Beta Galactosidasa

ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING
Asia-Pac. J. Chem. Eng. 2014; 9: 330348

Published online 22 April 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/apj.1801
Special theme review
Production, purication, characterization, immobilization,

and application of -galactosidase: a review
Arijit Nath,1 Subhoshmita Mondal,1 Sudip Chakraborty,1,2* Chiranjib Bhattacharjee1 and Ranjana Chowdhury1
1
2
Chemical Engineering Department, Jadavpur University, Kolkata, West Bengal 700032, India
Department of Chemical Engineering and Materials, University of Calabria, Cubo-44C, Rende 87036,CS, Italy
Received 20 June 2013; Revised 29 September 2013; Accepted 5 October 2013
ABSTRACT: Biopharmaceuticals are new categorizing biomolecules, which are the results of incredible proliferation in the
eld of biotechnology. One of the challenging biomolecule -galactosidase (-galactosidase galacto hydrolysase, trivially
lactase) catalyzes hydrolysis of lactose to produce glucose and galactose, and in some cases, it takes part in
transgalactosylation reaction that produces functional food galato-oligosaccharide. A wide variety of strategies had been
already attempted for production of -galactosidase through fermentative route. Beside the upstream process, downstream
technology towards purication and immobilization of target enzyme also create great attentions. Subsequently, its wide
applications in the eld of food, biopharmaceuticals, dairy, diagnosis, and waste treatment boost up biotechnological
economy as well as zero-efuent discharge. In dairy industry, -galactosidase has been used to degrade lactose, prevent
crystallization of lactose, improve sweetness, and increase the solubility of milk product, otherwise which would be an
environmental pollutant. In food and pharmaceutical industries, -galactosidase has been used to prepare low lactosecontaining food products for low lactose-tolerant people. Therefore, it is obvious to elucidate different technological aspects
of -galactosidase, which may provide a great knowledge in educational and industrial eld. Taking the enzyme into account,
a ready review has been made about its production, purication, characterization, and immobilization technology. The
review also addresses wide applications of -galactosidase in different elds. 2014 Curtin University of Technology
and John Wiley & Sons, Ltd.
Keywords: -galactosidase; production; purication; characterization; immobilization; application
INTRODUCTION
Emergence of biotechnology, which brings a boon to
biological origin, possesses a challenging revolution to
the categorical eld, biopharmaceuticals. There is
enormous range of therapeutics that involves
biopharmaceuticals, and it combines biomolecular
forms, which extensively give rise to the development
of the microbial synthesis of diverse enzymes and
metabolites. Intellectual revolution with new visions
and hopes by dispensation of any method of bioprocess
that deals with the design and development of
equipments for the manufacturing of various products,
such as food-beverages, sera, new medicines, semisynthetic organs, antibiotics, and enzymes from
biological sources creates a great attention. This is
responsible for the explosion of various biotechnological
processes used in industries for large-scale production of
*Correspondence to: Sudip Chakraborty, Chemical Engineering
Department, Jadavpur University, Kolkata, West Bengal 700032,
India. E-mail: zsudip.c@gmail.com
Authors have similar contribution.

2014 Curtin University of Technology and John Wiley & Sons, Ltd.
Curtin University is a trademark of Curtin University of Technology
biological products and optimization of their yields as

well as the qualities of end products.[1]
The lactose-hydrolyzing enzyme -galactosidase has
been accepted since long as an important ingredient in
dairy, food processing, and pharmaceutical industries.
-galactosidase catalyzes hydrolysis of lactose into
glucose and galactose and also takes part in
transgalactosylation reaction that produces galatooligosaccharide (GOS) (e.g., Gal (1 3) Gal
(1 4) Gal (1 6)).[2,3] One of the most common
disease hypolactasia (lactose intolerance) or lactose
maldigestion is caused by insufcient synthesis of
lactose-cleaving enzyme -galactosidase in the brush
border membrane of mucosa of the small intestine. In
most cases, this causes several symptoms, which may
include abdominal bloating and cramps, atulence,
diarrhea, nausea, rumbling stomach, or vomiting after
consuming signicant amounts of lactose.[4] In most
of the human races, -galactosidase is found to be lost
during the rst or second decade of life, and only some
isolates are seen among people of Northern European
origin and their overseas descendants. African and
Indian communities maintain a high intestinal lactase
Asia-Pacic Journal of Chemical Engineering
activity throughout life. According to reviews of

Scrimshaw, Murray, 1988 and Sahi, 1994, the global
prevalence of lactose maldigestion are above 50% in
South America, Africa, and Asia reaching almost
100% in some Asian countries such as China. In the
USA, prevalence is 15% among Whites, 53% among
Mexican-Americans, and 80% in the Black
population. In Europe, it varies from around 2% in
Scandinavia to about 70% in Sicily. Australia and
New Zealand have prevalence of 6% and 9%,
respectively.[5,6] -galactosidase has been used in
biopharmaceutical, food, and dairy industries to prevent
crystallization of lactose, to improve sweetness, to
increase the solubility of milk product, to prepare low
lactose-containing food products for low lactose-tolerant
people, and for the utilization of cheese whey, which
would otherwise be an environmental pollutant.[7,8]
Therefore, it is obvious to elucidate different
technological aspects of -galactosidase with special
interest in production, purication, characterization,
immobilization technology as well as its wide
applications in different process industries. The present
review may provide a great knowledge with ll-up gaps
of educational eld and industrial sector.
SYNTHESIS MECHANISM OF -GALACTOSIDASE

The control system of -galactosidase synthesis was rst
worked out by Jacob and Monod in1961, at the
molecular level. In prokaryotic type of gene expression,
the lac operon showed inducible system with the control
of enzymes that are produced in the presence of
lactose.[9] Presences of lac operon in Escherichia coli
and Bacillus sp. including lactic acid bacteria have
potential of synthesizing intracellular -galactosidase.
Meanwhile, lactose metabolism in E. coli synthesizes
several proteins, such as -galactosidase, which converts
lactose into glucose and galactose, -galactoside
permease that transports lactose into the cellular system,
-GALACTOSIDASE: A REVIEW
and the function of -galactoside transacetylase is yet

to be known. The construction of lac operon is
described in Fig. 1.
For any consortia, all the genes involved in controlling
this pathway are located next to each other on the
chromosome, and together they form an operon.
Generally, -galactosidase synthesis has been often
demonstrated considering E. coli as a model
microorganism. The genetic switches can also combine
positive and negative controls. The lac operon consists
of three structural genes and a promoter, a terminator,
regulator, and an operator. The three structural genes are
lacZ, lacY, and lacA. Regulation for the specic control
of the lac genes depends on the availability of the
substrate lactose. The proteins are not produced by the
bacterium when lactose is unavailable as a carbon source.
The lac genes are organized into an operon oriented in the
same direction, immediately adjacent on the chromosome
and are co-transcribed into a single polycistronic mRNA
molecule. The lac operon in E. coli, unlike trp operon,
works under both negative and positive transcriptional
controls by the lac repressor protein and catabolite
activator protein (CAP), respectively. CAP enables
bacteria to use alternative carbon sources such as lactose
in the absence of glucose. The CAP cannot induce
expression if lactose is not present, and the lac repressor
ensures that the lac operon is shutoff in the absence of
lactose. This arrangement enables the lac operon to
respond to and integrate two different signals, so that it
is expressed only when two conditions are met, i.e.,
lactose must be present and glucose must be absent.
Transcription of all genes starts with the binding of the
enzyme RNA polymerase (RNAP), a DNA-binding
protein that binds to a specic DNA binding site, the
promoter, immediately upstream of the genes. Binding
of RNA polymerase to the promoter is aided by the cyclic
adenosine monophosphate (cAMP)-bound CAP (also
known as the cAMP receptor protein). From this position,
RNAP proceeds to transcribe all three genes (lacZ, lacY,
and lacA) into mRNA.[11]
Figure 1. Construction of lac operon (Figure adapted from K. L. Anderson, G. Purdom
(2008)).[10]

DOI: 10.1002/apj
331
332
A. NATH ET AL.
Figure 2. Regulation of lac operon in (A) presence and (B) absence of lactose.[12]
Control mechanism of lac operon

