Production, purification, characterization, immobilization, and application of β -galactosidase: a...

Special theme review

Production, purification, characterization, immobilization,and application of β-galactosidase: a review

Arijit Nath,1† Subhoshmita Mondal,1† Sudip Chakraborty,1,2* Chiranjib Bhattacharjee1 and Ranjana Chowdhury1

1Chemical Engineering Department, Jadavpur University, Kolkata, West Bengal 700032, India2Department 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 thefield of biotechnology. One of the challenging biomolecule β-galactosidase (β-galactosidase galacto hydrolysase, triviallylactase) catalyzes hydrolysis of lactose to produce glucose and galactose, and in some cases, it takes part intransgalactosylation reaction that produces functional food galato-oligosaccharide. A wide variety of strategies had beenalready attempted for production of β-galactosidase through fermentative route. Beside the upstream process, downstreamtechnology towards purification and immobilization of target enzyme also create great attentions. Subsequently, its wideapplications in the field of food, biopharmaceuticals, dairy, diagnosis, and waste treatment boost up biotechnologicaleconomy as well as zero-effluent discharge. In dairy industry, β-galactosidase has been used to degrade lactose, preventcrystallization of lactose, improve sweetness, and increase the solubility of milk product, otherwise which would be anenvironmental pollutant. In food and pharmaceutical industries, β-galactosidase has been used to prepare low lactose-containing food products for low lactose-tolerant people. Therefore, it is obvious to elucidate different technological aspectsof β-galactosidase, which may provide a great knowledge in educational and industrial field. Taking the enzyme into account,a ready review has been made about its production, purification, characterization, and immobilization technology. Thereview also addresses wide applications of β-galactosidase in different fields. © 2014 Curtin University of Technologyand John Wiley & Sons, Ltd.

Keywords: β-galactosidase; production; purification; characterization; immobilization; application

INTRODUCTION

Emergence of biotechnology, which brings a boon tobiological origin, possesses a challenging revolution tothe categorical field, biopharmaceuticals. There isenormous range of therapeutics that involvesbiopharmaceuticals, and it combines biomolecularforms, which extensively give rise to the developmentof the microbial synthesis of diverse enzymes andmetabolites. Intellectual revolution with new visionsand hopes by dispensation of any method of bioprocessthat deals with the design and development ofequipments for the manufacturing of various products,such as food-beverages, sera, new medicines, semi-synthetic organs, antibiotics, and enzymes frombiological sources creates a great attention. This isresponsible for the explosion of various biotechnologicalprocesses used in industries for large-scale production of

biological products and optimization of their yields aswell as the qualities of end products.[1]

The lactose-hydrolyzing enzyme β-galactosidase hasbeen accepted since long as an important ingredient indairy, food processing, and pharmaceutical industries.β-galactosidase catalyzes hydrolysis of lactose intoglucose and galactose and also takes part intransgalactosylation reaction that produces galato-oligosaccharide (GOS) (e.g., Gal (β1→ 3) Gal(β1→ 4) Gal (β1→ 6)).[2,3] One of the most commondisease hypolactasia (lactose intolerance) or lactosemaldigestion is caused by insufficient synthesis oflactose-cleaving enzyme β-galactosidase in the brushborder membrane of mucosa of the small intestine. Inmost cases, this causes several symptoms, which mayinclude abdominal bloating and cramps, flatulence,diarrhea, nausea, rumbling stomach, or vomiting afterconsuming significant amounts of lactose.[4] In mostof the human races, β-galactosidase is found to be lostduring the first or second decade of life, and only someisolates are seen among people of Northern Europeanorigin and their overseas descendants. African andIndian communities maintain a high intestinal lactase

*Correspondence to: Sudip Chakraborty, Chemical EngineeringDepartment, Jadavpur University, Kolkata, West Bengal 700032,India. E-mail: [email protected]†Authors have similar contribution

© 2014 Curtin University of Technology and John Wiley & Sons, Ltd.Curtin University is a trademark of Curtin University of Technology

ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERINGAsia-Pac. J. Chem. Eng. (2014)Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/apj.1801

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activity throughout life. According to reviews ofScrimshaw, Murray, 1988 and Sahi, 1994, the globalprevalence of lactose maldigestion are above 50% inSouth America, Africa, and Asia reaching almost100% in some Asian countries such as China. In theUSA, prevalence is 15% among Whites, 53% amongMexican-Americans, and 80% in the Blackpopulation. In Europe, it varies from around 2% inScandinavia to about 70% in Sicily. Australia andNew Zealand have prevalence of 6% and 9%,respectively.[5,6] β-galactosidase has been used inbiopharmaceutical, food, and dairy industries to preventcrystallization of lactose, to improve sweetness, toincrease the solubility of milk product, to prepare lowlactose-containing food products for low lactose-tolerantpeople, and for the utilization of cheese whey, whichwould otherwise be an environmental pollutant.[7,8]

Therefore, it is obvious to elucidate differenttechnological aspects of β-galactosidase with specialinterest in production, purification, characterization,immobilization technology as well as its wideapplications in different process industries. The presentreview may provide a great knowledge with fill-up gapsof educational field and industrial sector.

