Applications of Enzyme

Immobilization

Submitted to Submitted by
Dr. S.R. Hashim Arun kumar Mishra
Professor M.Pharm 2nd sem
College of pharmacy College of pharmacy
IFTM,Moradabad IFTM,Moradabad
 ENZYME

IMMOBILISATION
Immobilization of enzyme is a process where an enzyme
makes use of carrier phase for safe homing.
 The use of enzyme in industrial application is limited because
enzyme are relatively unstable, of high cost isolation
,purification and recovery of active enzyme from reaction
mixture after the completion of catalytic process.
 For soluble enzyme in batch operation is uneconomical as
active enzyme is lost after each reaction.
 Some of enzyme are rapidly inactivated by heat and become
heat stable by attachment to inert polymeric supports.
Immobilized enzyme can be reused. process can be repeated continuously
and can be radically controlled.

Products can be easily separated and enzyme properties can be preserved.

METHOD OF IMMOBILISATION:

Depending upon physical relationship of catalyst to matrix.

Matrix chosen must enhance the operational stability of immobilized enzyme
purification.

Carrier may be porous or nonporous, matrices with organic natural or synthetic) or

Inorganic nature.
The catalyst may be covalently bonded to polymer, physically adsorbed onto the
Polymercross linked with itself and possibly another inert protein, entrapped inside
a polymer matrix or encapsulated in polymer bag.

1 cov alent bo nding

2 ionic b onbing
3 co pol ymeri za tion
4 poly mer e ntr apme nt
5 En caps ula tion
6 lipos ome entr apments
APPLICATION OF ENZYME IMMOBILISATION
Immobilization is beneficial for economic purpose and reduces the overall cost.
• PHARMACEUTICAL APPLICATION:
Immobilized biocatalyst are useful for pilot plant and fpr laboratory set up
1.Production of antibiotics
a) PENICILLINES:
i. 6 Aminopenicillanic acid
6-APA can be industrially produced on large scale from penicillin g or penicillin v
by deacylation with penicillin amidase these antibiotics are produced by
fermentation.
a).Partially purified amidase from E.coli trapped into cellulose triacetate fibers
can be used for production of 6APAfrom penicillin G,in a column as a part of
recirculation batch reactor .5%substrate solution hydrolyses to 99%.retention time
required is1.5 hrs.
Penicillin amidase immobilised by covalent binding to amberlite XDA-7 with glutaraldehyde,by
physical absorption to bentonite,or by ionic binding to DEAE-sephadex and by covalent
binding to a copolymer of acrylamide and maleic anhydride can be used for production of 6
APA from penicillin v.

ii) Penicillin g
Laboratory production from glucose involve the use of conidia,
mycelium and protoplast of penicillium chrysogenum Immoblised with
k -caragennen ,and calcium alginate.
A continuous flow bioreactor can be used. Conidia & P..chrysogenum
immobilized ,with K- carrageen operates for up to 16days.1.2mg\g
cells\hr yields can be obtained from glucose.(7.0mg\g glucose ). The
half life of production is greater than 15 days with a media contain
10.0g of glucose\l.
iii) Ampicilline and amoxicillin
Penicillin amidase from E.coli entrapped in cellulose triacetate fibre
can
be employed in continuous flow reaction to produce ampicilline or
amoxycilline from 6APA and d -phenylglycine -methyl ester
respectively.
Succinylated penicilline amidase adsorbed on a DEAEsephadexcan be used for the
Production of ampicilline with an yield of 67%in stirred batch reactor.

b)Cephalosporins
Cephalosporin can be obtained by fermentation with cephalosporium acremonium.
Cephalosporin amidase from various microorganism can be immobilised by different
Methods.
Enzymatic deacylation of cephalosporin can be carried out with compounds containing 7ADCA
nuclus.The substrate for reaction are usually 7phenylacetamidodesacetoxy cephalosporin
Acid and 7phenoxyacetamidodesacetoxy cephalosporin acid obtained by ring expansion
reaction from penicillin g and penicillin v.

CEPHALEXIN

The enzyme from.E. Coli entrapped in cellulose triacetate fibers can be

Used for production of cephalexin and Dphenylglycine methyl ester.
Immobilised acetone dried whole cells of Achromobacter spp adsorbed on a DEAE
Cellulose or hydroxylapatite can also be used.

c)BACITRACIN

Cells of bacillus spp ,producing bacitarcin immobilised in polyacrylamide gel lattice can
Be employed in batch and continous culture system.

D)TYLOSIN AND NIKKOMYCIN

Macrolide antibiotics tylosin and nikkomycin can be produced by living cells of streptomycin
spp Immobilised with calcium alginate .
2.PRODUCTION OF STEROIDS
Steroid transformation can be done by biocatalytic conversion cofactoers
are also needed

METHOD 1
Synthesis of hydrocartisone and prednisolone by immoblisation with
Polyacrylamide entrapement. OH
H2C
CO 11beta hydroxylation OH
OH
curvularia lunata H2C
CO
HO OH

H2C

dehydrogenation
corynebacterium simplex

OH
H2C
CO
OH

O
METHOD 2.
Whole cells of C.simplex entraped in collegen membrane can also be used
for production of prednisolone from hydrocortisone.