In the absence of lactose, the regulatory response to
lactose uses an intracellular regulatory protein, which
is called the lactose repressor to hinder the synthesis
of -galactosidase. The lacI gene coding for the
repressor lies nearby the lac operon and is always
expressed (constitutive). If lactose is absent in the
growth medium, the repressor binds very tightly to a
short DNA sequence just downstream of the promoter
near the beginning of lacZ called the lac operator.
The repressor binding to the operator interferes with
binding of RNAP to the promoter and therefore mRNA
encoding LacZ and LacY are only made at very low
levels. During the cells growth in the presence of lactose,
a lactose metabolite allolactose, which is a combination
of glucose and galactose, binds to the repressor, causing
a change in its shape. Thus, the altered repressor is
unable to bind to the operator, allowing RNAP to
transcribe the lac genes and thereby leading to higher
levels of the encoded proteins. The schematic diagrams
of the proposed phenomena are described in Fig. 2.
The second control mechanism is in response to
glucose, which is transported into the cell by the
phosphoenolpyruvate (PEP)-dependent phosphotransferase
system. The phosphate group of PEP is transferred via
a phosphorylation cascade, consisting of the general
phosphotransferase system (PTS) proteins HPr and
EIA, and the glucose-specic PTS proteins, EIIAGlc and
EIIBGlc, which are the cytoplasmic domain of the EII

glucose transporter. Transport of glucose is accompanied
by its phosphorylation by EIIBGlc through removing the
phosphate group from the other PTS proteins, including
EIIAGlc. The unphosphorylated form of EIIAGlc binds to
the lac permease and prevents it from bringing lactose into
the cell. Therefore, if both glucose and lactose are present,
the transport of glucose blocks the transport of the inducer
of the lac operon. This process is called inducer exclusion.[13]
In other way, 16 base pairs upstream site of the
promoter, which is used for a positive control of the
gene expression is known as CAP site or cAMP protein
site or catabolite gene activator (cga) site, because it is
utilized for binding of CAP to encourage gene
expression. CAP can bind to this site only when it is
bound with cAMP. Hence, a positive control is exerted
over the transcription process by the CAPcAMP
complex with an effect exactly opposite to that of
repressor binding to an operator. Moreover, the effector
molecule cAMP determines the effect of CAP on lac
operon transcription in the presence of glucose, it
inhibits the formation of cAMP and prevents it to bind
to CAP. The DNA-bound CAP is then able to interact
physically with RNA polymerase and essentially
increase the afnity of RNA polymerase for the lac
promoter. In this way, the catabolite repression system
contributes to the selective activation of the lac
operon.[14] The schematic diagram of mechanism of
carbon catabolic repression is described in Fig. 3.
DOI: 10.1002/apj
Figure 3. Mechanism of carbon catabolic repression (A) inducer exclusion and (B) induction
prevention (Figure adapted from B. Grke and J. Stlke (2008)).[13]
A mechanism of carbon catabolite repression by

which the activity of PTS-regulation domaincontaining transcription factors, which is inhibited in
the presence of preferred carbon sources, is called
Induction prevention. It proceeds with phosphorylation
of histidine protein (HPr) at Ser46 by HPr kinase/
phosphorylase (HPrK). This phosphorylation occurs
when the intracellular concentrations of fructose-1,
6-bisphosphate (FBP) and adenosine triphosphate are
high, which reects the presence of preferred carbon
sources. HPr(Ser-P) binds to CcpA protein, and this
interaction is enhanced by glycolytic intermediates, such
as FBP and glucose-6-phosphate. The complex of CcpA
and HPr(Ser-P) binds to cre sites on the DNA and
thereby represses the transcription of catabolic genes.
HPrK is also responsible for dephosphorylation of
HPr(Ser-P) under conditions of high inorganic
phosphate (Pi) and low adenosine triphosphate and
when there is poor nutritional supply of FBP. Also,
HPr(His-P) contributes to carbon catabolic repression:
in the absence of glucose, HPr(His-P) phosphorylates
glycerol kinase (GlpK), and transcriptional regulators
that contain PEPcarbohydrate phosphotransferase
system-regulatory domains, which acts as precursors
for their activity. Thus, in the presence of glucose,
activation of the phosphotransferase system-regulatory
domain regulators by their inducers is prevented.[13]
Research with this system is greatly added by the
availability of constitutive mutants. A constitutive
mutant of any consortia continuously produces gene
products where there is no control over its expression.
In these mutants, the aforementioned proteins are
produced all the time in comparison to the wild type
where the proteins only appear in the presence of lactose.
So in these mutants, the mutation must be a gene other
than those responsible for the structural gene.[15]
PRODUCTION OF -GALACTOSIDASE
-galactosidase belongs to the group of saccharides
converting enzymes, i.e., in the family of hydrolases.
They are widespread, distributed in numerous
biological systems, e.g., microorganisms (yeasts, fungi,
bacteria, and actinomycetes), plants, and animal
tissues. Compared with animal and plant sources,
microbial-synthesized enzyme provides higher yields,
which may decrease its production cost. Therefore,
production of -galactosidase through microbial route
creates a great attention. Although, the most studied
-galactosidase is produced by E. coli, possible toxic
factors associated with coliforms make it unlikely that
crude isolates of this enzyme, which may be permitted
in food processes.[1621] In industrial scale, production
of -galactosidases is carried out using generally
recognized as safe microorganism, yeast (mainly from
Kluyveromyces marxians, Kluyveromyces lactis, and
Kluyveromyces fragilis) and fungal (mainly from
Aspergillus niger and Aspergillus oryzae) consortia.
The detail works carried out in this direction have been
represented in Table 1.
PURIFICATION OF -GALACTOSIDASE
Different separation techniques, such as membranebased
separation,
ion
exchange
membrane
chromatography, gel permeation chromatography, zinc
chloride, protamine sulfate, and ammonium sulfate
precipitation; had already been attempted for
purication of -galactosidase from crude extract. The
detail works carried out in this direction have been
represented in Table 2.
DOI: 10.1002/apj
333
Bacillus licheniformis ATCC 12759

Lactobacillus acidophilus NRRL 4495
Bidobacterium animalis Bd12,
Lactobacillus delbrueckii ssp. bulgaricus
ATCC 11842
Bidobacterium animalis Bd12,

ATCC 11842
Lactobacillus plantarum Pi06
Lactobacillus acidophilus
Streptococcus thermophilus
ATCC 11842
Bacillus coagulans RCS3

Aspergillus avus
Penicillium chrysogenum NCAIM 00237
Aspergillus oryzae PTCC 5163
LacA from Aspergillus oryzae, expressing in

Saccharomyces cerevisiae NCYC869-A3/
pVK1.1
Rhizomucor sp.
Aspergillus niger
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Fungus
Fungus
Fungus
Fungus
Lactose-based chemically dened medium

Lactose-based yeast nitrogen base medium and semi-synthetic
lactose medium

Luria medium where individually galactose, glucose, mannitol,
lactose, and xylose were added
Luria medium
Acid whey
Chemically dened medium where individually glucose,
lactose, and galactose were used. Moreover, individually
peptone, yeast extract, casein hydrolysate, tryptone, or
ammonium sulfate were used
De-proteinated whey where protein was supplemented by
individually peptone, yeast extract, casein hydrolysate,
tryptone, and ammonium sulfate
de-Mann Rogosa and Sharpe medium
Whey-based medium
Skim milk, modied de-Mann Rogosa Sharpe medium, whey,
and whey permeate-based broth were used. Additionally, yeast
extract was added
Whey and chemically dened medium with different
carbohydrate, i.e., lactose, wheat barn, and soybean meal
Wheat bran and rice husk
Combination of skim milk powder, glucose, and yeast extract
Cheese whey
Cheese whey
Supplemented whey with cauliower waste
Whey
Whey
Lactose-based yeast-dened mineral medium
Lactose-based chemically dened medium, de-proteinated whey
Whey
Basal medium with IPTG
Substrate
Batch
Continuous
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch
Fed batch
Batch
Batch
Batch
Batch
Batch
Batch
Batch and fed batch
Batch
Fed batch
Batch
Batch
Operating mode of
bioreactor
[44]
[45]
[43]
[41]
[42]
[39]
[40]
[21]
[20]
[38]
[19]
[18]
[17]
[37]
[16]
[35]
[36]
[34]
[32]
[33]
[29]
[30]
[31]
[26]
[27]
[28]
[24]
[25]
[22]
[23]
Reference
A. NATH ET AL.
Fungus
Fungus
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Kluyveromyces marxians
Kluyveromyces marxians CBS 7894
Kluyveromyces marxians NCIM 3551
Kluyveromyces marxians CCT 7802
Kluyveromyces marxians MTCC 1388
Kluyveromyces fragilis NRRL-Y-1109
Recombinant Saccharomyces cerevisiae W303
Saccharomyces cerevisiae
Candida pseudotropicallis
Cloned Escherichia coli XL-1 Blue, XL-1
Blue FF, TB-1 and N4830
Lactobacillus sp. Bulgaricus CHR Hansen
Lb-12
Bacillus sp. MPTK 121
Bacillus sp.
Name of consortium
Yeast
Yeast
Yeast
Yeast
Yeast
Yeast
Yeast
Yeast
Yeast
Yeast
Yeast
Bacteria
Type of consortium
Table 1. Different types of consortium used in -galactosidase production.
334

