity.

The relevance of the pore and particle features was also demonstrated by the
comparison with an amorphous meso-macroporous siliceous material, where
electrostatic interactions were strengthen by the presence of amine groups. Despite

84
 Mesoporous silicas with tunable morphology for the immobilization of laccase

immobilization yield was higher in this material, leaching of the enzyme had no
restriction. However, the confinement in SBA-15 channels was more efficient to
retain enzyme.

Our results on the immobilization of laccase on ordered mesoporous materials

(SBA-15-L and SBA-15-S) demonstrate the importance of matching pore diameter
and enzyme dimensions and chemical affinity (MS-3030-N). Efficient
immobilization of laccase can be improved when a detailed understanding of the
enzyme properties is combined with a tailored design of mesoporous supports. The
aim should be now to synthesize ordered mesoporous materials with larger pore
diameter and to incorporate amine groups.

85
Capítulo 1

5. SUPPLEMENTARY INFORMATION

100 0.06

TGA

Derivative weight (%)/dT

95 0.04

90 0.02
Weight (%)

DTG 0.00
85

80 -0.02
SBA-15-S
75 SBA-15-L -0.04
MS-3030-N
70 -0.06
100 200 300 400 500 600 700
T (ºC)

Figure SI 1. TGA and DTG of SBA-15-S, SBA-15-L and MS-3030-N

100 SBA-15-L
SBA-15-S
I (a.u.)

110
200

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
2 (º)

Figure SI 2. Low-angle XRD patterns of SBA-15-L and SBA-15-S supports.

86
 Mesoporous silicas with tunable morphology for the immobilization of laccase

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silicates - Nanoscopic reactors coming of age, Adv. Mater., 12 (2000) 1403-
1419.

89
 Capítulo 1

[31] M.M. Bradford, A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding, Anal. Biochem., 72 (1976) 248-254.

[32] A. Leonowicz, K. Grzywnowicz, Quantitative estimation of laccase forms in

some white-rot fungi using syringaldazine as a substrate, Enzyme Microb.
Technol., 3 (1981) 55-58.

[33] A. Sanchez-Amat, F. Solano, A pluripotent polyphenol oxidase from the

melanogenic marine Alteromonas sp shares catalytic capabilities of
tyrosinases and laccases, Biochem. Biophys. Res. Commun., 240 (1997)
787-792.

[34] U.K. Laemmli, Cleavage of structural proteins during the assembly of the
head of bacteriophage T4, Nature, 227 (1970) 680-685.

[35] The PyMOL Molecular Graphics System, http://www.pymol.org/.

[36] R.M. Berka, P. Schneider, E.J. Golightly, S.H. Brown, M. Madden, K.M.
Brown, T. Halkier, K. Mondorf, F. Xu, Characterization of the gene encoding
an extracellular laccase of Myceliophthora thermophila and analysis of the
recombinant enzyme expressed in Aspergillus oryzae, Appl. Environ.
Microbiol., 63 (1997) 3151-3157.

[37] J. Fan, J. Lei, L.M. Wang, C.Z. Yu, B. Tu, D.Y. Zhao, Rapid and high-
capacity immobilization of enzymes based on mesoporous silicas with
controlled morphologies, Chem. Commun., (2003) 2140-2141.

[38] O. Fernandez, I. Diaz, C.F. Torres, M. Tobajas, V. Tejedor, R.M. Blanco,

Hybrid composites octyl-silica-methacrylate agglomerates as enzyme
supports, Appl. Catal., A, 450 (2013) 204-210.

[39] E. Weber, D. Sirim, T. Schreiber, B. Thomas, J. Pleiss, M. Hunger, R.

Glaser, V.B. Urlacher, Immobilization of P450 BM-3 monooxygenase on
mesoporous molecular sieves with different pore diameters, J. Mol. Catal. B:
Enzym., 64 (2010) 29-37.

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 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

CAPÍTULO 2

Efficient retention of laccase by non-covalent

immobilization on amino-functionalized ordered
mesoporous silica

Victoria Gascón, Carlos Márquez-Álvarez and Rosa M. Blanco

Published in Applied Catalysis A: General, 2014, vol. 482, pp. 116‐126.

DOI: 10.1016/j.apcata.2014.05.035

> 10 nm
NH3

NH3

SiO
NH3

SiO  Si-(CH2)3-NH3
SiO
NH3

NH3
NH3

NH3

91
Capítulo 2

ABSTRACT

The present work aims to be a step forward in the synthesis of siliceous ordered
mesoporous materials (OMM) as tailor made matrices to optimize the
immobilization and stabilization of enzymes. Based on a classic non-covalent
adsorption by electrostatic interactions we have developed the syntheses of materials
especially designed for this enzyme, in order to optimize the properties of the final
biocatalyst. Siliceous materials with a hexagonal arrangement of parallel mesoporous
channels (SBA-15 type of structure) have been synthesized, whose pore diameter has
been tuned according to the molecular dimensions of laccase. The synthesis
conditions used allowed to obtain pore sizes large enough to permit laccase entrance
and diffusion through the pore channels. Diffusion of the enzyme is crucial to obtain
high immobilization yield since most of the surface area of the particles is the
internal surface of the pores. A poor diffusion would involve retention of enzyme
molecules in the pore mouths preventing new ones to access the channel and leading
to a low enzyme loading of the catalyst. A micelle swelling agent has been used to
expand the supramolecular aggregates that generate the pore architecture of SBA-15
silica. The surfaces of the supports were functionalized with amino groups aiming to
strengthen electrostatic interactions between support and enzyme at a suitable pH.
Two strategies of surface functionalization of the large-pore ordered mesoporous
silica materials were followed: 1) anchoring of an amino-functional alkoxysilane on
mesoporous silica and 2) direct co-condensation of a silicon alkoxide and an amino-
functional alkoxysilane to obtain the functionalized material in one step. The
possibility to prepare carriers where each characteristic has been separately studied
and optimized has allowed to obtain biocatalysts with optimal properties. Enzyme
loading up to 187 mg per gram of catalyst and high activities were achieved with the
amino-functionalized large-pore supports. Furthermore, immobilization improved
enzyme stability in ethanol. Strong binding forces were capable of housing and
retaining the enzyme irreversibly, fully preventing leaching in aqueous medium.
Through the careful design of the support material, the biocatalysts obtained share
the advantages of enzyme-support covalent attachment regarding absence of leaching
and stability, while avoiding drawbacks like loss of activity and enabling the reuse of
the support.

KEYWORDS. Biocatalysts; enzyme immobilization; laccase; large pore; ordered

mesoporous materials.

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 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

1. INTRODUCTION
The field of ordered mesoporous materials (OMMs) has been progressively
growing in the last two decades, and a wide variety of new different types of
materials and structures have been obtained [1]. These mesostructured materials have
rapidly acquired significant attention in the field of materials science and in many
areas of chemistry and biotechnology [2-5]. Using cationic surfactants to template
the pore architecture, mesoporous silica materials with well-ordered pore networks
were first obtained by Mobil Corporation in 1991 [6, 7]. The possibility to obtain
silica with different pore network topologies and the ability to tailor pore size
boosted the rapid growth of research activity in this field of materials science. Pore
size control in OMMs can be achieved by modifying surfactant type and chain length
or by adding a swelling agent [8]. Silica with a pore size of approximately 3 nm can
be made using cationic surfactants, with reasonably short alkyl chains [6]. By
replacing the cationic head group by a larger non-ionic group, the size of the
surfactant micelles can be increased and materials with pore sizes in the range of 5-6
nm are obtained [9]. Even larger surfactants like polyethyleneoxide-
polypropyleneoxide-polyethyleneoxide triblock copolymers can produce materials
with pore sizes of 7 nm and higher such as SBA-15 silica, which possesses a two-
dimensional hexagonal structure of cylindrical mesopores [10]. These pore diameters
are however not sufficient for the immobilization of large enzymes with high
molecular weight. On the other hand, the synthesis of mesoporous silica with large
pore size tends to lead to less ordered structures. Making use of this approach, the
size of mesopores is limited by the dimensions of the micelle templates, and well-
ordered SBA-15 materials with pore size up to 10 nm have been obtained [11]. Other
mesostructures, named foam-like (MCF) or worm-like mesoporous molecular sieves,
have also been reported showing disordered mesoporous structures with a pore size
up to 10 nm, (HMS (Dp = 2-10 nm) [12]; MSU-J (Dp = 2.5-10 nm) [13-15]).

The addition of swelling agents which are dissolved inside the hydrophobic
regions of the surfactant micelles allows to further increase the pore diameter of the
solid [9, 16]. Hydrophobic swelling agents such as substituted aromatic hydrocarbons
(1,3,5-trimethylbenzene, 1,3,5-triisopropylbenzene) or other organic compounds like
alcohols (butanol, pentanol, hexanol), aliphatic hydrocarbons (dodecane), tertiary

93
Capítulo 2

amines, poly(propylene glycol), etc., have been used to expand the pore size of SBA-
15 silica up to 12-15 nm [8, 17, 18]. However, the pore diameter of these materials
has to be primarily controlled through the selection of the initial synthesis
temperature, and further adjusted through the selection of the hydrothermal treatment
temperature and time, as well as the type and amount of the swelling agent [18].
Otherwise, an increase in the amount of the swelling agent causes the formation of a
different structure, called a mesocellular foam (foamlike), which is poorly ordered or
disordered and features spherical mesopores of large diameter (20–40 nm) [19, 20].
In most cases it results in disordered mesostructures with the loss of the 2D
hexagonal ordering [4].

Mesostructured materials offer versatile features, such as high specific surface

area, large specific pore volume, narrow pore size distribution, mesopores
interconnected by micropores, tunable chemical properties and high chemical and
mechanical stability. These unique properties have been previously reported in our
group for lipases [21-23]. The aim on this work is to go further in the synthesis of
OMM with larger pore sizes which open new possibilities for immobilization of
molecules too large to fit into the pores of standard OMM. This is the case of laccase
from Myceliophthora thermophila (MtL), an enzyme with larger dimensions than
lipase.

Laccases (benzenediol:oxygen oxidoreductase, E.C. 1.10.3.2) belong to the group

of blue multicopper oxidases, an important class of enzymes found in many
organisms, including plants, insects, fungi and bacteria [24-27]. These enzymes
catalyse the reduction of molecular oxygen to water by oxidation of various phenolic
compounds. The fact that they only require molecular oxygen for catalysis makes
them suitable for different applications [26].

In last years, laccases have been used for many different biotechnological
processes, such as pulp delignification, oxidation of organic pollutants, textile and
petrochemical industries [28], development of biosensors [29], or wine and beverage
stabilization [30-34] among other uses [33, 35-37]. Some attempts to use laccase in
wine and beer industries have been done [30, 33, 34, 38] and this seems to be an
interesting application to study. However, the use of soluble laccase is not allowed in

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 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

these industries, which highlights the relevance of immobilized enzyme with

optimized properties.

Furthermore, the industrial application of free laccases is limited since their

stability and catalytic activity are considerably affected by a variety of environmental
conditions [35]. In fact, immobilization allows easy separation from the reaction
medium by simple filtration, potential reuse of the biocatalyst and sometimes more
resistance of the enzyme to a thermal or chemical inactivation [39].

In this work, a classic enzyme immobilization method on amino-containing

surfaces is used. Particularly, the design of ordered mesoporous materials with the
exact pore size required by laccase, and the synthesis conditions of this kind of
materials also containing amino groups are studied and presented. Pore size is maybe
the most relevant parameter; it must be wide enough to enable the enzyme to enter
and diffuse along the channels, leaving room for new molecules to enter as well. In
the opposite situation, if the enzyme does not diffuse, then the molecules would be
stacked in the pore mouths, acting as a stopper and preventing new laccase molecules
to enter the pore. As a consequence, most of the inner pore surface would not be
occupied by enzyme and the immobilization yield would be low.

Immobilization of laccases on OMM have been reported in the literature with

varying degrees of success, mainly due to denaturation processes and restricted
diffusion of the substrate [40-42]. We have chosen electrostatic interactions as the
driving forces for laccase immobilization. MtL has a low isoelectric point, so we
propose in this work the functionalization of the siliceous supports with high pKa
groups (amine) [4, 43] to interact with negative charges of the enzyme [44].

Many research efforts have focused on surface functionalization of mesoporous

silica materials with organic groups (such as amine) by the direct incorporation of
these functionalities through co-condensation of siloxane and organosiloxane
precursors [45, 46] or by post-synthesis grafting of organic groups onto the surface
of the mesoporous silica [2, 47]. The presence of these groups in mesoporous
materials additionally decreases pore sizes which again makes necessary wider
channels [48, 49].

95
Capítulo 2

In the present study, we extend the use of the swelling agent 1,3,5-
triisopropylbenzene (TIPB) to synthesize large pore ordered materials bearing amine
groups. We use the principle of large pore sizes in combination with surface
functionalization of mesoporous silicas with amino groups for improving the non-
covalent immobilization of laccase.

2. EXPERIMENTAL

2.1. Materials and Reagents

Chemicals. Triblock co-polymer poly(ethylene oxide)-poly(propylene oxide)-

poly(ethylene oxide) Pluronic P123 (PEO20PPO70PEO20), from Aldrich (USA), was
used as structure directing agent. Tetraethoxysilane (TEOS) (Merck, Germany) and
3-aminopropyltriethoxysilane (APTS) were provided by TCI (Belgium). Ammonium
fluoride (NH4F) and 1,3,5-triisopropylbenzene (TIPB) were from Alfa Aesar
(Germany). 2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
(ABTS) was from Sigma (USA). Amorphous silica MS-3030 was kindly donated by
Silica PQ Corporation (USA).

The extract of soluble laccase (Suberase) from Myceliophthora thermophila

expressed in Aspergillus oryzae was kindly donated by Novozymes (Denmark).
Bovine serum albumin (BSA, Sigma-Aldrich, USA) was used as protein standard for
protein content determination by the Bradford method [50]. The reagents for
electrophoresis (SDS-PAGE) and broad molecular weight standards were from Bio-
Rad (USA).

Potassium chloride, phosphoric acid, citric acid and toluene were purchased from
Sigma-Aldrich (USA). Sodium acetate was purchased from Scharlau (Spain).
Sodium di-hydrogen phosphate 1-hydrate, hydrochloric acid, ethanol, acetic acid and
acetone were purchased from Panreac (Spain). Tri-sodium citrate dehydrate was
from Analyticals Carlo Erba (Italy). Solvents were all analytical or HPLC grade and
salts were of high purity. All materials were used as obtained without further
purification. Water was grade Milli-Q.

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 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

2.2. Synthesis of supports

2.2.1. Synthesis of large-pore SBA-15 silica

The synthesis of ordered mesoporous silica with two-dimensional hexagonal

arrangement of pore channels (SBA-15) was carried out using 1,3,5-
triisopropylbenzene as micelle expander to obtain a large-pore material. The
procedure was based on a previously reported method [8, 18], which was slightly
modified. NH4F was used to facilitate hydrolysis and condensation of the silica
source [51]. Typically, 2.4 g of triblock co-polymer Pluronic P123 and 0.027 g of
NH4F were dissolved at room temperature in 84.0 mL of 1.30 M aqueous HCl
solution, in a closed container made of Teflon, using a magnetic stirrer. The
container was then transferred to a water bath kept at a temperature of 20 ºC and the
solution gently stirred for 1 hour to allow for temperature equilibration. Then, a
mixture of 5.5 mL of TEOS and 1.2 mL of micelle expander TIPB was added. The
white gel obtained was vigorously stirred for 24 h at 20 ºC and subsequently heated
at 100 ºC in the closed container under static conditions for 2 days. The mixture was
then filtered and the solid product was washed with water and dried at room
temperature. Finally, the sample was calcined in a furnace for 5 h at 550 ºC (heating
ramp: 2 ºC/min). Complete surfactant removal was verified by thermogravimetric
analysis (TGA). This material was labelled as OES (Ordered Expanded Silica).

2.2.2. Synthesis of aminopropyl-functionalized supports by grafting

Large-pore silica (OES) and commercial mesoporous amorphous silica MS-3030

(AS) were functionalized with aminopropyl groups by reaction of surface hydroxyls
with APTS. The silica powder (1.1 g) was introduced in a round bottom flask and
degassed at 80 ºC under vacuum for 18 h to remove adsorbed water. The dried
material was dispersed in 100 mL of dry toluene, then 0.96 mL of 3-
aminopropyltriethoxysilane were added and the mixture was refluxed at atmospheric
pressure under N2 for 24 h. The suspension was then filtered, washed twice with dry
toluene and three times with acetone and finally dried at room temperature for 24 h.
The functionalized nitrogen-bearing supports obtained were designated NGOES (N
Grafted Ordered Expanded Silica) and NAS (N Amorphous Silica).

97
Capítulo 2

2.2.3. Synthesis of aminopropyl-functionalized silica SBA-15 by co-

condensation

Amine-functionalized mesoporous SBA-15 silica obtained by a one-pot synthesis

method (co-condensation) was synthesized following the procedure previously
reported to incorporate other functional groups into mesostructured silica [23, 46,
52]. The synthesis was performed with the addition of the micelle expander TIPB.

Pluronic P123 (4 g) was dissolved at room temperature in 125 mL of 1.9 M HCl

aqueous solution. After cooling to 18 ºC, 8.21 mL of TEOS and 2 mL of TIPB were
added to the solution, and the mixture kept under stirring for 45 min. Then, 0.96 mL
of APTS were slowly added. The resulting mixture was stirred at 18 ºC for 20 h and
then aged at 100 ºC under static conditions for 24 h. The solid product was recovered
by filtration and dried at room temperature for 24 h. The surfactant and TIPB were
removed from the material by solvent extraction: 1 g of solid was added to 140 mL
of ethanol and refluxed for 24 h, followed by filtration, washing several times with
ethanol and drying at room temperature for 24 h. The resultant sample is denoted as
NCOES (Nitrogen-bearing Co-condensed Ordered Expanded Silica).

2.3. Characterization

Mesoscopic order was investigated by Low-angle X-Ray Diffraction (XRD).

Patterns of the samples were obtained with a Philips X´PERT diffractometer using
Cu K radiation.

Nitrogen adsorption-desorption isotherms were measured at -196 ºC using the

Micromeritics ASAP 2020 and ASAP 2420 sorptometers to determine textural
properties. Pure silicas were pretreated at 350 ºC for 16 h and the functionalized
supports, at 120 ºC for 16 h. The total pore volume, Vp, was determined from the
amount of nitrogen adsorbed at a relative pressure of 0.97. Pore size distributions
were determined from the adsorption branches of isotherms using the Barrett-Joyner-
Halenda (BJH) model with cylindrical geometry of the pores [53]. The BJH pore
diameter, Dp BJH, is defined as the position of the maximum of the pore size
distribution.

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 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

Quantitative determination of the nitrogen content of organo-functionalized

supports was performed using a LECO CHNS-932 Elemental Analyser with a Perkin
Elmer AD-4 autobalance.

Thermogravimetric analyses were carried out using a Perkin Elmer TGA 7

instrument Samples were heated in air atmosphere from 25 to 900 ºC at a rate of 20
ºC/min.

Transmission electron micrographs (TEM) were taken using a JEOL 2100

electron microscope operating at 200 kV. The samples for TEM analysis were
prepared by suspending a small amount of solid in acetone using sonication in an
ultrasonication water bath for 5 min. A drop of this suspension was then dispersed
onto a holey carbon film on a copper grid, followed by drying at room temperature.

Scanning electron microscopy (SEM) micrographs were collected with a FE-SEM

FEI Nova Nanosem 230 microscope with vCD detector. The samples were prepared
by placing material powder on double-sided graphite adhesive tape mounted on the
sample holder.

2.4. Laccase activity assay

Laccase activity was determined spectrophotometrically by measuring the

increase in absorbance at 405 nm caused by the oxidation of ABTS [36, 54, 55] at 25
ºC, using an Agilent 8453 UV-Vis spectrophotometer equipped with a stirring device
and temperature control. The reaction mixture consisted of a 1.6 mM ABTS solution
in 100 mM acetic acid/sodium acetate buffer at pH 4.5. To 1.9 mL of this solution in
the cuvette, 50 µL of enzyme solution or suspension were added under stirring and
the reaction was monitored continuously for 30 min by measuring the absorption at
405 nm. One unit of Laccase (U) was defined as the amount of enzyme required to
oxidize 1 µmol of ABTS per minute at 25 ºC (ε405 nm = 35,000 M-1·cm-1).

2.5. Protein determination

Protein content of solutions was determined with the Bio-Rad Protein Assay (Bio-
Rad, USA), based on the Bradford assay [50], using bovine serum albumin (BSA) as

99
Capítulo 2

protein standard. The Suberase commercial extract was found to contain a protein
concentration of 3.283 mg/mL.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for

identification of the enzyme and the purity of the commercial extract [56].

Bioinformatics analysis was used to determine the aminoacid sequence and 3D

structure of Myceliophthora thermophila laccase (MtL) because the crystal structure
is not resolved yet and its dimensions have not been reported. An existing aminoacid
sequence from MtL in the NCBI Protein Database [57] (accession number AEO
58496.1) was used as a template to identify homologous sequences of the laccase in
BLASTP algorithm [58]. The protein BLAST analysis for MtL showed that it shares
high identity (73 %) and high query coverage (99 %) with Melanocarpus albomyces
laccase (PDB: 1GWO-A) [59, 60].

The 3D structure of MtL was modeled applying the alignment mode on the Swiss
model server [58, 61-64] using the three-dimensional structure of the Melanocarpus
albomyces laccase. The modeled MtL structure has been visualized using Pymol
software [65].

The molecular analysis of the whole protein using the tools ProtParam [66],
UniProt KB (G2QFD0 and G2Q560) [67, 68] and Brenda [69] showed that it has a
molecular weight between 63 and 85 kDa. The predicted isoelectric point (pI) was
found to be 4.2. These data do not differ significantly from other authors [70], who
reported a molecular weight of 80 kDa for the glycosylated protein and 73 kDa upon
deglycosilation, and a pI of 4.2.

Laccase from Melanocarpus albomyces (1GWO) has been used to perform an

estimation of the molecular dimensions of Mt laccase used in this work. These
dimensions were calculated from the composition of the peptide chain by simulation
using the program PyMOL. From these data, approximate dimensions have been
estimated as 6.3 x 7.2 x 8.9 nm.

2.6. Immobilization of laccase on mesoporous silicates

Immobilization of laccase on purely siliceous supports, namely OES and AS, was
performed by suspending the materials in enzyme solutions at pH 3.5 (50 mM citric

100
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

acid/trisodium citrate buffer). Hybrid amino-silica materials NAS, NGOES and

NCOES were suspended in laccase solutions in 50 mM acetic acid/sodium acetate
buffer at pH 5.5.

To 10 mL enzyme solutions, 50 mg of the respective mesoporous material were

added and left in suspension under mild stirring at room temperature to permit
immobilization to occur by adsorption. Aliquots were withdrawn at given times, and
the enzymatic activity of the suspension and supernatant were analyzed by the ABTS
oxidation assay. The decrease in activity of the supernatant to a minimum and
constant value indicated the end point of the immobilization process. Protein content
of the supernatant was measured to calculate the immobilization yield. At this point,
the solid was separated by vacuum filtration using a quartz fritted disk and washed
with acetate buffer (50 mM, at the same pH as used for immobilization, 3.5 or 5.5).
No protein was detected in these washings. The solid samples were then dried under
vacuum and afterwards under dry nitrogen stream, and were stored at 4 ºC for later
analysis. For the determination of immobilized enzyme activity, 10 mg of the
respective biocatalysts were resuspended in 1 mL 50 mM acetate buffer (pH 3.5 for
biocatalysts prepared with purely siliceous supports or pH 5.5 for those on amino-
silica supports) and analyzed for remaining laccase activity by the ABTS oxidation
assay.

In order to determine the maximum enzyme loading attainable for each support,
adsorption isotherms were obtained by carrying out the same immobilization
procedure described above with various enzyme to support ratios in the range from
25 to 400 mg/g. Enzyme concentration in the solutions was changed but the final
volume was always 10 mL in order to keep a constant solution to support ratio.

2.7. Effect of pH on laccase activity

The activity assays of the free and immobilized laccases were performed as a
function of pH at 25 oC, following the procedure described in section 2.4. ABTS
solutions were prepared at pH ranging between 2.5 and 6.0 in 0.05 M phosphoric
acid/sodium dihydrogenphosphate (pH 2.0) and 0.05 M citric acid/trisodium citrate
(pH 3.0, 3.5, 4.0, 4.5, 5.0, 6.0) at 25 ºC (triplicated). In order to perform enzyme
spectrophotometric assays at different pH values, the isosbestic point of oxidized

101
Capítulo 2

ABTS (ABTS*) was determined. This value was found to be 430 nm. The molar
absorption coefficient of ABTS* measured at these wavelength was ε430 nm = 20,700
M-1 cm-1, independent of the pH.

2.8. Leaching

The resistance of the enzyme to leach from the support was studied under
conditions that presumably favor the release of the protein, namely high dilution and
low ionic strength. The catalysts were incubated in 50 mM acetic acid/sodium acetate
buffer at pH 4.5 and the solid/total volume ratio was 1.25 mg of solid per mL of
buffer. Enzyme-support interactions at this pH are weak and the effect of support
structure on leaching prevention can be observed. Enzyme leaching was calculated
by monitoring the appearance of enzyme in the supernatant with the Bradford assay
[50].

Enzyme molecules immobilized on the external surface of the particles or in the

mouths of the pore channels are released more easily than those supposedly
immobilized deep inside the pores. It is interesting to check if most of the enzyme is
located inside pore channels. Since solid samples are not suitable for electrophoresis,
the enzyme was forced to exit the pores. First, the laccase immobilized on different
materials (NGOES and NCOES) was suspended in buffered solutions as described
above to desorb as much enzyme as possible, especially to eliminate all protein
molecules adsorbed on the external surface of support particles. The supernatants
containing the released protein were separated, and the filtered solids were
suspended in electrophoresis sample buffer (containing Sodium Dodecyl Sulfate,
mercaptoethanol, bromophenol blue, Tris buffer pH 6.8 and glycerol) in the same
amounts as in electrophoresis samples, and boiled for 5 min. In such denaturing
conditions including the split of disulfide bonds favored by the presence of
mercaptoethanol, the tertiary structure of the protein should be lost and the random
coil chain should then be easily released from the pores. The supernatants of these
suspensions were withdrawn and analyzed by SDS-PAGE electrophoresis. The solids
corresponding to samples that did not show bands were again boiled in
electrophoresis buffer and filtrated and then additionally suspended for one to two
hours in a NaOH solution at pH 11.0. All amino groups from support and enzyme
should be deprotonated in these alkaline solution and repulsive forces should be

102
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

driving the release of protein from pore channels. These new supernatants were also
analyzed by SDS-PAGE electrophoresis.

2.9. Thermal stability test

Thermal stability was determined by incubating free or immobilized laccases in

50 mM acetic acid/sodium acetate buffer at pH 3.5 (for biocatalysts supported on
purely siliceous materials) or pH 5.5 (for amino-silica supported biocatalysts and free
laccase) at 50 ºC for different time periods. Incubations were performed in individual
vials for each aliquot to prevent evaporation. The pH values were selected to avoid or
minimize enzyme leaching and ensure that the measured activity corresponded only
to the immobilized enzyme. Aliquots of the suspensions of the catalysts were
withdrawn, cooled and immediately assayed spectrophotometrically in the oxidation
of ABTS (λ = 405 nm).

2.10. Evaluation of enzyme stability in the presence of

ethanol

The organic medium was selected on the basis of a possible application of the
laccase biocatalysts in controlled oxidation of wine polyphenols. Therefore the
stability of the enzyme was studied by incubating free and immobilized enzyme at 25
ºC for variable time periods in conditions similar to those of wine, i.e., 10 % v/v
ethanol/water solution at pH 3.5 (50 mM citric acid/trisodium citrate). Incubations
were performed in individual vials for each aliquot, thereby preventing evaporation.
Then the remaining laccase activity of the suspensions was assayed at 25 ºC in
standard conditions with ABTS (λ = 405 nm).

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Capítulo 2

3. RESULTS AND DISCUSSION

Various mesoporous silica and amino-functionalized silica materials with pore
diameter larger than 10 nm were synthesized to host the laccase. The pore size of
these mesoporous materials was tailored by the addition of a swelling agent (1,3,5-
triisopropylbenzene) to expand the Pluronic P123 micelles as described in the
experimental section [9, 20, 71, 72]. Commercial amorphous silica (AS) was also
used as support for comparison purposes.

3.1. Characterization of the supports

Figure 1 shows the XRD patterns of samples OES, NCOES and NGOES. The
diffractogram of sample OES shows three well-resolved peaks corresponding to the
100, 110 and 200 reflections characteristic of the p6mm hexagonal symmetry, which
confirms that this silica sample possesses a highly ordered SBA-15 type of structure
[10, 17]. The diffractogram of sample NGOES indicates that it also possesses a
highly ordered 2D hexagonal structure, indicating that grafting of aminopropyl
groups on sample OES do not affect the pore ordering. This is also supported by
TEM images of sample NGOES that evidence the well-ordered hexagonal
arrangement of mesopore channels (Figure 2 a, b). In the case of the amino-
functionalized silica obtained by co-condensation (sample NCOES), the
diffractogram (Figure 1) shows an intense peak at low angle that indicates that this
sample is mesostructured. Weak shoulders can also be observed at 2 angles around
1.5, which might be assigned to the 110 and 200 reflections of the p6mm space
group, although the fact that these reflections are not well resolved suggests that pore
ordering in sample NCOES might be restricted to smaller domains compared to
samples OES and NGOES. Nevertheless, TEM images (Figure 2 c, d) show that
sample NCOES possesses an ordered arrangement of uniform pore channels
corresponding to the SBA-15 structure. In contrast to the former samples, the
diffractogram of the commercial amorphous silica sample (AS) (not shown) does not
exhibit any diffraction peak at low angle, due the lack of pore ordering in this sample
[73].

104
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

100

I (a.u.)
110
200
NCOES
OES
NGOES

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

2 (degrees)

Figure 1. Low-angle XRD patterns of ordered mesoporous silica and amino-functionalized

silica supports.

a b

105
Capítulo 2

c d

Figure 2. TEM micrographs of samples NGOES (a, b) and NCOES (c, d).

Nitrogen adsorption-desorption isotherms at -196 ºC of the supports are plotted in

Figure 3, and their corresponding pore size distributions calculated by the Barrett-
Joyner-Halenda (BJH) method are shown in the inset. The isotherms of samples
OES, NGOES and NCOES (Fig. 3 A) show a steep adsorption step at high relative
pressure values and a hysteresis loop corresponding to the capillary condensation in
large mesopores [53]. The corresponding BJH plots show narrow pore size
distributions as expected for samples having uniform pore size. On the other hand,
the isotherms of sample AS and its amino-grafted counterpart (Fig. 3 B) show that
adsorption in mesopores takes place at higher relative pressure, indicating that these
samples contain larger mesopores. Furthermore, their BJH pore size distributions are
also much broader than those of the ordered mesoporous supports. Textural
properties obtained from the experimental isotherms are collected in Table 1.
Nitrogen content of amino-functionalized samples determined by elemental analysis
is also reported in Table 1. These values are given as mmol of nitrogen per gram of
silicon dioxide, which corresponds to the residual weight determined from the
thermogravimetric analyses. The pore diameter determined for sample OES was 15.0
nm, which represents a significant increase respect to the size obtained under similar
conditions in the absence of the swelling agent (7.8 nm [74]). Taking into account
the estimated dimensions of the enzyme, it is clear that only expanded materials
could be used to ensure efficient laccase diffusion inside the pore channels. This is
especially the case if pore surfaces of the support have to be functionalized to

106
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

improve enzyme immobilization, as grafting of the alkoxysilane precursor would

additionally decrease the pore opening. Indeed, the textural data of sample NGOES
(Table 1) show that grafting of the OES material with aminopropyl groups led to a
decrease of pore size close to 4 nm, along with a small decrease in total pore volume.
The decrease of both pore size and total pore volume are consistent with the
relatively high loading of aminopropyl moieties obtained, calculated in 1.2 mmol of
amine groups per gram of silica, according to the nitrogen content determined by
chemical analysis (Table 1).
2000
10
NCOES
A
dV/dlog(Dp) (cm /g)

1800
8 OES
3

1600 6
NGOES
1400
Vads (cm /g STP)

1200 2
3

1000 0
0 10 20 30 40
Dp (nm)
800

600

400

200

0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
P/Po

2600
10
2400 NAS B
dV/dlog(Dp) (cm /g)

8 AS
3

2200
2000 6

1800
Vads (cm /g STP)

1600 2
1400
3

0
1200 0 10 20 30 40 50 60
Dp (nm)
1000
800
600
400
200
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
P/Po

Figure 3. Nitrogen adsorption-desorption isotherms and pore size distributions of

107
 Capítulo 2

mesoporous silica and amino-silica supports. The isotherms have been shifted vertically for
clarity (A) NCOES +400; OES +200; NGOES +0. B) AS +700).

Compared to the two-steps grafting method, the synthesis of the amino-

functionalized silica sample by co-condensation allowed to obtain a support with
larger pore size and higher pore volume, and with a higher loading of amine groups
(Table 1). However, this material exhibits a broader pore size distribution (Figure 3
A), which is in agreement with its poorer pore ordering. The amorphous silica sample
(AS) exhibits textural properties notably different from those of the ordered
mesoporous supports. The BJH pore size distribution (Figure 3 B) shows that the
former sample possesses pores in the range of 10-60 nm, with the maximum of the
distribution around 30 nm. Therefore, it contains larger and less uniform pores. The
pore volume of sample AS is also much higher, reaching 2.5 cm3/g. This sample
allows for a large amount of aminopropyl species to be anchored on its surface by
grafting, as indicated by the nitrogen content determined for sample NAS (Table 1),
equivalent to 1.8 amine groups per gram of silica. As expected, this high level of
surface modification produces a substantial decrease of pore volume, which
nevertheless remains close to twice the pore volume of the amino-functionalized
ordered silica supports. However, the effect on pore size is negligible due to the large
pore dimensions.

Table 1. Textural properties and degree of functionalization of the supports, laccase

immobilization characteristics and catalytic performance.

Dp mmol Max. Biocatalyst Cat. Eff

Vp[b] SBET[c] tc[e]
Material BJH[a] N/g loading [f] activity [g] [h]
(cm /g) (m2/g)
3 (h)
(nm) SiO2[d] (mg/g) (U/g) (U/mg)

OES 15.0 1.2 427 - 24 14.2 0.56 0.04

NGOES 11.2 1.0 339 1.2 2.0 170.0 50.7 0.30
NCOES 17.6 1.2 385 1.5 3.5 173.8 56.0 0.32
AS ~30 2.5 296 - 24 15.8 0.52 0.03
NAS ~30 2.1 236 1.8 24 187.1 169.5 0.91

[a] BJH pore diameter (nm) calculated from adsorption branch.