SYNTHESISMECHANISMOF β-GALACTOSIDASE

The control system of β-galactosidase synthesis was firstworked out by Jacob and Monod in1961, at themolecular level. In prokaryotic type of gene expression,the lac operon showed inducible system with the controlof enzymes that are produced in the presence oflactose.[9] Presences of lac operon in Escherichia coliand Bacillus sp. including lactic acid bacteria havepotential of synthesizing intracellular β-galactosidase.Meanwhile, lactose metabolism in E. coli synthesizesseveral proteins, such as β-galactosidase, which convertslactose into glucose and galactose, β-galactosidepermease that transports lactose into the cellular system,

and the function of β-galactoside transacetylase is yetto be known. The construction of lac operon isdescribed in Fig. 1.For any consortia, all the genes involved in controlling

this pathway are located next to each other on thechromosome, and together they form an operon.Generally, β-galactosidase synthesis has been oftendemonstrated considering E. coli as a modelmicroorganism. The genetic switches can also combinepositive and negative controls. The lac operon consistsof three structural genes and a promoter, a terminator,regulator, and an operator. The three structural genes arelacZ, lacY, and lacA. Regulation for the specific controlof the lac genes depends on the availability of thesubstrate lactose. The proteins are not produced by thebacterium when lactose is unavailable as a carbon source.The lac genes are organized into an operon oriented in thesame direction, immediately adjacent on the chromosomeand are co-transcribed into a single polycistronic mRNAmolecule. The lac operon in E. coli, unlike trp operon,works under both negative and positive transcriptionalcontrols by the lac repressor protein and cataboliteactivator protein (CAP), respectively. CAP enablesbacteria to use alternative carbon sources such as lactosein the absence of glucose. The CAP cannot induceexpression if lactose is not present, and the lac repressorensures that the lac operon is shutoff in the absence oflactose. This arrangement enables the lac operon torespond to and integrate two different signals, so that itis 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 theenzyme RNA polymerase (RNAP), a DNA-bindingprotein that binds to a specific DNA binding site, thepromoter, immediately upstream of the genes. Bindingof RNA polymerase to the promoter is aided by the cyclicadenosine monophosphate (cAMP)-bound CAP (alsoknown 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]

A. NATH ET AL. Asia-Pacific Journal of Chemical Engineering

© 2014 Curtin University of Technology and John Wiley & Sons, Ltd. Asia-Pac. J. Chem. Eng. (2014)DOI: 10.1002/apj

Control mechanism of lac operon

In the absence of lactose, the regulatory response tolactose uses an intracellular regulatory protein, whichis called the lactose repressor to hinder the synthesisof β-galactosidase. The lacI gene coding for therepressor lies nearby the lac operon and is alwaysexpressed (constitutive). If lactose is absent in thegrowth medium, the repressor binds very tightly to ashort DNA sequence just downstream of the promoternear the beginning of lacZ called the lac operator.The repressor binding to the operator interferes withbinding of RNAP to the promoter and therefore mRNAencoding LacZ and LacY are only made at very lowlevels. During the cells growth in the presence of lactose,a lactose metabolite allolactose, which is a combinationof glucose and galactose, binds to the repressor, causinga change in its shape. Thus, the altered repressor isunable to bind to the operator, allowing RNAP totranscribe the lac genes and thereby leading to higherlevels of the encoded proteins. The schematic diagramsof the proposed phenomena are described in Fig. 2.The second control mechanism is in response to

glucose, which is transported into the cell by thephosphoenolpyruvate (PEP)-dependent phosphotransferasesystem. The phosphate group of PEP is transferred viaa phosphorylation cascade, consisting of the generalphosphotransferase system (PTS) proteins HPr andEIA, and the glucose-specific PTS proteins, EIIAGlc and

EIIBGlc, which are the cytoplasmic domain of the EIIglucose transporter. Transport of glucose is accompaniedby its phosphorylation by EIIBGlc through removing thephosphate group from the other PTS proteins, includingEIIAGlc. The unphosphorylated form of EIIAGlc binds tothe lac permease and prevents it from bringing lactose intothe cell. Therefore, if both glucose and lactose are present,the transport of glucose blocks the transport of the inducerof the lac operon. This process is called inducer exclusion.[13]