DOI: 10.1002/apj
Ammonium sulfate precipitation, hydrophobic

interaction chromatography, and afnity
chromatography
Gel permeation chromatography, afnity
chromatography, and ammonium sulfate
precipitation
Column chromatographies on resource Q and
sephacryl S-200 HR
Ammonium sulfate precipitation and gel
permeation chromatography
Protamine sulfate precipitation, ammonium
sulfate precipitation, and ion exchange
chromatography
Heat treatment, ammonium sulfate precipitation,
ion exchange, and gel ltration chromatography
Lactobacillus reuteri
Bacillus circulans
(Expressed in Escherichia coli)
Streptococcus lactis
Bacillus licheniformis (cloned

and expressed in Escherichia
coli)
Bacillus megaterium
Bacillus stearothermophilus
(expressed in Bacillus subtilis)
Sonication, ammonium sulfate precipitation,

desaltation, column chromatography using
DEAEsepharose fast ow column, and afnity
chromatography
Ion exchange chromatography on Ni Sepharose
6 fast ow column and ultraltration
ZnCl2 precipitation, ion exchange membrane

chromatography, hydrophobic interaction
chromatography and gel ltration
chromatography
Bacillus sp. 3088
Streptococcus thermophilus
Puried 292-fold by chromatography on

Ultrogel ACA 34, DEAESephadex A-50
columns and by afnity chromatography in
agarose-p-aminophenyl- -D-thiogalactoside
Purication procedure
Lactobacillus murinus
Source
Table 2. Different methodology for -galactosidase purication.
Intracellular, M.W. is 530 kg mol 1. Optimum pH is 7.1. Galactose

is competitive inhibitor (Ki 60 10 3 M), Km for ONPG and lactose
are 0.98 10 3 and 6.9 10 3 M, respectively.
Enzyme is most labile when it is suspended at cold (278.15 K)
phosphate buffer. In presence of high concentration ammonium
sulfate (0.85 M), enzyme is highly active and stabilizes.
Intracellular, M.W. is 70 kg mol 1. Isoelectric point is 5.1. Optimum
temperature and pH are 343.15 K and 7, respectively. Kinetics of
thermal inactivation and half life at 338.15 and 343.15 K are 18 104
and 324 102 s. Km and Vmax are 2.96 10 3 M and 0.11 10 3
kmol s 1 kg of protein, respectively. Inhibitors are Fe(2+), Zn(2+),
Cu(2+), Pb(2+), Sn (2+), and thiol agent.
M.W. is 118 kg mol 1. Optimum pH 7.58.0 and temperature 328 K.
Enzyme is stable at pH 6.09.0 and below 313 K. The Km and Vmax
values for ONPG and lactose are 9.5 mM, 16.6 mM.min 1 and
12.6 mM, 54.4 mM. min 1, respectively.
Homodimeric, optimum temperature, and pH are 323.15 K and
6.5, respectively. Km for lactose and ONPG are 169 10 3 and
13.7 10 3 M, respectively. Inhibitors are glucose and galactose.
Metal activators are Na+, K+, Mg+2, Mn+2, and Ca+2.
Subunits are same molecular weight. M.W. is 170 kg mol .

Maximum enzymatic activity at 318 K and pH 7 in presence of 50 mM
phosphate buffer. The Km for ONPG and ONPG + 20 10 3 M
of lactose are 480 10 6 and 870 10 6 M, respectively. Inhibitors
are 10 10 3 M CaCl2, glutathione, and cysteine, and stimulator is
10 10 3 M MgCl2. Activators are mercaptoethanol and dithiotreitol.
Enzymatic activity is not shown in presence of p--galactosidase.
M.W. is 484 kg mol 1, associate with subunits 115, 86.5, 72.5, 45.7,
and 41.2 kg mol 1. Optimum pH and temperature are 8 and
333.15 K, respectively. Isoelectric point is 6.2. Km is 6.34 10 3
and 6.18 10 3 M for ONPG and lactose, respectively.
Inhibitors are galactose divalent Hg, Cu, and Ag. Metal activator is
divalent Mg. EDTA do not affect the enzyme activity.
Heterodimer enzyme, M.W. is 105 kg mol 1 (monomers are 72 and
35 kg mol 1). Isoelectric points are 4.64.8 and 3.84.0 for L. reuteri
L103 and L. reuteri L461, respectively. Optimum pH is 68. Inhibitor
is D-glucose and activators are Na (+), Mn (+2), and K (+).
M.W. is 212 kg mol 1, composed by 145 and 86 kg mol 1. Km for
three subunits are 3.6 10 3, 5.0 10 3, and 3.3 10 3 M for ONPG,
respectively and those are 3.7 10 3, 2.94 10 3, and 2.71 10 3 M,
respectively considering lactose as a substrate.
Potential of GOS synthesis
Characteristics of puried enzyme
(Continues)
[55]
[54]
[53]
[52]
[51]
[50]
[49]
[48]
[47]
[46]
Reference


DOI: 10.1002/apj
335
Ultrasonic treatment in the presence of Triton

X-100
Ammonium sulfate precipitation, dialysis,

anion exchange column (Mono Q HR 5/5,
Pharmacia) using a FPLC system (Pharmacia).
Active -galactosidase fractions has been
collected from several chromatographic runs
and concentrated using the Centriplus system
(Millipore). The active fraction of galactosidase has been obtained from ion
exchange chromatography.
Column chromatographies by DEAESephadex

A-50,TSK gel Toyo Pearl HW-55S,
and TSK gel DEAE-5PW
Bidobacterium infantis
(expressed in Escherichia coli)
Cryptococcus laurentii OKN-4
Ion exchange chromatography on DEAE

sepharose,
afnity chromatography on PABTG-sepharose,
and gel ltration on Sephacryl S-300
Ultrasonication and subsequently puried
by Q Fast-Flow chromatography and gel
chromatography on a Superose 6 HR column
Bidobacterium bidum
Bidobacterium longum
CCRC 15708
Arthrobacter sp.
Source
Table 2. (Continued)
[56]
Intracellular, homodimeric, each subunit M.W. is 116 kg mol 1.