[b] Pore volume (cm3/g) estimated at a relative pressure of 0.97.

[c] BET surface area (m2/g).

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 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

[d] Amine groups incorporated in the silica framework = mmol N/g SiO2 = (mmol N/g
material) / %final weight (TGA)

[e] Time at which the maximum enzyme loading is achieved

[f] Maximum enzyme loading, expressed in milligrams of enzyme per gram of biocatalysts.

[g] Biocatalyst activity (Units/g biocatalysts) in ABTS test.

[h] Catalytic efficiency = Activity versus loading (Units/mg enzyme). The activity of
enzyme in solution was 1.25 U/mg.

SEM images of samples OES (Fig. S1 a) and NGOES (Fig. S1 b) show particles
with a fiberlike morphology, which consist of small agglomerated rod shaped
primary particles of several tens of microns in length and a thickness of about 1 μm.
This morphology is characteristic of the material SBA-15 [17]. In contrast, the SEM
image of NCOES (Fig. S1 c) displays predominantly agglomerated fibrous particles
and that of NAS (Fig. S1 d) shows fragmented spherical particles several tens of
microns in size.

3.2. Laccase immobilization

To carry out the immobilization of the enzyme on the supports by electrostatic

interactions, the solids should be suspended in an enzyme solution at a pH
intermediate between the isoelectric point of the support and that of the laccase. The
pI of purely siliceous supports is 2.0, while that of laccase is 4.2, therefore, the
selection of pH 3.5 may allow to establish electrostatic interaction between the
positively charged enzyme and the negatively charged support (Figure 4 A).
However, this pH is very close to pI of both the support and the enzyme, leading to a
weak driving force for adsorption, which in turn produces low enzyme loadings,
around 14-16 mg per gram of support for samples OES and AS (Table 1).

109
Capítulo 2

(A) (B)
SiO  H SiO
+
- SiO Si-(CH2)3-NH3 ---
SiO
SiO
SiO  H
-
SiO SiO +
SiO Si-(CH2)3 –NH3 ---
SiO  H
SiO
-
SiO
pI = 2 pI = 4.2 pKa = 10.5 pI = 4.2

Figure 4. Laccase immobilization on (A) siliceous materials without amino groups and (B)
supports with amino groups.

By introduction of primary amine groups (with pKa around 10) on the surface of
the support, the balance of charges can be reversed and immobilization may be
driven in a wider pH range, between 4 and 10, which allows to work at pH values far
from the pI of both the support and the enzyme, and thus higher charge density on
both species can be established and drive a stronger interaction (Figure 4 B).

Immobilization on amino-modified supports was therefore performed at pH 5.5, at

which the amino groups of the support are positively charged and the net charge of
the enzyme is negative as seen in Figure 4 B. Despite this is not the optimal pH for
laccase (as further shown), the weak nature of the chemical bonding ensures non or
minimal distortion of the enzyme molecule upon immobilization. The different
supports were suspended in solutions with increasing enzyme concentrations for
immobilization. Figure 5 shows the relationship between the initial amount of
enzyme in solution per gram of support in suspension and the actual enzyme loading
achieved at adsorption equilibrium.

110
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

250

Immobilized loading (mgE/g support)

225 NAS
NCOES
200
NGOES
175

150

125

100

75

50

25

0
0 50 100 150 200 250 300 350 400

Initial amount of enzyme in solution (mgE/g support)

Figure 5. Equilibrium loading of immobilized enzyme versus initial enzyme content in

solution at pH 5.5 for amino-modified supports.

For all the supports, the enzyme loading showed a continuous increase with the
total enzyme to support mass ratio until a plateau is reached, which means that the
maximum capacity of the support has been reached. The maximum adsorption
amounts determined were 187 mg/g for NAS and around 170 mg/g for NGOES and
NCOES (Table 1). These adsorption curves follow a Langmuir model, that is, their
shape indicates that immobilization takes place forming an enzyme monolayer. This
behavior is expected for the confinement of enzyme in the narrow pores of the two
ordered mesoporous supports, which does not permit the formation of a multilayer.
However, multilayer adsorption would be possible on the external surface of the
particles or within the large pores of the NAS support. The lack of multilayer
adsorption also in these cases can be attributed to electrostatic repulsion among
enzyme molecules, as the enzyme surface should possess a significant net negative
charge at the pH used for immobilization, well above the isoelectric point of the
enzyme.

The adsorption isotherm obtained for sample NAS shows a constant slope in the
low enzyme concentration region and an abrupt change of slope at the enzyme
concentration at which the plateau is reached. On the contrary, the isotherms of the

111
Capítulo 2

two OMM supports show a progressive decrease of slope with the increase of
enzyme concentration until the plateau is reached. This different behavior suggests
that diffusion of enzyme inside the narrow pore channels of the OMM supports is
progressively more restricted as the loading of enzyme increases, while the large
pores of the NAS support would allow enzyme diffusion to be independent of surface
coverage. Taking into account these considerations, it is worth determining whether
the maximum loading of enzyme obtained for the OMM supports corresponds to a
complete filling of pores by enzyme molecules. The estimated dimensions of the
enzyme are 6.3 x 7.2 x 8.9 nm, and its approximate volume is 402 nm3. Hence, the
maximum enzyme loading can be estimated in 2.5·1018 molecules per cm3 of pore
volume. According to its amino acid sequence, the molecular weight of the enzyme
would be around 73 kDa. Thus, the maximum loading achieved for the OMM
supports (around 170 mg of enzyme per cm3 of pore volume) would be equivalent to
60% of the theoretical maximum laccase loading. Indeed, the actual filling of pore
volume must be higher than 60%, as this calculation does not consider the
glycosylation of the protein [70]. Glycosylation explains that the band determined by
electrophoresis of the Suberase extract shows a molecular weight higher than 73
kDa, since the presence of covalently attached carbohydrates often results in
incorrect molecular weight values using the SDS-PAGE technique [70, 75-77]
(Figure S3).

The purely siliceous materials AS and OES yielded similar low enzyme loading
and catalytic efficiency values (Table 1), despite their large differences in pore sizes
and structures. The incorporation of amino groups on the surface of these materials
leads to a tenfold increase in enzyme loading on both, amorphous and ordered
materials. This indicates that the chemical affinity of the enzyme for the surface of
the materials is crucial for success in the immobilization of enzymes. Catalytic
efficiencies of biocatalysts based on purely siliceous materials OES and AS are some
10 times lower than their respective amine-functionalized analogues. In the case of
amorphous materials the difference is even more marked, maybe suggesting a better
substrate diffusion in this wider pore network.

The pores of the OES support are wide enough to accommodate the laccase
molecules even after anchoring propylamine groups on the silica surface as
confirmed by the high enzyme loading achieved on the NGOES support, very close

112
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

to that obtained with the NAS support, which possesses wider pores. Similar loading
capacity has been obtained with the amino-functionalized ordered mesoporous silica
obtained by co-condensation (NCOES). These results confirm that the tailored pore
dimension of the large-pore OMM materials (OES, NGOES and NCOES) allows for
diffusion of enzyme through the pore channels.

These results are especially encouraging considering that the Myceliophthora

thermophila laccase expressed in Aspergillus oryzae is hyper-glycosylated by
eukaryotic host so steric hindrances might prevent interactions between the surface
of the enzyme and the functionalized materials [78].

Immobilization of laccase on amine-containing supports is also faster than in

purely siliceous materials, as seen by the contact time required to reach the
respective maximum loadings (see Table 1). So, by controlling the affinity between
enzyme and ordered mesoporous silica, the uptake rate and the maximum loading
achieved can be drastically improved.

The orientation of the enzyme with regard to support surface is not an issue with
these materials, since confinement of the enzyme in the sufficiently narrow pore
channels ensures the same surrounding environment to all enzyme molecules.

3.3. Effect of pH on the activity of laccase

Once immobilized at pH 5.5, the biocatalysts can be suspended in solutions of

different pH values. Figure 6 shows relative activity of the laccase at different pH
values in the range 2.5-6.0 determined for the enzyme in solution and immobilized in
the different supports.

The activity of laccase shows very similar trend with pH for the enzyme in
solution and immobilized on amorphous silica (AS). In both cases, the maximum
activity is obtained at pH 3.0 and there is a strong decrease of activity at both higher
and lower pH values. On the other hand, for the enzyme immobilized on amino-
functionalized supports, the maximum activity is obtained at a higher pH (3.5). This
shift of the optimum pH might be attributed to the existence of a pH gradient
between the solution and the pores of the support due to the presence of anchored

113
Capítulo 2

amino groups. It can be also observed that, at higher pH, there is a strong decrease of
activity for the enzyme supported on the amino-functionalized amorphous silica
(NAS), in a similar way as it occurs for the enzyme supported on the unmodified
silica (AS). However, the pH increase has lower effect on activity for the enzyme
supported on NCOES and especially on NGOES, where the activity remains
constant. Pore size of NGOES is smaller than NCOES and much smaller than NAS.
This suggests that the microenvironment generated by amine groups in the enzyme
vicinity is more efficient as these groups are closer to the laccase. In the extreme
situations, the wide pore of NAS is not sufficient to maintain the effect over pH 3.5
whereas the effect of the presence of positive charges close to the enzyme in NGOES
remains even at pH 6.0. These results show that confinement in narrow pore sizes
permits to take advantage of the properties of the support.

110
a, b c, d, e
100

90

80

70
%Activity

60

50

40

30

20

10

0
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
pH

Figure 6. Effect of pH on the activity of a) free enzyme () and enzyme immobilized on b)
AS (), c) NGOES (), d) NCOES (), e) NAS (). Oxidation of ABTS at 25 C in 0.05
M phosphoric acid/sodium dihydrogenphosphate or 0.05 M citric acid/trisodium citrate
buffer solutions.

114
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

3.4. Enzyme leaching

The non-covalent nature of the enzyme bonding to support makes necessary an

evaluation of the potential enzyme leaching. Therefore, all the biocatalysts were
subjected to leaching experiments, in order to determine whether the confinement in
the pores and the enzyme-support electrostatic interaction allow to efficiently and
irreversibly entrap the laccase.

Figure 7 shows the relative amount of enzyme that was removed from the
different supports along the leaching experiment. Amorphous silica AS and its
corresponding amine functionalized form NAS showed a continuous release of
enzyme at a nearly constant rate during the whole leaching treatment (48 h). It can be
concluded that the wide pore size of these materials does not offer any restriction for
enzyme diffusion and, therefore, leaching can not be prevented under the conditions
used (high dilution and low ionic strength).

8
AS
7 NAS
OES
6 NGOES
NCOES
5
% Leaching

0
0 4 8 12 16 20 24 28 32 36 40 44 48

Time (h)

Figure 7. Leaching of enzyme from channel-like materials and amorphous mesoporous

silica, expressed as percent of the initial enzyme loading leached as a function of time.

115
Capítulo 2

In contrast the leaching profiles of OMM-supported samples, both purely

siliceous and amino-functionalized materials, show an initial desorption during the
first two hours of treatment, after which the profiles reach a plateau. Longer
incubation times showed no further increase of protein content in the supernatants
indicating that no more leaching occurred. These results suggest that confinement of
enzyme in narrow pores contributes to prevent leaching of the enzyme, even from the
support that was not modified with amino groups to increase its affinity with the
enzyme. The initial desorption observed is probably due mainly to removal of
enzyme molecules immobilized on the external surface of the OMM particles, which
is consistent with the maximum leaching level observed of only 4% of the enzyme
loading or lower. The density of amino groups seems to be less relevant than pore
size to determine resistance to leaching, although a lower enzyme leaching was
detected with NCOES, the material having the highest amine content (1.5 mmol/g)
among the OMM. Laccase was hardly desorbed (only 2%) from this material with
pore size slightly larger than enzyme dimensions, and a high density of amine groups
that should promote a strong interaction with laccase. It is worth emphasizing that
this strong retention of the enzyme inside the channels of OMM supports is
comparable to a situation of covalently bound enzymes, despite the nature of the
linkage is non-covalent.

In order to check the presence of enzyme inside particles, the catalysts previously
incubated under the conditions of leaching described above, were treated for SDS-
PAGE (polyacrylamide gel electrophoresis with Sodium Dodecyl Sulfate) in
standard conditions and their supernatants were assayed for electrophoresis (EF). The
incubation in 2-mercaptoethanol and SDS at 100 ºC should be severe enough to fully
denature the protein and thus enable its release from inner surfaces as a random coil.
Thus, a protein band was expected in the corresponding electrophoresis gels.
However, these bands were observed (Figure S2) only for the samples
corresponding to purely siliceous supports (OES and AS) and the amino-
functionalized amorphous silica (NAS). The lack of band in the lanes corresponding
to the amine-containing OMM supports (NGOES and NCOES) seems to suggest that
laccase was fully retained inside these supports, even as a linear aminoacid chain.
The high density of protonated amine groups seems to exert a strong electrostatic
interaction also on the denatured protein. In order to confirm the presence of the

116
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

enzyme inside these materials, the biocatalysts submitted to the leaching treatment
followed by the SDS-PAGE denaturing conditions were centrifuged and the
supernatant was replaced by the same volume of a NaOH solution at pH 11.0 to
ensure deprotonation of amine groups and, therefore, remove electrostatic interaction
between enzyme and support. The samples were incubated for 1-2 hours and then the
corresponding supernatants were assayed in EF. As seen in Figure S3 a band
corresponding to the same MW as laccase appeared in the electrophoresis gel in the
lanes corresponding to NCOES (lane 2) and NGOES (lane 3) supports.

These results confirm the immobilization of the enzyme inside pores and also
explain the absence of leaching of the enzyme. The adsorption process of the laccase
indicates a very strong interaction between the functionalized surface and the
enzyme, which remains when the protein is in the random coil conformation thus
preventing also the desorption of the denatured protein. Only when these interactions
disappear at high pH the leaching occurs.

The total absence of enzyme leaching from a non-covalent immobilization is a

promising result since advantages from both kind of bonding can be found in these
catalysts, and very interestingly, it permits support reutilization after enzyme
inactivation.

3.5. Thermal stability

Despite the strong interaction with the support, thermostability was not
significantly increased by immobilization. Suspension of the catalysts in aqueous
solutions at high temperature (50 ºC) did not show significant effects regarding
stability. All samples showed similar activity decay with time, comparable to that of
the enzyme in solution (Figure 8). Maybe the explanation is related to the fact that
laccase from Myceliophthora thermophila is thermoresistant in native state [68, 70].
Nonetheless it is also likely that partially unfolded and inactive conformation may be
stabilized by the proximity of the amino groups of the support.

117
Capítulo 2

100
50 ºC, pH 5.5
90

80

70
%Activity

60

50

40

30 NGOES
20 NAS
NCOES
10 Free enzyme
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
Time (h)

Figure 8. Thermostability profiles of free and immobilized laccase. Oxidation of ABTS at

50 ºC in 50 mM acetic acid/sodium acetate buffer at pH 5.5

3.6. Stability in ethanol-containing solution

Stability in organic solvent was also tested. Organic cosolvents are known to
interfere the hydrogen bonds and thus promote protein unfolding [79]. The solvent of
choice was ethanol because one of the possible applications of this enzyme is the
partial oxidation of wine polyphenols. Catalysts were incubated in a medium similar
to wine: 10% ethanol and acidic pH. Catalytic activity was assayed in ABTS
oxidation.

In all cases the immobilization protects from inactivation due to the distortion of
the protein structure driven by the organic medium (Figure 9). Confinement of the
enzyme within narrow pores and strong interaction with the surface contribute to
prevent enzyme unfolding and thus activity can be preserved for longer periods. In
all cases, immobilized enzyme showed higher stability than native one. The
amorphous and wide pore NAS showed the highest stability the catalysts. NGOES
and NCOES showed the same deactivation rate, with half-lives around double than
free laccase.

118
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

110
10% Ethanol, pH 3.5
100
90
80
70
%Activity

60
50
40
30 NAS
20 NCOES
NGOES
10
Free enzyme
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

Figure 9. Stability tests of free and immobilized laccase in acidic aqueous solution
containing ethanol (10 vol% ethanol in 50 mM citric acid/trisodium citrate buffer at pH 3.5).
Oxidation of ABTS was performed at 25 ºC.

4. CONCLUSION
Laccase from Myceliophthora thermophile (MtL) was readily and efficiently
immobilized in expanded-pore mesoporous silica supports functionalized with amino
groups by means of electrostatic interaction. The summary of the advantages of this
immobilization strategy are the following:

 Pore size is wide enough to permit access and diffusion of the enzyme.
 Pore size is narrow enough to prevent enzyme leaching and provide stabilization
against unfolding.
 Chemical affinity is efficient to increase enzyme loading.
 Chemical affinity, along with uniform pore size, is efficient to prevent enzyme
leaching.
 Ordered structure is efficient to limit diffusional restrictions of the substrates and
products.

119
Capítulo 2

Enzymes with large molecular dimensions are difficult to immobilize inside pores
of ordered mesoporous materials. The use of micelle expanders like TIPB has been
applied to overcome this handicap. Based on this methodology, a strategy of
synthesis of expanded pore ordered mesoporous materials (OMM) at low
temperature and surface functionalization with amine groups was successfully
developed. Laccase was transported through the channels and was effectively
immobilized on the inner surfaces with hardly any diffusional restriction. The
relatively large diameter of the pores also enabled to anchor functional groups to
increase the affinity between the silica support and the enzyme without
compromising the diffusion of the enzyme. The good pore connectivity common to
OMM facilitates diffusion of substrates and products throughout the porous network.
These features of the support materials enabled to obtain nanostructured biocatalysts
having high enzyme loading and high catalytic activity in terms of catalytic
efficiency, that is, minor loses of activity upon immobilization.

Confinement of enzyme molecules within pores having only slightly larger

diameter than enzyme dimension prevents unfolding of protein structure which
increases the stability of the biocatalyst in organic medium. The strong interaction
with the functional groups of the support contributes to additionally increase enzyme
stabilization.

As a result of the tuned pore size and high affinity provided by these materials,
the leaching of non-covalently anchored enzyme can be fully prevented.

Laccase immobilized on NGOES and NCOES displayed high enzyme loading,

good activity, good stability and no leaching which are promising properties for
industrial application in subsequent reaction cycles. Even after enzyme inactivation,
the non-covalent nature of the link may also permit to recover and reutilization of
supports, so their advantages can be further exploited with no additional cost.

120
 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

5. SUPPLEMENTARY INFORMATION

a b

5 µm 5 µm

c d

10 µm 50 µm

Figure S1. SEM micrograph of OES (a), NGOES (b), NCOES (c) and NAS (d).

121
Capítulo 2

116.2
97.4
66.2
45
31

21.5

14.4
6.5 1 2 3 4 5 6

Figure S2. Electrophoresis in standard conditions. 1: Protein standard (Broad range SDS-
Page standard stained with Coomassie G-250 stain), 2: OES, 3: NGOES, 4: AS, 5: NAS, 6:
NCOES.

*Due to the high glycosylation the band of the laccase do not correspond to the molecular
weight of the broad range molecular weight standard.

200
116
97.4
66.2
45

31

21.5

14.4
6.5 1 2 3 4

Figure S3. Electrophoresis at pH 11.0. 1: Protein standard; 2: NGOES, 3: NCOES, 4:

Soluble laccase.

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 Efficient retention of laccase by non-covalent immobilization on amino-functionalized
ordered mesoporous silica

6. REFERENCES
[1] Y. Han, D. Zhang, Ordered mesoporous silica materials with complicated
structures, Curr. Opin. Chem. Eng., 1 (2012) 129-137.

[2] A. Stein, B.J. Melde, R.C. Schroden, Hybrid inorganic-organic

mesoporous silicates - Nanoscopic reactors coming of age, Adv. Mater.,
12 (2000) 1403-1419.

[3] M. Hartmann, Ordered mesoporous materials for bioadsorption and

biocatalysis, Chem. Mater., 17 (2005) 4577-4593.

[4] H.H.P. Yiu, P.A. Wright, Enzymes supported on ordered mesoporous

solids: a special case of an inorganic-organic hybrid, J. Mater. Chem., 15
(2005) 3690-3700.

[5] X.S. Zhao, X.Y. Bao, W. Guo, F.Y. Lee, Immobilizing catalysts on porous
materials, Mater. Today, 9 (2006) 32-39.

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Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

CAPÍTULO 3

Hybrid periodic mesoporous organosilica

designed to improve properties of immobilized
enzymes

Victoria Gascón, Isabel Díaz, Rosa M. Blanco, Carlos Márquez-Álvarez

Published in RSC Advances, 2014, vol. 4, pp. 34356‐34368.

DOI: 10.1039/c4ra05362a

400nm 200nm 10nm

R: CH2-CH2 / (CH2)3-NH-(CH2)3

131
Capítulo 3

ABSTRACT

Two types of highly ordered periodic mesoporous organosilicas have been

synthesized as tailor-made supports to immobilize two different enzymes, lipase and
laccase. These materials provide an environment where abundant organic groups in
close vicinity of the enzyme surface generates a high chemical affinity, which results
in high values of enzyme loading, catalytic activity and stabilization. A hydrophobic
periodic mesoporous organosilica support (PMO) with a highly ordered hexagonal
arrangement of parallel pore channels with diameter around 7 nm, containing
framework hydrocarbon groups (ethylene), was used for lipase immobilization. A
novel periodic mesoporous aminosilica (PMA), containing secondary amine groups
in its framework and having expanded pores was synthesized and studied for
immobilization of a larger enzyme, namely laccase. The synthesis conditions were
adjusted, using bis[(3-trimethoxysilyl)propyl]amine and 1,2-
bis(trimethoxysilyl)ethane as framework co-precursors and 1,3,5-triisopropylbenzene
as micelle expander for producing large pores (> 10 nm). The properties of this
multifunctional PMA having hydrophilic amino groups and hydrophobic
ethylene/propylene groups within the framework were studied. This work compares
the confinement of lipase and laccase enzymes in the pores of these hybrid
organosilica materials and its effect on immobilization and stabilization parameters.
Laccase immobilized on PMA and lipase immobilized on PMO exhibited higher
stability in solvents (ethanol and methanol, respectively) compared to enzymes
supported on functionalized silica materials with pending organic groups on the
surface. High retention of enzymes inside the pores of these materials has been
achieved and leaching has been fully prevented. These results can be attributed to the
different interactions (hydrophobic, electrostatic and hydrogen bonding) established
between the surfaces of enzyme and the PMO/PMA support, which are enhanced by
an optimum pore size adjusted to the enzyme dimensions.

Keywords: Periodic mesoporous organosilica; periodic mesoporous

aminosilica; ordered mesoporous materials; expanded pores; enzyme
immobilization.

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

1. INTRODUCTION

Siliceous ordered mesoporous materials (OMM) have burst into the field of
enzyme immobilization as excellent supports to obtain nanostructured biocatalysts
with improved properties [1]. Three main parameters of these materials can be
controlled by adjusting the synthesis conditions: (a) pore connectivity through
regular porous network, (b) pore size, which can be fine-tuned to accommodate to
dimensions of the enzyme for optimal confinement, and (c) surface chemical
functionalization to enhance enzyme-support affinity. This control enables to obtain
biocatalysts with improved properties regarding enzyme loading, catalytic efficiency,
stability, and leaching prevention [2].

The development of hybrid periodic organo-bridged silicas with ordered

mesopores and large pore diameter is a breakthrough in the field of ordered
mesoporous materials. In these periodic mesoporous organosilicas (PMO), the
organic groups are not anchored to the surface but become part of the framework.
Using such hybrid supports allows to avoid the pore size reduction that occurs upon
grafting of the organic functionality on the surface of purely inorganic supports,
which might hinder diffusion of enzyme molecules [3]. However, literature regarding
bulky enzyme immobilization on these materials is rather scarce.

The synthesis of PMO materials is carried out using bridged alkoxysilane

molecules ((R′O)3Si-R-Si(OR′)3; R: bridging hydrocarbon group, R′: methyl or
ethyl) as precursors [4-6]. New hybrid organic-inorganic OMM offer more
possibilities for application, particularly in enzyme immobilization [4, 7, 8]. The
porous structures, surface, and framework properties of PMO materials can be finely
tuned by changing the bridging organic groups incorporated and the synthesis
conditions employed [9-13]. However, the lack of structural rigidity of the organic
moiety of the silsesquioxane may result in a disordered material, especially when
attempting to synthesize materials with large pore size.

Initially, research on PMOs had been focused on the incorporation of small

aliphatic moieties (methylene [14], ethylene [12, 15], ethenylene [16]) into the walls
of the channel-like structures, obtaining different morphologies [17, 18]. Later,
organic bridges in the framework were extended to more functional species ranging

133
Capítulo 3

from hydrocarbons and heteroaromatics to metal complexes. Recent developments in

novel PMO involve innovation in their components and structural designs.
Multifunctional PMO can be easily obtained by co-condensation of a mixture of
bridged organosilane precursors with different surface properties of the pore walls
[19-23]. For example, Morell et al., 2006 [24] incorporated aromatic bridging groups
(phenylene and thiophene) and obtained PMO materials with pore sizes in the range
of 4.8–5.4 nm employing the triblock copolymer Pluronic P123 as structure-directing
agent.

The limited pore size of PMO materials may restrict their applications in some
areas where large pores are essential for adsorption and immobilization of large
biomolecules [25], and only a few works have been published regarding the
immobilization of bulky enzymes, for which large pores are required [26, 27]. In
comparison with their purely siliceous counterparts, pore size is more difficult to
adjust in PMO [16, 28].

We have formerly described the immobilization of Candida antarctica lipase B

(CaLB) on a PMO material containing ethylene groups [29]. Lipases are known to
have a hydrophobic domain, which often constitutes a lid responsible for their
interfacial activation [30]. CaLB lacks this lid [31, 32], but still has the hydrophobic
domain on its surface. Supports with hydrophobic surfaces can interact with this
domain to drive lipase immobilization, therefore, PMO containing ethylene bridges
were obtained and tested as supports in our group.

Laccase is a larger enzyme than CaLB, therefore two challenges are to be faced.
One is the increase of chemical affinity and the other is obtaining a support with pore
size large enough to accommodate the enzyme. We have recently reported [33] the
immobilization of laccase on OMM with expanded pore size functionalized with
amine groups anchored on the silica surface. The large difference between the pI of
laccase (around 4) and the pKa value of these groups (close to 10) enabled strong
electrostatic interactions in a broad pH range. Based on these results we report here
the synthesis of large-pore periodic mesoporous organosilica materials containing
amino groups within the framework as supports for immobilization of laccase. Few
attempts to introduce nitrogen-containing groups into the walls of hybrid
organosilica materials have been reported. Asefa et al, 2003 [34] prepared periodic

134
 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

mesoporous aminosilica (PMA) materials that contained amine functional groups

within the framework via thermal ammonolysis of PMOs under a flow of ammonia
gas. The pore diameter of these materials was around 3-4 nm. It has also been
reported the synthesis of PMA using silane precursors with amine groups. However,
it has to be noticed that the order of the structure is an issue. The increase in
concentration of the bridged alkoxysilane with non-rigid organic bridges may lead to
a decrease in the order of the material, as reported by Wahab et al., 2004 [4] even for
materials with pore size as small as 3.5 nm. An interesting transition of the
mesostructure of this system was observed with increasing content of bis[(3-
trimethoxysilyl)propyl]-amine (BTMSPA) in the starting mixture for the co-
condensation of BTMSPA and 1,2-bis(trimethoxysilyl)ethane (BTME) in the
presence of octadecyltrimethylammonnium chloride [35]. When the ratio of
BTME/BTMSPA was changed from 90:10 to 55:45, a change from a 2D hexagonal
(p6mm) to a cubic mesophase took place. Higher BTMSPA concentrations in the
reaction mixture led to the collapse of the structure. Tan et al., 2006 [36] obtained
uniform mesopores in a material made with BTMSPA using
hexadecyltrimethylammonium bromide (CTAB) as template. When BTMSPA was
used, the hydrophilic chain helped to promote co-assembly of the precursor with
CTAB micelles, limiting bulk condensation. The reported material had relatively low
pore volume and specific surface area, as well as a reduced pore diameter (3.1 nm)
but it had a combination of uniform pore size and amine groups integrated in the pore
walls. In all these reported works, the pore sizes obtained were always smaller than
those attainable for pure silica and did not exceed 6 nm. This factor limits the
potential applications of PMAs as supports for the immobilization of large enzymes.

On the basis of previous works with laccase immobilized on OMM with SBA-15
structure and expanded pores, functionalized with primary amine groups anchored to
the siliceous surface, we report here the synthesis of a novel hybrid material: periodic
mesoporous aminosilica with large pore size. The aim of this work is to study and
compare the behaviour of two model enzymes, laccase and lipase confined within the
pores of two different periodic mesoporous organosilica supports.

135
Capítulo 3

2. EXPERIMENTAL

2.1. Materials and Reagents

Triblock copolymer PEO20PPO70PEO20 (Pluronic P123) and 1,2-

bis(trimethoxysilyl)ethane (BTME) were from Aldrich (USA). 3-
aminopropyltriethoxysilane (APTES) and bis[3-(trimethoxysilyl)propyl]amine
(BTMSPA) were purchased from TCI (Belgium). 1,3,5-triisopropylbenzene (TIPB)
and n-octyltriethoxysilane (OTES) were from Alfa Aesar (Germany). Amorphous
silica MS-3030 was kindly donated by Silica PQ Corporation (USA).

The extracts of soluble laccase from Myceliophthora thermophila (Suberase) and

lipase from Candida antarctica B (Lipozyme CaLB·L) both expressed in Aspergillus
oryzae were kindly donated by Novozymes (Denmark). 2’-azino-bis-(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), p-nitrophenyl
acetate (p-NPA), tributyrin (TB), mercaptoethanol, bromophenol blue and glycerol
were from Sigma (USA). Bovine serum albumin (BSA, Sigma-Aldrich, USA) was
used as protein standard for protein content determination by the Bradford method
[37]. The reagents for electrophoresis SDS-PAGE (sodium dodecyl sulfate (SDS),
ammonium persulfate, N,N,N',N'-tetramethylethylenediamine (TEMED), 40 %
acrylamide/bis solution, 10x Tris base/glycine/SDS buffer and bio-safeTM coomasie
G-250 stain) and broad molecular weight standards were from Bio-Rad (USA).

Citric acid, potassium chloride, phosphoric acid, sodium carbonate, sodium

bicarbonate and toluene were purchased from Sigma-Aldrich (USA). Acetonitrile
and sodium acetate were purchased from Scharlau (Spain). Sodium dihydrogen
phosphate 1-hydrate, hydrochloric acid, ethanol, acetic acid, potassium dihydrogen
phosphate, disodium hydrogen phosphate, sodium hydroxide, potassium phosphate
and acetone were purchased from Panreac (Spain). Trisodium citrate dehydrate was
from Analyticals Carlo Erba (Italy). Tris(hydroxymethyl)aminomethane (Tris base)
was purchased from Fluka Analytical (USA). Solvents were all analytical or HPLC
grade, salts were of high purity and water was Milli-Q grade. All materials were used
as obtained without further purification.

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
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2.2. Synthesis of hybrid periodic mesoporous materials

Ethylene-bridged periodic mesoporous organosilica (PMO) was synthesized as

previously reported [29, 38] with few modifications. 3.19 g of Pluronic P123 were
dissolved at room temperature in 126.84 mL of 0.174 M HCl aqueous solution in a
flask with slow stirring. Once the surfactant was dissolved, 9.38 g of KCl were
added. Both, the low acid concentration and the presence of inorganic salts play an
important role in the formation of highly ordered materials [12, 18, 38-40]. When the
resulting solution was homogenized, it was heated in a thermostated water bath to a
constant temperature of 40 ºC. Then, 3.97 mL of bis-functional alkoxysilane, BTME,
were added at once with rapid stirring. The resulting mixture was stirred at 40 ºC for
24 h and then aged at 100 ºC under static conditions for 24 h. Subsequently, the solid
product was recovered by filtration, washed with ethanol and dried at room
temperature for 24 h. The surfactant was removed from the material by two
successive reflux extractions in ethanol/HCl (1.5 g of as-made PMO in a solution
made with 20 mL of 35 wt % HCl and 205 mL of ethanol) for 24 h. The resulting
solid was recovered by filtration, washed with ethanol, and dried in air.

2.3. Aminodipropyl-bridged periodic mesoporous aminosilica

(PMA)

Materials synthesized using only the highly flexible aminodipropyl-bridged silane

collapsed upon surfactant extraction due to the lack of structural rigidity of the
framework [4, 34]. Therefore, in order to obtain well-ordered pore structures PMA
materials were synthesized by co-condensation of ethylene- and aminodipropyl-
bridged silanes.

For the standard synthesis of periodic mesoporous aminosilica (PMA), 3.19 g of

P123 were dissolved in 126.8 mL of a 0.17 M HCl aqueous solution. To this
homogeneous mixture, 9.38 g KCl were added. The mixture was slowly stirred for
24 h. Then, 3.49 g of 1,2-bis(trimethoxysilyl)ethane (BTME) and 0.95 g of bis(3-
trimethoxysilyl)propylamine (BTMSPA) were added, and the solution was stirred at
40 ºC for 24 h. The mixture was aged at 100 ºC under static conditions for 24 h. The
solution was filtered and the solid product was washed with ethanol and air-dried at
room temperature. The surfactant was extracted from the sample by two successive

137
Capítulo 3

reflux extractions in ethanol/HCl, as indicated above for the PMO sample. The
solution was filtered, and the solid product was washed with ethanol and dried under
ambient conditions.

The synthesis of pore-expanded PMA (E-PMA) followed similar procedure with

some modifications. 1.595 mL of the micelle expander agent TIPB were added to the
surfactant solution and the mixture cooled down to 18 ºC prior to addition of BTME
and BTMSPA. The suspension was kept under stirring at 18 ºC for 24 h. The
obtained solid was suspended in 200 mL toluene and refluxed for 24 hours before
being filtered and washed with ethanol. The solid was then submitted to reflux
extraction in ethanol/HCl and dried in the same way as PMA.

2.4. Functionalization of mesoporous materials by grafting

For comparative purposes, commercial mesoporous amorphous silica (AS) was

functionalized with both amine [33] and hydrophobic groups [41] to immobilize
laccase and lipase, respectively.

The parent material (1.1 g) was degassed at 80 ºC under vacuum for 18 h and then
dispersed in a solution containing the organoalkoxysilane selected for the
functionalization process (22 mmol of 3-aminopropyltriethoxysilane (APTES) or 29
mmol of n-octyltriethoxysilane (OTES)) in 100 mL of toluene. The mixture was
refluxed under N2 stream for 24 h. The suspension was filtered, washed twice with
dry toluene, three times with acetone and finally was dried at room temperature for
24 h. These supports are named NAS (amine-functionalized amorphous silica) and
OAS (octyl-functionalized amorphous silica).