METHOD 3.
The two step tranformation (cortexolone) to its dehydro 11-hydroxy derivative)
Can also be performed by combined used of C.lunata mycelia and
immobilized arthobector simplex.

3.PRODUCTION OF AMINO ACID:-

Many large scale process using immobilized enzyme are in operation.
Amino acid production by enzymatic resolution using amino acids acylase and
fructose syrups form glucose isomerase are few among them.
A.. opti cal res olut ion of D L ami no acid
Synthesised acyleDL amino acids are assymetrycally hydrolysed by aminoacylase
to give L-aminoacid and unhydrolysed acyle D aminoacid.
Immbolized aminoacylase of Aspergillus oryzae is used for continous .
optical resolution of DL- amino acid.
L-aminoacid crystlises from concentrated effulent. Acyle Daminoacid contained in
Mother liquor is recimized by heating and can be used for optical resolution
B…. OPTICALLY ACTIVE AMINOACID.
L-aspartic acid
Can be produced by fermetation / enzymatic bathe proceses from
fumaric acid and ammonia using aspartase. E.coli immobilised with
K.Carragennan tearted with glutraldehyde and hexameyhylenediamine .
Column packed with immobilsed cells of E.coli with higest prodictivity has half
Life period of 680 days at 37. degree.

4. PR OD UC TION OF ACIDS .
For improvement of productivty of organic acids immobilised enzyme are used.
Process is developed by Acetobecter aceti immbiliesd porous ceramics,
Citric acid by A.niger with calciun alginate,lactic acid by L.casei with
Polyacrylamide, Lmalic acid by B.flavum with carageenan,12 ketocheno
Deoxycholic acid B.fuscum with carageenan,2keto gluconic acid by
S.Marcesmens with colagen.
5.OTHER ORGANIC COMPOUNDS.
• Coenzyme A…. It is produced from pentothenic acid, l cystein , and ATP
can be carried out with Bravibacterium ammoniagenes cells immobilised on
Polyacryleamind gel.
• Athraquinones Denovo synthsis of anthraquninoes can be performed by
Immobilised plants cells of Morindacitrifolia with calcium alginate from
Secologanin.
iii. Azmalicine isomers. Plant cells catharnthus roesus immobilised with
Calcium alginate were used for synthsis of Azmalicine isomers from tryptamine
iv. Prostglandine. Using arachidonic acid and ram seminal microsomes
Immobilisd on photocrose linkable resin.
v. Proinsuline B.subtiles cells carrying plasmide encoding for rat proinsuline
Were immbilised in agrose beeds.
vi. FAD(Flavine adenine dinucletide). With FMD and ATP by whole cells
of orthobecteroxydans.
vii. Pyridoxile 5phaphate it is porduced with highenzyme activity formpyridoxine
5-phaphate by whole cells P.fluorescnes immblised on film of polyvinyl alcohal
Crosed linked with tetraethyl silicates.
6.RECENT APPLICATION IN CLINICAL ANALYSIS…….

For diagnostic and industrial monitoring programs immobilization is

employed .
In clinical analysis enzymes are used as reagents to measure specific
Metabolites and are measured as such in body fluids and tissues as an
Indicator of pathological condition or metabolic disturbances

9. BIOSENSORS enzyme electrodes

commercial devices has been developed
To measure sugar amino acid urea, cholesterol penicillin. In this nylon coil
to which hexokinase G6PD are immobilized. Glucose is estimated by rate of
formation of NADH. For this poly aryl amide gel and oxygen sensor are used.

2.LIPID SENSOR..
Cholesterol conc. In serum indicates abnormality of lipid metabolism,
Hypertension for this an immobilized enzyme reactor containing cholesterol
Oxidases is coupled with ampearometric detector system based on platinum
electrode.
3. L LACTATE DEHYRO GENASE…
The amperometric determination of pyruvate can also be carried with pyruvate
Sensor for analyzing LDH level. In consisted of pyruvate oxydase and an
Oxygen electrode.

4. ELISA..

•It rapidly replaces RIA. For hormones, by coupiling their catalytic ability
specific immunoglobulin. In ELISA enzyme activity lable tagged on to antigen
antibody molecules. In this plastic tube coated known quantity of antigen to
Which is added the sample antigen and a known quantity of enzyme labeled
Antibody.
•The enzyme can be linked to proteins that bind spcifically to various subclass
Of immunoglobulin G. this enzyme protein reagent can employed to bind to
Any bound immunoglobulin instead of enzyme labeled immunoglobins.
Enzymes used in such a way includes peroxidase,alkaline phosphatase, and beta
Galactosidases.
5. ENZYME THERMISORTS;

Enzyme thermistors have been developed in which a substrate is feed to an enzyme ,a product is formed
with liberation of heat..A thermistors is used to monitor this
Heat of reaction whIch can be co related with concentration of substrate.
 REFERENCES

1.DIXIT V K,VYAS S P.PHARMACEUTICAL BIOTECHNOLOGY, CBS

PUBLISHER AND DISTRIBUTERS,PP.13-119,2002.
2.KORI S.S.,HALKANI M.A.,PHARMACEUTICAL BIOTECHNOLOGY,VALLAB
PUBLICATION,PP 260-278,1999.