Optimum pH and temperature are 68 and 298.15 K, respectively.
Activators are thiol compounds, Na (+), and K (+). Inactivated by
4-chloromercuribenzoic acid, Pb (2+), Zn (2+), and Cu (2+)
Finally 15.7-fold, a yield of 29.3%, and a specic activity of
168.6*106 U kg-1 protein (-galactosidase) has been achhived. The
MW was 357 kg.mol-1 as determined from Native-PAGE. The pH
and temperature optima of the puried -galactosidase were 7.0 and
323 K, respectively considering ONPG as a substrate,. The enzyme
was stable at a temperature up to 313 K and at pH values of 6.5-7.0.
Values of K m and V max for this puried enzyme were found to be
0.85 mM and 70.67 *106 U kg-1, respectively. It was found that
Na+ and K+ stimulated the enzyme up to 10-fold, while Fe3+, Fe2+,
Co2+, Cu2+, Ca2+, Zn2+, Mn2+ and Mg2+ inhibited the activity of galactosidase. Furthermore it was found that glucose, galactose,
maltose, or rafnose exerted little or no effect on the -galactosidase
activity, lactose and fructose inhibited the enzyme activity. The
effect of lactose on the enzyme activity for ONPG is probably a case
of competitive inhibition.
Enzyme has different subunits, such as 163, 170, 178, and
190 kg mol 1. The M.W. is 362 kg mol 1 and isoelectric point is
5.25. The puried enzyme is stable at temperatures below 318 K and
over the pH ranges from 6.58. Lactose hydrolysis by the puried
enzyme take places at pH 6.5 and temperature 310 K.
The enzyme is tetramer, with an M.W. of about 470 kg mol 1, and
its subunit is 115 kg mol 1. The optimum temperature and pH for
ONPG and lactose are 333 K, pH 7.5 and 323 K, pH 7.5, respectively.
The enzyme is stable over a pH range of 5.08.5 and remains active
for more than 4800 s at pH 7.0, 323 K. The enzyme activity has been
signicantly increased by reducing agents. Maximum activity has
been shown in presence of both Na+ and K+ at a concentration of
10 10 3 M. The enzyme is strongly inhibited by
p-chloromercuribenzoic acid, divalent metal cations and Cr3+, and
to a lesser extent by EDTA and urea. The hydrolytic activity using
lactose as a substrate has been signicantly inhibited by galactose.
The Km and Vmax values for ONPG and lactose are 2.6 10 3 M,
262 106 U kg 1 and 73.8 10 3 M, 1.28 106 U kg 1, respectively.
The enzyme possesses strong transgalactosylation activity. The
production rate of GOS from 20% lactose at 303 and 333 K is
120 10 3 kg L 1, and this rate increases to 190 10 3 kg L 1 when
30% lactose has been used as a substrate.
M.W. is 200 kg mol 1. homodimeric, optimum pH is 4.3, and enzyme
is stable at pH between 2.8 and 9.3. The optimum temperature is
333 K and enzyme is stable at temperature below 330.5 K for 600 s
(Continues)
A. NATH ET AL.
[60]
[59]
[58]
[57]
Reference
336

DOI: 10.1002/apj
Precipitation with ammonium sulfate, ion

exchange chromatography on DEAESephadex,
afnity chromatography, and chromate focusing
Gel ltration chromatography, ion exchange
chromatography, and afnity chromatography
Gel ltration on Superose 12 PC 3.2/30 column
and ion exchange
Kluyveromyces lactis
Protein precipitation by Pectinex Ultra SP followed

by desalting and column chromatography using
DEAESepharose fast ow.
Aspergillus aculeatus
Penicillium chrysogenum
Gel ltration, anion exchange chromatography

on DEAESepharose CL-6B, hydrophobic
chromatography on octyl-SepharoseCL-4B and
cation exchange
chromatography on CMSepharose CL-6B
Metal-ion afnity chromatography (IMAC)

followed by size-exclusion separation
2-propanol fractionation, column

chromatography on DEAESephadex
A-50, and Sephadex G-200
Aspergillus niger
Aspergillus oryzae
Source
incubation. The Km values of the enzyme are 18.2 10 3 M and

11.4 10 3 M, and the values of Vmax are 1.28 10 3 kmol s 1 kg
of protein, and 0.09 10 3 kmol s 1 kg of protein for ONPG and
lactose, respectively. The enzyme is strongly inhibited by Hg2+, Ag+,
2-mercaptoethanol, glucose, maltose, and maltotriose. Enzyme is
potential of GOS synthesis.
Extracellular, optimum pH for ONPG and lactose are 4.5 and 4.8,
respectively. Optimum temperature is 319.15 K. Km are 7.2 10 4
and 1.8 10 2 M for ONPG and lactose respectively. Inhibitors are
divalent Hg, Cu, N-bromosuccinimide, and sodium laurylsulfate.
Apparent M.W. is 105 kg mol 1.
Extracellular, M.W. is 113 kg mol 1. Mutant enzyme has ve times
higher catalytic activity on the synthetic substrate ONPG compared
with the wild-type enzyme. Moreover, the mutant enzyme is more
thermo resistant compared with the wild type.
Glycoprotein in nature. Associates with three subunits, and each M.
W. are 124, 150, and 173 kg mol 1. Isoelectric point is 4.6.
Optimum pH lies between in 2.5 to 4.0. Heat stable up to 333.15 K.
Km value varies from 85 10 3 to 125 10 3 M for lactose. Km
value varies 2.4 10 3 M for ONPG. Vmax values are 104 103 unit
enzyme kg of protein and 121 103 unit enzyme kg of protein at
303.15 K for ONPG and lactose, respectively.
The M.W. is 120 kg mol 1 (approximately), isoelectric point lies
between 5.3 and 5.7 and is optimally active at pH 5.4 and
temperature 328333 K. Based on the N-terminal amino acid
sequence, the enzyme probably belongs to family 35 of the glycosyl
hydrolases. Enzyme is potential of synthesizing GOS.
Enzyme has activity towards two exo-poly-saccharide (EPSs),
having lactosyl side chains attached to different backbone
structures. The enzyme degraded O-deacetylated EPS B891 faster
than EPS B39. Moreover, the presence of acetyl groups
in EPS B891 slows down the hydrolyzing rate but the enzyme is
still able to release all terminally linked galactose.
Intracellular, Specic activity is 5.84 103 U kg of protein. Optimum
temperature and pH are 303.15 K and 4, respectively. Km and
isoelectric point are 1.81 10 3 M and 4.6 for ONPG. Multimeric
enzyme, M.W. is 270 kg mol 1, and M.W. of single monomer is
66 kg mol 1
Afnity chromatography is shown better results than gel permeation
chromatography, and ion exchange chromatography. Similar
molecular weight subunit. Enzyme is conrmed by specic antigenantibody reaction (ELISA test).
Intracellular, M.W. of single monomer is 124 kg mol 1
(Continues)
[66]
[65]
[41]
[64]
[63]
[62]
[61]
Reference


DOI: 10.1002/apj
337
Gel ltration chromatography (Ultrogel AcA

34), anion exchange (Mono Q), and gel
ltration (Superose-12)
Centrifugation, cell disruption, heat treatment,

DEAEToyopearl chromatography,
salting out, Butyl-Toyopearl chromatography,
Chromatofocusing chromatography, and PATG
chromatography
Ammonium sulfate fractionation, column

chromatography on Sephadex G-100, and
DEAESephadex A-50, 78-fold purication
FPDA 13 column chromatography, ammonium
sulfate fractionation, Ultrogel AcA34 column
chromatography, DEAESepharose CL-6B
column chromatography, TSK gel DEAEToyopearlpak 650S High-pressure liquid
chromatography (HPLC)
chromatography on Mono Q PC 1.6/5 column

using a FPLC system
Cell disruption, DEAESephadex ion exchange
chromatography,
and chromatography on hydroxylapatite
Extraction with 2% chloroform, acetone, and
ammonium sulfate precipitation
Extracellular thermostable, relative M.W. of 145 000 and s20,w of

7.1 s. Michaelis constant Km is 0.75 10 6 kM and molecular
activity (kcat) is 63.1 s 1 at pH 7.2 and 328 K for ONPG where as Km
is 0.04 10 6 kM and kcat is 55.8 s 1 for p-NPG. Enzyme has a high
transgalactosylation activity. The enzyme reacts
with 1.75 M lactose at 333 K and pH 7.0 for 7.92 104 s to obtain
maximum yield oligosaccharides (41% (w/w)). The general structure
for the major transgalactosylic products can be expressed as (Gal)cGlc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal.
M.W. is 170 and 86 kg mol 1 for denatured enzyme, isoelectric
point 4.1. The optimal temperature is 358 K, and it is stable at
temperatures up to 353 K for 3 600 s. The optimal pH range for the
enzyme is 4.5 to 5.0 and the enzyme is stable at pH 2.5 to 7.0.
Ezyme is inhibited by Hg2+. The Km values for ONPG and l
actose are 9.5 10 3 and 2.4 10 3 M, respectively. The Vmax for
these substrates are 96 mol s 1 kg of protein and 4 mol s 1 kg of
protein, respectively. The enzyme possesses a high level of
transgalactosylation activity (higher yield 39% from 200 g L 1
lactose), which produces GOS including tri- and tetrasaccharides.
M.W. is 700 kg mol 1 and subunit is 59 1 kg mol 1. Isoelectric
point is 4.9. The optimum temperature and pH for enzyme activity
are 353 K and 5.5, respectively. The enzyme is stable over a wide
pH range (pH 312), and the thermostability of the enzyme is
enhanced by CaCl2. The enzyme is signicantly activated by alkali
and alkaline-earth-metal salts. Inhibitors are thiol-binding agents,
glucose, and galactose. The enzyme specic for b-D anomeric
linkages and the identity of the aglycone moiety also inuenced
enzyme activity dramatically. Enzyme is potential of GOS
synthesis.
M.W. is 135 kg mol 1. Optimum pH is 7.25. Km is 12 10 3 to