2.5. Characterization of supports

Mesoscopic order was investigated by low-angle X-ray diffraction (XRD) using a

PANalytical X´Pert diffractometer with Cu K radiation.

Nitrogen adsorption-desorption isotherms were measured at -196 ºC using two

Micromeritics sorptometers (ASAP 2020 and ASAP 2420) to determine textural
properties. Pure silicas were pretreated at 350 ºC for 16 h and the functionalized
supports, at 120 ºC for 16 h. The specific surface area, SBET, was calculated from
nitrogen adsorption data in the relative pressure range from 0.04 to 0.2 using the

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

Brunauer-Emmet-Teller (BET) method [42]. The total pore volume, Vp, was
determined from the amount adsorbed at a relative pressure P/Po of 0.97 [42]. Pore
size distributions were determined from the adsorption branch of the isotherms using
the Barrett-Joyner-Halenda (BJH) model with cylindrical geometry of pores. The
BJH pore diameter, Dp BJH, is defined as the position of the maximum in the pore size
distribution.

Quantitative determination of the nitrogen content of amino-functionalized

supports (NAS, PMA and E-PMA) was performed using a LECO CHNS-932
Elemental Analyser with a Perkin Elmer AD-4 autobalance.

Thermogravimetric analyses of the supports were carried out using a Perkin Elmer
TGA 7 instrument. Samples were heated under synthetic air flow (60 mL/min) from
25 to 900 ºC at a rate of 20·ºC/min.

Transmission electron micrographs (TEM) were taken using a JEOL 2100

electron microscope operating at 200 kV. The samples for TEM analysis were
prepared by suspending a small amount of solid in acetone by sonication in an
ultrasonic water bath for 10 min. A drop of this suspension was then poured onto a
copper grid coated with a holey carbon film and the solvent allowed to evaporate at
room temperature.

2.6. Protein determination

The crystal structure of Candida antarctica lipase B, CaLB (PDB: 1TCA) [43]
was taken from the Protein Data Bank [44]. CaLB has a molecular weight of 33 KDa
and an isoelectric point (pI) of 6.0. CaLB is a globular α/β type protein with
approximate dimensions of 3 nm x 4 nm x 5 nm.

Bioinformatic analysis was used to determine the aminoacid sequence and 3D

structure of Myceliophthora thermophila laccase (MtL) because the crystal structure
is not resolved. The amino acid sequence of MtL was taken from the NCBI Protein
Database (accession number AEO 58496.1) [45]. This amino acid sequence was used
as a template to identify homologous sequences of the laccase in BLASTP algorithm
[46]. The protein BLAST analysis for MtL showed that it shares high identity (73 %)
and query coverage (99 %) with the Melanocarpus albomyces laccase (PDB: 1GWO-
A) [44, 47].

139
Capítulo 3

The 3D structure of MtL was modelled applying the alignment mode on the Swiss
model server [46, 48-50] using the three-dimensional structure of the Melanocarpus
albomyces laccase. The modelled MtL structure has been visualized using Pymol
software [51]. The molecular analysis of the whole protein using the tools ProtParam
[52], UniProt KB (G2QFD0 and G2Q560) [53] and Brenda [54] showed that it has a
molecular weight between 63 and 80 KDa. The predicted isoelectric point (pI) was
found to be 4.2. These data are in agreement with those reported by other authors
[55, 56]. MtL has approximate dimensions of 6.3 nm x 7.2 nm x 8.9 nm.

Protein content of solutions was determined with the Bio-Rad Protein Assay (Bio-
Rad, USA), based on the Bradford assay [37], using bovine serum albumin (BSA) as
protein standard. The commercial extracts of laccase and lipase were found to
contain a protein concentration of 3.3 mg/mL and 3.0 mg/mL, respectively.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for

identification of the enzymes and determination of the purity of the commercial
extracts [57].

Proteins structural changes by organic solvents were evaluated

spectrophotometrically by the changes in the UV-Vis spectra of enzyme solutions
with different solvent concentration: laccase in 10 % ethanol and 50 mM sodium
dihydrogenphosphate/disodium hydrogenphosphate buffer pH 7.0, and lipase in 50 %
methanol and 50 mM sodium dihydrogenphosphate/disodium hydrogenphosphate
buffer pH 7.0 compared to the spectra registered by identical enzyme concentrations
in their respective buffers.

2.7. Enzymes immobilization

Lipase immobilization was carried out according to a protocol previously reported

[41, 58, 59]. Enzyme solutions of different concentrations were prepared in buffered
solutions to carry out immobilization at selected pH values. The buffer solutions used
and their corresponding pH were: 50 mM glycine/hydrochloric acid (pH 3.5), 50 mM
sodium acetate/acetic acid (pH 5.0), 50 mM potassium dihydrogen
phosphate/disodium hydrogen phosphate (pH 7.0) and 50 mM sodium
carbonate/sodium bicarbonate (pH 9.0). To carry out the immobilization, 100 mg of
the support were impregnated with 0.5 mL ethanol (to facilitate its dispersion in

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

aqueous solutions, due to the hydrophobic character of supports), and added to 10

mL enzyme solution. The suspension was kept under gentle stirring at room
temperature. Aliquots of the suspension and supernatant were withdrawn at different
times and assayed for catalytic activity measurement (pNPA test). The activity of a
control enzyme solution was used as reference to evaluate the degree of enzyme
immobilization with time. The time at which the residual activity of the supernatant
reached a constant value was taken as the end of the immobilization process. The
suspensions were then filtered, washed with 200 mM of the respective buffer at the
same pH as used for immobilization and subsequently with acetone and allowed to
dry at room temperature. High concentration of buffers was preferred for washing in
order to preserve hydrophobic interactions and thus preventing enzyme leaching in
this step. Dried biocatalysts were stored at 4 ºC. Activity of the supported
biocatalysts was determined by tributyrin hydrolysis assay. Suspensions with a lipase
to support weight ratio between 20 and 600 mg/g were used to obtain adsorption
isotherms (see Supplementary Information Figure S1 a) and determine the
maximum enzyme loading capacity of the support under the best conditions of
immobilization.

Laccase immobilization on the different supports was carried out at pH 5.5 (and
also pH 6.0 for E-PMA) in 50 mM acetic acid/sodium acetate buffer solutions
containing different enzyme concentrations. To the enzyme solution, 50 mg of the
support were added and kept in suspension under mild stirring at room temperature.
Aliquots were withdrawn at given times and the enzymatic activities of suspension
and supernatant were assayed (ABTS assay). The decrease of the supernatant activity
to a minimum and constant value indicated the end point of the immobilization
process. At this point, the solids were filtered off and washed with acetate buffer (50
mM at the same pH as used for immobilization). The solid samples were first dried
under vacuum and then under nitrogen stream and stored at 4 ºC for later analysis.
Immobilizations were performed using different enzyme to support weight ratios in
the range between 25 and 350 mg/g in order to determine adsorption isotherms and
the maximum loading capacity for all supports (see Figure S1 b for enzyme
adsorption isotherms). To determine the catalytic activity of the supported
biocatalysts, 10 mg of the solid were suspended in 1 mL of 50 mM acetate buffer (at
the same pH as used for immobilization) and assayed in the ABTS oxidation test.

141
Capítulo 3

2.8. Enzyme leaching study

The resistance to enzyme leakage from the supports was studied under conditions
that presumably favour the release of the protein, namely high dilution and low ionic
strength. Lipase catalysts were suspended in 50 mM potassium dihydrogen
phosphate/disodium hydrogen phosphate at pH 7.0. The laccase catalysts were
incubated in 50 mM acetic acid/sodium acetate buffer at pH 4.5. Suspensions were
prepared with 1.25 mg of solid per mL of buffer solution and were incubated at 25
ºC. Enzyme leaching was calculated at different incubation times by measuring the
amount of enzyme present in the supernatant using the Bradford assay [37]. After the
leaching treatment, the solids were separated by filtration, suspended in
electrophoresis sample buffer (containing SDS, mercaptoethanol, bromophenol blue,
Tris buffer pH 6.8 and glycerol) and boiled for 5 min. After this treatment, proteins
trapped in the solids would be denatured and the lineal polypeptide chains should be
easily released from the pores. The supernatants of these suspensions were
withdrawn and analysed by SDS-PAGE electrophoresis.

2.9. Enzyme activity assays

Routine laccase activity tests were carried out spectrophotometrically by

measuring the increase in absorbance at 405 nm caused by the oxidation of ABTS
[60]. The reaction mixture consisted of a 1.6 mM ABTS solution in 100 mM acetic
acid/sodium acetate buffer at pH 4.5. To 1.9 mL of this solution in the cuvette, 50oµL
of enzyme solution or suspension were added under stirring and the reaction was
monitored continuously at 25 ºC for 30 min. One unit of laccase activity (UABTS) was
defined as the amount of enzyme required to oxidize 1 μmol of ABTS per minute at
25 ºC (the molar absorption coefficient of oxidized ABTS at 405 nm was taken as
ε405 nm = 35,000 M-1·cm-1).

In order to perform enzyme spectrophotometric assays at different pH values, the

isosbestic point of oxidized ABTS (ABTS*) was determined and established at 430
nm, and the molar absorption coefficient of ABTS* was calculated (ε430 nm = 20,700
M-1·cm-1). The activities of the free and immobilized laccases (on PMA and NAS)
were determined at this wavelength as a function of pH, in 50 mM phosphoric

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

acid/sodium dihydrogenphosphate (pH 2.5) and 50 mM citric acid/trisodium citrate

(pH 3.0 to 6.0) buffer solutions at 25 ºC (triplicated).

Hydrolysis of p-NPA was used as a routine test to measure the nonspecific

esterase activity for monitoring the immobilization of lipase. An aliquot of 50 µL of
enzyme solution (control or supernatant) or immobilized enzyme suspension was
added to a cuvette with 1.9 mL of 0.4 mM p-NPA aqueous solution (pH 7.0). The
activity was determined at 25 ºC by measuring the rate of increase of absorbance at
348 nm due to the release of p-nitrophenol (the molar absorption coefficient was
taken as ε348 nm = 5,150 M-1·cm-1).

Spectrophotometric assays of free enzyme and immobilized enzyme suspensions

were performed using an Agilent 8453 UV-Vis spectrophotometer equipped with a
stirring device and temperature control.

Lipase biocatalysts stability tests were performed using the hydrolysis of

tributyrin as test reaction to evaluate hydrolytic activity of lipase. The activity was
determined by titration of the butyric acid released by the hydrolysis of tributyrin.
1.47 mL of tributyrin were added under stirring to 48.5 mL of 10 mM potassium
phosphate buffer at pH 7.0 in a thermostated titration vessel at 25 °C. Once
equilibrated at pH 7.0, a weighted amount of biocatalyst was added, and the rate of
addition of a 0.1 M NaOH solution required to neutralize the acid product and
maintain a constant pH of 7.0 was measured. One unit of activity (UTB) was defined
as the amount of lipase converting 1 µmol of tributyrin per minute.

Titrimetric determination of lipase activity was performed using a Mettler Toledo

DL-50 pH-state.

2.10. Stability tests

The thermal stability of free and immobilized enzymes was determined by

incubation at 55 ºC in 50 mM potassium dihydrogen phosphate/disodium hydrogen
phosphate buffer at pH 7.0 (for lipase) and at 60 ºC in 50 mM acetic acid/sodium
acetate buffer at pH 5.5 (for laccase).

143
Capítulo 3

Stability of free and immobilized enzymes in organic solvents was evaluated by

incubating samples at 25 ºC in 50 % v/v methanol/water and pure methanol (lipase
samples) or 10 % v/v ethanol at pH 3.5 (laccase samples).

Incubations were performed in different vials for each aliquot to prevent solvent
evaporation during sampling. Aliquots were withdrawn at different times, cooled
down and their activities were assayed in the hydrolysis of tributyrin (lipase) or in
the oxidation of ABTS (laccase).

3. RESULTS

3.1. Characterization of the supports

TEM images of solvent-extracted hybrid organosilicas (Figure 1) show large

domains with two-dimensional hexagonal arrangements of parallel channels of
uniform size. This is consistent with X-ray diffraction (XRD) patterns (see
Supplementary Information Figure S2), which exhibit an intense reflection and two
relatively well resolved weak peaks in the low angle region (0.5-2º), that can be
indexed as the 100, 110 and 200 reflections of p6mm hexagonal symmetry. These
patterns indicate that the samples possess high degree of mesostructural order. In
contrast with the hybrid organosilica supports, the XRD pattern of the commercial
amorphous silica (not shown) does not exhibit any diffraction peak at low angle,
evidencing the lack of mesoscopic order and, hence, the non-uniform pore structure
of this material.

Figure 2 a shows nitrogen adsorption-desorption isotherms and the corresponding

pore size distributions calculated by the BJH method for the solvent-extracted hybrid
aminosilica samples. The isotherms are type IV, with H1 type hysteresis loop at high
relative pressure, which is characteristic of mesoporous materials. The calculated
pore size distributions (Figure 2 a, inset) indicate that the three samples possess
uniform mesopores, in agreement with the regular pore structure shown by XRD and
TEM.

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

The diameter of pore channels estimated by the BJH method is reported in Table
1. The pore size estimated for PMO and PMA supports is around 7 nm. This value
increases up to more than 10 nm when the synthesis of hybrid aminosilica was
carried out using the swelling agent (sample E-PMA). In the case of sample E-PMA
the nitrogen isotherm (Figure 2 a) shows that, besides the main steep nitrogen
uptake at a relative pressure around 0.8, corresponding to the regular mesopore
channels, an additional increase of nitrogen adsorption occurs at relative pressures
above 0.9. This is also apparent in the pore size distribution (Figure 2 a, inset) that
evidences the presence of secondary mesoporosity, with a relatively broad pore size
distribution ranging from 15 to 30 nm, which can be attributed to interparticle spaces.
The three samples possess high surface area and pore volume (Table 1), although
these parameters differ significantly among the three samples. Taking into account
the unit cell parameters (a0) calculated from the XRD patterns (11.6, 12.4 and 15.9
for PMO, PMA and E-PMA, respectively) and the diameter of the pore channels
determined from the nitrogen isotherms (Table 1), it can be estimated that the pore
wall thickness of PMO, PMA and E-PMA is 4.5, 5.2 and 5.5 nm, respectively.
Therefore, the differences in textural properties might be attributed to the different
pore size and pore wall thickness of the three samples.

a b

50 nm 50 nm

145
Capítulo 3

c d

50 nm 50 nm

e f

50 nm 50 nm

Figure 1. TEM micrographs of PMO (a, b), PMA (c, d) and E-PMA (e, f).

800
9
a 8 PMO
dV/dlog(Dp) (cm /g)

700
3

7
PMA
6
600 5 E-PMA
4
Vads (cm /g STP)

3
500
2
1
3

400 0
4 8 12 16 20 24 28
300 Dp (nm)

200

100

0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

P/Po

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

b 1800 10
9 AS
1600 8 NAS

dV/dlog(Dp) (cm /g)

7

3
1400 6 OAS
5

Vads (cm /g STP)

4
1200
3
2
3
1000
1
0
800 10 100
Dp (nm)
600

400

200

0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

P/Po

Figure 2. Nitrogen adsorption isotherms and pore size distributions of: a) hybrid
organosilica supports and b) parent and functionalized amorphous silica supports.

Figure 2 b shows the N2 adsorption-desorption isotherms of the parent (AS) and

functionalized amorphous silica supports (OAS and NAS). These supports show type
IV isotherms corresponding to mesoporous materials [29, 41]. In contrast with hybrid
organosilicas, the commercial amorphous silica has a wide pore size distribution,
indicating heterogeneity in the porous structure, and larger pores, with the maximum
of the pore size distribution around 30 nm (Figure 2 b, inset). Functionalization of
the AS support has a negligible effect on pore size (Figure 2 b, inset and Table 1), as
can be expected due to the large pore size. Nevertheless, grafting with aminopropyl
or octyl groups on the silica surface produces a small decrease of both surface area
and pore volume (Table 1).

Thermogravimetric analysis (TGA) profiles show the incorporation of organic

groups in functionalized silica and hybrid organosilica supports (Figure S3). All
materials show a small weight loss at temperatures below 150 ºC that can be
attributed to desorption of water or residual organic solvents. The main weight loss
occurs in the temperature range 150-600 ºC, which corresponds to decomposition
and combustion of the organic groups. At temperatures higher than 600 ºC a further
small weight loss is observed that that might be attributed to dehydroxylation of the
silica surface. Therefore, the amount of hydrophobic groups (octyl chains in sample
OAS and ethylene bridges in sample PMO) have been calculated from the weight

147
Capítulo 3

loss in the temperature range 150-600 ºC. The results, expressed as mmol C per gram
of silica are given in Table 1. It can be seen that the total amount of carbon of the
hybrid organosilica material is around four times that of the amorphous silica
functionalized with octyl groups. In the case of supports containing amino groups
(PMA and E-PMA), the weight loss assigned to removal of organic groups
corresponds to both aminodipropyl and ethylene bridges. Therefore, the
concentration of amino groups was determined by chemical analysis. All the
supports obtained contain a relatively high amount of amine, ranging from 1 to 1.8
mmol per gram of silica (Table 1).

Table 1. Textural properties and organic groups content of supports.

mmol
Dp BJH[a] SBET[b] Vp[c] mmol N/g
Material C/g
(nm) (m2/g) (cm3/g) SiO2[e]
SiO2[d]
AS 30 285 2.2 - -
OAS 30 212 1.6 4.2 -
NAS 30 236 2.1 - 1.8
PMO 7.1 882 1 16.1 -
PMA 7.2 594 0.7 - 1.5
E-PMA 10.4 264 0.6 - 1

[a] BJH pore diameter (nm).

[b] BET surface area (m2/g).
[c] Total pore volume (cm3/g).
[d] Hydrophobic (aliphatic carbon) groups content determined from TG analysis (weight
loss in the temperature range 150-600 °C).
[e] Concentration of amine groups determined by chemical analysis.

3.2. Lipase immobilization on PMO at different pH values

The loading capacity of PMO for lipase immobilization was determined at pH 5.0
by the adsorption isotherm at room temperature (Supplementary Information, Figure
S1). Maximum enzyme loading remained nearly constant (about 90 mg enzyme per
gram of support) at a content of enzyme in the liquid phase over 100 mg/g. This can
be attributed to the relatively small pore size that would prevent multilayer
adsorption of enzyme. In contrast, on the octyl-functionalized amorphous silica
support OAS, lipase loadings up to 400 mg enzyme per gram of support were
obtained [29] due to its larger pore size. However, this high enzyme loading leads to

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

lower catalytic efficiency (90 UTB per mg of enzyme, compared to 200 UTB/mg for
the PMO-supported catalyst [29]). This is probably due to the intense interaction
with the highly hydrophobic octyl groups. Despite each single interaction is mild, the
high content in ethylene groups and the high contact surface in PMO due to the
confinement of enzyme within the pore, makes the overall interaction intense. But
the mild interactions permit the catalyst to preserve high catalytic activity [29].

Suspensions with 90 mg enzyme per gram of support were prepared to evaluate

the effect of pH on lipase immobilization on PMO. The maximum amount of lipase
slightly decreased as the pH increased from 3.5 to 9.0 (Table 2). In the whole pH
range used for lipase immobilization, silanol groups present on the support should be
deprotonated, as the point of zero charge (pzc) of silica is around 2. Taking into
account that the isoelectric point (pI) of lipase is around 6.0, electrostatic attractions
or repulsions may also be established with the remaining siloxane groups on the
surface of PMO [22] when the immobilization is performed at pH 3.5-5.0 or pH 7.0-
9.0 respectively. Although maximal loading was achieved at pH 3.5 and 5.0, as
expected, the values are rather close indicating that the driving forces of the
immobilization are hydrophobic interactions.

Table 2. Immobilization of lipase on PMO at different pH values from suspensions

containing 90 mg lipase per gram of support.

Max. Biocatalyst
Cat. Eff.[d]
pH tc[a] (h) Load[b] activity[c]
(UTB/mg)
(mg/g) (UTB/g)
3.5 1.25 77.05 2377.8 30.9
5 1.25 70.18 4020 57.3
7 1.5 69.85 3958 56.7
9 1.75 49.14 1952.4 39.7

[a] Time at which the maximum loading is achieved.

[b] Maximum enzymatic loading, expressed in milligrams of lipase per gram of support.
[c] Biocatalyst activity expressed in UTB per g of support.
[d] Catalytic efficiency expressed in UTB per mg of lipase.

3.3. Laccase immobilization on PMA

The low isoelectric point of laccase (4.2) permits a wide pH range for electrostatic
interactions between negatively charged enzyme and positively charged amine

149
Capítulo 3

groups of the hybrid aminosilica supports (pKa around 11.0). Thus, immobilization
was tested at pH 5.5. Because of the similar size between the dimensions of laccase
(6.3 x 7.2 x 8.9) and the pore diameter of PMA (7.2 nm), maximal loading in this
support is low, as well as activity and efficiency of the biocatalyst (Table 3). The
enzyme molecules are probably absorbed only onto the external surface of PMA
particles.

Loading capacity of our expanded pore material E-PMA with 10.2 nm pore
diameter was tested. Table 3 shows the loading capacities of the three amine-coated
supports. The rise in pore size enabled a twofold increase in the enzyme loading of
E-PMA compared to PMA. Also, amine-functionalized amorphous silica was tested
for comparative purposes. Similarly to lipase immobilization, the highest laccase
loading was achieved in amorphous silica with much larger pore size. However, it is
worth noting that adsorption equilibrium was reached more rapidly for the supports
with uniform pore size.

It was also observed that increasing the pH of laccase solution to 6.0 led to a
faster adsorption and higher enzyme loading on E-PMA: 119 mg/g vs 88.0 mg/g at
pH 5.5. However the catalytic efficiency was significantly decreased at pH 6.0.

Table 3. Immobilization of laccase on different supports.

Max. Biocatalyst
Cat. Eff.[e]
Support pH[a] tc[b] (h) Load[c] activity[d]
(UABTS/mg)
(mg/g) (UABTS/g)
NAS 5.5 24 187 170 0.91
PMA 5.5 2 42 4.7 0.11
E-PMA 5.5 1.5 88 29 0.33
E-PMA 6 1.3 119 19 0.16

[a] pH of immobilization.
[b] Time at which the maximum loading is achieved.
[c] Maximum enzymatic loading, expressed in milligrams of laccase per gram of
supported biocatalyst.
[d] Biocatalyst activity expressed in UABTS per g.
[e] Catalytic efficiency expressed in UABTS per mg of laccase.

Figure 3 shows the pH/activity profiles of laccase in soluble and immobilized

estates. It can be observed that for laccase supported on NAS, the optimum pH was
0.5 units higher than that of soluble laccase. This result might be explained assuming

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

that amino groups incorporated into materials generate a new environment which
may result in a pH gradient from inside the particle towards the external aqueous
medium in which catalyst particles are suspended. This trend had been previously
found with expanded-pore SBA-15 materials functionalized with primary amine
groups, where optimum pH was shifted 0.5 pH units, as well as NAS [33]. The shift
of optimum pH underwent by laccase immobilized on E-PMA was much more
pronounced: from pH 3.0 to pH 5.0. As noted, laccase immobilized in the supports
showed higher activity than the free enzyme in the pH range 4.0-6.0. This effect can
be attributed to the microenviromental conditions [61] in this material, with
secondary amines in the close vicinity of enzyme.

110
a, b c d a
100
b
90 c
d
80

70
%Activity

60

50

40

30

20

10

0
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5
pH

Figure 3. Effect of pH on the activity of a) free enzyme, b) AS, c) NAS and d) E-PMA.

3.4. Leaching and electrophoresis

In former works we had determined the amount of lipase leached from OAS and
PMO after two hour incubation under conditions of charge repulsion and high
dilution, where the release of the enzyme is favoured and expected [29]. We present
here a 24 h time course of enzyme leaching (Figure 4 a). Lipase in PMO only
undergoes a 15·% initial leaching that may be due to removal of enzyme molecules
immobilized on the outer surface of PMO particles, and then no more leaching was
detected. Leaching of lipase from OAS is higher and growing with incubation time.

151
Capítulo 3

70 10
65 OAS NAS
a 9
E-PMA
b
60 PMO
55 8

50 7
45
6

% Leaching
% Leaching

40
35 5
30 4
25
3
20
15 2
10
1
5
0 0
0 2 4 6 8 10 12 14 16 18 20 22 24 0 4 8 12 16 20 24 28 32 36 40 44 48

Time (h) Time (h)

Figure 4. a) Leaching of lipase from ordered material (PMO) and amorphous mesoporous
silica (OAS). b) Leaching of laccase from ordered material (E-PMA) and amine amorphous
silica (NAS). Expressed as percent of the initial enzyme leached on each material as a
function of time.

Lipase tightly fitting into pores of PMO and fixed by ethylene groups is highly
retained. In contrast, stronger interaction with octyl groups of OAS does not prevent
enzyme from leaching through the 30 nm with pores of amorphous silica. These
results seem to suggest that it both, chemical affinity and confinement in the pore
contribute to minimize enzyme leaching.

The same trend is observed for laccase leaching from catalysts (Figure 4 b). NAS
catalyst shows a continuous release of enzyme. Again, the affinity of laccase with
amine groups is not enough to prevent leaching from pores with an average diameter
of around 30 nm, with no diffusion restrictions. As shown in Figure 4 b, leaching
profile in E-PMA showed also initial desorption, probably due to enzyme molecules
immobilized on external surfaces. But percent values of laccase leached are much
lower than in the case of lipase (4 %). The uniform pore size close to the enzyme
dimensions along with the increased affinity of the laccase for secondary amine
groups make this material more efficient to retain the enzyme inside the inner
surfaces.

An attempt to verify the location of enzymes inside the porous network of the
materials was made. Since solid samples of the biocatalysts are not suitable for
electrophoresis, the enzymes were forced to exit the pores. First, biocatalysts were

152
 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

suspended as described above for leaching tests to desorb as much enzyme as

possible, especially to eliminate all protein molecules adsorbed on the external
surface of support particles. Then the supernatants were removed and the filtered
solids were boiled in sample electrophoresis buffer. In such denaturing conditions
including the split of disulphide bonds, the tertiary structure of the protein should be
lost and the random coil chain should then be easily released from the pores.
However, no protein band could be seen from the E-PMA supernatant
electrophoresis (Fig. S4 a). Then, E-PMA samples after the same SDS-PAGE
treatment were centrifuged and suspended at pH 11 and 14 respectively and the
supernatants were analysed again. Protein band only appeared in the supernatant of
the sample suspended at pH 14.0. These results confirm the presence of the enzyme
inside the pores, in agreement with previous results based on advanced TEM
techniques [62] and evidence the strong binding of the enzyme to the inner surface of
the support.

The results obtained with lipase catalysts are shown in Figure S4 c. In this case
no further incubation was necessary, and the band corresponding to the immobilized
lipase appeared from both, PMO and OAS.

3.5. Thermal stability

The thermal stability of enzymes catalysts is important for some industrial

applications [63] because bioreactors are sometimes operated at elevated
temperatures to improve productivity and to avoid microbial contamination.

Free and immobilized lipases were incubated in buffer at 55 ºC and their

inactivation courses are shown in Figure 5. Lipase on PMO was inactivated at a
faster rate than free enzyme, while maximal stabilization was achieved in OAS.

Thermal stabilities of free and immobilized laccase on NAS and E-PMA materials
were evaluated at 60 ºC in the same way. Again, a faster inactivation rate was
underwent by laccase in E-PMA and immobilization in NAS resulted in enzyme
stabilization (Fig. 6).

153
Capítulo 3

100
T = 55 ºC OAS
90
Free lipase
80 PMO
70

Activity (%)
60

50

40

30

20

10

0
0 20 40 60 80 100 120 140 160 180 200 220 240

Time (min)

Figure 5. Thermostability profiles at 55 ºC of free and immobilized lipase on PMO and octyl
amorphous silica (OAS).

110

100
T = 60 ºC, pH 5.5

90

80
NAS
70 E-PMA
%Activity

60 Free Laccase
50

40

30

20

10

0
0 20 40 60 80 100 120 140 160 180 200 220 240

Time (min)

Figure 6. Thermostability profiles at 60 ºC of free and immobilized laccase. Runs were

performed at pH 5.5.

3.6. Stability in organic solvents

Lipase can be used in the synthesis of biodiesel to catalyse both, the hydrolysis of
triacylglycerols to release fatty acids and the methanolysis of these fatty acids [64-
67]. The biocatalytic pathway is attractive due to the easier purification of products
and environmental advantages [8, 68]. However, the necessary presence of methanol
involves severe losses of enzymatic activity because of structural and inhibitory

154
 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

damages on the enzyme [66, 69]. This solvent was chosen to study the structural
protection that PMO may provide.

PMO-lipase incubated in 50 % methanol remained fully active after 24 hours,

although the stability of the soluble enzyme was also high, as well as the one on OAS
(Figure 7 a). Severe effects on the activity were registered by incubation in 100 %
methanol, where soluble lipase and OAS-lipase kept only 25 % and 32 % residual
activity after 1 hour respectively. In the same conditions PMO-lipase still kept 75 %
activity (Figure 7 b). But the activity was almost completely lost at longer
incubation time for the three samples, probably because of inhibitory effect of
methanol.

Laccase has been used to partially oxidize wine polyphenols [70-72]. Therefore,
in order to evaluate the potential application of PMA-laccase biocatalysts in wine
stabilization, the stability of biocatalysts was tested by incubation in a low ethanol
concentration. Free laccase, NAS and E-PMA were incubated in a medium similar to
wine: 10 % ethanol and acidic pH (Fig. 8). Inactivation was faster in the native
enzyme, while NAS preserved around 85 % activity after 6 hours incubation and
laccase immobilized on PMA remained fully active during the same period of time.

100
100 PMO
90
90 b OAS
80 80 Free Lipase
70 70

60
%Activity

60
%Activity

50 50

40 40

30 PMO 30

20
OAS 20
Free Lipase
10 10

0
50% Water : 50% Methanol. T = 25 ºC a 0 0% Water : 100% Methanol. T = 25 ºC
0 2 4 6 8 10 12 14 16 18 20 22 24 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Time (h) Time (h)

Figure 7. Stability of lipase in solution and supported on OAS and PMO in: a) 50 %
aqueous solution: 50 % methanol; b) 0 % aqueous solution: 100 % methanol. Runs were
performed at 25 ºC.

155
Capítulo 3

110
10% Ethanol, pH 3.5
100
90
80
70
%Activity 60
50
40
30
20 E-PMA
10
NAS
Free laccase
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

Figure 8. Stability of free and immobilized laccase in ethanol : water solution at acidic pH.
Runs were performed at 25 ºC

Figure 9 shows the UV-Vis spectra of soluble enzymes: lipase in buffer and 50 %
methanol (a) and laccase in buffer and 10 % ethanol (b). Proteins absorbance at 280
nm is due to the presence of aromatic side chain of aminoacids, especially
tryptophan. Spectra of the enzyme in the presence of cosolvents show higher peaks
of absorbance at 280 nm, which suggests a higher amount of these aromatic
aminoacids on the surface of the protein.

2.00
1.50
1.75
a 50 % Methanol b 10 % Ethanol
0 % Methanol 0 % Ethanol
1.25
1.50
Absorbance (AU)
Absorbance (AU)

1.25 1.00

1.00
0.75

0.75
0.50
0.50

0.25
0.25

0.00 0.00
250 300 350 400 450 500 550 600 650 700 750 800 250 300 350 400 450 500 550 600

Wavelength (nm) Wavelength (nm)

Figure 9. UV-Vis spectra of lipase in aqueous buffer and 50 % methanol (a) and laccase in
aqueous buffer and 10% ethanol (b).

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 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

4. DISCUSSION

The main characteristics of periodic mesoporous organosilica as support for

enzyme immobilization are the confinement and the presence of chemical
functionalities. These chemical groups are not anchored on the internal surface of
pores but become part of it. These two features mean that there is close vicinity
between the side chain groups of the enzyme and the functional groups of the support
with no steric hindrances. Apart from the specific organic moieties introduced in the
synthesis of the PMO (ethylene bridges) and PMA (dipropylamine and also ethylene
bridges), silanol groups are present on the surface of silica that can also play their
part.

The same loading of lipase on PMO was achieved at pH 5.0 and 7.0 despite
repulsion should be established at the higher pH between negative charges on both,
enzyme and support. What this probably means is that hydrophobic interactions are
the driving forces of the process, over electrostatic ones. The only difference is found
in the contact time: immobilization is faster at pH 5.0 than at pH 7.0. Also, the
highest values of catalytic efficiency were found at pH 5.0 and 7.0. At pH 3.5 higher
loading was achieved as a result of the additional electrostatic attraction (the enzyme
should have a net positive charge while the support is negatively charged).
Nevertheless, the catalytic efficiency was lower probably because an excessive
interaction is established with a distortional effect on the protein structure.

The immobilization of laccase showed higher values of enzyme loading and

catalytic efficiency on the amorphous and wide pore support NAS. Although this
amine-functionalized amorphous silica has lower surface area than E-PMA, the
available surface in E-PMA is much lower because only one molecule of enzyme
occupies the whole pore section, as the pore size is close to enzyme dimensions.
Thus, the maximal capacity for enzyme immobilization determined for NAS was
around four times higher than on E-PMA (Table 3). The intensity of the interaction
with laccase is not identical for both supports since there are primary propylamine
groups (pKa around 10) in NAS and secondary dipropylamine groups in E-PMA
(pKa around 11). The interaction of negative charges of enzyme with these high pKa
should be more intense and this is probably the reason why the catalytic efficiency is
lower. As discussed for lipase-PMO, the stonger interaction may distort protein

157
Capítulo 3

structure. This may also explain that immobilization at pH 6.0 displays higher
enzyme loading and lower catalytic efficiency than at pH 5.0, because of the higher
density of negative charges on the enzyme.

This high pKa in E-PMA is also responsible for a higher density of positive
charges at the same pH, and consequently a higher pH gradient between the inner
pores of the particles and the external medium. As a consequence, there is a large
optimum pH shifting in the activity vs. pH profile, and it is likely that the
immobilization inside the particle is occurring at a lower pH than measured in the
bulk of the solution.

The absence of enzyme leaching from E-PMA samples can also be explained by
this property. The protein unfolded under denaturing conditions does not leave the
pores at pH below 11, probably because electrostatic attractions remain even in the
random coil configuration. Only at pH extremely high (pH 14.0) all amino groups
from support should be deprotonated and repulsive forces would be driving the
release of protein from pore channels. These experiments reveal that leaching only
occurs in very harsh conditions, showing that this non-covalent immobilization is
nearly as irreversible as a covalent one.