17 10 3 M for lactose. Km is 1.6 10 3 M for ONPG. Metal
activators are Na(+) and Mn(+2).
M.W. is 280 kg mol 1. Optimum temperature and pH are 318.15 to
325.15 K and 6.2, respectively. Inactivates in 240 s at 329.15 K. Km
are 3.1 10 3 and 25 10 3 M for ONPG. Inhibition constants are
58 10 3, 110 10 3, 111 10 3, and 52 10 3 M for ONPG,
galactose, ribose, and lactose. Inhibitors are also
P-chloromercuribenzoate and dithiothreitol.
Optimum pH is 7.2. Metal activator divalent magnesium, Km is
1.18 10 3 M for ONPG.
[72]
[71]
[70]
[69]
[68]
[67]
Reference
A. NATH ET AL.
Thermus aquaticus
Sterigmatomyces elviae
Saccharopolyspora rectivirgula
Saccharomyces lactis
Kluyveromyces marxianus
Source
338

DOI: 10.1002/apj
CHARACTERIZATION OF -GALACTOSIDASE
Apart from catalyzing the hydrolysis of -galactosides
into monosaccharides by lactase, the enzyme may also
cleave fucosides and arabinosides with much lower
efciency. Lactase is often confused as an alternative
name for -galactosidase, but it is merely a sub-class
of -galactosidase. In other words, -galactosidase is
an exoglycosidase, which hydrolyzes the -glycosidic
bond formed between galactose and its organic
moiety.[73] Molecular weight, amino acids chain length,
position of the active site, pH, and optimum thermal
stability are signicantly differed by the microbial
sources.[74] The choice of suitable -galactosidase source
depends on the condition of reaction. For example, dairy
yeasts with optimum pH (6.57.0) are habitually used for
the hydrolysis of lactose in milk or sweet whey.[75] On the
other hand, the fungal -galactosidases with optimum pH
(3.05.0) are more suitable for acidic whey hydrolysis.[76]
The activity of different -galactosidases also depends
on presence of ions. The fungal -galactosidases
are active without presence of ions as cofactors; where
-galactosidase isolated from K. lactis shows its higher
activity in presence of Mn2+ and Na+. -galactosidase
synthesized from K. fragilis are mostly active in presence
of Mn 2+ , Mg 2+ , and K + . [77] On the contrary, Ca 2+
and heavy metals inhibit the enzyme activity of all
-galactosidases.[78] Properties of -galactosidase
synthesized by different microorganisms are described
in Table 3.
In 1970, 1024 amino acids of -galactosidase of E.
coli were rst sequenced.[93] After 24 years, four chains
comprising the protein were discovered to be 464 kg
mol 1 tetramer with 222-point symmetry. Every unit
of -galactosidase contains ve domains; whereas the
active site persists in the third domain. This enzyme
can be split into two peptides, LacZ and LacZ, none
of them is active but both spontaneously reassemble a
functional enzyme. This characteristic is used for many
cloning vectors to achieve -complementation in
specic laboratory strains of E. coli, where the plasmid
encodes the small LacZ while the large LacZ is
encoded by the bacterial chromosome. Aftermath,
when DNA fragments inserted in the vector,
production of LacZ disrupted, the cells revealed no
-galactosidase activity, were subjected to the blue/white
screening of recombinant clones further.[94] The active
site of -galactosidase catalyzes the hydrolysis of
disaccharide substrate via shallow and deep binding.
The beta-linkage of the substrate was cleaved by a
terminal carboxyl group on the side chain of glutamic
acid.[95] It has been determined that the DNA fragment
of thermophilic bacterium Thermoanaerobacter
ethanolicus contains three open reading frames. One of
the open reading frames corresponded to the LacA gene
for a thermostable s-galactosidase and the native
recombinant LacA showed the highest activity at
348 K353 K. Immobilized on aldehyde silochrome,

LacA was even more thermo stable and retained high
activity.[96] It has been found that individual molecules
of -galactosidase from the crystallize enzyme as well
as the original enzyme displayed a range of activity of
20-fold or greater. Molecules obtained from two diverse
crystals have identical activity distributions, i.e.,
31 600 1100 and 31 800 1100 reactions per minute
per enzyme molecule. This activity of the enzyme was
found to be drastically different from that of the enzyme,
which was used to grow the crystals (showed an activity
distribution of 38 500 900 reactions per minute per
enzyme molecule).[97] Induced -galactosidase in E. coli
wild-type strains ATCC 8677 and 35321 in the presence
of various protease inhibitors have been studied. The
presence of the protease inhibitors had least effect on
the average distribution of single molecule activities,
and the relative activities of the enzyme for the diverse
substrates differed between the strains.[98] Averagecombined turnover numbers of the enzyme from wildtype E. coli strains ATCC 35321 and 8677 in vivo and
in vitro conditions in the presence and absence of His6
tag differed considerably. This indicated that synthesized
enzymes in both conditions (vivo and vitro) were not
alike and presence of a C-terminal His6 tag altered the
activity of s-galactosidase.[99] Moreover, it was found
that electrophoretic mobility and catalytic activity of
individual molecules of -galactosidase synthesized by
E. coli were different, although they had potentiality to
act on the same substrate molecule.[100]
IMMOBILIZATION OF -GALACTOSIDASE
Immobilization has shown to improve the stability of
-galactosidase, reusages, and reduces the processing
time in food and other industries. For example, the
immobilization of -galactosidase of Thermus sp. T2
was performed using ionic adsorption by a new anionic
exchanger resins (based on coating of Sepabeads
internal surfaces with polyethylenimine) and
conventional DEAEagarose. Immobilization was
carried out in both cases, but the adsorption strength
showed much greater in the case of PEISepabeads than
in DEAE supported at both pH 5.0 and 7.0. Also, the
PEISepabeads remained wholly active, and after several
weeks of incubation at 323 K, it showed the lactose
hydrolysis in milk.[101] Also a new hetero functional
epoxy supports were used for immobilization of
-galactosidase. The capability of a standard
Sepabeads-epoxy supports to immobilize -galactosidase
from Thermus sp. strain T2 that equalized with other
Sepabeads-epoxy, which supports partially modication
using boronate, iminodiacetic, metal chelates and
ethylenediamine. Here, immobilization was depended
on the support, ranging from 95% to 5% using
DOI: 10.1002/apj
339
Archae
Fungus
Arthrobacter psychrolactophilus
Bacillus circulans
Bacillus megaterium
Bidobacterium adolescentis
Bidobacterium bidum
Cryptococcus laurentii
Enterobacter agglomerans
Streptococcus pneumonia
Alicyclobacillus acidocaldarius
Caldicellulosiruptor
saccharolyticus
Geobacillus stearothermophilus
Thermotoga maritima
Thurmus sp.
Thurmus aquaticus
Kluyveromyces fragilis
Sporobolomyces singularis
Bullera singularis
Aspergillus oryzae
Aspergillus aculeatus
Sulfolobus solfataricus
Name of consortium
8
56
69
7
6
6.5
7.5
4.3
7.58
6.58
68
5.57.5
5.5
6
6.5
6.57.2
6.5
6.5
5.5
6.5
6.67
4
4.55
5
4.8
5.4
6.5
338
333
353358
343
353
303
310313
303
358
323
303
328333
348
pH
333
323
313
343
323
310
323333
331
310313
328
323
303
338
353
Temperature (K)
20
5.1
60
125
526
569
12
230
180
592
211
0.5
70
5.7
8.7
20
56
40
24
116
1.44 104 (333 K)

960 (363 K)
7.2 104 (343 K)
1.8 104 (313 K)
3600 (353 K)
7200 (318 K)
600 (343 K)
1.08 104 (358 K)
Specic
activity (U mg 1)
3600 (303 K)
3.24 104 (343 K)

600 (323 K)
1.44 104 (318 K)
7200 (333 K)
600 (331 K)
1.728 105 (310 K)
360 (353 K)
1.728 105 (343 K)
Half life (s)
[92]
[62]
[64]
[90]
[71]
[91]
[88]
[89]
[87]
[88]
[72]
[86]
[70]
[84]
[85]
[82]
[48]
[83]
[60]
[81]
[80]
[58]
[59]
[54]
[53]
[79]
[50]
Reference
A. NATH ET AL.
Yeast
Bacteria
Type of consortium
Table 3. Properties of -galactosidase synthesized by different microorganisms.
340

DOI: 10.1002/apj
Sepabeads-epoxy-chelate, Sepabeads-epoxy-amino, or
Sepabeads-epoxy-boronic using Sepabeads-epoxy-IDA.
Amazingly, the immobilized -galactosidase derivatives
showed outstandingly good result but different stabilities
had been notied after favoring multipoint covalent
attachment by long-term alkaline incubation. The
enzyme immobilized on Sepabeads-epoxy-boronic
was found to be the steadiest. The crosslinking with
aldehyde-dextran allowed the stabilization of the
quaternary structure of the enzyme. The optimal
derivative was extremely active in lactose hydrolysis
even at 343 K (over 1000 IU g 1), maintaining its
activity after extended incubation times with no risk
of product contamination with enzyme subunits.[102]
Protocol for immobilization of -galactosidase
synthesized by E. coli using diverse supports (glyoxyl,
epoxy, BrCN groups, or by glutaraldehyde crosslinking
on matrix, containing primary amino groups), and
strategies have been studied. In each case, the
immobilization yield showed 100% with active recoveries
between 50% and 100% (using ortho-nitrophenyl-galactoside as substrate). Ratio of synthetic activity
to hydrolytic activity (Vs/Vh) was lower than 0.1 when
soluble enzyme and the Eupergit 250 L enzyme
were immobilized on BrCN at 277 K and pH 7.0,
resulting 0.46 and 0.8, respectively.[103] Immobilization
of -galactosidase producing permeabilized dead cells
of K. lactis ATCC 8583 into gelatin using
glutaraldehyde as crosslinker was performed, where
30% activity obtained by immobilized cells relative to
free disrupted cells.[104] The usage of calcium alginate,
-carrageenan, and gellan-xanthan gel beads for
entrapment of -galactosidase synthesized by
Streptococcus thermophilus, enhanced the stability of
enzyme at higher temperatures (>298 K).[105] Solid state
fermentation with co-immobilized -galactosidases
synthesized by K. lactis, Aspergillus oryzae, and yeasts
in polyvinyl alcohol hydrogel lens-shaped capsules have
been performed. In the process the enzyme, synthesized
from Kluyveromyces lactis and Saccharomyces
cerevisiae showed the highest activity (galactose output
increased from 3 to 4.1 g l 1 h 1), which reduced the
Different
reduction
of
processing
time.[106]
methodologies for immobilization of -galactosidases
have been described in Table 4.
INDUSTRIALIZED APPLICATIONS OF
-GALACTOSIDASE
Microbial -galactosidase plays a tremendously
essential role in the production of various industrial
relevant products such as biosensor, lactosehydrolyzed milk, ethanol, and GOS, also it has been
used in the eld of bioremediation, diagnosis, and in
treatment of lactose digestion disorder etc.
Use of -galactosidase in biosensor

A biosensor associated with two distinct enzymatic
activities (-galactosidase and glucose peroxidase) has
been developed for quantitative detection of lactose in
commercial samples of milk. To avoid interferences
with glucose, a degree of different mode of
measurement was done using biosensor.[146]
Foresighting this technique, presumed -galactosidase
from Streptococcus mitis with a choline-binding
domain was identied recently. This remarkable
property makes it differentially functional property for
biotechnological applications.[147]
Intended as lactose-hydrolyzed milk production
The coldstable properties of Arthrobacter sp. 32c
synthesized -galactosidase could be useful for
commercial, industrial conversion of lactose into
galactose and glucose in milk products.[56] It has been
reported that rudimentary -galactosidase extract
produced by Lactobacillus ssp. bulgaricus CHR
Hansen Lb-12 was applied in sterile milk, which has
been processed through ultra-high temperature method
(UHT milk) for hydrolyzing lactose. Optimum amount
of crude -galactosidase extract and Maxilact enzyme
for producing lactose-hydrolyzed milk was observed
to be 0.418 and 0.512 U mL 1 respectively during 6 h
of processing. Using more than 418 U L 1 of crude
-galactosidase extract showed undesirable acidity of
lactose-hydrolyzed milk that signicantly increased at
temperature of between 288 and 290 K, while
enrichment of acidity in lactose-hydrolyzed milk
produced through Maxilact enzyme was not signicant.
Total count of lactose-hydrolyzed milk by 418 U L 1
of crude -galactosidase extract, after 2.16 104 s of
processing was signicant high (5 to 30 Colony Forming
Unit). Sensory estimation of lactose-hydrolyzed milk
and ordinary ultra-high temperature milk (controlled)
did not show any major differences with respect to
acceptability of sweetness, taste, and color.[33] The
difculty in enzyme extraction and poor permeability
of cell membrane to lactose was solved when
permeabilized Kluyveromyces marxianus NCIM 3465
cells were used for the production of lactose-hydrolyzed
milk. The ethanol-permeabilized yeast cells showed 89%
of hydrolysed lactose under optimized conditions.[148]
Role of -galactosidase in whey utilization
The execution of profound environmental legislation
for recycling and reuse of waste materials is grabbing
lots of headlines at present century. Since many years,
dairy products have been used as valuable ingredients
by the confectionery industry, being a huge economical
source of capital, especially in the tropical and
subtropical countries. The large volume of byproduct
DOI: 10.1002/apj
341
342
A. NATH ET AL.
Table 4. Different techniques for immobilization of -galactosidases.
Immobilization method
Source of -galactosidase
Covalent binding
Covalent binding
Entrapment
Physical adsorption
Physical adsorption
Covalent binding
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Aspergillus
Covalent binding
Covalent binding
Entrapment
Physical adsorption
Covalent binding
Physical adsorption
Covalent binding
Entrapment
Physical adsorption
Physical adsorption
Aspergillus oryzae
Aspergillus oryzae
Aspergillus oryzae
Aspergillus oryzae
Aspergillus oryzae
Aspergillus niger
Kluyveromyces bulgaricus
Kluyveromyces fragilis and
Kluyveromyces lactis, Aspergillus
oryzae and Saccharomyces
cerevisiae
Thermus aquaticus YT-1
Thermus sp. T2
Penicillium expansum F3
Saccharomyces anamensis
Pisum sativum
Lactobacillus bulgaricus
Bacillus circulans
Bacillus circulans
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli (Recombinant
-galactosidase)
Escherichia coli
Escherichia coli
Physical adsorption
Physical adsorption
Covalent binding
Physical adsorption
Entrapment
Covalent binding
Covalent binding
Covalent binding
Covalent binding
Entrapment
Physical adsorption
Entrapment
Covalent binding
Physical adsorption
Covalent binding
Physical adsorption
Physical adsorption
Covalent binding
Entrapment
Covalent binding
Physical adsorption
Covalent binding
Covalent binding
Covalent binding
oryzae
oryzae
oryzae
oryzae
oryzae
oryzae
of the dairy industry, namely, whey, creates severe

disposal problem. Concerning about environmental
aspects, research has been emphasized on membrane
separation technology, providing remarkable new
opportunities for large-scale protein and lactose
fractionation. They intrude proteins that are multifunctional food ingredients of high nutritional value
and offer a wide range of functional properties allowing
development of new products and optimization of
existing products within considerable low cost, but
presence of high concentration of lactose in aqueous
medium after separation of proteins from whey creates
severe disposal problem.[149153] Different strategies
Immobilization matrix
References
Amino-epoxy Sepabead
Chitosan bead and nylon membrane
Nylon-6 and zeolite
Phenol-formaldehyde resin
Polyvinyl chloride and silica gel membrane
Polyvinyl alcohol hydrogel and magnetic
Fe3O4-chitosan as supporting agent
Silica
Silica gel activated with TiCl3 and FeCl3
Spongy polyvinyl alcohol Cryogel
Celite and chitosan
Cotton cloth and activated with tosyl chloride
Porous ceramic monolith
Magnetic polysiloxane-polyvinyl alcohol
Alginate using BaCl3
Cellulose beads
Chitosan
[107]
[108]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
[116]
[117]
[118]
[119]
[120]
[121]
[3]
Chitosan
Chitosan bead
Cotton fabric
CPC-silica and agarose
Poly(vinylalcohol) hydrogel
[122]
[123]
[124]
Corn grits
Thiosulnate/thiosulfonate
Graphite surface
Silica-alumina
Agarose bead
PEISepabeads, DEAEagarose
Calcium alginate
Calcium alginate
Sephadex G-75 and chitosan beads
Egg shells
Chitosan
Polyvinyl chloride and silica
Eupergit C (Spherical acrylic polymer)
Polyacrylamide gel
Polyvinyl alcohol
Chromosorb-W
Cyanuric chloride-activated cellulose
[127]
Hen egg white