Unfolding of the protein is promoted by both, high temperature and organic

solvents. These conformational changes may drive the formation of new interactions
of side chain amino acids with the support, but the processes do not occur identically.

At high temperature the close vicinity of amines (or ethylenes) and silanol groups
promotes abundant new interactions with amino acids exposed upon partial
unfolding of the peptide chain. In our experiments, incubation of the catalysts
suspensions or enzyme solutions were performed at high temperatures, and cooled
down to the assay temperature (25 ºC for lipase and laccase). The fact that in both
cases: PMO-lipase and E-PMA-laccase resulted the least stable ones may be related
to confinement. Probably the interactions established between the enzyme structure,
partially unfolded upon heating and the “close-fitting-around” support, are preserved
after cooling. Thus, refolding to a more active conformation is prevented and the
activity would be irreversibly lost in PMO/E-PMA biocatalysts. Neither enzymes in
the wide-pore amorphous silica (OAS, NAS) nor the free enzymes have this
restriction from the support, and this absence of steric hindrance would still permit

158
 Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

some margin for reversibility. As a consequence the enzymes can partially recover
their native conformation at lower temperatures.

Only the lipases naturally contain a hydrophobic domain on their surface. With
this exception, hydrophobic side chains of aminoacids are usually placed inside the
protein globule, avoiding contact with aqueous medium. Cosolvents miscible in
water are known to interfere hydrogen bonds and to unfold proteins, thus forcing
some hydrophobic amino acids to arise to the external surface. Spectra in aqueous
and organic media of both lipase and laccase (Figure 9) show a moderate higher
absorbance at 280 nm in the organic solvents, which is usually attributed to
tryptophan residues (hydrophobic) and confirm this effect of the solvents on both
enzymes. PMO and E-PMA surfaces are contain ethylene bridges capable to
establish new interactions with these arising hydrophobic groups. The stabilization
results confirm that PMO and E-PMA are highly efficient to stabilize lipase and
laccase versus methanol and ethanol respectively (Figures 7 and 8). This higher
presence of hydrophobic aminoacids on the external protein surface increases the
bonding intensity with the close support surface containing ethylene groups. In the
case of E-PMA-laccase, new hydrophobic interactions are added to the former
electrostatic ones.

Aminoacids involved in unfolding promoted by high temperature and solvents are

different, thus the new interactions and the structure of the partially unfolded proteins
are also different. If the change does not lead to inactive conformations (as it happens
by heating) the result is the stabilization of an active conformation, which becomes
strengthened by abundant non-covalent bonds with the support.

The close match of sizes of enzyme and pore channels is the key to obtain this
stabilization since the tertiary structure of the protein is physically prevented to keep
on unfolding. However, laccase immobilized on ordered mesoporous materials
(OMM) with similar structure and pore size bearing aminopropyl groups anchored on
the surface had proven less stable than laccase-E-PMA catalyst in the same
conditions [33]. The difference between both kinds of materials is that the organic
moiety becomes part of the wall of the channel pores in the PMO/E-PMA materials,
at the same level as silanols. Aminoacids can easily interact with either the
negatively charged siloxanes or the ethylene bridges or dipropylamine groups. It is

159
Capítulo 3

noticeable that in the case of E-PMA interactions can be established with the three of
them. In the OMM materials where organic moieties are anchored to the siliceous
surfaces, the silanol groups of the wall are not as accessible, as they are partially
hindered by the propylamine or alkyl chains. Thus, additional interactions are not
easily established.

5. CONCLUSIONS

We have synthetized a novel periodic mesoporous aminosilica with a pore size

that matches with the dimensions of the enzyme laccase. The effects of presence of
ethylene bridges and dipropylamine groups becoming part of the surface on laccase
immobilization and the behaviour of the catalyst have been studied and compared to
lipase immobilized on an ethylene-bridged PMO. The PMO materials display some
specific properties which enable the immobilization with high enzyme loading while
retaining high catalytic activity and providing protection against inactivation in
organic solvents. This is possible because these materials integrate organic and
inorganic components in a restricted space where the interaction with the enzyme
takes place all around the enzyme molecules and the mobility is prevented.
Interactions are also feasible with silanol (or siloxane) groups which are not hindered
by organic groups since these are also part of the support framework. Moreover, the
number of contact points increase in the presence of an organic cosolvent, where
additional hydrophobic interactions are established. The highly ordered structure
contributes to keep high catalytic efficiency because it favours pore connectivity.

Laccase undergoes an especially strong interaction with secondary amines of E-

PMA, which fully prevents leaching at pH below 14. This is a relevant result since
the non-covalent nature of the linkage permits to recover the support after enzyme
inactivation, namely by suspending it at elevated pH. Lipase immobilization on
ethylene-bridged periodic mesoporous organosilica (PMO) appeared to be the most
promising approach, since it occurred with high efficiency, maintained enzyme
activity, and provided enzyme stability.

160
Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

6. SUPPLEMENTARY INFORMATION

200

Immobilized loading (mgE/g support)

180 PMO
160

140

120

100

80

60

40

20

0
0 50 100 150 200 250 300 350 400 450 500 550 600

Initial amount of enzyme in solution (mgE/g support)

Figure S1 a. Immobilized loading versus initial enzyme loading at pH 5.0 for PMO.

250
Immobilized loading (mgE/g support)

225 E-PMA (pH 6.0)

E-PMA (pH 5.5)
200

175

150

125

100

75

50

25

0
0 50 100 150 200 250 300

Initial amount of enzyme in solution (mgE/g support)

Figure S1 b. Immobilized loading versus initial enzyme loading at pH 5.5 and 6.0 for E-
PMA.

161
Capítulo 3

100

110
200
E-PMA

I (a.u.)

PMA

PMO

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

2 (degrees)

Figure S2. Low-angle XRD patterns of hybrid organosilica supports.

100

95
NAS
90
OAS
Weight (%)

85

80

PMO
75

70
PMA

65
E-PMA
100 200 300 400 500 600 700 800

T (ºC)

Figure S3. TGA profiles of hybrid organosilica and functionalized amorphous silica
supports.

162
Hybrid periodic mesoporous organosilica designed to improve properties of immobilized
enzymes

Figure S4. Electrophoresis.

1 2 3

a) 1: Protein standard (high range SDS-Page standard stained with coomassie G-250
stain), 2: E-PMA at pH 6.5 and 3: NAS at pH 6.5.

1 2 3

b) 1: Protein standard, 2: E-PMA at pH 14.0 and 3: soluble laccase.

1 2 3 4

c) 1: Protein standard, 2: soluble lipase, 3: PMO, 4: OAS (all in standard conditions).

163
 Capítulo 3

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 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

CAPÍTULO 4

Advanced Electron Microscopy applied to

biocatalysts with enzymes immobilized on
functionalized mesoporous materials

Álvaro Mayoral[a], Raúl Arenal[a, b], Victoria Gascón[c], Carlos Márquez-

Álvarez[c], Rosa M. Blanco[c] and Isabel Díaz[c]

Published in ChemCatChem, 2013, vol. 5, pp. 903‐909.

DOI: 10.1002/cctc.201200737

Ordered mesoporous materials can be customized for the desired

applications, being the optimization of enzyme loading a real grounded
scientific field with solid uses. Functionalized OMM have been produced for
the immobilization of lipase and laccase and an exhaustive structural
analysis has been performed by means of the most advanced electron
microscopy techniques

171
Capítulo 4

ABSTRACT

One of the widely accepted uses of ordered mesoporous materials is as supports of

enzymes for biotechnological applications. Enzymes have been trapped, anchored,
or encapsulated in organized porous networks of the mesoporous range (2–50
nm). The reactivity of the surface of mesoporous materials has enabled the
synthesis of various supports by using different forces for the immobilization
process. To design catalysts for specific applications, we have developed
functionalized mesoporous materials with tunable hydrophobicity for the
immobilization of lipase. More recently, we moved to the immobilization of
laccase with amino-functionalized ordered mesoporous materials. In this case, it is
required to use pore expanders along with optimized functionalization techniques.
Advanced TEM techniques have been applied to locate not only the functional
groups but also the macromolecules inside the silica matrix.

Keywords: electron microscopy; enzymes; enzyme immobilization; functionalized

mesoporous materials; laccase; lipase.

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 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

1. INTRODUCTION

Ordered mesoporous materials (OMMs) are no longer a new class of porous

solids with great potential, but a grounded scientific field with solid uses. One of the
widely accepted uses of OMMs is as supports of enzymes for biotechnological
applications. Enzymes have been trapped, anchored, or encapsulated in organized
porous networks of the mesoporous range (2–50 nm) [1]. The reactivity of silica and
the introduction of organosilanes have allowed for a wide spectrum of supports using
various forces for the immobilization process [2]. The incorporation of functional
groups into OMMs for catalytic applications has yielded optimum materials in the
past [3]. The porous space engineering however brings OMMs to a scenario in which
material science, biotechnology, and engineering bind together in a multidisciplinary
field [4]. This scenario fits perfectly with the aim of this issue “Chemical
Functionalities on Mesoporous Materials: Methods and Catalytic Applications”.

Herein, two cases are exemplified: immobilization of lipase by hydrophobic

forces using alkyl functionalities on OMMs and immobilization of laccase by either
hydrogen bonding or electrostatic interaction between amino groups and carboxylic
acids on the surface of the enzyme. Interesting reviews can be found regarding
enzyme immobilization [5] and particularly immobilization on mesoporous materials
[6]. However, the location of enzymes inside the pores cannot be confirmed easily
and usually a combination of indirect techniques is used for the characterization of
these materials, such as nitrogen adsorption or thermogravimetry. The only direct
and most complete method for the characterization of mesoporous solids is based on
high-resolution TEM [7] combined with electron crystallography, [8] which allows
solving all kinds of structures and gives a complete image of not only the pore
distribution but also the pore connectivity. If the subject of interest is guest materials,
scanning transmission electron microscopy (STEM) combined with high-angle
annular dark field (HAADF) detector is more suitable because the contrast is related
to the atomic number Z [9]. With the modern electron microscopes, which
incorporate spherical aberration (Cs) correctors, probes down to sub-Angstrom
resolution can be achieved, which facilitates an exhaustive analysis of the pores for
OMMs. Herein, a detailed and conclusive analysis is performed on different
functionalized OMMs loaded with lipase and laccase. From the ultrahigh-resolution

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Capítulo 4

images of perfectly oriented thin particles combined with electron energy loss
spectroscopy (EELS), the location of the enzymes inside the pores has been
confirmed. All analyses were performed at an accelerating voltage of 80 kV, which
proves a good stability of the mesoporous materials at this beam energy.

2. EXPERIMENTAL SECTION
2.1. Synthesis of pure silica SBA-15, expanded SBA-15, and
functionalized OMM, PMO, and PMA

All samples were prepared in acid media with triblock copolymeric (Pluronic)
surfactants and tetraethoxysilane as a silica source. Details of the synthesis of each
material are available in the Refs. [10] and [11]. In addition, methylated analogues of
the materials were synthesized through the co-condensation of tetraethoxysilane and
methyltriethoxysilane. The amino-functionalized SBA-15 sample was prepared
through grafting with aminopropyltrimethoxysilane (1 %). PMO was synthesized as
described in the literature [11] with 1,2-bis(triethoxysilyl)ethane as a silica source;
PMA was synthesized from 1,2-bis(trimethoxysilyl)ethane (82 % molar) and bis[3-
(trimethoxysilyl) propyl]amine (18 % molar) [12]. The surfactant was removed
through calcination at 550 ºC under consecutive N2/air streams from pure silica
samples and through extraction with ethanol/HCl from the methylated, amino-
functionalized PMO and PMA.

2.2. Characterization of the OMM

All the samples were characterized extensively with a combination of several

techniques. XRD was performed with a Seifert XRD 3000P diffractometer operating
at a low angle. Nitrogen adsorption–desorption isotherms were determined at 77 K
with a Micromeritics TriStar 3000 apparatus. Specific surface areas were calculated
by using the BET method and the pore size distribution by applying the BJH protocol
to the adsorption branch of the isotherm. Thermogravimetric analysis thermograms
were recorded on a Perkin–Elmer TGA 7 equipment, scanning a temperature range
of 20–900 ºC with a heating rate of 20 ºC/min. SEM images were recorded on a

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 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

JEOL JSM-6400 Philips XL30 operating at 20 kV. TEM analyses of the enzyme-
loaded OMM were performed in the bright-field mode of TEM coupled with selected
area electron diffraction in an FEI Tecnai G2 F30 FEG microscope by achieving a
point-to-point resolution of 1.9 Å when operated at 300 kV. A more exhaustive
analytical investigation was performed in an XFEG FEI Titan 60–300 kV
microscope operated at 80 kV equipped with a monochromator (not excited for the
current experiments), a Cs probe corrector from CEOS Company (which enables the
formation of an electron probe of 0.08 nm mean size), and a Gatan Energy Filter
Tridiem 866 ERS energy filter for the EELS experiments. The geometric aberrations
of the probe-forming system were controlled so that a beam convergence of 24.9
mrad half-angle could be selected. The alignment of the microscope at 80 kV was
verified through the CETCOR software. A focus/tilt tableau was acquired by
measuring defocus and twofold astigmatism as a function of both radial and
azimuthal tilt angles. For the EELS measurements, the collection semiangle was set
to 145 mrad with an energy resolution of 1 eV.

2.3. Enzyme immobilization

Lipase from Candida antarctica fraction B (CaLB) and laccase from

Myceliphthora thermophila were kindly donated by Novozymes. Different amounts
(between 0.25 and 2.5 mL) of the commercial extract of CaLB with a protein content
of 8 mg/mL determined by using Bradford assay were dissolved in phosphate buffer
(50 mM) at pH 5 up to a total volume of 20 mL. To that solution the OMM (100 mg)
was added and left in suspension with a helical stirrer. Aliquots were taken at given
times, and the enzyme activity of the suspension and supernatant was determined
spectrophotometrically by the increase of absorbance at 348 nm along with the
hydrolysis of p-nitrophenyl acetate. The supernatant was obtained by centrifuging the
aliquots for 2 min at 13,000 rpm. Once the enzyme activity of the supernatant was
constant, the suspension was filtered off and washed twice with acetone. Finally, the
solid was dried at room temperature, collected, and weighed.

Different amounts of the commercial extract of laccase with a protein content of

3.28 mg/mL were dissolved in acetate buffer (50 mM) at pH 5.5 up to a total volume
of 15 mL. To that solution, the amino-containing mesoporous material (50 mg) was
added and maintained in suspension with mild stirring. When the OMM was purely

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Capítulo 4

siliceous (SBA-15), the material was suspended in citrate buffer at pH 4.5. Aliquots
of the suspension and supernatant were taken, and their catalytic activities were
assayed spectrophotometrically by the increase of absorbance at 405 nm caused by
2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonate) oxidation. When the constant
values of supernatant activities were obtained, the suspension was filtered off,
washed with distilled water, and dried in N2 stream with use of molecular sieves.

In both cases, a control sample was also assayed to confirm that no enzyme
inactivation occurred during immobilization. The immobilization yield was
calculated from the difference between the initial catalytic activity of the respective
enzyme solutions before suspension with supports and the final activity of the
respective supernatants. Catalytic efficiency is defined as the ratio between the
activity of the catalyst and its enzyme loading, which thus indicates the activity per
milligram of immobilized enzyme.

2.4. Reproducibility

Catalytic activities of the obtained enzyme-immobilized samples were measured

to determine catalytic efficiencies. Triplicate experiments were performed, and the
results provided here are the average data of enzyme loading and activity.

2.5. Leaching and stability

Leaching tests with lipase were performed as published elsewhere [8, 13] by
suspending soluble lipase and samples immobilized on SBA-15, Me-SBA-15, and
PMO at pH 7 (at which the lipase presented negative net charge) as well as the silica
surfaces. Therefore, electrostatic repulsions were established, and so the only forces
keeping the enzyme linked to the support were hydrophobic interactions.

To measure the leaching of lipase from the supports, a sample of the solid
containing the enzyme was suspended in phosphate buffer (50 mM) at pH 7. The
solid/liquid ratio was 1.25 mg/mL. At different times, aliquots of the suspensions
were taken and centrifuged and the protein content of the supernatants was
determined by using Bradford assay [14].

A study of the leaching of some of the lipase catalysts in aqueous medium was
described earlier [10, 11], which depended on the structure of the porous network: 10

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 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

and 15 % was leached from Me-SBA-15 and PMO, respectively. The possible
contribution of the enzyme leached during the assay to the final activity measured
was negligible. The retention was also related to the hydrophobic interaction with the
protein because leaching from SBA-15 increased linearly with time.

In contrast to lipase immobilization, laccase immobilization was driven only by

electrostatic interactions; therefore, incubation could not be performed at pH values
at which charge repulsions were established. Incubation was performed under the
same conditions as for the reaction, but for longer time and in triplicate, to ensure
that leaching occurred and the contribution of soluble enzyme to the activity
measured. Laccase catalysts were incubated at pH 4.5 (NH2-SBA-15exp and PMA)
or pH 3.5 (SBA-15exp). The total reaction time was 30 min. To study laccase
leaching from supports during the reaction, catalysts were incubated in the same
buffers at a lower concentration (50 mM) for 1.5 h. After this time, suspensions were
centrifuged and the protein content of the supernatants was determined by using
Bradford assay. Leaching after 1.5 h was 1 % of laccase from NH2-SBA-15exp, 2.8
% from SBA-15exp, and 3.35 % from PMA; therefore, the contribution of the
leached enzyme to the catalytic activity measured in the assays could be neglected.

Leaching tests were performed at the same pH as that for the immobilization
process (SBA-15exp), or at the same pH of the assay of catalytic activity (NH2-SBA-
15exp and PMA); thus, the results are reliable.

Preliminary studies on enzyme stability were performed through incubation in

buffer at 50 ºC (laccase samples) and 65 ºC (lipase samples). Both are thermophile
enzymes.

2.6. Laccases

Samples of soluble enzyme immobilized on SBA-15-exp and NH2-SBA-15-exp

and incubated at 50 ºC showed similar inactivation curves in which the time required
to decrease activity by 50 % (t50) was approximately 150 min in the three cases.
However, this time was approximately 300 min for the PMA sample incubated under
the same conditions (data not shown). The pore diameter of PMA is 1 nm smaller
than that for both SBA-15 preparations, which means a higher confinement in this

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Capítulo 4

material that prevents unfolding of the enzyme and, therefore, contributes to increase
its stability.

2.7. Lipases

Soluble enzyme maintained 50 % activity after 50 min of incubation at 65 ºC.

Lipases immobilized on Me-SBA-15 and PMO were less stable with respective t50
values of 42 and 37 min. Only SBA-15 was more stable with a t50 value of 67 min. In
the case of lipases, the stability depended not only on the pore size but also on the
chemistry of the silica surface and the incubation medium.

Given the thermophile nature of the soluble enzymes, no increased stability was
achieved under the conditions tested through immobilization, except for laccase-
PMA and lipase-SBA-15 catalysts, which, together with low leaching during
catalytic activity assays, means that a hypothetical contribution of the desorbed
enzyme to the activity measured was negligible.

3. RESULTS AND DISCUSSION

3.1. Immobilization of lipase

The lipase from Candida antarctica fraction B (1TCA in Protein Data Bank 62 x
46 x 92 [13] can be immobilized easily on pure silica mesoporous materials of
regular pore size (e.g., SBA-15 with 9 nm pore channel diameter) by electrostatic
interactions. The isoelectric point of this enzyme is 6.2; thus, optimum
immobilization is performed at pH 5, which is above the isoelectric point of silica,
which is between 2 and 3. However, enzyme loading under these conditions is poor,
and therefore, low activity is achieved [10]. In contrast, lipase is usually better
immobilized on hydrophobic supports by using their property to possess a
hydrophobic domain on the surface [15]. In this direction, several researchers have
developed OMMs functionalized with hydrophobic groups. Han et al. functionalized
SBA-15 with butyl groups; however, the decrease in pore size led to an
immobilization process involving high pressure to facilitate the access of the enzyme
in the framework [16]. We have managed to tune the surface properties of OMMs to
an optimum hydrophobicity [11]. We have grafted the SBA-15 surface with methyl

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 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

groups to increase hydrophobicity. Although the surface coverage with these small
alkyl groups produced only a slight decrease in pore size (Table 1), the enzyme
loading was not favored. However, the enzyme immobilized inside the channels was
more active than that on the surface of pure silica SBA-15.

To achieve higher enzyme loading by gaining in affinity while maintaining the

pore diameter, periodic mesoporous organosilica (PMO) was synthesized with
ethanediyl bridges as organic groups. On one hand, these ethanediyl radicals are
more hydrophobic than methyl groups, and on the other hand, the PMO material
contains a higher amount of organic groups (100 % vs. 6 %). Thus, the PMO
material is more hydrophobic than methyl-SBA-15. The hydrophobic groups are not
anchored but protrude on the siliceous surface and become part of it. This way the
hydrophobic moiety could be a part of the material without decreasing the pore
channel dimensions. The powder XRD patterns and nitrogen adsorption–desorption
isotherms of the samples are shown in Figure 1. The symmetry of the materials is
exactly the same in the three cases, which show the p6mm space group. In addition,
the geometry of the mesopores is the same, given the type IV isotherms obtained in
all the cases. Despite the smaller pore size shown by the PMO material, the enzyme
loading was much higher (91 mg/g), which indicates the crucial role of the increase
in affinity by the hydrophobic groups. Moreover, the catalytic efficiency was much
higher in the PMO material, which indicates that the hydrophobic linkage does not
involve significant conformational changes in the enzyme molecule and the catalytic
activity is better preserved than in the case of electrostatic interaction.

Table 1. Textural and catalytic results of lipase immobilized on OMM.

Max. Load.[b] Cat. Eff.[c]

Biocatalyst Pd[a] (nm) % Hydroph.[d]
(mg/g) (U/mg)

SBA-15 8.8 44 60 -

CH3-SBA-15 7.9 23 88 6

PMO 7.1 91 202 100

[a] Pore diameter; [b] Maximum enzyme loading; [c] Catalytic Efficiency = activity
versus loading (Units/mg); [d] Percent of Si atoms linked to CH- chain.

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Capítulo 4

I (a.u.)

PMO

CH3-SBA-15

SBA-15

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

2 º

1200
B
1000

800
Vads (cm /g STP)

600
PMO
3

400 CH3-SBA-15

200 SBA-15

0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

P/P0

Figure 1. (A) Powder XRD patterns and (B) N2 adsorption-desorption of functionalized

ordered mesoporous materials with increasing hydrophobic surface properties.

3.2. Immobilization of laccase

To extend the method to other enzymes, the properties of each protein must be
studied previously. Laccase (from Myceliphthora thermophila) has a potential use in
the bioremediation field (wastewater treatment) as well as in many other fields [17].
The dimensions of this enzyme are 9.8 x 9.8 x 15 nm, which is too large to fit in the
9 nm diameter channels of the SBA-15 support. Therefore, the first thing to do is to

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 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

synthesize a material with larger pore size. However, in this expanded SBA-15, the
immobilization of laccase by electrostatic interactions renders poor immobilization
yields (Table 2). Because pore size is large enough (11.9 nm), the challenge must be
the low affinity between the laccase and the pore surface of pure silica SBA-15. This
protein has a low isoelectric point (pH 4). Therefore, the useful pH range for
immobilization by electrostatic interaction is very narrow (ca. 3.0–3.5) and close to
the isoelectric points of both the support and the enzyme. Under these circumstances,
the introduction of positive charges on the OMM surface at a pH value high enough
to ensure deprotonation of carboxylic groups on the enzyme surface would be the
way to favor the interactions. Therefore, the functionalization with amine groups was
performed on the SBA-15 support, with extended pores aiming to increase the
affinity between the enzyme and the support. The large pores obtained with use of
micelle expanders enable the introduction of propylamine groups without
compromising the dimensions of the channels. The presence of amines (1.07 mmol
N/g) led to a significant increase in enzyme loading, from 14 to as high as 170 mg/g
(see Table 2).

To design an optimum OMM for the immobilization of laccase, periodic

mesoporous aminosilica (PMA) was synthesized with the intention of increasing the
affinity without decreasing the pore size. The XRD patterns with p6mm symmetry in
the three cases are shown in Figure 2, which suggest that the expanded micelle
method also works for the PMA materials [12, 18]. Furthermore, the isotherms
correspond to those of functionalized mesoporous materials, with bulkier functional
groups producing a broader hysteresis loop owing to a complex mechanism of
adsorption and desorption given by a rougher surface. The PMA sample shows a
second step at higher relative pressure owing to the small particle size obtained in
this material, which leads to agglomerates as observed by using SEM (data not
shown). Nevertheless, the textural properties of the samples enable the
immobilization of laccase, even in the case of PMA for which a smaller pore size
was obtained following the tendency observed in PMO. Despite a slightly smaller
pore size of PMA (10.2 nm; Table 2), the dimensions are still wide enough to
allocate laccase molecules in the proper orientation. The nitrogen concentration in
PMA (0.58 mmol N/g) is 54 % smaller than in NH2-SBA-15exp, and the enzyme
loading achieved is 52 % lower. The higher affinity of laccase for NH2-SBA-15exp

181
Capítulo 4

can, therefore, be attributed to its higher nitrogen content. Thus, the chemical affinity
of the enzyme for the OMM surface is crucial for the success of enzyme
immobilization if the pore size is at least large enough to let enzyme molecules fit
inside.

Table 2. Textural and catalytic results of laccase immobilized on the ordered mesoporous
material.

Max. Load.[b] Cat. Eff.[c]

Biocatalyst Pd[a] (nm) mmol N/g[d]
(mg/g) (U/mg)

SBA-15exp 11.9 14.2 0.04 -

NH2-SBA-15exp 11.2 170 0.30 1.07

PMAexp 10.2 88 0.33 0.58

[a] Pore diameter; [b] Maximum enzyme loading; [c] Catalytic Efficiency = activity
versus loading (Units/mg); [d] mmol of nitrogen per gram of the ordered
mesoporous material.

A
I (a.u.)

SBA-15exp

NH2-SBA-15exp

PMA exp

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
2 º

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 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

1400
B
1200

1000
Vads (cm /g STP)
800
3

600 SBA-15exp

400 NH2-SBA-15exp

200
PMA exp
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

P/P0

Figure 2. (A) Powder XRD patterns and (B) N2 adsorption-desorption of functionalized

ordered mesoporous materials with increasing amount of amine groups on the surface.

3.3. TEM studies

TEM is used conventionally to characterize mesoporous materials (ordered and

nonordered) because it is the only technique that allows obtaining complete
information of the materials [10]. From high-resolution TEM images, the pore
distribution of purely siliceous SBA-15 can be observed easily, which confirms the
good ordering of the material produced [19]. As previously described, a group of
materials for enzyme immobilization is OMMs that contain HC groups in their
frameworks.

These are novel materials, and a complete characterization before enzyme

introduction must be undertaken to confirm good crystallinity and a nice pore
distribution. The TEM images of the as-synthesized PMO material are shown in
Figure 3. The hexagonal array of the channels that corresponds to the p6mm
symmetry with a unit cell value of a = 104.49 Å is shown in Figure 3 A, whereas the
perpendicular image of the pores is shown in Figure 3 B. The same preliminary
analysis was performed for the PMA material. A typical image of the porous solid is
shown in Figure 3 C, in which a large region with uniform pore distribution can be
observed. In this case, a crystal of approximately 1 mm size is perfectly oriented,

183
Capítulo 4

with its pores parallel to the electron beam. The ordering of this material with the
pore arrangement extended over particles of several micrometers is remarkable. The
electron diffraction pattern (Figure 3 C, inset) can also be indexed as the p6mm
space group with a unit cell value of a = 135.05 Å, which is 30 Å larger than that for
the PMO material. A magnified image of the sample is shown in Figure 3 D, in
which the last layer of pores is observed. This image clearly shows an “open-pore
ending” with steps of one pore size, which are denoted by white arrows.

Figure 3. TEM micrographs (A) of periodic mesoporous organosilica (PMO) and (B) taken
along the [001] and [110] orientations, respectively. (C) Periodic mesoporous aminosilica
crystal oriented on the [001] and (D) magnified image of sample C in which steps of one
pore size can be observed on the particle surface.

By using conventional TEM imaging (using a parallel illumination inside the

microscope), the identification and location of guest solids, such as enzymes or
metals, is unfortunately not possible in most cases. An in-depth study must be
conducted by using a STEM-HAADF detector. In the STEM-HAADF detector, the
beam is converged into a very fine probe that is scanned over the material and only
the electrons that are scattered at very high angles are used to form the image. With
these kinds of detectors, data can be interpreted easily because the contrast depends

184
 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

on the atomic number Z [9] and is less affected by multiple scattering or different
defocus values, as commonly occurs in the TEM mode. For light elements
introduced into the pores, a combination of STEM-HAADF and EELS, with the
spectrum-image acquisition mode [20, 21], is necessary to obtain a conclusive
confirmation of the presence of enzymes inside the pores.

The [001] STEM-HAADF image is shown in Figure 4, which is parallel to the

mesopores of the lipase–PMO sample. The organosiliceous matrix is shown in
Figure 4 A, in which the cavities appearing in black represent a hexagonal array
(p6mm symmetry). The fast Fourier transform (upper left corner of the inset) proves
the good crystallinity of the material; a perfectly oriented six pore distribution
(subject to analysis) with respect to the electron beam is shown in the upper right
corner; crystals perfectly aligned with respect to the electron beam is an
indispensable requirement for a reliable analysis of the pores and mesoporous walls.
The STEM-HAADF image of the area in which a spectrum image was recorded is
shown in Figure 4 B. The dimensions used were 30 x 26 pixels, which give a spatial
resolution of 0.89 nm, and the time for analysis was 0.6 s/pixel. The relative
carbon/oxygen composition is presented in Figure 4 C, which is the most crucial
information for the enzyme location. As expected, the relation between carbon and
oxygen is different irrespective of whether the beam illuminates the pore within the
enzyme or the walls that contain SiO2+(-CH2-CH2) groups; red areas correspond to
the higher carbon content. This difference confirms the presence of lipase inside the
pores. Because the nitrogen per molecule is very low, it is impossible to construct a
map based on the nitrogen signal; however, a punctual analysis (black dashed square
in Figure 4 C) of 3 s enabled the observation of the nitrogen K edge at 401 eV. The
results of the EELS analysis of the pore is shown after background subtraction in
Figure 4 D, in which oxygen and nitrogen edges can be seen clearly.

185
Capítulo 4

Figure 4. (A) Aberration-corrected scanning transmission electron microscopy combined

with high-angle annular dark field (STEM-HAADF) image of a periodic mesoporous
organosilica (PMO) crystal along the [001] orientation, showing the hexagonal array. The
fast Fourier transform (upper left corner of the inset) confirms the sixfold axis. A magnified
image of the pores is presented in the upper right corner. (B) STEM-HAADF signal of the
area analyzed. (C) Map obtained from the EELS signal of the relative carbon/oxygen
composition (in red). (D) Sum of the EEL spectra, after background subtraction, recorded in
the dashed square marked in image C.

Because of different particle morphologies of PMOs and PMAs, the latter allowed
a better tilting of larger crystals. Therefore, although the enzyme loading is similar in
both cases, it was easier to locate pores with higher enzyme content. A low-
magnification image is shown in Figure 5 A. A magnified image of the pore system
is shown in Figure 5 B, in which a spectrum image collection analysis was
performed inside one of the pores (marked by green square) by analyzing 8 x 6 pixels
with an exposure time of 2.5 s/pixel. The extracted spectrum profile (raw data;
Figure 5 C) shows the oxygen and nitrogen edges corresponding to the enzyme.

186
 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

Figure 5. (A) Cs-corrected scanning transmission electron microscopy combined with high-
angle annular dark field (STEM-HAADF) image of the periodic mesoporous aminosilica
(PMA) loaded with laccase. (B) High-resolution image of the pores. The green rectangle
represents the area in which the EELS analysis was performed. (C) EELS profile collected
from image B, showing the three edges that confirm the presence of laccase.

The nitrogen maps extracted from the spectra collected in areas perpendicular and
parallel to the pores are depicted in Figure 6. The green square (Figure 6 A and C)
represents the area in which the map was acquired by using 1.5 s/pixel in both cases.
The nitrogen maps are shown in light blue color (Figure 6 B and D). As a
consequence of the amino functionalization, nitrogen was detected in the framework
and in certain pores, in which less contrast was observed (Figure 6 A and B); this
confirms that some pores were not filled with laccase or that the nitrogen content was
too low to be detected. In Figure 6 C, a different contrast, much “whiter”, appears
for which the analysis indicates higher nitrogen content attributed to the enzyme
immobilized within the pores, as clearly observed in Figure 6 D.

187
Capítulo 4

Figure 6. (A) Cs-corrected scanning transmission electron microscopy combined with high-
angle annular dark field (STEM-HAADF) image of the pore system of periodic mesoporous
aminosilica (PMA). (B) Nitrogen map extracted from the green square in image A. (C) Cs-
corrected STEM-HAADF image of the channels of PMA and (D) the corresponding nitrogen
map.

A relatively strong electron beam was needed to gain enough counts for
identifying the species. The total dose was 0.48 eA-12/s, which remained on each
pixel during data collection. The radiation damage is due to radiolysis if the energy
from an electron of the beam is transferred to an electron of the specimen, which
causes an increase in the electron energy and thus bond breakage [22, 23]. This effect
depends directly on the beam energy, which makes the materials more stable if
higher accelerating voltages (in kilovolts) are applied as the cross section is
decreased and the temperature in the sample is also kept to lower values. However,
“knock-on” damage (which occurs if an incoming electron from the beam interacts
with the core of an atom and displaces it from its original position) cannot be
discarded completely [24], especially in the current study in which carbon chains are
present in the structure. For both PMOs and PMAs, knock-on damage could have
increased its importance in the mesoporous decomposition as C-C bonds remained
stable at 80 kV. On the contrary, working at this voltage can increase the temperature
of the crystals analyzed, which is an important contributor to specimen damage [22].

188
 Microscopy applied to biocatalysts with enzymes immobilized on functionalized
mesoporous materials

For the case of analytical microscopes in which the beam is “converted” to a very
fine spot of approximately 1 Å (as a result of the Cs corrector present in the
microscope), the temperature heating can be reduced significantly with respect to
conventional TEM, which illuminates the entire crystal.