Gelatine
[144]
[125]
[126]
[128]
[129]
[130]
[131]
[132]
[133]
[134]
[135]
[136]
[137]
[138]
[139]
[140]
[141]
[142]
[143]
[145]
have been attempted for whey lactose conversion, those

are related to the synthesis of nutraceuticals GOS, as well
as alcohol from whey permeates would ensure ultimate
consumption and utilization of casein whey.[154,155]
Function of -galactosidase in ethanol
fermentation
The idea of kinetic analysis of alcoholic fermentation
of lactose using a recombinant occulent strain of
Saccharomyces
cerevisiae
NCYC869-A3/T1,
expressing both the LAC4 (coding for -galactosidase)
and LAC12 (lactose permease) genes of
Kluyveromyces Lactis conceded. The lactose was
DOI: 10.1002/apj
completely utilized in all fermentative processes, and it

has been observed that with increase of initial lactose
concentration (5 to 200 g L 1), the level of ethanol
production increased linearly.[156] Meticulously, kinetic
model with respect to biomass growth, lactose
hydrolysis, and ethanol production using -galactosidase
synthesized by Kluyveromyces sp. was performed
considering whey permeate and cheese whey powder
as growth medium.[157159] Moreover, Saccharomyces
cerevisiae has also been reported for production of
ethanol considering concentrated deproteinized whey,
cheese whey powder, and salted cheese whey as feed
stock.[160162] Ethanol fermentation of cheese whey
powder solution using the pure culture of Kluyveromyces
marxianus (DSMZ 7239) was studied in packed column
bioreactor using olive pits as sustaining particles for
cell attachment.[163] Researches have also been
conducted for ethanol production by membrane
recycle bioreactor and using co-immobilized S.
cerevisiae strain and -galactosidase in semicontinuous fermentation process considering whey
permeate and whey medium, respectively.[164,165]
Thus, whey utilization by -galactosidase reduces the
burden of water pollution and establishes the concept
of recycling and reuse of waste materials.
Galato-oligosaccharide production by
-galactosidase
In recent years, many investigations have been carried
out in the eld of prebiotics, considered as functional
food. Among them, one of the recognized functional
food ingredients is oligosaccharides. They are
carbohydrates containing three to ten sugar units bound

with glycosidic bonds. It has been seen that there are a
number of classes of oligosaccharides, but among
them, GOS has attracted particular attention because
of their presence in human breast milk. GOS are nondigestible, carbohydrate-based food ingredient,
responsible for human and animal nutrition. Production
of GOS by transgalactosylation activity results the
formation of 4- or 6-galactosylactose, longer
oligosaccharides, transgalactosylated disaccharides,
and nonreducing oligosaccharides in presence of
-galactosidases. Depending on the source of enzyme
and conditions of reaction, various glycosidic linkages,
such as (1,2), (1,3), (1,4), and (1,6), are formed
in the end product (GOS).[166] It has been found that
the amount of GOS synthesis from lactose depends
on the initial concentrations of lactose present in
the reaction mixture instead of the concentration of
-galactosidase. There are different phenomena that
have been reported to elucidate the synthesis
mechanism of GOS, which are depicted in Figs 46.
It is believed that the presence of GOS in human
milk inuences the growth of bidobacteria in the
gastrointestinal tract of newly born and breast-fed
infants. GOS fraction (referred to as bidus factor) in
cows milk also provides several health benets.[170]
GOS is stable under acidic conditions during food
processing as well as maintain excellent taste quality,
which makes them popular as an active ingredient in
a wide variety of food products. They pass through
the small intestine without being digested for low
caloric value. In addition, GOS is not metabolized by
Figure 4. Schematic diagram of galato-oligosaccharide synthesis from lactose,
(A) engineering approach and (B) biochemical approach (Figure adapted from
Boon et al. (1999)).[167]

DOI: 10.1002/apj
343
344
A. NATH ET AL.
Figure 5. Schematic diagram of galato-oligosaccharide

synthesis from lactose (Figure adapted from Neri et al.
(2008)).[168]
Figure 6. Schematic diagram of galato-oligosaccharide

synthesis from lactose (Figure adapted from Palai et al.
(2012)).[169]
microorganisms in the oral cavity.[171] The detail works

carried out in this direction have been represented in
Table 5.
Applications -galactosidase in medical and
immunology research
Clostridium perfringens ATCC 10543 synthesized
endo -galactosidase, which is capable of liberating
benecial A trisaccharide and B trisaccharide from
glycoconjugates containing blood group A and B
glycotopes, respectively, was isolated by Anderson
et al., 2005. Recombinant EABase damages the blood
group A and B antigenicity of human type A and B
erythrocytes with the release of A-Tri and B-Tri from
blood group A + and B + containing glycoconjugates.
Here, the incomparable specicity of -galactosidase was
useful for studying the structure and function of blood
group A-containing and B-containing glycoconjugates.[193]
The recombinant endo--galactosidase (ABase), which
releases A/B antigen was developed in 2009. It
removed 82% of A antigen and 95% of B antigen from
human A/B red blood cells and concealed anti-A/B
antibody binding, also the complement activation
effectively. In vivo infusion into a blood type A
demonstrated the reduction of A antigen expression
in the glomeruli of kidney (85% at 3600 s, 9% at
1.44 104 s, and 13% at 8.64 104 s) and the sinusoids
of liver (47% at 3600 s, 1% at 1.44 104 s, and 3% at
8.64 104 s) without any grave adverse effects. This
substitute approach remains useful for minimizing
antibody removal and anti-B cell immunosuppression

as an adjuvant therapy of ABO incompatible kidney,
liver, and possibly heart transplantation.[194]
Expression of -galactosidase, synthesized by E. coli
within muscle bers has been demonstrated by Liu
and Rofer, 2006. They conclude that repeated
intramuscular injections of -galactosidase could
encourage strong immune responses among
immuno-competent animals and cause elimination
of transduce muscle bers by inammatory cells.[195]
Recently, -galactosidase from the mesoacidophilic
fungus Bispora sp. MEY-1 under simulated gastric
conditions has shown greater stability (100%) and
hydrolysis ratio (>80%) toward milk lactose than
commercially available -galactosidase from Aspergillus
oryzae ATCC 20423. Thus, this -galactosidase may be
a better digestive supplement for alleviating
symptoms associated with lactase deciency.[196]
Recombinant -galactosidases in cooperating one or
two different peptides from the foot-and-mouth
disease virus (FMDV) nonstructural protein 3B per
enzyme monomer, granted specied differentiation
between sera of FMDV-infected pigs, cattle, and
sheep, and those of native and conventionally
vaccinated animals. These FMDV infection-specic
biosensors can provide effectual and versatile
alternatives for the serological differentiation of
FMDV-infected animals.[197]
SCOPE FOR FURTHER STUDY