4. CONCLUSIONS

The current study proves the feasibility of designing functionalized ordered

mesoporous materials for enzyme immobilization. A rational design of the surface
properties, pore size, and connectivity of ordered mesoporous materials is nowadays
straightforward. Furthermore, Cs-corrected scanning transmission electron
microscopy combined with electron energy loss spectroscopy analysis allows
imaging the functionalities of the mesopores of silica structures with ultra-high
resolution as well as ambiguously determining the presence of enzyme molecules
inside the channels of the material. The presence of lipase has been determined in
periodic mesoporous organosilica (PMO) by observing the carbon/oxygen and
nitrogen edges and by mapping the carbon/oxygen relative composition, which
clearly differs from the wall to the pores because of the presence of organic
molecules. Meanwhile, owing to the different morphologies of amino-functionalized
periodic mesoporous aminosilica, nitrogen maps can be collected by locating
enzymes inside the selected pores.

189
Capítulo 4

5. REFERENCES

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silica for bioanalytical applications, Anal. Bioanal. Chem., 393 (2009)
543-554.

[2] H.H.P. Yiu, P.A. Wright, Enzymes supported on ordered mesoporous

solids: a special case of an inorganic-organic hybrid, J. Mater. Chem., 15
(2005) 3690-3700.

[3] I. Diaz, C. Marquez-Alvarez, F. Mohino, J. Perez-Pariente, E. Sastre,

Combined alkyl and sulfonic acid functionalization of MCM-41-type
silica - Part 1. Synthesis and characterization, J. Catal., 193 (2000) 283-
294.

[4] X.S. Zhao, X.Y. Bao, W. Guo, F.Y. Lee, Immobilizing catalysts on porous
materials, Mater. Today, 9 (2006) 32-39.

[5] U. Hanefeld, L. Gardossi, E. Magner, Understanding enzyme

immobilisation, Chem. Soc. Rev., 38 (2009) 453-468.

[6] M. Hartmann, D. Jung, Biocatalysis with enzymes immobilized on

mesoporous hosts: the status quo and future trends, J. Mater. Chem., 20
(2010) 844.

[7] Y. Sakamoto, M. Kaneda, O. Terasaki, D.Y. Zhao, J.M. Kim, G. Stucky,

H.J. Shim, R. Ryoo, Direct imaging of the pores and cages of three-
dimensional mesoporous materials, Nature, 408 (2000) 449-453.

[8] M.W. Anderson, T. Ohsuna, Y. Sakamoto, Z. Liu, A. Carlsson, O.

Terasaki, Modern microscopy methods for the structural study of porous
materials, Chem. Commun., (2004) 907-916.

[9] A. Mayoral, T. Carey, P.A. Anderson, A. Lubk, I. Diaz, Atomic Resolution

Analysis of Silver Ion-Exchanged Zeolite A, Angew.Chem. Int. Ed., 50
(2011) 11230-11233.

[10] E. Serra, A. Mayoral, Y. Sakamoto, R.M. Blanco, I. Diaz, Immobilization

of lipase in ordered mesoporous materials: Effect of textural and
structural parameters, Microporous Mesoporous Mater., 114 (2008) 201-
213.

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mesoporous materials

[11] E. Serra, E. Diez, I. Diaz, R.M. Blanco, A comparative study of periodic

mesoporous organosilica and different hydrophobic mesoporous silicas
for lipase immobilization, Microporous Mesoporous Mater., 132 (2010)
487-493.

[12] V. Gascón, I. Díaz, R.M. Blanco, C. Márquez-Alvarez, Hybrid periodic

mesoporous organosilica designed to improve properties of immobilized
enzymes, RCS Adv., 4 (2014) 34356-34368.

[13] J. Uppenberg, M.T. Hansen, S. Patkar, T.A. Jones, The sequence,

crystal structure determination and refinement of two crystal forms of
lipase B from Candida antarctica, Structure, 2 (1994) 293-308.

[14] M.M. Bradford, A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding, Anal. Biochem., 72 (1976) 248-254.

[15] R.M. Blanco, P. Terreros, N. Munoz, E. Serra, Ethanol improves lipase

immobilization on a hydrophobic support, J. Mol. Catal. B: Enzym., 47
(2007) 13-20.

[16] Y. Han, S.S. Lee, J.Y. Ying, Pressure-Driven Enzyme Entrapment in

Siliceous Mesocellular Foam, Chem. Mater., 18 (2006) 643-649.

[17] N. Duran, M.A. Rosa, A. D'Annibale, L. Gianfreda, Applications of

laccases and tyrosinases (phenoloxidases) immobilized on different
supports: a review, Enzyme Microb. Technol., 31 (2002) 907-931.

[18] V. Gascón, C. Márquez-Álvarez, R.M. Blanco, Efficient retention of

laccase by non-covalent immobilization on amino-functionalized ordered
mesoporous silica, Appl. Catal. A-Gen., 482 (2014) 116-126.

[19] F. Hoffmann, M. Cornelius, J. Morell, M. Froeba, Silica-based

mesoporous organic-inorganic hybrid materials, Angew. Chem. Int. Ed.,
45 (2006) 3216-3251.

[20] C. Jeanguillaume, C. Colliex, Spectrum-image - The next step in EELS

digital acquisition and processing, Ultramicroscopy, 28 (1989) 252-257.

[21] R. Arenal, F. de la Pena, O. Stephan, M. Walls, M. Tence, A. Loiseau, C.

Colliex, Extending the analysis of EELS spectrum-imaging data, from

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elemental to bond mapping in complex nanostructures, Ultramicroscopy,

109 (2008) 32-38.

[22] C.F. Blanford, C.B. Carter, Electron radiation damage of MCM-41 and
related materials, Microsc. Microanal., 9 (2003) 245-263.

[23] O. Terasaki, T. Ohsuna, V. Alfredsson, J.O. Bovin, D. Watanabe, K.

Tsuno, The study of zeolites by HVHREM, Ultramicroscopy, 39 (1991)
238-246.

[24] R. Csencsits, R. Gronsky, Damage of zeolite-Y in the TEM and its

effects on TEM images, Ultramicroscopy, 23 (1987) 421-431.

a. Dr. A. Mayoral. Laboratorio de Microscopias Avanzadas, Instituto de Nanociencia

de Aragon. Universidad de Zaragoza. Edificio I+D, Mariano Esquillor, 50018,
Zaragoza, Spain.
b. Dr. Raul Arenal. Fundacion Araid, 50004, Zaragoza, Spain
c. V. Gascón, Dr. C. Márquez-Álvarez, Dra. R. M. Blanco, Dra. I. Diaz. Instituto de
Catálisis y Petroleoquímica. ICP-CSIC. C/ Marie Curie 2, 28049 Madrid, Spain.

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 Location of laccase in ordered mesoporous materials

CAPÍTULO 5

Location of laccase in ordered mesoporous

materials

Álvaro Mayorala, Victoria Gascónb, Rosa M. Blancob, Carlos

Márquez-Álvarezb and Isabel Díazb

Published in APL Materials, 2014, vol. 2, pp. 113304-113311

DOI: 10.1063/1.4897281

Location of laccase
inside the pores

193
Capítulo 5

ABSTRACT

The functionalization with amine groups was developed on the SBA-15, and its
effect in the laccase immobilization was compared with that of a Periodic
Mesoporous Aminosilica (PMA). A method to encapsulate the laccase in situ has
now been developed. In this work, spherical aberration (Cs) corrected scanning
transmission electron microscopy (STEM) combined with a high angle annular dark
field detector (HAADF) and electron energy loss spectroscopy (EELS) were applied
to identify the exact location of the enzyme in the matrix formed by the ordered
mesoporous solids.

194
 Location of laccase in ordered mesoporous materials

1. INTRODUCTION

Mesoporous silica materials have received increasing attention due to their

optimum to encapsulate enzymes within the pores providing a more stable
environment in comparison to that at a soluble enzyme [1-3]. The advantage of using
porous supports for immobilization of enzymes is evident, the large internal surface
area can provide a safe haven for the enzyme [4, 5]. The most important factor in this
use is that the pore diameter must be sufficiently large to accommodate the enzyme
[6, 7]. Recent progress has been achieved in the preparation of silica devices
materials which can be used in enzyme nanoarchitectures with soft functions [8-10].
This work focusses on one particular type of enzyme, laccase from Myceliophthora
thermophila (Figure 1).

Figure 1. Average dimensions of laccase measured in PyMOL (~6.1 nm × 5.0 nm × 4.9 nm).

Laccases are attractive biocatalysts for manufacturing pharmaceutical

intermediates, specialty chemicals, and for bioremediation [11-20]. They exhibit
excellent catalytic activity even with water-insoluble substrates such as lignin. One
of the most important aspects of using these enzymes is the sensitivity to phenolic
compounds that are considered very toxic [21-23]. In these reactions the oxygen is
reduced directly to water without the intermediate formation of toxic hydrogen
peroxide. In this direction, the immobilization of laccase in OMM provides the
optimal control of the stabilization of the enzyme towards different media.

So far in our group we have successfully immobilized laccase in ordered

mesoporous materials such as SBA-15 [24] via grafting approach and PMA [25].
Enzyme encapsulation is an attractive method among the different immobilization

195
Capítulo 5

strategies to improve the reusability and stability of enzymes; however, current

encapsulation methods have limitations including enzyme leakage. Silica based sol-
gel encapsulation is the most used technique for enzyme encapsulation without the
need of surfactants. OMM, which are prepared using surfactants, can provide a high
specific surface area, controllable pore diameter, ordered porous network and large
pore volume. Many of the classical OMM are formed using acidic pH, high
temperature processes and hydrothermal synthesis reaction, which are severe
conditions and are not compatible with the presence of enzymes. The goal in this
work is to encapsulate laccase into a shell structure, with the silica shell serving as a
robust and stable encapsulation layer to protect the enzymes where the encapsulated
enzymes are physically confined inside the siliceous material. Until recently, it was
impossible to corroborate the presence of the enzyme within the pores, only indirect
method such as comparisons of XRD patterns and or pore volume before and after
adsorption were applied [1, 3]. Evidences for the encapsulation of an enzyme in the
pores of mesoporous silica were obtained in our group by combining spherical
aberration (Cs) corrected scanning transmission electron microscopy (STEM) with a
high angle annular dark field detector (HAADF) and electron energy loss
spectroscopy (EELS) to show that lipase and laccase were present inside the pores of
the host [26, 27]. In this work the presence of individual molecules of laccase within
the pores could be directly observed.

The molecular weight estimated for the laccase is around 80 kDa, obtained from
SDS-PAGE [28, 29] and the average molecular size was found to be approximately
6.1 nm high, 5.0 nm wide and 4.9 nm deep [29]. According to the modelling studies
and the textural properties of the supports it is expected that the enzyme dimensions
enable its adsorption inside the pores of OMM materials.

This protein has an isoelectric point rather low (pI 4). Hence, the useful pH range
of immobilization via electrostatic interaction is very narrow (around 3.0-3.5) and
close to the isoelectric points of both support and enzyme. Under these
circumstances, the introduction of positive charges on the surface of the ordered
mesoporous materials at a pH value high enough to ensure deprotonation of
carboxylic groups on the surface of the enzyme would be the way to favor the
interactions. Therefore, the functionalization with amine groups was developed on
the SBA-15 with extended pore aiming to increase the affinity between the enzyme

196
 Location of laccase in ordered mesoporous materials

and the support [24]. Figure 2, right side, shows a scheme of the functionalization
process and location of the amino groups protruding the walls in SBA-15 and
incorporated in the walls in PMA. Following, the materials are put in contact with
the enzyme solution for immobilization via grafting method or post-synthesis
approach. The large pores obtained with the use of micelle expanders allow
introducing propylamine groups without compromising too much the dimensions of
the channels (SBA-15-NH2).
Pendant-groups

F127, KCl,
In Situ
H2O, TMOS

In situ Encapsulation
SBA-15-NH2 (Amino grafting ordered
expanded silica)
Framework

Surfactant
In Situ-E
removal

PMA (Expanded periodic mesoporous aminosilica)

electrostatic attraction
Immobilization by

Figure 2. Left: Incorporation of amine groups in SBA-15 and PMA materials and laccase
immobilization post-synthesis. Right: Scheme of the in situ laccase encapsulation.

197
 Capítulo 5

2. METHODOLOGY AND RESULTS

As seen in Table 1, the presence of amines (1.07 mmol N/g) leads to a significant
increase in enzyme loading, as high as 170 mg/g. Following with our aim of
designing an optimum ordered mesoporous material for the immobilization of
laccase, Periodic Mesoporous Aminosilica (PMA) was prepared with the aim to
design an ordered mesoporous material with increasing affinity but without
decreasing the pore size [25]. Figure 3 a shows the XRD patterns corresponding to
p6mm symmetry in the case of SBA-15-NH2 and PMA suggesting that the expanded-
micelles method is also working for the PMA materials [24, 25]. Figure 3 b plots the
isotherms corresponding to those of functionalized mesoporous materials: SBA-15-
NH2 and PMA with functional groups giving broader hysteresis loop due to a
complex mechanism of adsorption and desorption given by a rougher surface. PMA
sample shows a second step at higher relative pressure due to the textural porosity,
porosity between particles of small size that tend to agglomerate. This trend was
corroborated by SEM (data not shown). Nevertheless, the textural properties of the
samples allow for immobilization of laccase (Table 1). Despite of its slightly smaller
pore size (10.2 nm) the dimensions are still wide enough to allocate laccase
molecules in the proper orientation. The concentration of N in PMA (0.58 mmol/g) is
54 % smaller than in SBA-15-NH2, and the enzyme loading achieved is 52 % lower.
The higher affinity of laccase for SBA-15-NH2 can therefore be attributed to its
higher N content. This leads again to the conclusion that the chemical affinity of the
enzyme for the surface of the OMM is crucial to succeed in enzyme immobilization,
when the pore size is at least large enough to let enzyme molecules fit inside.

Table 1. Textural properties, laccase immobilization and catalytic performance.

Max. Biocatalyst Catalytic

SBET Vp
Material Dp BJH (nm) loading activity Efficiency c
(m2/g) (cm3/g)
(mg/g)a (U/g)b (U/mg)

SBA-15-NH2 11.2 339 1.0 170 50.7 0.30

PMA 10.4 264 0.6 88 29 0.33
In Situ 19.5 40 0.2 24.6 8.9 0.36
In Situ-E 20.8 465 1.1 24.6 3.6 0.15

a
Maximum enzyme loading, expressed in milligrams of enzyme per gram of biocatalysts.
b
Biocatalyst activity (Units/g biocatalysts) in ABTS test.
c
Catalytic efficiency = Activity versus loading (Units/mg enzyme)

198
 Location of laccase in ordered mesoporous materials

800
a b In Situ-E
700
SBA-15-NH2
600 PMA

Vads (cm /g STP)

In Situ
500
I (a.u.)

3
400

300

SBA-15-NH2 200

100
PMA
0
1 2 3 4 5 0.0 0.2 0.4 0.6 0.8 1.0

2 (º) P/Po

Figure 3. a) Low angle XRD patterns of amino-functionalized silica supports; b) N2
adsorption-desorption isotherms at -196 ºC.

On the other hand, the right side of Figure 2 shows an alternative path to
encapsulate enzymes in ordered mesoporous materials [30], a novel route developed
to provide the enzyme with hydrophobic moieties that may help to drive the
encapsulation in only one step. At neutral pH carboxylic groups of laccase (with pI
around 4) should be deprotonated, which in the presence of n-butylamine, it should
interact through positively charged amino groups, so that the alkyl chains would be
oriented outwards. In the end, the “protected” enzyme would be surrounded by
surfactant, and thus by silica in a bottle around the ship approach. This synthesis,
denoted as in situ (In Situ), was prepared using Pluronic F127 as template,
tetramethoxysilane (TMOS) as silica source and mild conditions (neutral pH and 35
ºC). As a result, a foam like of mesoporous material is formed, with large cages of
regular size proving the capacity of these butyl chains on laccase surface to drive a
correct arrangement of micelles to give rise to siliceous ordered structures [31].

During the synthesis process, aliquots were taken at given times, and the laccase
activity of the suspension and supernatant were determined spectrophotometrically
towards the oxidation of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
diammonium salt (ABTS). The activity of supernatant decreases as the enzyme
molecules get encapsulated into the support. When the decrease in the activity
reaches a minimum and constant value, it means that no more enzyme molecules can
be immobilized. The suspension was then filtered off and washed. The solid sample

199
Capítulo 5

was first dried under vacuum and then under nitrogen stream, collected, weighted
and finally stored at 4 ºC. Surfactant removal (In Situ-E for “extracted”) was
achieved by mild treatment with 10 % ethanol and acidic pH (3.5 in phosphoric acid
solution).

The biocatalyst prepared in this manner, showed the maximum leaching of the
laccase at 4 h and then remained constant proving that the remaining enzyme
molecules are successfully encapsulated in the silica mesoporous material. The
leaching observed before 4 h is probably due to the enzyme molecules loosely
trapped in the material. As shown in Figure 4 and Table 1 only 10 % is lost in the
leaching test. The biocatalyst activity and catalytic efficiency also decrease after the
treatment for surfactant removal probably due to the above mentioned loss of active
enzyme molecules. For the other two materials (PMA and SBA-15-NH2) only 4 % of
the enzyme is lost in the leaching test corroborating that the incorporation of amino
groups enhances the affinity of the laccase for the mesoporous supports. Besides, in
the case of the PMA, the pore size closer to the size of enzyme, results in a
confinement effect that favors the versatility of this material as support. In summary,
as observed from the data collected in Table 1, despite the lower loading of laccase
in the In Situ material, it shows the highest catalytic efficiency regardless of the
removal of the surfactant.

14

12
In Situ

10
% Leaching

PMA
4
SBA-15-NH2
2

0 2 4 6 8 10 12 14 16 18 20 22 24
Time (h)

Figure 4. Leaching of enzyme from channel-like materials (SBA-15-NH2 and PMA) and in
situ mesoporous silica, expressed as percent of the initial enzyme loading leached as a
function of time.

200
 Location of laccase in ordered mesoporous materials

In order to corroborate the results discussed above, our aim in this work is to
develop a reliable method to prove that the location of enzymes is actually inside the
pores. In the past twenty years, the only direct method for the characterization of
mesoporous materials has been based on high-resolution transmission electron
microscopy [32] combined with electron crystallography [33] which have allowed
solving all kinds of structures, giving a three dimensional reconstruction of the
porous structure. When the subjects of interest are guest materials, scanning
transmission electron microscopy (STEM) combined a HAADF detector is more
suitable as the contrast is related to the atomic number Z. In the current manuscript
Cs-corrected STEM-HAADF combined with electron energy loss spectroscopy
(EELS) were the solely methods for the location and the structural determination of
the enzyme and mesoporous solids. A FEI X-FEG Titan 60-300 kV, operated at 80
kV, equipped with a monochromator (not excited for the current experiments), a
CEOS Cs-probe corrector (allowing forming an electron probe of 0.12 nm mean size
at 80 kV) and a Gatan Energy Filter Tridiem 866 ERS was used for the experiments.
The geometric aberrations of the probe-forming system were controlled to allow a
beam convergence of 24.9 mrad half-angle to be selected. The alignment of the
microscope at 80 kV was verified through the CETCOR software. A focus/tilt
tableau was acquired measuring defocus and two-fold astigmatism as a function of
both radial and azimuthal tilt angles. Concerning the EELS measurements, the
collection semi-angle was of ~100 mrad, the energy resolution ~ 1.2 eV and the data
were collected using the spectrum-imaging mode.

Figure 5 a shows an image of the pore system in the PMA-laccase system. A

collection of spectrum has been taken in the same image inside one of the pores
(green square) analyzing 8 × 6 pixels with an exposure time of 2.5 seconds per pixel.
The extracted spectrum profile (raw data), Figure 5 b, exhibits the O-K and N-K
edges corresponding to the enzyme [26]. The nitrogen map, extracted from the
spectrum-image collected from the green rectangle in Figure 5 c is also presented,
using a pixel time of 1.5 seconds in an image formed by 26 × 26 pixels. In light blue
the correspondent N map is shown. As a consequence of the amino functionalization
N was detected in the framework and in certain pores, evidencing that some pores
where not filled with laccase or that its content was too low to be detected. A
relatively strong electron beam was necessary in order to gain enough counts to

201
Capítulo 5

identify the species. The total dose was 0.48 e/A2s which stayed on each pixel during
the data collection.

20 nm 5 nm

Figure 5. a) Cs-corrected STEM-HAADF image of the pore system. The green rectangle
represents the area in which the EELS analysis was performed. b) EELS profile collected
from image a, showing the N-K and O-K edges that confirm the presence of laccase. c) Cs-
corrected STEM-HAADF image showing the hexagonal pore arrangement, the green square
denoted as spectrum image corresponds to the area of analysis; the correspondent extracted
N signal (blue color) is also displayed. Figure adapted from reference 26.

In the case of the material prepared in situ (In Situ-E-laccase), the presence of
butylamine together with the surfactant yielded to a cage-type of materials although
no ordered structure could be identified (Figure 6 a). Nevertheless, the presence of
enzymes inside the cages was proven by the N and C content [27]. Figure 6 b shows
the Cs-corrected HAADF image of certain pores, taken at 80 kV, where the spectrum
image was acquired. The compositional map formed by Si-L1 signal in green and N-
K signal in red corroborates the presence of the enzyme in some of these cavities
(Figure 6 c). The EEL spectrum is depicted in Figure 6 d, after background

202
 Location of laccase in ordered mesoporous materials

subtraction, proving the presence of all elements coming from the mesoporous silica
and the enzyme.

Figure 6. a) Cs-corrected STEM-HAADF images of the IS-laccase, a closer observation of

the pores is shown inset. b) Area of EELS analysis, green rectangle. c) EELS compositional
map, formed by Si (green) and N (red). d) EEL spectrum profile of the area analyzed.

3. CONCLUSION

In summary, current studies in our laboratory prove the feasibility of designing

functionalized ordered mesoporous materials for enzyme immobilization. A rational
design of the synthesis conditions, surface properties, pore size and connectivity of
ordered mesoporous material is nowadays straightforward. Furthermore Cs corrected
STEM microscopy simultaneously combined with EELS analysis allows imaging the
functionalities of the mesopores of silica structures with ultra-high resolution and to
unambiguously determine the presence of enzyme molecules inside the mesopores of
the hybrid biocatalysts.

203
Capítulo 5

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[17] O.V. Morozova, G.P. Shumakovich, S.V. Shleev, Y.I. Yaropolov, Laccase-
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[18] A. Kunamneni, F.J. Plou, A. Ballesteros, M. Alcalde, Laccases and their

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[19] K. Brijwani, A. Rigdon, P.V. Vadlani, Fungal laccases: production,

function, and applications in food processing, Enzyme Res., 2010 (2010)
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[20] J.F. Osma, J.L. Toca-Herrera, S. Rodriguez-Couto, Uses of laccases in

the food industry, Enzyme Res., 2010 (2010) 918761.

[21] Y. Li, G. Jiang, J. Niu, Y. Wang, L. Hu, Laccase-Catalyzed Oxidation of

Organic Pollutants in Water, Prog. Chem., 21 (2009) 2028-2036.

[22] A. Salis, M. Pisano, M. Monduzzi, V. Solinas, E. Sanjust, Laccase from

Pleurotus sajor-caju on functionalised SBA-15 mesoporous silica:
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[24] V. Gascón, C. Márquez-Álvarez, R.M. Blanco, Efficient retention of

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[25] V. Gascón, I. Díaz, R.M. Blanco, C. Márquez-Alvarez, Hybrid periodic

mesoporous organosilica designed to improve properties of immobilized
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Diaz, Designing Functionalized Mesoporous Materials for Enzyme
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hybrid biocatalyst, J. Mol. Catal. B: Enzym., 90 (2013) 23-25.

[28] R.M. Berka, P. Schneider, E.J. Golightly, S.H. Brown, M. Madden, K.M.
Brown, T. Halkier, K. Mondorf, F. Xu, Characterization of the gene
encoding an extracellular laccase of Myceliophthora thermophila and
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Appl. Environ. Microbiol., 63 (1997) 3151-3157.

[29] V. Gascón, I. Díaz, C. Márquez-Alvarez, R.M. Blanco, Mesoporous silica

with tunable morphology for the immobilization of lacase, Molecules, 19
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[30] S. Urrego, E. Serra, V. Alfredsson, R.M. Blanco, I. Díaz, Bottle-around-

the-ship: A method to encapsulate enzymes in ordered mesoporous
materials, Microporous Mesoporous Mater., 129 (2010) 173-178.

[31] V. Gascón, C. Márquez-Álvarez, R.M. Blanco, In-Situ encapsulation of

laccase and β-glucosidase in mesoporous silicas, Paper under
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[32] Y. Sakamoto, M. Kaneda, O. Terasaki, D.Y. Zhao, J.M. Kim, G. Stucky,

H.J. Shim, R. Ryoo, Direct imaging of the pores and cages of three-
dimensional mesoporous materials, Nature, 408 (2000) 449-453.

[33] M.W. Anderson, T. Ohsuna, Y. Sakamoto, Z. Liu, A. Carlsson, O.

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Dr. A. Mayoral. Laboratorio de Microscopias Avanzadas, Instituto de Nanociencia de Aragon. Universidad

de Zaragoza. Edificio I+D, Mariano Esquillor, 50018, Zaragoza, Spain.

V. Gascón, Dr. C. Márquez-Álvarez, Dra. R. M. Blanco, Dra. I. Diaz. Instituto de Catálisis y Petroleoquímica.
ICP-CSIC. C/ Marie Curie 2, 28049 Madrid, Spain.

206
 6. RESUMEN DE
RESULTADOS

> 10 nm
Inmovilización

pH 5.5
Lacasa de Myceliophthora
thermophila (MtL)
SBA-15-LP Interacción
electrostática

NH3 NH3 NH3 NH3 SiO

SiO Si-(CH2)3–NH3
SiO
NH3 NH3 NH3
 Resumen de resultados

6. RESUMEN DE RESULTADOS

6.1. ANTECEDENTES

Las enzimas poseen características de gran interés para su aplicación en catálisis.

Es de destacar que son muy activas en condiciones suaves, con el consiguiente
ahorro energético. Además, se pueden aprovechar sus propiedades intrínsecas como
son la gran especificidad de sustrato, lo que reduce la proliferación de productos
secundarios indeseables. Sin embargo, son muy inestables especialmente si se
utilizan de forma soluble, por ello, se requiere su inmovilización en un soporte
adecuado que preserve sus propiedades y permita su reutilización abaratando además
su coste. Entre las ventajas que presenta la inmovilización de enzimas, cabe destacar
el aumento de la estabilidad de la misma: mediante una técnica de inmovilización
apropiada que consiga que la enzima se encuentre unida a un soporte evitando que su
estructura terciaria se altere, se desnaturalice y se inactive. Además, debido a su
carácter insoluble, los biocatalizadores heterogéneos pueden recuperarse y separarse
fácilmente de los productos de reacción. Sin embargo, a pesar de las claras ventajas
que presentan, el empleo de estos biocatalizadores muestra limitaciones como son la
disminución de actividad en condiciones extremas de pH y temperatura y en
presencia de disolventes orgánicos, o la liberación de la enzima por lixiviado. Por
otro lado, el confinamiento de las enzimas en el interior de las partículas de soportes
da lugar a limitaciones difusionales de sustratos hacia el centro activo de la enzima y
de los productos hacia el exterior a través de los entramados porosos de las
partículas. Para solventar todos estos problemas se requiere un diseño adecuado del
soporte.

La aplicación comercial de los biocatalizadores depende del desarrollo de

métodos eficaces de inmovilización. La inmovilización de enzimas en materiales
mesoporosos ordenados (MMO) es un campo de investigación de gran interés, que
enlaza la catálisis inorgánica y la biológica, y se beneficia de la interacción
multidisciplinar entre las dos áreas. Los MMO poseen varias características
ventajosas frente a otros soportes convencionales, convirtiéndolos en soportes de
enzimas potencialmente ideales. Poseen tamaños de poro uniformes, grandes áreas
superficiales, se pueden funcionalizar con grupos orgánicos activos, y además

209
Resumen de resultados

presentan alta estabilidad térmica y química. Desde los trabajos pioneros de Balkus y
col. en 1996 [1], se han incorporado numerosos tipos de enzimas en los MMO con
diferente éxito; tanto post-síntesis (es decir, mediante la síntesis previa de soporte y
posterior inmovilización de la enzima), como in-situ o encapsulación de la enzima en
el interior de la estructura durante la propia síntesis del material. Resultados previos
logrados en el grupo de investigación en el que se ha realizado la presente Tesis
Doctoral, mostraban biocatalizadores de lipasa activa tanto cuando se realizaba la
inmovilización post-síntesis como in-situ [2-6]. Dichos resultados previos han
llevado a una investigación más exhaustiva en la presente Tesis Doctoral con otro
tipo de enzima, una lacasa, así como un estudio más profundo del comportamiento de
lipasa en soportes de tipo PMO.

6.2. DISCUSIÓN DE RESULTADOS

Esta Tesis Doctoral ha abordado el diseño de soportes silíceos mesoporosos

ordenados para la inmovilización de enzimas, principalmente lacasa (EC 1.10.3.2.,
bencenodiol : oxígeno oxidorreductasa), en concreto la lacasa de Myceliophthora
thermophila, un hongo ascomiceto de podredumbre blanca, termófilo (crecimiento
óptimo entre 30-50 ºC), que pertenece al grupo de las enzimas multicobre de color
azul [7, 8] (Figura 6.1). Se trata de una glicoproteína monomérica extracelular con
un peso molecular de unos 110 KDa, un contenido de carbohidratos en torno al 40-
60%, 573 aminoácidos y un punto isoeléctrico de 4,2 [9, 10]. Esta oxidorreductasa
cataliza la oxidación de diversos compuestos, principalmente aromáticos (mono, orto
y para difenoles, metoxifenoles, polifenoles, poliaminas, lignina y arildiaminas),
utilizando el oxígeno molecular como aceptor final de electrones que se reduce a
agua [11]. Esta enzima es de gran interés para la preparación de biocatalizadores
activos con potenciales aplicaciones en biorremediación, síntesis química,
bioblanqueo de pasta de papel, biosensores, acabado textil, en la estabilización del
vino, y en el tratamiento de los tapones de corcho para botellas de vino [12-14]. La
lacasa, en forma de extracto líquido enzimático, con la que se ha trabajado en esta
Tesis Doctoral es la de nombre comercial Suberase®, cedida por Novozymes.

210
 Resumen de resultados

Figura 6.1. Estructura terciaria tridimensional modelizada de la enzima lacasa de

Myceliophthora thermophila. Sus dimensiones aproximadas calculadas utilizando el
programa Pymol son 6,1 x 5,0 x 4,9 nm. Los átomos de cobre se representan como esferas
rosas. El modelo se ha creado mediante Pymol [15] y Swiss-Model Server [16-19] a partir de
la lacasa de Melanocarpus Albomyces (73 % de identidad de secuencia con MtL ATCC
42464).

Siguiendo metodologías descritas en bibliografía se han sintetizado con éxito dos

soportes mesoporosos puramente silíceos con simetría hexagonal plana tipo SBA-15
con canales paralelos (ver Figura 3.4 a de la Introducción), con alta superficie
específica, diámetros de poro similares al de la enzima lacasa y con diferente tamaño
y morfología de partícula, siguiendo dos procedimientos distintos como se explica en
el Capítulo 1. A ambos soportes (SBA-15-L y SBA-15-S) se les puede diferenciar
por el tipo de surfactante, precursor silíceo empleado y condiciones de síntesis, que
se resumen en la Tabla 6.1 junto con sus principales características estructurales y
texturales.

En cuanto a sus propiedades texturales existen varios métodos para llevar a cabo
aproximaciones en la caracterización de la estructura porosa de los materiales
mediante isotermas de adsorción-desorción de nitrógeno. En el caso de los materiales
mesoporosos, resulta especialmente adecuado el método desarrollado por Barret,
Joyner y Halenda (BJH) [20], que ha sido hasta ahora uno de los más utilizados y
permite obtener una buena estimación de la distribución de tamaño de poro y del
diámetro de poro medio. Sin embargo, este método subestima en cerca de un
nanómetro el tamaño de poro para materiales con poros menores de 10 nm, debido a
que la ecuación de Kelvin, en la que se basa el modelo, no tiene en cuenta las

211
Resumen de resultados

interacciones adsorbente-adsorbato. No obstante, existen correcciones a este método,

como la corrección propuesta por Kruk, Jaroniec y Sayari (KJS), que permiten
obtener una mejor aproximación a la distribución de tamaño de poro [21]. Sin
embargo, en este trabajo la corrección no fue empleada y se ha utilizado el método
BJH, para determinar los diámetros de poro (Dp BJH) de todos los materiales.

De acuerdo con el procedimiento de síntesis, en ambos soportes la vía de

eliminación del surfactante ha sido mediante calcinación en atmósfera de aire en una
mufla a 550 ºC. El material SBA-15-L (L, long channel) está compuesto de
agregados de partículas alargadas de varias decenas de micras de longitud (~1000
nm) y un grosor de aproximadamente 2oµm (Figura 6.2 a y b), en el que los canales
se disponen longitudinalmente. En cambio, el material SBA-15-S (S, short channel)
está compuesto por partículas con una morfología bien definida de placas apiladas y
un tamaño de partícula homogéneo con un diámetro de disco de aproximadamente 7
µm y un espesor de unos 170 nm (Figura 6.2 c y d), en el que los canales se
disponen perpendicularmente a la superficie de las placas (Figura 6.2 d).

Tabla 6.1. Características de las síntesis, propiedades estructurales y texturales de los

materiales silíceos SBA-15-L y SBA-15-S.

Fuente de ao c Dp BJH d SBET e Vp f wt g

Soporte Surfactante a
sílice b (nm) (nm) (m2/g) (cm3/g) (nm)

P123
SBA-15-L (EO)20(PO)70(EO)20 TEOS 11,1 7,8 609 1,0 3,3

PE-10400
SBA-15-S (EO)25(PO)56(EO)25 TMOS 11,1 6,4 550 0,7 4,7

a) surfactante empleado; b) fuente de sílice: TEOS (tetraetilortosilicato) y TMOS

1/2
(tetrametilortosilicato); c) parámetro de celda (ao = 2·d100/3 ); d) diámetro de poro BJH calculado a
partir de la rama de adsorción de la isoterma de adsorción-desorción de nitrógeno; e) área superficial
calculada empleando el método BET (Brunauer, Emmett y Teller); f) volumen de poro estimado a la
presión relativa P/Po de 0,97; g) espesor de pared calculado como la diferencia entre ao y Dp BJH.

212
 Resumen de resultados

a b

500 nm 50 nm

c d

300 nm 50 nm

Figura 6.2. Micrografías SEM y TEM de los materiales SBA-15-L (a, b) y SBA-15-S (c, d).