-galactosidase belongs to lactase group, which
hydrolyzes -glycosidic bond formed between
galactose and its organic moiety. Moreover, in some
cases, it takes part in transgalactosidase reaction. The
disadvantage of wild-type -galactosidase is its lower
activity as well as it is inhibited by hydrolyzed product,
glucose, and galactose. Therefore, research regarding
transgenic -galactosidase synthesis will boost up
hydrolytic activity as well as GOS synthesis. Although,
synthesis mechanism of -galactosidase is well established
for E. coli, still much more information is needed for other
microorganism. Although, it is recommended that
probiotic consortium (lactic acid bacteria) is much more
acceptable for food grade -galactosidase synthesis, but
because of product inhibition by lactic acid, research
on bioprocess design is in demand. More than 90-fold
purication of -galactosidase is yet to achieved. In this
circumstances, suitable purication device as well as
development of purication protocol for high throughput
is required. Research in the line of process intensication
of industrial -galactosidase production and purication
should be a great challenge. It is obvious, immobilized
-galactosidase provides several benets for GOS
synthesis but due to lower activity at immobilize
condition; studies on the active site of enzyme and
DOI: 10.1002/apj
Table 5. Different microorganisms for galato-oligosaccharide synthesis and parameters of transgalactosylation

reaction.
Nature of enzyme
Crude
Crude
Crude
Crude
Recombinant
Crude
Crude
Puried
Puried
Puried
Puried
Recombinant
Recombinant
Recombinant
Whole cells
Toluene-treated cells
Immobilized
Immobilized
Immobilized
Immobilized
Immobilized cells
Immobilized cell
Crude
Crude
Puried
Puried
Recombinant
Recombinant
Immobilized
Immobilized
Immobilized
Immobilized cell
Temperature
(K)
Enzyme source
Aspergillus oryzae
Bacillus circulans
Bidobacterium longum
Geobacillus stearothermophilus
Geobacillus stearothermophilus R109W
Talaromyces thermophilus
Bullera singularis
Enterobacter agglomerans
Penicillium simplicissimum
Sulfolobus solfataricus
Thermotoga maritima
Bidobacterium bidum
Rhodotorula minuta
Aspergillus candidus
Bacillus circulans
Bullera singularis
Cryptococcus laurentii
Penicillium sp.
Pyrococcus furiosus
Thermus sp.
Sirobasidium magnum
Sirobasidium magnum
Aspergillus oryzae
Sporobolomyces singularis
choice of suitable matrix are required. Without any

contradiction, -galactosidase plays several roles in
biopharmaceutical, food, and dairy industries. Research
for more applications in several elds particularly cancer
detection and pharmaceutical research will boost up
immunological research and biotechnological economy.
CONCLUSION
In this review, authors have tried their best to
accumulate all the information regarding production,
purication, characterization, immobilization, and
application of -galactosidase. -galactosidase is one
of the important enzyme that not only offers nutritional applications but also used for waste treatment.
-galactosidase used in food, pharmaceuticals, and
dairy industries for producing low lactose-containing
food product, sweeteners, GOS, and also used for dairy
waste treatment. It is also used for ethanol production,
which is a challenging step in present century. Several
microbes, especially probiotic consortia are
recommended for enzyme production using casein whey
as a growth medium. Puried -galactosidase will ensure
313
313
318
310
310
303
313
323
323
303
323
333
353
353
312
333
313
313
318
313
313
313
313
328
333
333
353
343
323
323
313
328
pH
4.5
6
6.8
6.5
6.5
6.5
6.5
5
7.5
6.5
6.5
7.5
6
6
6.8
6
6.5
6
3.7
7
6.5
4.3
6.5
4
6.5
5
5
7
4.5
5.0
Synthesis rate
(g L 1 h 1)
24
2.2
13
0.4
6.9
3.9
13
3.9
3.9
7.9
11
13
5.6
18
65
3.2
87
4.2
4.8
25
18.75
3.6
3.2
3.2
5.8
106
8.7
Reference
[172]
[173]
[174]
[86]
[86]
[175]
[176]
[91]
[81]
[82]
[177]
[59]
[178]
[179]
[180]
[181]
[182]
[183]
[184]
[185]
[186]
[187]
[186]
[188]
[70]
[71]
[189]
[190]
[191]
[191]
[117]
[192]
the high activity as well as poses better catalysis

properties. Enzyme immobilization provides enzyme
reutilization, also the immobilized enzyme showed high
level of hydrolysis and thus it can be applied successfully
for hydrolyzing lactose in milk and whey. The isolation of
pyschrophilic bacteria with cold active -galactosidase
has opened up the possibility of processing of GOS at
low temperatures. On the other hand, thermostable
enzyme has the unique ability to retain their activity at
higher temperature for prolonged periods, and the process
is less prone to microbial contamination due to higher
operating temperature. Therefore, it may be concluded
that utilization of -galactosidase is a twofold solution of
biotechnology economy and waste treatment.
Furthermore, this work will provide a ready reference
for future scientic research regarding -galactosidase.
Acknowledgements
Arijit Nath acknowledges Council of Scientic and
Industrial Research (CSIR), New Delhi for nancial
DOI: 10.1002/apj
345
346
A. NATH ET AL.
support as an SRF. The reported work is a part of a

University Grants Commission (UGC) Major project,
entitled Production and Purication of galactosidase from Milk Whey-based Lactic Acid
Bacteria using Fermentation and Membrane-based
Separation Techniques. The contribution of UGC is
gratefully acknowledged.
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ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING

Asia-Pac. J. Chem. Eng. 2014; 9: 330348

Special theme review

Production, purication, characterization, immobilization,

Received 20 June 2013; Revised 29 September 2013; Accepted 5 October 2013

Authors have similar contribution.

biological products and optimization of their yields as

Asia-Pacic Journal of Chemical Engineering

activity throughout life. According to reviews of

SYNTHESIS MECHANISM OF -GALACTOSIDASE

and the function of -galactoside transacetylase is yet

Figure 1. Construction of lac operon (Figure adapted from K. L. Anderson, G. Purdom

Asia-Pac. J. Chem. Eng. 2014; 9: 330348

Asia-Pacic Journal of Chemical Engineering

Control mechanism of lac operon

EIIBGlc, which are the cytoplasmic domain of the EII

Asia-Pacic Journal of Chemical Engineering

prevention (Figure adapted from B. Grke and J. Stlke (2008)).[13]

A mechanism of carbon catabolite repression by

Bacillus licheniformis ATCC 12759

Bidobacterium animalis Bd12,

Bacillus coagulans RCS3

LacA from Aspergillus oryzae, expressing in

Lactose-based chemically dened medium

Lactose-based chemically dened medium

Combination of skim milk powder, glucose, and yeast extract

Table 1. Different types of consortium used in -galactosidase production.

Asia-Pac. J. Chem. Eng. 2014; 9: 330348

Ammonium sulfate precipitation, hydrophobic

(Expressed in Escherichia coli)

Bacillus licheniformis (cloned

Sonication, ammonium sulfate precipitation,

ZnCl2 precipitation, ion exchange membrane

Bacillus sp. 3088

Puried 292-fold by chromatography on

Table 2. Different methodology for -galactosidase purication.

Intracellular, M.W. is 530 kg mol 1. Optimum pH is 7.1. Galactose

Subunits are same molecular weight. M.W. is 170 kg mol .

Characteristics of puried enzyme

Asia-Pacic Journal of Chemical Engineering

Asia-Pac. J. Chem. Eng. 2014; 9: 330348

Ultrasonic treatment in the presence of Triton

Ammonium sulfate precipitation, dialysis,

Column chromatographies by DEAESephadex

Cryptococcus laurentii OKN-4

Ion exchange chromatography on DEAE

Intracellular, homodimeric, each subunit M.W. is 116 kg mol 1.

Characteristics of puried enzyme

Asia-Pac. J. Chem. Eng. 2014; 9: 330348

Precipitation with ammonium sulfate, ion

Protein precipitation by Pectinex Ultra SP followed

Gel ltration, anion exchange chromatography

Metal-ion afnity chromatography (IMAC)

2-propanol fractionation, column

incubation. The Km values of the enzyme are 18.2 10 3 M and

Characteristics of puried enzyme

Asia-Pacic Journal of Chemical Engineering

Asia-Pac. J. Chem. Eng. 2014; 9: 330348

Gel ltration chromatography (Ultrogel AcA

Centrifugation, cell disruption, heat treatment,

Ammonium sulfate fractionation, column

chromatography on Mono Q PC 1.6/5 column

Extracellular thermostable, relative M.W. of 145 000 and s20,w of

M.W. is 135 kg mol 1. Optimum pH is 7.25. Km is 12 10 3 to