Estos soportes se han utilizado para inmovilizar lacasa por interacción iónica con
los grupos siloxano superficiales (Si-O-) mediante fuerzas electrostáticas atractivas,
para lo cual enzima y soporte deben mostrar cargas opuestas. Por tanto, el pH de
inmovilización debe ser intermedio entre el punto isoeléctrico de los soportes
puramente silíceos (~ 2,0) y el de la lacasa (con un pI de 4,2). La región de trabajo es
relativamente estrecha. A un pH de 3,5 la enzima está cargada positivamente (+) y el
soporte negativamente (-) (Figura 6.3), pero debido a la relativamente pequeña
diferencia entre el pH del medio y ambos puntos isoeléctricos, cabe esperar que la
densidad de cargas no sea muy grande tanto en la superficie de la enzima como en la
del soporte. Los resultados obtenidos del cálculo de la actividad enzimática frente al
sustrato siringaldazina pueden verse en la Tabla 6.2. En el soporte SBA-15-L con un
diámetro de poro de 7,8 nm, queda retenido el 63 % de la proteína ofrecida (para una
carga inicial de 50 mg/g sílice) y el 77 % para SBA-15-S (Dp = 6,4 nm).

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Resumen de resultados

pHinmov. = 3,5
SiO  H
SiO-
SiO  H

SiO-
SiO  H
SiO-

pI = 2 pI = 4,2

Figura 6.3. Inmovilización de lacasa sobre soportes puramente silíceos. El pH de

inmovilización debe estar comprendido entre los puntos isoeléctricos de la enzima y el
soporte. A un pH de 3,5 ambos presentan cargas opuestas, pudiéndose establecer
interacciones electrostáticas.

La conclusión que se desprende es que a pesar de que estos materiales poseen alta
superficie específica (~ 600 m2/g), el similar tamaño de poro de los materiales y la
enzima (~ 6,1 x 5,0 x 4,9 nm, dimensiones calculadas mediante el programa
PyMOL) dificulta una buena difusión de la enzima, que sería responsable de las
bajas cargas alcanzadas. Aun así, la morfología de partícula de la SBA-15 juega un
papel significativo en la eficiencia del proceso de inmovilización de enzimas. El
soporte SBA-15-S con un sistema de canales de poros de menor longitud ha
demostrado ser más eficiente en cuanto a carga enzimática y actividad específica que
la SBA-15-L, con canales más largos. Probablemente se deba a que la enzima
difunde con mayor facilidad para acceder a las cavidades del poro del soporte SBA-
15-S, lo que también se corrobora por la mayor actividad específica de la enzima,
posiblemente también porque el acceso de los sustratos al biocatalizador y la difusión
de los productos hacia el medio externo esté facilitada por situarse las moléculas de
enzima hacia los extremos de los canales (Figura 6.4). Sin embargo, se observa una
retención de la enzima ligeramente mayor (o menor lixiviado) en el biocatalizador de
canales más largos como era de esperar (Tabla 6.2).

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 Resumen de resultados

Tabla 6.2. Inmovilización de lacasa a pH 3,5 sobre dos soportes SBA-15 con diferente
longitud de canal, para una carga enzimática ofrecida de 50 mg de enzima por g de soporte.

Carga Actividad
% Lix.
Soporte tc (h) a % Inmov. b enzimática específica
(tras 24 h) e
(mg/g) c (U/mg) d
SBA-15-L 3 62,56 31,28 0,134 16,4
SBA-15-S 3 76,82 38,41 0,159 17,5

a) Tiempo de contacto enzima-soporte; b) Porcentaje de enzima inmovilizada para una carga inicial de
50 mg/g; c) Carga enzimática expresada en miligramos de enzima por gramo de soporte; d) Actividad
específica en Unidades por miligramo de enzima inmovilizada, siendo la unidad U la cantidad de
enzima capaz de transformar 1 µmol de siringaldazina en producto en 1 minuto; e) Porcentaje de
enzima lixiviada tras incubar el biocatalizador durante 24 h en condiciones de alta dilución y baja
fuerza iónica a pH 4,5.
SBA-15-L

1000 nm
SBA-15-S

170 nm

Figura 6.4. Inmovilización de lacasa en los mesocanales de los soportes SBA-15-L y SBA-
15-S.

Por otro lado, se podría mejorar la carga enzimática aumentando la afinidad

química con el soporte, funcionalizando la superficie con organosilanos ya que los
materiales silíceos mesoporosos contienen grupos silanol superficiales que se pueden
funcionalizar con una variedad de grupos orgánicos que incorporan aminas,
carboxilatos, fenilo, y grupos alquilo. Sin embargo, debido a la escasa diferencia de
tamaños entre las dimensiones de la enzima y los diámetros de los poros, una
funcionalización de estos materiales de tipo SBA-15 con grupos orgánicos
voluminosos reduciría adicionalmente el diámetro de poro y dificultaría aún más el
acceso de la enzima a su interior. Por eso, se ha funcionalizado un material
puramente silíceo mesoporoso, MS-3030 (denominado como AS (Amorphous Silica)
en los Capítulos 2 y 3 de la Tesis), que carece de una estructura de poros ordenada y

215
Resumen de resultados

presenta una distribución de tamaño de poro amplia, con un diámetro medio de poro
de unos 30 nm. Este soporte, se utiliza como modelo ya que se elimina la variable
“efecto del diámetro de poro”. Se ha seleccionado como organosilano el
3-aminopropiltrietoxisilano (APTES) para funcionalizar la sílice comercial MS-3030
mediante el método de anclaje. La funcionalización se lleva a cabo por reacción de
silanización de los grupos silanol superficiales con el organosilano APTES, de
acuerdo con el esquema mostrado en la Figura 6.5, resultando en el soporte MS-
3030-N (denominado como NAS (Aminopropyl-functionalized Amorphous Silica) en
los Capítulos 2 y 3 de la Tesis)).

-Si-O-H SiO
-Si-O-H SiO Si-(CH2)3-NH2
Reflujo en tolueno
-Si-O-H SiO
-Si-O-H SiO
-Si-O-H SiO Si-(CH2)3-NH2
-Si-O-H SiO

Sílice mesoporosa amorfa:

MS-3030 APTES MS-3030-N

Figura 6.5. Funcionalización del material silíceo amorfo comercial MS-3030 mediante
anclaje. El grupo orgánico incorporado es el aminopropilo (N).

El punto isoeléctrico del soporte aumenta al incorporarse de forma covalente el

aminopropilo a su superficie (pKa alrededor de 10): a un pH inferior a 10, el amino
está protonado. A valores de pH superiores al pI de la enzima, la superficie de ésta
presentará carga neta negativa, de tal manera que el soporte MS-3030-N permite
trabajar en un intervalo de pH de inmovilización más amplio (entre
aproximadamente 4,2 y 10), lo que puede potenciar las atracciones electrostáticas
(Figura 6.6). De esta manera se invierte la naturaleza de las cargas y además se
amplía el rango de pH de trabajo en el que se dan cargas opuestas.

El mayor tamaño de poro de este material, MS-3030-N, permite la presencia de

los grupos orgánicos sin que se resienta el acceso a la enzima, aunque presenta
menor superficie específica (Tabla 6.3). En cuanto a las condiciones de
inmovilización, el pH óptimo de inmovilización de lacasa en este material es pH 5,5,

216
 Resumen de resultados

al que queda anclada en el soporte el 99,3 % tras 3 horas de inmovilización cuando

se ofrecen 50 mg de proteína por g de soporte (Tabla 6.3 y Figura 6.6).

pHinmov. = 5,5
SiO
SiO Si-(CH2)3-NH3+---
SiO
SiO
SiO Si-(CH2)3 –NH3+---
SiO

pKa = 10,5 pI = 4,2

Figura 6.6. Inmovilización de lacasa en soportes con grupos amino. A un pH de

inmovilización de 5,5 (intermedio entre el punto isoeléctrico de la enzima y el soporte), la
enzima se encuentra cargada negativamente y los grupos amino del soporte positivamente,
potenciando las atracciones electrostáticas.

La inmovilización por interacciones electrostáticas en un mayor rango de pH

favorece una interacción mucho más intensa a un pH intermedio donde las
densidades de carga de enzima y soporte son más altas que en los sistemas con el
soporte sin funcionalizar puramente silíceos (SBA-15-L, SBA-15-S y MS-3030). La
interacción con estos grupos no sólo contribuye positivamente para inmovilizar la
enzima, sino también para limitar su desorción o lixiviado (Tabla 6.3).

En la Tabla 6.3 se compara el soporte MS-3030 sin y con grupos amino. Se

muestra el efecto de las interacciones entre la enzima y el soporte modificado
superficialmente con grupos amino, ya que como se observa en el soporte puramente
silíceo MS-3030 la carga enzimática y la eficiencia es muy baja. El material con
grupos amino (MS-3030-N) presenta una química superficial óptima dándose el
proceso de adsorción de la enzima de forma rápida y sencilla. Este soporte no
presenta una estructura ordenada y el diámetro de poro es mucho mayor que el
tamaño de la enzima por lo que no se impide su lixiviado, como se ha demostrado
sometiendo el biocatalizador a condiciones en las que se favorece el lixiviado de la
enzima a lo largo del tiempo (tras 72 h se lixivia el 20 % con un crecimiento lineal
con el tiempo).

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Resumen de resultados

Tabla 6.3. Propiedades texturales de los materiales silíceos MS-3030 y MS-3030-N y

resultados de inmovilización de lacasa a pH 3,5 sobre MS-3030 y pH 5,5 sobre MS-3030-N.

mmol Carga
Dp BJH SBET Vp Act. Esp. %Lix.
Soporte N/g % Inm.e enzim.
(nm)a (m2/g)b (cm3/g)c (U/mg) g h
SiO2d (mg/g) f
MS-3030 ~30 296 2,5 - 31,76 15,88 0,014 90
MS-3030-N ~30 236 2,1 1,81 99,28 49,64 0,981 4,4

a) Diámetro de poro BJH calculado a partir de la rama de adsorción de la isoterma de adsorción-

desorción de nitrógeno; b) Área superficial calculada empleando el método BET (Brunauer, Emmett y
Teller); c) Volumen de poro estimado a la presión relativa P/Po de 0,97; d) mmol de aminopropilo por
gramo de sílice; e) Porcentaje de enzima inmovilizada para una carga inicial de 50 mg/g; f) Carga
enzimática expresada en miligramos de enzima por gramo de soporte; g) Actividad específica en
Unidades por miligramo de enzima inmovilizada, siendo la unidad U la cantidad de enzima capaz de
transformar 1 µmol de siringaldazina en producto en 1 minuto; h) Porcentaje de enzima lixiviada tras
incubar el biocatalizador durante 24 h en condiciones de alta dilución y baja fuerza iónica a pH 4,5.

Las conclusiones que derivaron de este trabajo llevaron a pensar en la necesidad

de sintetizar materiales mesoporosos ordenados de mayor diámetro de poro, que
permitieran la incorporación de grupos amino sin comprometer el tamaño de poro
adecuado para la inmovilización de la enzima lacasa (Capítulo 2). De esta manera se
empleó un agente expansor de micelas, el 1,3,5-triisopropilbenceno (TIPB), sales
(NH4F) y bajas temperaturas durante las etapas de hidrólisis y condensación de la
síntesis de los soportes para conseguir mayores diámetros de poro y una estructura
final ordenada (Figura 6.7).

TIPB

Surfactante Fuente sílice

P123
NH4F
1,3,5-triisopropilbenceno
OES

Figura 6.7. Obtención de estructuras ordenadas, uniformes y con tamaños de poro diseñados
a medida, mediante el uso de expansores de micelas. Se añade TIPB durante la etapa de
formación de las micelas para conseguir MMO con mayor diámetro de poro.

El estudio de las condiciones de síntesis condujo a la obtención de un material

puramente silíceo ordenado tipo SBA-15 (OES: Ordered Expanded Silica), y con un
diámetro de poro de unos 15 nm. Es decir, suficientemente ancho para que tras una

218
 Resumen de resultados

funcionalización con grupos amino siga siendo apto para alojar la enzima lacasa de
grandes dimensiones. En la Figura 6.8 se muestran sus imágenes SEM, con una
morfología tipo fibrosa (Fig. 6.8 a y b), que consisten en pequeños aglomerados de
partículas más pequeñas. En la Figura 6.8 c y d, se aprecia incluso la mesoporosidad
de estos aglomerados.

a b

5 μm 1 μm

c d

0.5 μm 250 nm

Figura 6.8. Micrografías SEM de SBA-15 con diámetro de poro expandido (OES).

Sobre este soporte se han incorporado grupos amino mediante el método de

anclaje (soporte denominado NGOES: N-Grafted Ordered Expanded Silica), de
representado en la Figura 6.9. Asimismo, se ha obtenido un soporte, NCOES (N-
Cocondensation Ordered Expanded Silica), funcionalizado en la propia etapa de
síntesis mediante el método de cocondensación (Figura 6.10). Este procedimiento
involucra la hidrólisis y condensación simultánea del precursor silíceo
tetraetilortosilicato (TEOS) y el precursor orgánico 3-aminopropiltrietoxisilano
(APTES). Además, se ha utilizado como expansor de micelas TIPB.

219
Resumen de resultados

TEOS

P123 TIPB

Eliminación
surfactante APTS

OES NGOES

Figura 6.9. Funcionalización del material silíceo OES, con diámetro de poro previamente
expandido, mediante anclaje. El grupo orgánico incorporado es el aminopropilo (N).

TEOS

Eliminación
surfactante
P123 TIPB
APTS NCOES

Figura 6.10. Funcionalización del material silíceo SBA-15 mediante cocondensación en

presencia del agente expansor de micelas TIPB.

Los materiales resultantes NGOES (Dp 11,2 nm, 1,2 mmol N/g SiO2) y NCOES
(Dp 17,6 nm, 1,5 mmol N/g SiO2) poseen una estructura ordenada (imágenes TEM
Figura 6.11), un diámetro de poro adecuado, e incorporan grupos amino. En las
imágenes se pueden apreciar las dimensiones de los poros así como la estructura
ordenada que presentan.

220
 Resumen de resultados

a b

5 0 n m

c d

Figura 6.11. Micrografías TEM de las muestras NGOES (a, b) and NCOES (c, d).

Una vez puesto a punto el ensayo de actividad utilizando ABTS como sustrato, se
ha inmovilizado la lacasa a un pH de 3,5 en los soportes puramente silíceos OES y
MS-3030 (denominado en el Capítulo 2 como AS) y a pH 5,5 en los soportes
NGOES, NCOES y MS-3030-N (ó NAS) (Figura 6.12).

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Resumen de resultados

a b

+
SiOH H3N-(CH2)3-Si-
SiO- pI 4.2 +
H3N-(CH2)3-Si-
SiO- +
H3N-(CH2)3-Si-
SiO- +
H3N-(CH2)3-Si-
SiOH +
H3N-(CH2)3-Si-
SiO-

Protonada, pH < pI Desprotonada, pH > pI

Figura 6.12. a) A pH 3,5, la enzima está cargada positivamente (la superficie positiva se
muestra en verde: residuos His + Lys + Arg protonados) y el soporte silíceo negativamente
debido a los grupos siloxano de la superficie del soporte. b) A pH 5,5, la enzima está cargada
negativamente (la superficie negativa se muestra en rojo: residuos Asp + Glu desprotonados)
y el soporte cargado positivamente debido a la protonación de los grupos amino.

En la Tabla 6.4 se muestran los resultados de inmovilización y catalíticos. A

partir de los soportes con estructura ordenada y que incorporan grupos amino
(NGOES y NCOES), se han obtenido biocatalizadores con altas cargas enzimáticas
(~ 170 mg/g), altas actividades catalíticas (~ 50 U/g) y actividades específicas (0,30
U/mg en NGOES y 0,33 U/mg para NCOES). En cuanto a la sílice amorfa comercial
con grupos amino con gran diámetro de poro los valores de carga enzimática y
actividad de la lacasa también fueron muy altos (MS-3030-N (denominado NAS en
los Capítulos 1 y 2): 187 mg/g; 169 U/g; 0,91 U/mg), aunque como se verá
posteriormente, presentan importantes inconvenientes.

A diferencia de los materiales puramente silíceos (OES y MS-3030), en los que la

carga enzimática sigue siendo baja, en los materiales funcionalizados con aminos la
carga enzimática, la actividad del biocatalizador, y la actividad específica aumentan
notablemente. Esto indica de nuevo que la afinidad química de la enzima por la
superficie de los materiales mesoporosos es crucial para tener éxito en la
inmovilización de enzimas (Figura 6.13).

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 Resumen de resultados

Tabla 6.4. Propiedades texturales, grado de funcionalización de los soportes con grupos
amino, resultados de inmovilización de lacasa y actividad catalítica.

Carga Actividad Actividad

Dp BJH Vp mmol tc
Soporte enzim. Máx. biocat. f específica
(nm)a (cm3/g)b N/g SiO2c (h)d e (mg/g) (U/g) (U/mg)g

OES 15 1,2 - 24 14,2 0,56 0,04

NGOES 11,2 1 1,2 2 170 50,7 0,3
NCOES 17,6 1,2 1,5 3,5 173,8 56 0,32
MS-3030 ~30 2,5 - 24 15,8 0,52 0,03
MS-3030-N ~30 2,1 1,8 24 187,1 169,5 0,91

a) Diámetro de poro BJH calculado a partir de la rama de adsorción de la isoterma de adsorción-

desorción de nitrógeno; b) Volumen de poro estimado a la presión relativa P/Po de 0,97; c) mmol de
aminopropilo por gramo de sílice; d) Tiempo de contacto enzima-soporte hasta alcanzar el equilibrio
de adsorción; e) Carga enzimática máxima expresada en miligramos de enzima por gramo de soporte;
f); Actividad del biocatalizador en Unidades por gramo de biocatalizador, utilizando como sustrato
ABTS en el ensayo de actividad catalítico; g) Actividad específica en Unidades por miligramo de
enzima inmovilizada, siendo la unidad U la cantidad de enzima capaz de transformar 1 µmol de ABTS
en producto en 1 minuto.

NH3 NH3 NH3 NH3 SiO

SiO Si-(CH2)3 –NH3
SiO

NH3 NH3 NH3

Figura 6.13. Representación esquemática de la enzima lacasa inmovilizada en el interior del

poro de un soporte ordenado (NGOES o NCOES).

Los biocatalizadores se han sometido a condiciones que favorecen el lixiviado de

la enzima (Figura 6.14). Como se observa en los soportes amorfos MS-3030 y MS-
3030-N (AS y NAS respectivamente en el Capítulo 2), la enzima se libera de forma
continua en el tiempo, no alcanzando un máximo en el rango de tiempo estudiado. El
carácter no permanente de las uniones que se establecen, junto con un diámetro de
poro muy amplio (~30 nm), permite el lixiviado de la proteína. Estos
biocatalizadores mostraron buenos resultados de carga y actividad enzimática, que se
debían a la ausencia de limitaciones de la enzima para entrar en los poros.
Precisamente esta ausencia de restricción de movimiento permite la fácil liberación
de enzima al exterior.

223
Resumen de resultados

Sin embargo, en el caso de los soportes mesoestructurados (NGOES y NCOES),

con un tamaño de poro adaptado a las dimensiones moleculares de la enzima, y con
alta afinidad química se consiguió que la enzima apenas lixiviara de los soportes (< 4
%), a pesar de no existir una unión covalente entre la enzima y el soporte (Figura
6.14). Como se observa, muestran perfiles de lixiviado con saturación. El lixiviado
inicial se debe probablemente a las moléculas de enzima inmovilizadas en la
superficie externa de las partículas del material.

8
MS-3030
7 MS-3030-N
OES
6 NGOES
NCOES
5
% Lixiviado

0
0 4 8 12 16 20 24 28 32 36 40 44 48

Tiempo (h)

Figura 6.14. Perfiles de lixiviado de los biocatalizadores de lacasa que han sido suspendidos
en condiciones en las que se favorece el lixiviado de la enzima del interior de los materiales:
Baja fuerza iónica y alta dilución. La concentración de enzima lixiviada se ha medido
mediante el ensayo de Bradford.

También el alto confinamiento de las moléculas de enzima dentro de los poros de

estos materiales hace que se evite el desplegamiento de la estructura de la proteína lo
que mantiene la estabilidad del biocatalizador en medio orgánico a pH ácido (10 %
de etanol y pH 3,5) con respecto a la enzima libre (Figura 6.15).

224
 Resumen de resultados

110
10% Etanol, pH 3.5
100
90
80
70

%Actividad
60
50
40
30 MS-3030-N
20 NCOES
10
NGOES
Enzima soluble
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Tiempo (h)

Figura 6.15. Incubación de los biocatalizadores en 10 % de etanol y a un pH ácido de 3,5.

Se ha ensayado la actividad enzimática con el sustrato ABTS.

Los biocatalizadores obtenidos por inmovilización de lacasa sobre NGOES y

NCOES presentan características muy ventajosas, ya que la enzima se retiene
permanentemente, como si de un enlace covalente se tratara. Pero con la ventaja de
que la inmovilización mediante adsorción se realiza de forma más sencilla y rápida
que la covalente. Además, se mantienen altas cargas enzimáticas y altas actividades
catalíticas. Incluso después de la inactivación de la enzima, la naturaleza no
covalente del enlace también puede permitir la recuperación y reutilización de los
soportes, con el consiguiente ahorro, ya que el soporte en algunos sistemas puede
llegar a ser tan costoso como la enzima. Por todo ello, estos biocatalizadores tienen
propiedades prometedoras para posibles aplicaciones industriales.

Se quiso llegar más allá en el diseño de los soportes y se diseñaron materiales

híbridos orgánicos-inorgánicos donde el grupo orgánico forma parte de la propia
pared de material, y el tamaño de poro final no se reduce al incorporar los grupos
funcionales (Capítulo 3). De esta manera, se han desarrollado soportes para la
inmovilización de la enzima lipasa, denominados PMO (Periodic Mesoporous
Organosilica), donde el material incorpora grupos hidrófóbicos (grupos etileno)
dentro de la propia pared. Estos grupos pueden interaccionar con la superficie
hidrofóbica de la enzima consiguiendo altas cargas de lipasa. Asimismo se han

225
Resumen de resultados

desarrollado soportes para la enzima lacasa (PMA: Periodic Mesoporous

Aminosilica) que integran grupos amino en su estructura.

Biocatalizadores PMO-lipasa

En este trabajo se ha empleado etileno como grupo funcional orgánico dentro de

la matriz mesoporosa ordenada, para ello se ha utilizado el alcoxisilano BTME
(bis(trimetoxisililetano)), obteniendo un soporte de naturaleza hidrofóbica,
denominado PMO. En la Figura 6.16 se muestran imágenes SEM; como se aprecia
se trata de partículas con una morfología fibrosa. Mediante difracción de rayos X
(Fig. S2, Capítulo 3), isotermas de adsorción-desorción de nitrógeno a -196 ºC (Fig.
2 a, Capítulo 3), análisis químico elemental y termogravimetría se han determinado
sus propiedades estructurales, texturales y composición química (Tabla 6.5). Para el
soporte PMO, se ha alcanzado un diámetro de poro (Dp de 7.1 nm) suficientemente
grande para inmovilizar la enzima lipasa (con unas dimensiones de 3 x 4 x 5 nm), y
mayor grado de hidrofobicidad (con un contenido de carbono de 16,1 mmol de C/g
SiO2), en comparación con el soporte OAS (Octyl-amorphous silica), que incorpora
mediante el método de anclaje 4,2 mmol de C/g SiO2.

a b

5 μm 2 μm

Figura 6.16. Micrografías SEM de los materiales PMO (Ethylene-bridged periodic

mesoporous organosilica).

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 Resumen de resultados

Tabla 6.5. Propiedades estructurales, texturales y composición química de los soportes

silíceos.

a0 a Dp BJH b SBET c Vp d mmol C/g mmol N/g

Soporte
(nm) (nm) (m2/g) (cm3/g) SiO2 e SiO2 f

AS - 30 285 2.2 - -
OAS - 30 212 1.6 4.2 -
NAS - 30 236 2.1 - 1.8
PMO 11.7 7.1 882 1.0 16.1 -
PMA 12.4 7.2 594 0.7 - 1.5
E-PMA 15.9 10.4 264 0.6 - 1.0

a) parámetro de celda (ao = 2·d100/31/2); b) Diámetro de poro BJH calculado a partir de la rama de
adsorción de la isoterma de adsorción-desorción de nitrógeno; c) Área superficial calculada
empleando el método BET (Brunauer, Emmett y Teller); d) Volumen de poro estimado a la presión
relativa P/Po de 0,97; e) mmol de carbono por gramo de sílice; f) mmol de aminopropilo por gramo de
sílice.

CH2 H2C H2C H2C

CH2 CH2 CH2 CH2

-
Si Si Si Si
O O
O O
Región O O O O
hidrofóbica
- Si Si Si Si
O O O O
CH2 CH2 CH2 CH2

CH2 CH2 CH2 CH2

-
Si Si Si
O Si
O O O
H2C H2C H2C H2C

CH2
CH2 CH2 CH2

Región Si
Lipasa inmovilizada -
Si Si Si
hidrofílica O O O O
en PMO

Figura 6.17. Inmovilización de lipasa de Candida antarctica B en PMO que incorpora

grupos etileno.

Mediante el control de la composición y naturaleza de los componentes orgánicos

de las paredes silíceas se ha podido modular el carácter hidrofílico/hidrofóbico de las
estructuras de PMO sintetizadas. De este modo se ha inmovilizado lipasa por

227
Resumen de resultados

adsorción no covalente sobre estos soportes, ya que estas enzimas tienen alta afinidad
hacia superficies hidrofóbicas (Figura 6.17).

Los resultados de inmovilización (Tabla 6.6) muestran como la sílice amorfa

hidrofobizada con grupos octilo (OAS) puede alojar una carga enzimática muy alta,
de unas cuatro veces mayor que en PMO, que puede explicarse por su mayor tamaño
de poro. El biocatalizador PMO-lipasa muestra propiedades específicas destacables,
como es la alta carga enzimática, alta actividad catalítica, y sobretodo mayor
actividad específica que en el soporte OAS.

Tabla 6.6. Inmovilización de lipasa en OAS y PMO. La carga enzimática inicial ofrecida es
de 600 mg de lipasa por gramo de soporte.

Actividad Actividad
Carga enzim.
Biocatalizador tc a (h) biocatalizador c específica d
Máx. b (mg/g)
(UTB/g) (UTB/mg)

OAS 24 400 37000 92,5

PMO 1,5 94 18984 202,3

a) Tiempo de contacto enzima-soporte hasta alcanzar el equilibrio de adsorción; b) Carga enzimática

máxima expresada en miligramos de enzima por gramo de soporte; c) Actividad del biocatalizador en
Unidades por gramo de biocatalizador, utilizando como sustrato tributirina en el ensayo de actividad
hacia la hidrólisis de tributirina; g) Actividad específica en Unidades por miligramo de enzima
inmovilizada, siendo la unidad U la cantidad de enzima capaz de transformar 1 µmol de tributirina en
producto en 1 minuto.

Un parámetro importante a comprobar es la resistencia de la lipasa al lixiviado, ya

que las uniones enzima-soporte no son covalentes. El lixiviado de la enzima
inmovilizada en los soportes se estudia suspendiendo los biocatalizadores en medido
acuoso de alta dilución y a un pH por encima del pI tanto de la enzima como del
soporte, de modo que ambas especies tienen una carga neta negativa y hay repulsión
entre ellas (Figura 6.18). El perfil de lixiviado de la sílice amorfa funcionalizada con
grupos octilo (OAS) muestra una liberación continua y rápida de la enzima, que se
mantiene a lo largo de todo el tiempo estudiado. La difusión de la enzima no se ve
limitada porque el diámetro de poro medio de estos materiales es de 30 nm (5 veces
superior a la enzima). Por el contrario, el material PMO muestra un perfil de
lixiviado con saturación, alcanzándose un máximo inferior al 18 % de lipasa
desorbida. El lixiviado inicial se puede deber a moléculas de enzima inmovilizadas

228
 Resumen de resultados

en la superficie externa de las partículas del material. Esto sugiere que el tamaño de
poro adaptado y la alta afinidad de la lipasa por las superficies interiores hidrofóbicas
del soporte debido a la presencia de grupos etileno evitan el lixiviado,
estabilizándose la enzima en el interior de los canales y quedando altamente retenida
sin recurrir al enlace covalente.

70
65 OAS
60 PMO
55
50
45
% Lixiviado

40
35
30
25
20
15
10
5
0
0 2 4 6 8 10 12 14 16 18 20 22 24

Tiempo (h)

Figura 6.18. Proteína lixiviada del soporte organosilíceo periódico mesoporoso (PMO) y de
la sílice amorfa con grupos octilo en medio acuoso.

La presencia del enzima en el interior de los poros se ha comprobado

indirectamente por electroforesis de los biocatalizadores previamente sometidos a
lixiviado para eliminar la enzima inmovilizada sobre la superficie externa de las
partículas. Como se aprecia en la Figura 6.19, en el caso de los biocatalizadores de
lipasa (OAS y PMO), se observa una banda mayoritaria correspondiente al peso
molecular de la lipasa (32 KDa). Esto es una evidencia de que la enzima estaba
dentro de los poros.

229
Resumen de resultados

1 2 3 4

Figura 6.19. Electroforesis SDS-PAGE. Gel de poliacrilamida al 12 % (p/v), teñido con azul
de Coomasie G-250, tampón de ruptura en condiciones estándar (pH 6,5). Carril 1: Patrón
estándar de proteínas; Carril 2: lipasa soluble; Carril 3 proteína forzada a salir del soporte
PMO; Carril 4 proteína forzada a salir del soporte OAS.

En la Figura 6.20 a y b se muestra la estabilidad de los soportes frente a metanol.

Se observa alta estabilidad del soporte PMO, esto es posible porque como se ha
indicado estos materiales integran componentes orgánicos e inorgánicos en su
estructura formando una red híbrida, de modo que el canal rodea las moléculas de
enzima, creando un espacio restringido, y limitando su movilidad, su inactivación y
el desplegamiento de la proteína. En un medio orgánico (metanol), se produce una
reorientación de las cadenas laterales de aminoácidos hidrofóbicos de la proteína
hacia su superficie. Estos grupos pueden a su vez interaccionar con la pared híbrida
orgánica-inorgánica del PMO, con lo que se establecen nuevas interacciones
hidrófobicas adicionales con los grupos etileno. Es decir, el número de puntos de
contacto con la enzima aumenta, lo que estabiliza la estructura semidesplegada de la
enzima. Esto explica la alta estabilidad del biocatalizador lipasa-PMO en presencia
de metanol, que no se daría nunca sin la protección del soporte, ya que la enzima
libre se inactiva rápidamente en este medio.

230
 Resumen de resultados

a 100 b 100
PMO
90 90 OAS
80 80 Lipasa soluble
70 70
%Actividad

%Actividad
60 60

50 50

40 40

30 PMO 30

20
OAS 20
Lipasa soluble
10 10

0
50% Agua : 50% Metanol. T = 25 ºC 0 0% Agua : 100% Metanol. T = 25 ºC
0 2 4 6 8 10 12 14 16 18 20 22 24 0.0 0.5 1.0 1.5 2.0 2.5 3.0

Tiempo (h) Tiempo (h)

Figura 6.20. Estabilidad de lipasa soluble e inmovilizada sobre OAS y PMO en: a) 50%
agua: 50% metanol; b) 100 % metanol. Ensayos realizados a 25 ºC.

Biocatalizadores PMA-Lacasa

Ha habido algunos intentos en la literatura para introducir grupos que contienen

nitrógeno en las paredes del PMO. Asefa et al, 2003 [22] preparó materiales híbridos
mesoporosos amino-silíceos que contenían grupos funcionales de tipo amina en la
estructura de la red mesoporosa. Para ello, utilizó la amonolisis térmica de PMO bajo
un flujo de gas amoníaco. Determinó que la cantidad de grupos amino introducidos
en los materiales dependía fuertemente de la temperatura de la amonolisis. El
diámetro de poro de los soportes fue de alrededor de 3-4 nm.

Wahab et al., 2004 [23] publicó un nuevo intento de preparar materiales

mesoporosos organosilíceos que incorpora grupos amina puente. Para la
funcionalización utilizó 1,2-bis (trietoxisilil) etano (BTEE) y bis [(3-trimetoxisilil)
propil] amina (BTMSPA) en presencia de bromuro de cetiltrimetilamonio (CTAB)
en condiciones básicas. Desafortunadamente, cuando el contenido de BTMSPA se
incrementaba en el medio de síntesis el grado de ordenamiento de los materiales
disminuía. El tamaño de poro obtenido fue de unos 3,5 nm.

Hofmann et al., 2006 [24] observó una transición interesante de la mesoestructura

de este sistema al aumentar el contenido BTMSPA en la mezcla de partida durante la
cocondensación de 1,2-bis (trimetoxisilil) etano (BTME) y BTMSPA, en presencia
de cloruro de octadeciltrimetilamonio (OTCA) [24]. Cuando se cambiaba la relación

231
Resumen de resultados

de BTME / BTMSPA de 90:10 a 55:45, se producía un cambio de simetría hexagonal

2D (p6mm) a una mesofase cúbica. Con mayores concentraciones de BTMSPA en la
mezcla de reacción se producía el colapso de la estructura.

Tan et al., 2006 [25] obtuvo materiales con mesoporos uniformes cuando utilizaba
una mezcla de BTMSPA con el surfactante CTAB. Cuando se utiliza BTMSPA, la
cadena hidrófila ayuda a promover el co-ensamblaje del precursor de micelas,
CTAB. El material resultante tenía unas propiedades texturales poco destacables
(reducido volumen de poro, área de superficie específica baja y un diámetro de poro
pequeño (3,1 nm)), pero poseía una combinación óptima de mesoporosidad
uniforme, y las aminas estaban integradas en las paredes de los poros.

Los tamaños de poro obtenidos que se han encontrado en bibliografía fueron

siempre inferiores a los que se consiguen con materiales puramente silíceos, y no
exceden de 6,2 nm. Este factor limita las posibles aplicaciones de PMA como
soportes para la inmovilización de enzimas de grandes dimensiones.

Aprovechando la experiencia adquirida a la hora de sintetizar materiales en

presencia de expansores de micelas (tanto puramente silíceos como mediante
cocondensación con la dificultad añadida de funcionalizar en un único paso), se
realizó un profundo estudio de las condiciones de síntesis. De esta manera se
consiguió obtener un novedoso material en el que, además de los grupos etileno, se
incorporan los grupos amino secundarios en la pared del soporte, formando una red
híbrida orgánica-inorgánica, y además, mediante el empleo de expansores de
micelas, el diámetro de poro es suficiente para albergar y retener a la enzima lacasa
en el interior de sus canales (Figura 6.21). En la Figura 6.22 se muestran unas
micrografías obtenidas mediante SEM donde se observa la morfología de las
partículas tipo placas apiladas.

232
 Resumen de resultados

BTME

Eliminación
P123 y TIPB surfactante

BTMSPA E-PMA

Figura 6.21 Esquema del procedimiento de síntesis de E-PMA (Pore-Expanded Periodic

Mesoporous Aminosilica).

En la Tabla 6.5 se muestran las propiedades texturales y el contenido de grupos

orgánicos tanto para PMA sin expansores, como E-PMA (Expanded-Periodic
Mesoporous Aminosilica) en el que se han empleado expansores de micelas. En el
caso de lacasa, como se observa en la Tabla 6.7 se inmoviliza tanto en PMA como
en E-PMA, pero se alcanzan mejores cargas y actividad específica sobre el soporte
de mayor diámetro de poro. Como se muestra en la Figura 6.23, es de destacar la
elevada afinidad química enzima-soporte en el material E-PMA, que elimina
prácticamente el lixiviado pese a la naturaleza no covalente de la unión.

a b

4 μm 1 μm

Figura 6.22. Micrografías SEM de la muestra E-PMA.

233
Resumen de resultados

Tabla 6.7. Inmovilización de lacasa en diferentes soportes.

Carga enzim. Actividad Actividad

Soporte pH a tc b (h) Máx. c biocat. d específica
(mg/g) (UABTS/g) (UABTS/mg)e
NAS 5.5 24 187 170 0.91
PMA 5.5 2 42 4.7 0.11
E-PMA 5.5 1.5 88 29 0.33
E-PMA 6 1.3 119 19 0.16

a) pH de inmovilización; b) Tiempo de contacto enzima-soporte hasta alcanzar el equilibrio de

adsorción; c) Carga enzimática máxima expresada en miligramos de enzima por gramo de soporte; d);
Actividad del biocatalizador en Unidades por gramo de biocatalizador, utilizando como sustrato
ABTS en el ensayo de actividad catalítico; e) Actividad específica en Unidades por miligramo de
enzima inmovilizada, siendo la unidad U la cantidad de enzima capaz de transformar 1 µmol de ABTS
en producto en 1 minuto.

10
NAS
9
E-PMA
8

6
% Lixiviado

0
0 4 8 12 16 20 24 28 32 36 40 44 48

Tiempo (h)

Figura 6.23. Proteína lixiviada del soporte organosilíceo periódico mesoporoso (E-PMA) y
de la sílice amorfa con grupos amino en medio acuoso.

Se ha verificado la presencia de la enzima en el interior de los poros de los

soportes a través de estudios directos mediante técnicas de microscopía electrónica
avanzada (Capítulos 4 y 5) e indirectos. El método indirecto ha consistido en
recuperar el biocatalizador después de los ensayos de lixiviado, en el momento en
que no se desorben más moléculas de enzima de las partículas del biocatalizador
(especialmente de las superficies externas), y se han sometido a condiciones de

234
 Resumen de resultados

electroforesis SDS-PAGE. El tampón de ruptura para hacer la electroforesis está

compuesto principalmente por SDS que es un agente desnaturalizante de proteínas, y
mercaptoetanol que rompe los enlaces disulfuro. Además, la muestra de
biocatalizador junto con el tampón de ruptura (a pH 6,5) se ha hervido durante 5
minutos. Después de este tratamiento, la estructura terciaria de la enzima se pierde y
sólo consiste en una cadena lineal de aminoácidos que pueden ser fácilmente
liberadas de las partículas del material de soporte. Los sobrenadantes después de este
tratamiento drástico han sido estudiados en la electroforesis (Figura 6.24).

En el caso de los biocatalizadores de lacasa, se observa en la Figura 6.24 a la

presencia de banda en el soporte de sílice amorfa funcionalizada con grupos amino
mediante anclaje (NAS), pero no así en el soporte E-PMA. De hecho, solo se detectó
lacasa en el sobrenadante cuando el biocatalizador fue incubado a pH 14 tras el
tratamiento en las condiciones desnaturalizantes de SDS-PAGE (Figura 6.24 b), es
decir, en condiciones en las que los aminos del soporte están desprotonados. Con
estos materiales se han combinado las ventajas de un poro regular y de tamaño
adecuado, con una composición química de la superficie del soporte que interacciona
con la superficie de la enzima y, por tanto, influye favorablemente para alcanzar altos
rendimientos de inmovilización y retención de la enzima.

a) 1 2 3 b)
1 2 3

Figura 6.24. Electroforesis SDS-PAGE. Gel de poliacrilamida al 12 % (p/v), teñido con azul
de Coomasie G-250. A) Tampón de ruptura en condiciones estándar (pH 6,5). Carril 1:
Patrón estándar de proteínas; Carril 2: Proteína forzada a salir del soporte E-PMA; Carril 3
Proteína forzada a salir del soporte NAS; B) Carril 1: Patrón estándar de proteínas; Carril 2:
Proteína forzada a salir del soporte E-PMA a pH 14,0; Carril 3: lacasa soluble.

235
Resumen de resultados

La Figura 6.25 muestra la estabilización de los biocatalizadores lacasa-PMA y

lacasa-NAS en 10 % etanol. Al estar compuesta la pared también por grupos etileno
hidrófobos, la reorientación de las cadenas laterales de aminoácidos hidrofóbicos en
presencia de etanol da lugar a nuevas uniones enzima-soporte. De modo análogo al
descrito para la lipasa-PMO en metanol, este refuerzo de la interacción es lo que
estabiliza la conformación parcialmente desplegada (pero activa) de la enzima.

110
10 % Ethanol, pH 3.5
100
90
80
70
%Actividad

60
50
40
30
20 E-PMA
10
NAS
Lacasa soluble
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Tiempo (h)

Figura 6.25. Estabilidad de lacasa, soluble e inmovilizada en E-PMA y NAS, en disolución

ácida a pH 3,5 y en presencia de 10 % de etanol.

Este trabajo ha demostrado que adecuando el diámetro de poro con el tamaño de

la enzima, y estableciendo una intensa afinidad química se mejora la estabilidad de la
enzima inmovilizada y se reduce su lixiviado prácticamente hasta la supresión, sin
necesidad de que la inmovilización sea por unión covalente.

La localización de la enzima después de la inmovilización es una cuestión clave.

Es fundamental constatar que las enzimas se sitúan principalmente dentro de la
estructura porosa y no adsorbidas en la superficie de la partícula externa [26, 27], a
pesar de que los resultados descritos así lo indicaban.

En un intento de localizar directamente a la enzima dentro de las cavidades y

canales del propio soporte se han utilizado técnicas avanzadas de microscopía
electrónica de transmisión. Utilizando imágenes de ultra alta resolución obtenidas
con un microscopio con corrección de la aberración esférica y combinando un
detector STEM-HAADF (imágenes de campo oscuro obtenidas con electrones

236
 Resumen de resultados

dispersados a ángulos altos) con espectroscopia EELS (pérdida de energía de

electrones), se ha conseguido la localización precisa de la enzima en el interior de los
poros del soporte (Capítulo 4).

La determinación de la concentración de enzima en la suspensión y sobrenadante

durante el proceso de inmovilización (determinada por ensayos Bradford o ensayos
de actividad espectroscópicos) pueden mostrar que la enzima se une a las partículas
porosas, pero no prueban que la enzima está realmente en los poros.

Las técnicas indirectas más comúnmente usadas para demostrar que las enzimas
están dentro de la poros son la adsorción de N2, difracción de rayos X (XRD),
espectroscopia infrarroja con transformada de Fourier (FTIR) y el análisis
termogravimétrico (TGA). A continuación se comenta cada una de ellas, así como
sus limitaciones:

La adsorción de N2 es un método estándar de caracterización de los materiales

porosos para conocer el tamaño de poro, área superficial específica, y volumen de
poro. También es común el uso de esta técnica para evaluar el grado de ocupación de
los poros mediante la medición de la adsorción de N2 antes y después de la
inmovilización de proteínas [28]. La reducción de poro que se mide después de la
inmovilización, no se debe necesariamente a proteínas que ocupan los poros, sino
que también puede ser explicada por las proteínas que bloquean las aberturas de los
poros, que a su vez pueden dificultar la entrada del nitrógeno gaseoso. Por lo tanto,
esta técnica no asegura que las proteínas se encuentran en los poros, además tampoco
es posible utilizar la adsorción de nitrógeno para determinar la cantidad de moléculas
de proteínas inmovilizadas. Ya que las proteínas son muy grandes en comparación
con las moléculas de nitrógeno, el área superficial total calculada con el método BET
rara vez corresponde a la superficie accesible para las proteínas dentro de los poros.
Sobre todo en los materiales tipo SBA-15, donde las propias paredes de los poros
contienen además microporos, inaccesibles para las enzimas.

La difracción de rayos X aporta información directa de la estructura del material.

Para los materiales mesoporosos ordenados, los difractogramas son el reflejo de la
simetría que gobierna la ordenación de los poros, no de los átomos de la estructura
que es amorfa. La regularidad que ofrecen los poros permite obtener difractogramas
característicos, permitiendo identificar simetrías, asignar los índices de Miller de
cada familia de planos y dilucidar estructuras formadas por los sistemas de canales.

237
Resumen de resultados

Los patrones de difracción presentan picos sólo a bajos ángulos, reflejando los
grandes parámetros de red que presentan, debido al tamaño mesoporoso.

Cuando se adsorbe una sustancia dentro de la estructura porosa se observa, por lo

general, una disminución en la intensidad de los picos debido a la presencia de
medios con diferente poder dispersor de los rayos X, dentro de los poros en
comparación con los poros vacíos, dando como resultado un contraste reducido entre
adsorbente y adsorbato [28]. Así por ejemplo, la inmovilización de lacasa en SBA-
15-L y SBA-15-S da como resultado una disminución en las intensidades de los
picos para ambos soportes, lo que podría indicar que los poros se han llenado con la
proteína (Figura 6.26). Otra cuestión a considerar es que la propia preparación de la
muestra influye en las intensidades de los picos a bajo ángulo.

100 SBA-15-L 100 SBA-15-S

SBA-15-L-Lacasa SBA-15-S-Lacasa
I (a.u.)

I (a.u.)

110
200
110
200

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
2 (º) 2 (º)

Figura 6.26. Difractogramas a bajo ángulo de los soportes SBA-15-L (L: long channel) y
SBA-15-S (S: short channel) antes y después de la inmovilización de la enzima lacasa.

Mediante la comparación de los espectros FTIR de la proteína inmovilizada en el

soporte con los espectros de la proteína libre y del soporte por separado es posible
determinar si la proteína se une al soporte o no. El espectro FTIR de sílice contiene
una banda de absorción fuerte que representa la abundancia de grupos silanol en la
superficie. Hay estudios de FTIR para la inmovilización de lipasa [29, 30], que
muestran que después de la adsorción de la lipasa, la banda se redujo drásticamente
lo que sugiere la formación de enlaces de hidrógeno entre los grupos silanol y los
grupos amino de la lipasa.

Los resultados obtenidos a partir de mediciones de FTIR dan información acerca

de la presencia de enlaces de hidrógeno, pero no dan información sobre la

238
 Resumen de resultados

abundancia de estos enlaces. Por lo tanto, FTIR puede ser una técnica adecuada para
determinar que los enlaces se forman entre la proteína y las partículas del soporte,
pero no confirma que las proteínas están en el interior de los poros.

TGA es un método para caracterizar materiales mediante la medición de cambios

en las propiedades físicas y químicas, tales como transiciones de fase, deshidratación
y descomposición, como una función de la temperatura. TGA también se puede
utilizar para determinar la cantidad de material orgánico en una muestra. Al aumentar
la temperatura, se induce la degradación de la proteína, que conduce a la
descomposición de la materia orgánica del soporte inorgánico. La pérdida de peso
que experimenta el biocatalizador con la temperatura corresponde a la cantidad de
proteína adsorbida en el soporte. Aunque el análisis termogravimétrico es una técnica
adecuada para determinar la cantidad de proteína adsorbida al soporte, no revela la
ubicación de la proteína.

Por tanto, la adsorción de nitrógeno, DRX, FTIR y TGA sólo dan información
indirecta con respecto a la presencia de proteínas dentro de los poros y se debe tener
cuidado al afirmar que una de estas técnicas prueba que las proteínas se encuentran
en los poros.

En cuanto a las técnicas directas para localizar a las enzimas inmovilizadas,

principalmente existen: 1) técnicas de microscopía de fluorescencia; 2) técnica de
microscopía electrónica de transmisión en combinación con tinción en oro (TEM-
IGS); y 3) la técnica que se ha empleado en la presente Tesis, y que se trata de
STEM-HAADF-EELS.

La primera técnica consiste en marcar una proteína con un colorante fluorescente

antes de la inmovilización. Posteriormente, se pueden detectar las proteínas marcadas
con microscopía de fluorescencia. Los estudios publicados muestran, mediante
perfiles de intensidad de fluorescencia e imágenes confocales, que la mayoría de las
proteínas se adsorben en la superficie exterior, y en las entradas de los poros de las
partículas [31-33]. Un inconveniente de esta técnica es que no es posible visualizar
moléculas individuales de proteínas o incluso poros individuales debido a la limitada
resolución (máximo 200 nm).

En la técnica TEM-IGS se combina la microscopía electrónica de transmisión

(TEM) con la post tinción IGS (ImmunoGold Staining) que tiñe las proteínas [34].

239
Resumen de resultados

Después de la inmovilización, las partículas de sílice deben ser cortadas en películas

delgadas. La combinación de IGS y TEM tiene suficiente resolución para dar una
imagen de la distribución de las moléculas de proteínas individuales en un soporte
poroso. Sin embargo, sólo es adecuada para la determinación de la ubicación de las
proteínas y no es adecuada para la cuantificación. Las condiciones drásticas que se
necesitan para el procedimiento inmunoquímico, además del proceso de corte de la
película en la sección transversal pueden causar un cambio conformacional de la
proteína, lo que puede conducir a la pérdida del sitio de reconocimiento del
anticuerpo. Además, como las películas han de ser muy finas (~ 60-80 nm), las
proteínas localizadas en la superficie interna de las secciones pueden no ser
accesibles para la tinción de oro, lo que conduce a una subestimación de la carga de
proteínas.

Las técnicas utilizadas en la presente Tesis pertenecen a lo que denominamos

Microcopia Electrónica Avanzada, es decir, en un Microscopio TEM de aberración
corregida, se emplea el modo STEM (Scanning Transmission Electron Microscopy)
y el detector de ángulo alto HAADF (High Angle Annular Dark Field) para obtener
imágenes de alta resolución en las que el contraste depende del número atómico, lo
que facilita la interpretación de las imágenes. Por otra parte, se combina esta imagen
con la adquisición de espectros EELS (Electron Energy Loss Spectroscopy) que
permite hacer una análisis químico local con resolución nanométrica.

Estas técnicas son muy novedosas en el campo de la inmovilización de enzimas

en materiales mesoporosos ordenados, y permiten detectar los grupos funcionales del
soporte, la presencia de macromoléculas en el interior de los poros, así como su
interacción con la matriz silícea. La dificultad radica en que los materiales son
altamente inestables bajo el haz de electrones, y en que el carbono y el nitrógeno
(componentes mayoritarios de las enzimas) tienen baja capacidad para dispersar
electrones y generar contraste.

Mediante un adecuado control de las técnicas, se ha podido resolver el contraste

C-Si alcanzando niveles de resolución del análisis químico por debajo del
nanómetro, y se ha mantenido la estructura del material sin alterar gracias al control
preciso de la dosis de electrones. Combinando imágenes STEM-HAADF con la
adquisición de espectros EELS se ha podido confirmar la localización de la enzima
dentro de los poros. En la Figura 6.27 se muestran para el biocatalizador PMO-

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 Resumen de resultados

lipasa, imágenes de STEM-HAADF y el análisis EELS de la muestra. El espectro

EELS en la región de los poros muestra la señal de nitrógeno, que identifica
inequívocamente la presencia del enzima. Sin embargo, debido a que el contenido de
carbono del enzima es muy superior al de nitrógeno, para mejorar la sensibilidad del
análisis, el mapa de colores de la Figura 6.27 c se ha obtenido a partir de la
intensidad relativa de la señal de carbono. El color rojo muestra las zonas ricas en
carbono y confirma la presencia de enzima en el interior de los poros.

Figura 6.27. a) Imagen STEM-HAADF con aberración corregida de la muestra PMO-Lipasa

en la orientación [001] de la estructura porosa con simetría p6mm y b) ampliación de un área
seleccionada, donde las cavidades aparecen en negro. c) Mapa de proporción relativa C/O
obtenido a partir de la señal EELS que muestra en rojo zonas ricas en carbono, confirmando
la presencia de lipasa en los poros. d) Espectro EELS en la zona del recuadro marcado en (c)
mostrando el borde K de nitrógeno que identifica a la enzima.

Utilizando la experiencia previa del grupo en encapsulación de lipasa, se han

desarrollado biocatalizadores por un procedimiento in-situ, en el que se sintetizan los
materiales en presencia de la enzima. La enzima lacasa carece de superficie
hidrofóbica (a diferencia de la lipasa), por lo que se ha probado a encapsular la
enzima en presencia de aditivos (n-butilamina) que provean a la enzima de suficiente
hidrofobicidad para promover su afinidad con la región apolar que forma la parte

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Resumen de resultados

interna de las micelas de surfactante que dirigen la formación de la estructura porosa

del material silíceo, quedando la enzima previsiblemente atrapada en su interior. Para
ello, se han modificado las condiciones y el medio de síntesis altamente agresivo de
los materiales mesoporosos a unas condiciones suaves para mantener la actividad
enzimática de la lacasa tras la encapsulación: pH neutro y temperatura de síntesis
fijada en 35 ºC, además de adicionar sales (KCl) que ayuden al ordenamiento de la
estructura y aditivos (n-butilamina) para generar un medio suficientemente hidrófobo
para que la enzima interaccione con las colas hidrófobas del surfactante (Figura
6.28). Esta estrategia ha servido para obtener materiales en los que se ha comprobado
que la enzima está retenida y encapsulada de forma activa en su interior (Capítulo 5)
mediante técnicas bioquímicas, de microscopía (STEM-HAADF), y en ensayos de
actividad. Si bien estos materiales no resultaron ser altamente ordenados, ya que las
condiciones de síntesis no son favorables al ordenamiento de la estructura.

pKa 10.78
pI 4.2 F127
KCl

H2O
pH neutro
T= 35ºC

TMOS
T= 35ºC

In-situ-Lacasa

Figura 6.28. Estrategia de inmovilización in-situ de lacasa. La unión no covalente del

compuesto orgánico añadido con la superficie de la enzima se promueve a través de un grupo
amino que, en las condiciones de síntesis, presenta alta afinidad con la superficie de la
enzima. Las cadenas apolares del aditivo orgánico favorecen la incorporación del complejo
enzima-aditivo en la región interior, hidrofóbica, de las micelas de surfactante.

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 Resumen de resultados

Trabajos pendientes de publicación y trabajo futuro:

El desarrollo de la labor descrita en la presente Memoria ha tenido continuidad en

numerosos trabajos todavía en curso, y que por ello no han podido ser recogidos en
la misma. No obstante, y dada su estrecha relación con esta Tesis Doctoral se
describen brevemente a continuación.

Aplicación de los biocatalizadores de lacasa en una reacción de oxidación de un

compuesto fenólico presente en vino, el ácido cafeico. Como se ha mostrado en los
Capítulos 2 y 3 de la Tesis, todos los biocatalizadores obtenidos presentan alta
estabilidad en medio orgánico. Por ello, se han ensayado en una reacción de
oxidación de ácido cafeico a pH 3,5, tanto en presencia, como en ausencia de etanol
(10 % etanol). Se ha obtenido con estos biocatalizadores una oxidación suave y
controlada de este compuesto fenólico modelo, que permite el mantenimiento de una
alta capacidad antioxidante en la mezcla de reacción, y alto contenido en compuestos
fenólicos. Estas características podrían permitir evitar o al menos limitar la adición
de sulfitos como reductores en el proceso de maduración del vino (para prevenir los
procesos de oxidación (maderización) del vino), lo que abre una futura y
prometedora vía de aplicación.

También se ha probado a inmovilizar otro tipo de enzimas en los soportes silíceos.

Para ello, se ha empleado β-glucosidasa (EC 3.2.1.21). Esta enzima puede recibir
diferentes nombres entre ellos: β-D-glucosidasa, β-1,6-glucosidasa y celobiasa [35].
Se encarga de catalizar la hidrólisis del enlace O-glicosídico de los hidratos de
carbono liberando moléculas de glucosa. Su origen es principalmente de hongos; la
que se emplea en este trabajo es producida por el organismo Aspergillus niger [36].
Esta enzima no está aún cristalizada por lo que no aparece en la base de datos del
Protein Data Bank (PDB), por ello se ha modelizado utilizando β-Glucosidasa de
Aspergillus aculeatus (4IIB.pdb) que tiene una secuencia de 841 aminoácidos (aa.)
frente a 860 aa. de la β-Glucosidasa de Aspergillus niger. En la Figura 6.29 se
muestra su estructura modelizada. Como se observa se trata de una enzima dimérica
formada por dos subunidades de mismo peso molecular, siendo sus dimensiones
aproximadas de ~ 12.3 nm x ~10.7 nm x ~ 8.1 nm, su peso molecular es de unos 240
KDa y su punto isoeléctrico de 4,0 [37], además está glicosilada.

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Resumen de resultados

Figura 6.29. Estructura modelizada de β-Glucosidasa de Aspergillus niger.

En el caso de esta enzima, con grandes dimensiones moleculares y alto peso

molecular, las cargas enzimáticas que se consiguen mediante inmovilización post-
síntesis son limitadas (tanto por adsorción como por enlace covalente). En cambio,
mediante la encapsulación in-situ de la enzima en el interior de la estructura
mesoporosa del soporte inorgánico se pueden conseguir mayores cargas, y disminuir
la pérdida de actividad enzimática, y el lixiviado de la misma. Para ello, tras un
exhaustivo estudio de las condiciones de síntesis, modificando las mismas para
hacerlas más suaves, se ha desarrollado una novedosa estrategia in-situ para
encapsularla. Se han sintetizado materiales con estructura mesoporosa tipo caja-
ventana (Figura 6.30) en presencia de la enzima, sales, agentes expansores de
micelas y bajas temperaturas.

In-situ-β-glucosidasa

Figura 6.30. Esquema de la encapsulación in-situ de β-Glucosidasa en materiales tipo caja-

ventana.

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 Resumen de resultados

Esta estrategia se ha aplicado también para encapsular in-situ lacasa, además se

dispone de una batería de resultados en los que se han ensayado y probado diferentes
cantidades de aditivos, de surfactantes y de condiciones de síntesis, que formarán
parte de una publicación en preparación. Además, se han sintetizado soportes tipo
caja-ventaja FDU-12 con grandes cavidades, y se han funcionalizado con grupos
amino, lo que nos va a permitir inmovilizar post-síntesis y estudiar el
comportamiento de la enzima en estos otros nuevos soportes.

También se está explorando una nueva línea de investigación basada en la

encapsulación de enzimas en Metal-Organic Frameworks (MOF). Hasta el momento
están dando buenos resultados, y se está preparando una patente.

Estudios preliminares de inmovilización sobre MOF: Obtención de

biocatalizadores en los que las enzimas lacasa y glucosidasa se encuentren
adsorbidas o atrapadas en la mesoporosidad intercristalina de MOF. Se espera que la
conectividad de los poros contribuya a la difusión de sustratos y productos, y el
confinamiento de la enzima contribuya a su estabilidad. Se espera encapsular estos
enzimas tanto mediante aproximaciones post-síntesis e in-situ, con la ayuda de
aditivos y aglomerantes que contribuyan a la estabilización de los biocatalizadores.
De esta manera se pretende extender la aplicación de MOF en un campo poco
extendido, en la inmovilización de enzimas, siempre con vistas a su aplicación
catalítica.

Para la inmovilización post-síntesis: Se preparan MOF en condiciones adecuadas

para que presenten cationes metálicos con coordinación insaturada (por ejemplo,
Zn2+), que podrán actuar como centros de coordinación de grupos ácidos (carboxilos)
presentes en las cadenas laterales del enzima (aspártico y glutámico) para el anclaje
de la enzima en la superficie de los cristales de MOF. La implicación de numerosos
grupos carboxilos por parte del enzima en este tipo de anclaje es, en principio,
bastante probable. La consecución de este objetivo sería de gran interés para evitar el
lixiviado del enzima.

Para la inmovilización in-situ: Resultados recientes de nuestro Grupo han

demostrado que la síntesis de este tipo de MOF se puede realizar en condiciones
suaves de pH y temperatura, por lo que en principio debe ser compatible con el
mantenimiento de la actividad catalítica de los enzimas. Los requerimientos de
medio orgánico de reacción (dimetilformamida) harán necesarios estudios previos de

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Resumen de resultados

estabilidad de los enzimas para encontrar las condiciones óptimas para evitar su
inactivación.

Como estos últimos trabajos están actualmente pendientes de publicar y/o

patentar, no se han podido incluir en esta memoria que se presenta como compendio
de publicaciones.

De forma global en la presente Tesis, se han diseñado y optimizado materiales

silíceos mesoestructurados híbridos organo-inorgánicos en los que se controla el
tamaño de poro, la morfología de partícula y la funcionalización de la superficie para
incorporar moléculas de enzima (lacasa, lipasa y β-glucosidasa) en su interior. Se han
obtenido biocatalizadores con elevadas cargas enzimáticas, altas actividades y
actividades específicas. Además, se ha podido localizar a las moléculas de enzima en
el interior de los poros de los soportes. El diámetro de poro diseñado a medida y la
alta afinidad enzima-soporte producen alta retención de la enzima mediante uniones
no covalentes, y en algunos casos se consigue evitar el lixiviado. La inmovilización
y/o encapsulación de la enzima protege de la inactivación frente a disolventes
orgánicos y se mejora la estabilidad térmica. Por todo ello, estos biocatalizadores
podrían cumplir potencialmente con los requisitos para ser utilizados
industrialmente.

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 Resumen de resultados

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250
7. CONCLUSIONES
Conclusiones

7. CONCLUSIONES

La presente Tesis Doctoral se ha desarrollado en un campo multidisciplinar que abarca

los ámbitos de la Ciencia de Materiales y la Biocatálisis. Se han desarrollado con éxito
materiales mesoporosos ordenados con diferentes características químicas y texturales
diseñadas a medida para inmovilizar la enzima lacasa. Este planteamiento se ha realizado
desde distintos puntos de vista, dando lugar a excelentes resultados en todos los casos,
que nos permiten extraer las siguientes conclusiones:

1. A partir de la inmovilización de lacasa sobre materiales puramente silíceos de poro

tipo canal se ha podido demostrar que la longitud de los canales es decisiva para la
capacidad de carga enzimática del material. Canales más cortos permiten cargas más
altas.

2. El desarrollo de estrategias para obtener canales de poro más anchos ha permitido la

introducción de grupos orgánicos funcionales (tipo amino) en la superficie de los poros
de las sílices mesoporosas ordenadas, ya sea por anclaje o por cocondensación. La
presencia de estos grupos ha resultado ser fundamental para alcanzar una alta afinidad
química enzima-soporte. Esta afinidad química ha permitido mejorar las características
de los biocatalizadores en distintas facetas:

 Alta carga enzimática

 Alta actividad específica
 Alta resistencia al lixiviado

3. Se han planteado y desarrollado estrategias de síntesis de materiales híbridos

organosilíceos periódicos mesoporosos (de tipo PMO) en los que el grupo funcional
se incorpora como puente entre dos átomos de silicio, formando parte de la propia
pared de soporte. Se han obtenido dos tipos de materiales diseñados específicamente
para dos enzimas distintas:

 PMO con grupos etileno, sobre los que se ha inmovilizado lipasa a través de
interacciones hidrofóbicas, con alta carga y alta eficiencia catalítica.

 PMA con diámetro de poro expandido y con doble funcionalización: grupos

amino y grupos etileno. Esta doble funcionalización ha permitido obtener

253
 Conclusiones

estructuras ordenadas (gracias a una mayor rigidez estructural aportada por los
grupos etileno puente) y con alta afinidad química por la lacasa (gracias al
grupo amino).

Los biocatalizadores obtenidos mediante esta estrategia, han resultado ser altamente
estables en medios orgánicos gracias a la presencia de los grupos etileno que
refuerzan la estructura semidesplegada de las enzimas en estos medios.

Los biocatalizadores de lacasa resultan ser totalmente resistentes al lixiviado. Incluso

las estructuras desplegadas de la enzima, una vez sometidas a procesos de
desnaturalización en condiciones drásticas, quedan retenidas por interacción
electrostática en el interior de los poros hasta pH 14, en el que los grupos amino están
desprotonados y se puede producir la liberación de la proteína.

4. Se ha conseguido encapsular lacasa directamente en materiales silíceos mediante el

uso de aditivos que favorecen la interacción de la enzima con el surfactante, y
mediante el estudio y desarrollo de unas condiciones de síntesis del material silíceo
compatibles con la actividad catalítica de la enzima.

5. Los biocatalizadores obtenidos a partir de los diversos planteamientos y estrategias

han demostrado propiedades muy ventajosas desde distintos puntos de vista:

 Elevada actividad específica, gracias a la unión no covalente enzima-soporte,

que no ejerce un efecto distorsionante sobre la estructura proteica.

 Elevadas cargas enzimáticas, gracias a la optimización de las propiedades del

soporte: tamaño de poro, tamaño de partícula y afinidad química.

 Estabilización frente a medios orgánicos de reacción, gracias a las

características de los materiales híbridos orgánico-inorgánicos en los que se
refuerzan las uniones enzima-soporte en estos medios.

 Minimización del lixiviado enzimático, gracias a la alta afinidad química y el

tamaño de los poros ajustado a las dimensiones de la enzima. Es de destacar el
caso de lacasa inmovilizada sobre PMA, donde se elimina por completo la
posibilidad de lixiviado enzimático en un biocatalizador basado en unión no
covalente de la enzima.

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Conclusiones

6. Los exhaustivos estudios de caracterización de los biocatalizadores con diversas

técnicas fisicoquímicas han permitido determinar sus características texturales y
estructurales, en las que se basan las conclusiones de esta Tesis.

Hemos podido verificar la estructura regular de los materiales obtenidos por

difracción de rayos X (DRX) y microscopía de transmisión (TEM), y la morfología de
las partículas mediante microscopía electrónica de barrido (SEM). Asimismo,
mediante isotermas de adsorción-desorción de nitrógeno a -196 °C, se ha determinado
la uniformidad en la distribución de tamaños de poro.

Se han aplicado también técnicas de microscopía electrónica avanzada con las que,
por vez primera, se ha podido observar la presencia de la enzima en el interior de los
poros ordenados del material utilizando imágenes de ultra alta resolución obtenidas
con un microscopio con corrección de la aberración esférica y combinando un
detector STEM-HAADF con espectroscopia EELS.

Los resultados obtenidos permiten concluir que se ha conseguido dar un paso más en el
desarrollo de biocatalizadores mesoestructurados. Suponen el sentar nuevas bases de
trabajo sobre las que continuar explorando este campo multidisciplinar con potenciales
aplicaciones industriales.

Con esta Tesis Doctoral se ha podido responder en parte a la pregunta que se planteaba
en el primer párrafo de la Introducción, “Inmovilización de enzimas: ¿Ciencia o arte?”.
Efectivamente este campo de la ciencia es un arte, y como cualquier arte, se necesita no
sólo de inspiración, sino también de mucho trabajo.

255
8. ANEXOS
 Aportaciones científicas

I. APORTACIONES CIENTÍFICAS
PUBLICACIONES CIENTÍFICAS
(Etapa 2010-2014)

259
Aportaciones científicas

260
 Aportaciones científicas

261
Aportaciones científicas

262
 Aportaciones científicas

263
Aportaciones científicas

TRABAJOS PRESENTADOS EN CONGRESOS

(Etapa 2010-2014)
1.
Denominación del evento. XXIV Congreso Iberoamericano de Catálisis (CICAT 2014)
Lugar de celebración y año. Medellín, Colombia 2014 (15-19 Septiembre)
Título. Inmovilización no covalente de lacasa sobre sílice mesoporosa ordenada
funcionalizada con grupos amino
Autores. V. Gascón, E. Sastre, C. Márquez-Álvarez, R. M. Blanco
Tipo de participación. Póster
2.
Denominación del evento. 6th FEZA Conference. Porous Systems: From Novel
Materials to sustainable solutions
Lugar de celebración y año. Leipzig, Alemania 2014 (8-11 Septiembre)
Título. Large-pore periodic mesoporous organosilica (PMO and PMA) for lipase and
laccase immobilization
Autores. V. Gascón, I. Díaz, C. Márquez-Álvarez, R. M. Blanco
Tipo de participación. Comunicación oral
3.
Denominación del evento. ANQUE-ICCE-BIOTEC science, the key for a better life
Lugar de celebración y año. Madrid, España 2014 (1-4 Julio)
Título. Direct encapsulation of enzymes in sol-gel derived ordered mesoporous silica
hosts
Autores. V. Gascón, C. Márquez-Álvarez, R. M. Blanco
Tipo de participación. Comunicación oral
4.
Denominación del evento. I Encuentro de Jóvenes Investigadores de la SECAT (JJ.II
SECAT’14)
Lugar de celebración y año. Málaga, España 2014 (22-24 Junio)
Título. Diseño de materiales silíceos mesoporosos ordenados “a medida” para
inmovilizar enzimas
Autores. V. Gascón, C. Márquez-Álvarez, R. M. Blanco
Tipo de participación. Flash oral + Póster
5.
Denominación del evento. 10th International Conference on Protein Stabilization
(ProtStab 2014)
Lugar de celebración y año. Stresa, Italia 2014 (7-9 Mayo)
Título. Efficient immobilization of laccase by non-covalent interactions on amino-
functionalized mesostructured silica
Autores. V. Gascón, C. Márquez-Álvarez, R. M. Blanco
Tipo de participación. Póster
6.
Denominación del evento. 3rd Multistep enzyme catalyzed processes congress (MECP
2014)
Lugar de celebración y año. Madrid, España 2014 (7-10 Abril)
Título 1. Optimization of enzyme immobilization in ordered mesoporous materials as
an approach to efficient multi-enzyme system
Autores. V. Gascón, I. Martín de Lucía, C. Márquez-Álvarez, R. M. Blanco

264
 Aportaciones científicas

Tipo de participación. Comunicación oral y póster

Título 2. Controlled caffeic acid oxidation catalyzed by laccase immobilized on

ordered mesoporous materials. An approach to wine oxidation processes
Autores. V. Gascón, I. Martín de Lucía, C. Márquez-Álvarez, R. M. Blanco
Tipo de participación. Póster
7.
Denominación del evento. 1st CZM Workshop on Catalysis
Lugar de celebración y año. TU Clausthal-Zellerfeld, Alemania 2013 (22-23
Noviembre)
Título. Polyphenols oxidation with supported laccase biocatalysts
Autores. V. Gascón, I. Martín de Lucía, C. Márquez-Álvarez, R. M. Blanco
Tipo de participación. Comunicación oral
8.
Denominación del evento. Catalizadores y Reactores Estructurados (SECAT 2013)
Lugar de celebración y año. Sevilla, España 2013 (26-28 Junio)
Título. Biocatalizadores de lacasa soportada en sílices mesoestructuradas:
Estabilidad y lixiviado
Autores. V. Gascón, I. Martín de Lucía, C. Márquez-Álvarez, R. M. Blanco
Tipo de participación. Comunicación oral
9.
Denominación del evento. IV Iberian Symposium on Hydrogen, Fuel Cells and
Advanced Batteries (HYCELTEC 2013)
Lugar de celebración y año. Estoril, Portugal 2013 (26-28 Junio)
Título. Operando Raman-MS study of thermal decomposition of NH3BH3 supported
into nanoporous materials for hydrogen storage
Autores. M. J. Valero-Pedraza, V. Gascón, M. A. Carreón, F. Leardini, J. R. Ares, M.
Sánchez-Sánchez, M. A. Bañares
Tipo de participación. Comunicación oral
10.
Denominación del evento. 5th Czech-Italian-Spanish Conference on Molecular Sieves
and Catalysis (CIS-5)
Lugar de celebración y año. Segovia, España 2013 (16-19 Junio)
Título. Stability and enzyme leaching of laccase biocatalysts supported on
mesostructured silica
Autores. V. Gascón, I. Martín de Lucía, R. M. Blanco, C. Márquez-Álvarez
Tipo de participación. Póster
11.
Denominación del evento. 8th International Mesostructured Materials Symposium
(IMMS 2013)
Lugar de celebración y año. Awaji Island, Hyogo, Japón 2013 (20-24 Mayo)
Título. Location of enzymes in ordered mesoporous materials using aberration
corrected STEM
Autores. I. Díaz, V. Gascón, R. M. Blanco, C. Márquez-Álvarez, A. Mayoral
Tipo de participación. Comunicación oral
12.
Denominación del evento. 69th Annual Meeting of the Japanese Society of Microscopy
(JSM 2013)
Lugar de celebración y año. Osaka, Japón 2013 (20-22 Mayo)

265
Aportaciones científicas

Título. Location of enzymes in ordered mesoporous materials using Advanced

Electron Microscopy Techniques
Autores. V. Gascón, C. Márquez-Álvarez, R. M. Blanco, A. Mayoral, I. Díaz
Tipo de participación. Comunicación oral
13.
Denominación del evento. Forum on innovation in zeolites and ordered porous
materials (Zeoforum 2012)
Lugar de celebración y año. Valencia, España 2012 (3-4 Diciembre)
Título. Designing functionalized mesoporous materials for enzyme immobilization
Autores. V. Gascón, A. Mayoral, C. Márquez-Álvarez, R. M. Blanco, I. Díaz
Tipo de participación. Póster
14.
Denominación del evento. 6th International Meeting on Biotechnology (Biotec 2012/
Biospain 2012)
Lugar de celebración y año. Bilbao, España 2012 (19-21 Septiembre)
Título. Nanostructured biocatalysts. Tuning siliceous ordered mesoporous materials
improves properties of supported enzymes
Autores. V. Gascón, E. Serra, I. Díaz, C. Márquez-Álvarez, R. M. Blanco
Tipo de participación. Comunicación oral y póster
15.
Denominación del evento. 8th International Symposium Effects of Surface
Heterogeneity in Adsorption and Catalysis on Solids (ISSHAC-8)
Lugar de celebración y año. Krakow, Polonia 2012 (27-31 Agosto)
Título. Adsorption of lead(II) and cadmium(II) from aqueous solution by amino-
functionalized mesostructured silica SBA-15
Autores. J. M. Arsuaga, J. Aguado, A. Arencibia, V. Gascón
Tipo de participación. Póster
16.
Denominación del evento. I Jornada de Jóvenes Investigadores 2012
Lugar de celebración y año. Cantoblanco (Madrid), España 2012 (5 Julio)
Título. Inmovilización de lacasa en materiales silíceos mesoporosos funcionalizados
con grupos amino
Autora. V. Gascón, R. M. Blanco, C. Márquez-Álvarez
Tipo de participación. Comunicación oral
17.
Denominación del evento. ANQUE International Congress of Chemical Engineering
Lugar de celebración y año. Sevilla, España 2012 (24-27 Junio)
Título. Adsorption of aqueous cadmium by mesostructured Silica SBA-15
functionalized with amino groups
Autores. J. Aguado, J. M. Arsuaga, A. Arencibia, E. García, V. Gascón, M. S. López
Tipo de participación. Comunicación oral
18.
Denominación del evento. 9th International Conference on Protein Stabilisation
(ProtStab2012)
Lugar de celebración y año. Lisboa, Portugal 2012 (2-4 Mayo)
Título. Immobilization of laccase on amino-functionalized mesoporous silica
Autores. V. Gascón, R. M. Blanco, C. Márquez-Álvarez
Tipo de participación. Póster

266
 Aportaciones científicas

19.
Denominación del evento. La Catálisis ante las crisis energética y ambiental (SECAT
2011)
Lugar de celebración y año. Zaragoza, España 2011 (29 Junio-1 Julio)
Tipo de participación. Asistencia
20.
Denominación del evento. XVI International Zeolite Conference joint with VII
International Mesostructured Materials Symposium (IZC16-IMMS 2010)
Lugar de celebración y año. Sorrento, Italia 2010 (4-9 Julio)
Título. Amine-functionalization of mesostructured silica for aqueous Pb(II)
adsorption: Optimization of hydration-dehydration conditions
Autores. V. Gascón, A. Arencibia, J. Aguado, J. M. Arsuaga
Tipo de participación. Póster
21.
Denominación del evento. XXXV Reunión Ibérica de Adsorción (XXXV RIA 2010)
Lugar de celebración y año. Lisboa, Portugal 2010 (8-10 Septiembre)
Título. Optimización del proceso de funcionalización de SBA-15 para la adsorción de
plomo y cadmio acuosos
Autores. A. Arencibia, J. Aguado, V. Gascón, J. M. Arsuaga
Tipo de participación. Comunicación oral

TRABAJOS EDITADOS Y PUBLICADOS

1.
Autora. Victoria Gascón Pérez
Título. Influencia de la química superficial y topología de materiales mesoporosos
silíceos en la inmovilización de enzimas de interés biotecnológico
Premio/Reconocimiento. Edición y difusión del mejor proyecto de Fin de Máster en
Química Aplicada por su relevancia científica y su calidad técnica (Curso 2010-2011)
Fecha de edición. 2013
Organismo o Institución que lo concede. Universidad Autónoma de Madrid (UAM) y
UAM Ediciones/Servicio de Publicaciones UAM
Depósito Legal. M-28019-2013
ISBN. 978-84-8344-379-8

267
 Índice de figuras

II. INDICE DE FIGURAS

3. INTRODUCCIÓN Página

Figura 3.1. Esquema del diseño de la inmovilización de una enzima en un

nanomaterial mediante la estrategia post-síntesis. 14
Figura 3.2. Proceso de reorganización de micelas en forma cilíndrica. 21

Figura 3.3. Comparación entre el tamaño de poro de diferentes materiales silíceos

mesoporosos y el tamaño de las enzima inmovilizadas. 23

Figura 3.4. Representación esquemática de los materiales MCM-41 y MCM-48

correspondiente a la familia M41S. 24

Figura 3.5. Esquema del mecanismo de síntesis para obtener el material silíceo de
tipo SBA-15, utilizando como surfactante P123 y TEOS (tetraetilortosilicato) como
fuente de sílice en medio ácido. 25

Figura 3.6. Esquema de canales de mesoporos y microporos que conforman el

material SBA-15. 26

Figura 3.7. Modelización de la estructura de SBA-16 utilizando como surfactante

F127. 27

Figura 3.8. Interacción del agente expansor TIPB (1,3,5-triisopropilbenceno) con las
micelas de surfactante P123. 28

Figura 3.9. Metodología de funcionalización en materiales mesoporosos: a) Anclaje,

b) cocondensación. 31

Figura 3.10. Funcionalización del material silíceo amorfo comercial MS-3030

mediante anclaje. El grupo orgánico incorporado es el n-octilo. 32
Figura 3.11. Funcionalización del material silíceo SBA-15 mediante cocondensación. 33

Figura 3.12. Esquema del procedimiento de síntesis de PMO donde las unidades
orgánicas R, correspondientes al grupo funcional, se anclan dentro de las paredes
del poro. 34

Figura 3.13. Factores a tener en cuenta a la hora de realizar una inmovilización de

enzimas en materiales porosos. 36
Figura 3.14. Efecto del diámetro de poro sobre la inmovilización de una enzima. 38

Figura 3.15. Estructura modelizada de lacasa de Melanocarpus albomyces. Los

aminoácidos aspártico y glutámico están resaltados en rojo y azul respectivamente.
Sus dimensiones moleculares son 6,3 x 7,2 x 8,9 nm. 41
Figura 3.16. Estructura modelizada de la lipasa de Candida antartica B. 42

Figura 3.17. Representación de una enzima adsorbida mediante interacciones

electrostáticas sobre un soporte silíceo. 44

Figura 3.18. Inmovilización de la enzima lipasa en SBA-16 mediante la estrategia

post-síntesis. 46

269
Índice de figuras

CAPÍTULO 1 Página
Figure 1. SEM micrographs of mesoporous silicas: SBA-15-L (A–C), SBA-15-S (D–F). 75

Figure 2. TEM images of calcined SBA-15-L in the direction perpendicular to the

pore axis (A) and SBA-15-S in the direction of the pore axis (B). 76
Figure 3. SEM micrographs of amorphous silicas: MS-3030 (A) and MS-3030-N (B). 76

Figure 4. N2 adsorption-desorption isotherms and pore size distributions: (A) SBA-

15-L and SBA-15-S; (B) MS-3030 and MS-3030-N. 77

Figure 5. (A) Average dimensions of laccase measured in PyMOL (~6.1 nm × 5.0

nm × 4.9 nm). (B) Surface distribution of amino acids in laccase constructed in
PyMOL (blue, basic amino acids; red, acidic amino acids; orange, polar amino acids;
white, nonpolar amino acids; pink, copper atoms). 78

Figure 6. Schematic illustration of the laccase immobilization on SBA-15-L

mesochannels (A) and SBA-15-S mesochannels (B). 80

Figure 7. Adsorption capacity of different supports. Solid bars: immobilization at

pH 3.5. Dashed bar: immobilization at pH 5.5. 81

Figure 8. Leaching of enzyme from materials, expressed as percent of the initial

enzyme loading released as a function of time. 83

Figure 9. Electrophoresis in standard conditions. 1: Protein standard (high range

SDS-Page standard stained with coomassie G-250 stain; 2: Soluble laccase; 3: SBA-
15-L; 4: SBA-15-S; 5: MS-3030; 6: MS-3030-N. 84
Figure SI 1. TGA and DTG of SBA-15-S, SBA-15-L and MS-3030-N 86
Figure SI 2. Low-angle XRD patterns of SBA-15-L and SBA-15-S supports. 86

CAPÍTULO 2 Página

Figure 1. Low-angle XRD patterns of ordered mesoporous silica and amino-

functionalized silica supports. 105
Figure 2. TEM micrographs of samples NGOES (a, b) and NCOES (c, d). 106

Figure 3. Nitrogen adsorption-desorption isotherms and pore size distributions of

mesoporous silica and amino-silica supports. The isotherms have been shifted
vertically for clarity (A) NCOES +400; OES +200; NGOES +0. B) AS +700). 107

Figure 4. Laccase immobilization on (A) siliceous materials without amino groups

and (B) supports with amino groups. 110

Figure 5. Equilibrium loading of immobilized enzyme versus initial enzyme content

in solution at pH 5.5 for amino-modified supports. 111

Figure 6. Effect of pH on the activity of a) free enzyme (!) and enzyme immobilized
on b) AS (b), c) NGOES (,), d) NCOES (8), e) NAS (y). Oxidation of ABTS at 25 °C in
0.05 M phosphoric acid/sodium dihydrogenphosphate or 0.05 M citric
acid/trisodium citrate buffer solutions. 114

270
 Índice de figuras

Figure 7. Leaching of enzyme from channel-like materials and amorphous

mesoporous silica, expressed as percent of the initial enzyme loading leached as a
function of time. 115

Figure 8. Thermostability profiles of free and immobilized laccase. Oxidation of

ABTS at 50 ºC in 50 mM acetic acid/sodium acetate buffer at pH 5.5 118

Figure 9. Stability tests of free and immobilized laccase in acidic aqueous solution
containing ethanol (10 vol% ethanol in 50 mM citric acid/trisodium citrate buffer at
pH 3.5). Oxidation of ABTS was performed at 25 ºC. 119
Figure S1. SEM micrograph of OES (a), NGOES (b), NCOES (c) and NAS (d). 121

Figure S2. Electrophoresis in standard conditions. 1: Protein standard (Broad range

SDS-Page standard stained with Coomassie G-250 stain), 2: OES, 3: NGOES, 4: AS,
5: NAS, 6: NCOES. 122

Figure S3. Electrophoresis at pH 11.0. 1: Protein standard; 2: NGOES, 3: NCOES, 4:

Soluble laccase. 122

CAPÍTULO 3 Página
Figure 1. TEM micrographs of PMO (a, b), PMA (c, d) and E-PMA (e, f). 146

Figure 2. Nitrogen adsorption isotherms and pore size distributions of: a) hybrid
organosilica supports and b) parent and functionalized amorphous silica supports. 147
Figure 3. Effect of pH on the activity of a) free enzyme, b) AS, c) NAS and d) E-
PMA. 151

Figure 4. a) Leaching of lipase from ordered material (PMO) and amorphous

mesoporous silica (OAS). b) Leaching of laccase from ordered material (E-PMA)
and amine amorphous silica (NAS). Expressed as percent of the initial enzyme
leached on each material as a function of time. 152

Fig. 5. Thermostability profiles at 55 ºC of free and immobilized lipase on PMO and

octyl amorphous silica (OAS). 154

Figure 6. Thermostability profiles at 60 ºC of free and immobilized laccase. Runs

were performed at pH 5.5. 154

Figure 7. Stability of lipase in solution and supported on OAS and PMO in: a) 50 %
aqueous solution: 50 % methanol; b) 0 % aqueous solution: 100 % methanol. Runs
were performed at 25 ºC. 155

Fig. 8. Stability of free and immobilized laccase in ethanol : water solution at acidic
pH. Runs were performed at 25 ºC 156

Figure 9. UV-Vis spectra of lipase in aqueous buffer and 50 % methanol (a) and
laccase in aqueous buffer and 10% ethanol (b). 156
Figure S1 a. Immobilized loading versus initial enzyme loading at pH 5.0 for PMO. 161

Figure S1 b. Immobilized loading versus initial enzyme loading at pH 5.5 and 6.0
for E-PMA. 161
Figure S2. Low-angle XRD patterns of hybrid organosilica supports. 162

Figure S3. TGA profiles of hybrid organosilica and functionalized amorphous silica
supports. 162

271
Índice de figuras

Figure S4. Electrophoresis. a) 1: Protein standard (high range SDS-Page standard

stained with coomassie G-250 stain), 2: E-PMA at pH 6.5 and 3: NAS at pH 6.5; b) 1:
Protein standard, 2: E-PMA at pH 14.0 and 3: soluble laccase; c) 1: Protein standard,
2: soluble lipase, 3: PMO, 4: OAS (all in standard conditions). 163

CAPÍTULO 4 Página
Figure 1. (A) Powder XRD patterns and (B) N2 adsorption-desorption of
functionalized ordered mesoporous materials with increasing hydrophobic surface
properties. 180
Figure 2. (A) Powder XRD patterns and (B) N2 adsorption-desorption of
functionalized ordered mesoporous materials with increasing amount of amine
groups on the surface. 183
Figure 3. TEM micrographs (A) of periodic mesoporous organosilica (PMO) and (B)
taken along the [001] and [110] orientations, respectively. (C) Periodic mesoporous
aminosilica crystal oriented on the [001] and (D) magnified image of sample C in
which steps of one pore size can be observed on the particle surface. 184

Figure 4. (A) Aberration-corrected scanning transmission electron microscopy

combined with high-angle annular dark field (STEM-HAADF) image of a periodic
mesoporous organosilica (PMO) crystal along the [001] orientation, showing the
hexagonal array. The fast Fourier transform (upper left corner of the inset) confirms
the sixfold axis. A magnified image of the pores is presented in the upper right
corner. (B) STEM-HAADF signal of the area analyzed. (C) Map obtained from the
EELS signal of the relative carbon/oxygen composition (in red). (D) Sum of the EEL
spectra, after background subtraction, recorded in the dashed square marked in
image C.
186

Figure 5. (A) Cs-corrected scanning transmission electron microscopy combined

with high-angle annular dark field (STEM-HAADF) image of the periodic
mesoporous aminosilica (PMA) loaded with laccase. (B) High-resolution image of
the pores. The green rectangle represents the area in which the EELS analysis was
performed. (C) EELS profile collected from image B, showing the three edges that
confirm the presence of laccase. 187

Figure 6. (A) Cs-corrected scanning transmission electron microscopy combined

with high-angle annular dark field (STEM-HAADF) image of the pore system of
periodic mesoporous aminosilica (PMA). (B) Nitrogen map extracted from the
green square in image A. (C) Cs-corrected STEM-HAADF image of the channels of
PMA and (D) the corresponding nitrogen map. 188

CAPÍTULO 5 Página
Figure 1. Average dimensions of laccase measured in PyMOL (~6.1 nm × 5.0 nm ×
4.9 nm). 195
Figure 2. Left: Incorporation of amine groups in SBA-15 and PMA materials and
laccase immobilization post-synthesis. Right: Scheme of the in situ laccase
encapsulation. 197

272
 Índice de figuras

Figure. 3. a) Low angle XRD patterns of amino-functionalized silica supports; b) N2

adsorption-desorption isotherms at -196 ºC. 199
Figure 4. Leaching of enzyme from channel-like materials (SBA-15-NH2 and PMA)
and in situ mesoporous silica, expressed as percent of the initial enzyme loading
leached as a function of time. 200

Figure 5. a) Cs-corrected STEM-HAADF image of the pore system. The green

rectangle represents the area in which the EELS analysis was performed. b) EELS
profile collected from image a, showing the N-K and O-K edges that confirm the
presence of laccase. c) Cs-corrected STEM-HAADF image showing the hexagonal
pore arrangement, the green square denoted as spectrum image corresponds to the
area of analysis; the correspondent extracted N signal (blue color) is also displayed.
Figure adapted from reference 26.
202
Figure 6. a) Cs-corrected STEM-HAADF images of the IS-laccase, a closer
observation of the pores is shown inset. b) Area of EELS analysis, green rectangle. c)
EELS compositional map, formed by Si (green) and N (red). d) EEL spectrum
profile of the area analyzed. 203

6. RESUMEN DE RESULTADOS Página

Figura 6.1. Estructura terciaria tridimensional modelizada de la enzima lacasa de

Myceliophthora thermophila. Sus dimensiones aproximadas calculadas utilizando el
programa Pymol son 6,1 x 5,0 x 4,9 nm. Los átomos de cobre se representan como
esferas rosas. El modelo se ha creado mediante Pymol [15] y Swiss-Model Server
[16-19] a partir de la lacasa de Melanocarpus Albomyces (73 % de identidad de
secuencia con MtL ATCC 42464).
211
Figura 6.2. Micrografías SEM y TEM de los materiales SBA-15-L (a, b) y SBA-15-S
(c, d). 213

Figura 6.3. Inmovilización de lacasa sobre soportes puramente silíceos. El pH de

inmovilización debe estar comprendido entre los puntos isoeléctricos de la enzima
y el soporte. A un pH de 3,5 ambos presentan cargas opuestas, pudiéndose
establecer interacciones electrostáticas.
214
Figura 6.4. Inmovilización de lacasa en los mesocanales de los soportes SBA-15-L y
SBA-15-S. 215
Figura 6.5. Funcionalización del material silíceo amorfo comercial MS-3030
mediante anclaje. El grupo orgánico incorporado es el aminopropilo (N). 216

Figura 6.6. Inmovilización de lacasa en soportes con grupos amino. A un pH de

inmovilización de 5,5 (intermedio entre el punto isoeléctrico de la enzima y el
soporte), la enzima se encuentra cargada negativamente y los grupos amino del
soporte positivamente, potenciando las atracciones electrostáticas.
217

Figura 6.7. Obtención de estructuras ordenadas, uniformes y con tamaños de poro

diseñados a medida, mediante el uso de expansores de micelas. Se añade TIPB
durante la etapa de formación de las micelas para conseguir MMO con mayor
diámetro de poro.
218
Figura 6.8. Micrografías SEM de SBA-15 con diámetro de poro expandido (OES). 219

273
Índice de figuras

Figura 6.9. Funcionalización del material silíceo OES, con diámetro de poro
previamente expandido, mediante anclaje. El grupo orgánico incorporado es el
aminopropilo (N). 220
Figura 6.10. Funcionalización del material silíceo SBA-15 mediante cocondensación
en presencia del agente expansor de micelas TIPB. 220

Figura 6.11. Micrografías TEM de las muestras NGOES (a, b) and NCOES (c, d).
221

Figura 6.12. a) A pH 3,5, la enzima está cargada positivamente (la superficie

positiva se muestra en verde: residuos His + Lys + Arg protonados) y el soporte
silíceo negativamente debido a los grupos siloxano de la superficie del soporte. b)
A pH 5,5, la enzima está cargada negativamente (la superficie negativa se muestra
en rojo: residuos Asp + Glu desprotonados) y el soporte cargado positivamente
debido a la protonación de los grupos amino.
222
Figura 6.13. Representación esquemática de la enzima lacasa inmovilizada en el
interior del poro de un soporte ordenado (NGOES o NCOES). 223

Figura 6.14. Perfiles de lixiviado de los biocatalizadores de lacasa que han sido
suspendidos en condiciones en las que se favorece el lixiviado de la enzima del
interior de los materiales: Baja fuerza iónica y alta dilución. La concentración de
enzima lixiviada se ha medido mediante el ensayo de Bradford.
224
Figura 6.15. Incubación de los biocatalizadores en 10 % de etanol y a un pH ácido
de 3,5. Se ha ensayado la actividad enzimática con el sustrato ABTS. 225
Figura 6.16. Micrografías SEM de los materiales PMO (Ethylene-bridged periodic
mesoporous organosilica). 226
Figura 6.17. Inmovilización de lipasa de Candida antarctica B en PMO que incorpora
grupos etileno. 227
Figura 6.18. Proteína lixiviada del soporte organosilíceo periódico mesoporoso
(PMO) y de la sílice amorfa con grupos octilo en medio acuoso. 229

Figura 6.19. Electroforesis SDS-PAGE. Gel de poliacrilamida al 12 % (p/v), teñido

con azul de Coomasie G-250, tampón de ruptura en condiciones estándar (pH 6,5).
Carril 1: Patrón estándar de proteínas; Carril 2: lipasa soluble; Carril 3 proteína
forzada a salir del soporte PMO; Carril 4 proteína forzada a salir del soporte OAS.
230
Figura 6.20. Estabilidad de lipasa soluble e inmovilizada sobre OAS y PMO en: a)
50% agua: 50% metanol; b) 100 % metanol. Ensayos realizados a 25 ºC. 231
Figura 6.21. Esquema del procedimiento de síntesis de E-PMA (Pore-Expanded
Periodic Mesoporous Aminosilica). 233
Figura 6.22. Micrografías SEM de la muestra E-PMA. 233
Figura 6.23. Proteína lixiviada del soporte organosilíceo periódico mesoporoso (E-
PMA) y de la sílice amorfa con grupos amino en medio acuoso. 234

274
 Índice de figuras

Figura 6.24. Electroforesis SDS-PAGE. Gel de poliacrilamida al 12 % (p/v), teñido

con azul de Coomasie G-250. A) Tampón de ruptura en condiciones estándar (pH
6,5). Carril 1: Patrón estándar de proteínas; Carril 2: Proteína forzada a salir del
soporte E-PMA; Carril 3 Proteína forzada a salir del soporte NAS; B) Carril 1:
Patrón estándar de proteínas; Carril 2: Proteína forzada a salir del soporte E-PMA a
pH 14,0; Carril 3: lacasa soluble.
235
Figura 6.25. Estabilidad de lacasa, soluble e inmovilizada en E-PMA y NAS, en
disolución ácida a pH 3,5 y en presencia de 10 % de etanol. 236

Figura 6.26. Difractogramas a bajo ángulo de los soportes SBA-15-L (L: long
channel) y SBA-15-S (S: short channel) antes y después de la inmovilización de la
enzima lacasa. 238

Figura 6.27. a) Imagen STEM-HAADF con aberración corregida de la muestra

PMO-Lipasa en la orientación [001] de la estructura porosa con simetría p6mm y b)
ampliación de un área seleccionada, donde las cavidades aparecen en negro. c)
Mapa de proporción relativa C/O obtenido a partir de la señal EELS que muestra
en rojo zonas ricas en carbono, confirmando la presencia de lipasa en los poros. d)
Espectro EELS en la zona del recuadro marcado en (c) mostrando el borde K de
nitrógeno que identifica a la enzima.
241

Figura 6.28. Estrategia de inmovilización in-situ de lacasa. La unión no covalente

del compuesto orgánico añadido con la superficie de la enzima se promueve a
través de un grupo amino que, en las condiciones de síntesis, presenta alta afinidad
con la superficie de la enzima. Las cadenas apolares del aditivo orgánico favorecen
la incorporación del complejo enzima-aditivo en la región interior, hidrofóbica, de
las micelas de surfactante.
242
Figura 6.29. Estructura modelizada de β-Glucosidasa de Aspergillus niger. 244
Figura 6.30. Esquema de la encapsulación in-situ de β-Glucosidasa en materiales
tipo caja-ventana. 244

275
 Índice de tablas

III. INDICE DE TABLAS

3. INTRODUCCIÓN Página
Tabla 3.1. Características de diferentes estructuras mesoporosas. 17
Tabla 3.2. Métodos aplicados para la inmovilización de enzimas en MMO. 20

CAPÍTULO 1 Página

Table 1. Laccase immobilization characteristics and catalytic results for initial

amount of enzyme solution of 50 mg/g support. 82

CAPÍTULO 2 Página

Table 1. Textural properties and degree of functionalization of the supports, laccase

immobilization characteristics and catalytic performance. 108

CAPÍTULO 3 Página
Table 1. Textural properties and organic groups content of supports. 148

Table 2. Immobilization of lipase on PMO at different pH values from suspensions

containing 90 mg lipase per gram of support. 149
Table 3. Immobilization of laccase on different supports. 150

CAPÍTULO 4 Página
Table 1. Textural and catalytic results of lipase immobilized on OMM. 179

Table 2. Textural and catalytic results of laccase immobilized on the ordered

mesoporous material. 182

CAPÍTULO 5 Página
Table 1. Textural properties, laccase immobilization and catalytic performance. 184

6. RESUMEN DE RESULTADOS Página

Tabla 6.1. Características de las síntesis, propiedades estructurales y texturales de
los materiales silíceos SBA-15-L y SBA-15-S. 212

Tabla 6.2. Inmovilización de lacasa a pH 3,5 sobre dos soportes SBA-15 con
diferente longitud de canal, para una carga enzimática ofrecida de 50 mg de enzima
por g de soporte. 215

Tabla 6.3. Propiedades texturales de los materiales silíceos MS-3030 y MS-3030-N y

resultados de inmovilización de lacasa a pH 3,5 sobre MS-3030 y pH 5,5 sobre MS-
3030-N. 218

Tabla 6.4. Propiedades texturales, grado de funcionalización de los soportes con

grupos amino, resultados de inmovilización de lacasa y actividad catalítica. 223

Tabla 6.5. Propiedades estructurales, texturales y composición química de los

soportes silíceos. 227

277
Índice de tablas

Tabla 6.6. Inmovilización de lipasa en OAS y PMO. La carga enzimática inicial

ofrecida es de 600 mg de lipasa por gramo de soporte. 228
Tabla 6.7. Inmovilización de lacasa en diferentes soportes. 234

278
 Lista de abreviaturas y acrónimos

IV. LISTA DE ABREVIATURAS Y ACRÓNIMOS

Abreviaturas y
Significado
Acrónimos

a0 Parámetro de celda unidad

2,2´-azino-bis(3-etil-benzotiazolin-6-sulfonato), sal
ABTS diamónica (2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic
acid)
A ABTS* ABTS oxidado
APTES 3-Aminopropiltrietoxisilano (3-aminopropyltriethoxysilane)
APTS 3-Aminopropiltrietoxisilano (3-aminopropyltriethoxysilane)
AS Sílice amorfa comercial (Amorphous silica)

Método de Brunauer, Emmet y Teller: evaluación del área

BET
superficial
Método de Barret, Joyner y Halenda: Evaluación del
BJH
diámetro de poro
B BSA Albúmina de suero bovino (Bovine Serum Albumin)
BTEE 1,2-bis(trietoxisilietano) (1,2-bis(triethoxysilyl)ethane)
BTME 1,2-bis(trimetoxisilietano) (1,2-bis(trimethoxysilyl)ethane)
bis(3-trimetoxisilil)propilamina (bis(3-
BTMSPA
trimetoxysilyl)propylamine)
CALB /CaLB Lipasa de Candida antarctica
Concentración Micelar Crítica (Critical Micelle
CMC
Concentration)
C CMT Temperatura Micelar Crítica (Critical Micelle Temperature)
Cs Spherical aberration correctors
CTAB Bromuro de cetiltrimetilamonio

DRX Difracción de rayos X

D
Dp Diámetro de poro (Pore diameter)

ε Coeficiente de extinción molar

E EELS Electron Energy Loss Spectroscopy
E-PMA Expanded-Periodic Mesoporous Aminosilica
Copolímero de tres bloques de tipo Pluronic
F127
(EO106PO70EO106)
F FDU Fudan University (Shanghai) materials
FSM Folded Sheet Mesoporous Materials

HAADF High angle Annular Dark Field

H HCl Ácido clorhídrico
HMM Hiroshima Mesoporous Materials

279
Lista de abreviaturas y acrónimos

HMS Hexagonal Mesoporous Silica

HRP Peroxidasa de rábano

IGS ImmunoGold Staining

I
IUPAC International Union of Pure and Applied Chemistry

KiloDalton. Unidad de masa atómica. 1 KDa equivale a

KDa
1000 Da (1 Da = 1g/mol)
K KIT-n Korea Advanced Institute of Science and Technology materials
Método Kruk, Jaroniec y Sayari: Evaluación del diámetro
KJS
de poro

MtL Lacasa de Myceliophthora thermophila

M41S Familia de materiales mesoporoso ordenados
MCF Espuma Mesocelular Silícea (Mesostructured cellular Foam)
MCM-n Mobil Composition of Matter-number n
Soporte SBA-15 funcionalizada con grupos metilo
Me-SBA-15
mediante cocondensación
M MMO Materiales mesoporosos ordenados
MMSO Materiales silíceos mesoporosos ordenados
MOF Metal-Organic Frameworks
MS-3030 Sílice amorfa comercial (Amorphous silica)
MS-3030-G-N Aminopropyl (N)-Functionalized Amorphous Silica by Grafting
MS-3030-N Aminopropyl (N)-Functionalized Amorphous Silica by Grafting
MSU-n Michigan State University materials

NAS Aminopropyl (N)-Functionalized Amorphous Silica by Grafting

NCOES N-Cocondensation ordered expanded silica
NGOES N-Grafted ordered expanded silica
N
NH4F Fluoruro de amonio (Ammonium fluoride)
NaOH Hidróxido de sodio (Sodium hidroxide)
NH2-SBA15exp N-Grafted ordered expanded silica

OES Ordered expanded silica

O OTCA Cloruro octadeciltrimetilamonio
OTES n-octiltrietoxisilano (n-octyltriethoxysilane)

PDB Banco de datos de proteínas (Protein Data Bank)

E-PMA Pore-Expanded PMA
P/P0 Presión relativa
P Copolímero de tres bloques de tipo Pluronic
P123
(EO20PO70EO20)
Copolímero de tres bloques de tipo Pluronic
PE-10400
(EO25PO56EO25)
PEO Poly(ethylene oxide)

280
 Lista de abreviaturas y acrónimos

pI Punto isoeléctrico
pKa Constante de disociación ácida
PMA Periodic Mesoporous Aminosilica
PMO Ethylene bridged PMO
PMOs Periodic Mesoporous Organosilicas
PPO Poly(propylene oxide)
PPO Polifenol oxidasa
Pymol Molecular Graphics System
p-NPA p-nitrofenil acetato (p-nitrophenyl acetate)

SBA-15-L Santa Barbara Amorphous-15 (L= Long Channel)

SBA-15-S Santa Barbara Amorphous-15 (S= Short Channel)
SBA-15exp Ordered Expanded Silica
SBA-n Santa Barbara Amorphous-number n

S Electroforesis en gel de poliacrilamida con dodecilsulfato

SDS-PAGE
sódico (Sodium dodecyl sulfate polyacrylamide gel)
Microscopía electrónica de barrido (Scanning Electron
SEM
Microscopy)
STEM Scanning Transmission Electron Microscopy
SBET Área específica BET (Specific surface area)

Tiempo de contacto enzima-soporte tras alcanzar el

tc
equilibrio de adsorción
TB Tributirina (Tributyrin)
Microscopía electrónica de transmisión (Transmission
TEM
Electron Microscopy)
TEOS Tetraetilortosilicato (Tetraethyl ortosilicate)
TG Análisis termogravimétrico
T
TGA Thermogravimetric analysis
TIPB 1,3,5 triisopropilbenceno (1,3,5-triisopropylbenzene)
TMB 1,3,5-tetrametilbenceno (1,3,5-Trimethylbenzene)
TMOS Tetrametilortosilicato (Tetramethyl orthosilicate)
TPOS Tetrapropilortosilicato
TMED N,N,N',N'-tetramethylethylenediamine
Tris base Tris(hydroxymethyl)aminomethane

Unidad de actividad enzimática. Se define como la

U cantidad de enzima que cataliza la conversión de un
U micromol de sustrato en un minuto
Actividad del biocatalizador. Se define como el número de
U/g
unidades (U) por gramos de biocatalizador

281
Lista de abreviaturas y acrónimos

Actividad específica. Se define como el número de

U/mg unidades (U) de enzima por miligramo de proteína
inmovilizada
Actividad específica. Se define como el número de
U/mL
unidades (U) de enzima por mililitros de proteína
UV Espectro ultravioleta
USA United States of America

Vp Volumen de poro (Pore volume)

V
Vis Espectro visible

X XRD X-Ray Diffraction

282