In other way, 16 base pairs upstream site of thepromoter, which is used for a positive control of thegene expression is known as CAP site or cAMP proteinsite or catabolite gene activator (cga) site, because it isutilized for binding of CAP to encourage geneexpression. CAP can bind to this site only when it isbound with cAMP. Hence, a positive control is exertedover the transcription process by the CAP–cAMPcomplex with an effect exactly opposite to that ofrepressor binding to an operator. Moreover, the effectormolecule cAMP determines the effect of CAP on lacoperon transcription in the presence of glucose, itinhibits the formation of cAMP and prevents it to bindto CAP. The DNA-bound CAP is then able to interactphysically with RNA polymerase and essentiallyincrease the affinity of RNA polymerase for the lacpromoter. In this way, the catabolite repression systemcontributes to the selective activation of the lacoperon.[14] The schematic diagram of mechanism ofcarbon catabolic repression is described in Fig. 3.

Figure 2. Regulation of lac operon in (A) presence and (B) absence of lactose.[12]

Asia-Pacific Journal of Chemical Engineering β-GALACTOSIDASE: A REVIEW


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A mechanism of carbon catabolite repression bywhich the activity of PTS-regulation domain-containing transcription factors, which is inhibited inthe presence of preferred carbon sources, is calledInduction prevention. It proceeds with phosphorylationof histidine protein (HPr) at Ser46 by HPr kinase/phosphorylase (HPrK). This phosphorylation occurswhen the intracellular concentrations of fructose-1,6-bisphosphate (FBP) and adenosine triphosphate arehigh, which reflects the presence of preferred carbonsources. HPr(Ser-P) binds to CcpA protein, and thisinteraction is enhanced by glycolytic intermediates, suchas FBP and glucose-6-phosphate. The complex of CcpAand HPr(Ser-P) binds to cre sites on the DNA andthereby represses the transcription of catabolic genes.HPrK is also responsible for dephosphorylation ofHPr(Ser-P) under conditions of high inorganicphosphate (Pi) and low adenosine triphosphate andwhen there is poor nutritional supply of FBP. Also,HPr(His-P) contributes to carbon catabolic repression:in the absence of glucose, HPr(His-P) phosphorylatesglycerol kinase (GlpK), and transcriptional regulatorsthat contain PEP–carbohydrate phosphotransferasesystem-regulatory domains, which acts as precursorsfor their activity. Thus, in the presence of glucose,activation of the phosphotransferase system-regulatorydomain regulators by their inducers is prevented.[13]

Research with this system is greatly added by theavailability of constitutive mutants. A constitutivemutant of any consortia continuously produces geneproducts where there is no control over its expression.In these mutants, the aforementioned proteins areproduced all the time in comparison to the wild typewhere the proteins only appear in the presence of lactose.So in these mutants, the mutation must be a gene otherthan those responsible for the structural gene.[15]

PRODUCTION OF β-GALACTOSIDASE

β-galactosidase belongs to the group of saccharidesconverting enzymes, i.e., in the family of hydrolases.They are widespread, distributed in numerousbiological systems, e.g., microorganisms (yeasts, fungi,bacteria, and actinomycetes), plants, and animaltissues. Compared with animal and plant sources,microbial-synthesized enzyme provides higher yields,which may decrease its production cost. Therefore,production of β-galactosidase through microbial routecreates a great attention. Although, the most studiedβ-galactosidase is produced by E. coli, possible toxicfactors associated with coliforms make it unlikely thatcrude isolates of this enzyme, which may be permittedin food processes.[16–21] In industrial scale, productionof β-galactosidases is carried out using generallyrecognized as safe microorganism, yeast (mainly fromKluyveromyces marxians, Kluyveromyces lactis, andKluyveromyces fragilis) and fungal (mainly fromAspergillus niger and Aspergillus oryzae) consortia.The detail works carried out in this direction have beenrepresented in Table 1.

PURIFICATION OF β-GALACTOSIDASE

Different separation techniques, such as membrane-based separation, ion exchange membranechromatography, gel permeation chromatography, zincchloride, protamine sulfate, and ammonium sulfateprecipitation; had already been attempted forpurification of β-galactosidase from crude extract. Thedetail works carried out in this direction have beenrepresented in Table 2.

Figure 3. Mechanism of carbon catabolic repression (A) inducer exclusion and (B) inductionprevention (Figure adapted from B. Görke and J. Stülke (2008)).[13]



Table

1.Differenttypes

ofco

nso

rtium

usedin

β-galactosidaseproduction.

Typ

eof

consortiu

mNam

eof

consortiu

mSub

strate

Operatin

gmod

eof

bioreactor

Reference

Yeast

Kluyverom

yces

marxian

sCheesewhey

Fed

batch

[22]

Yeast

Kluyverom

yces

marxian

sCheesewhey

Batch

[23]

Yeast

Kluyverom

yces

marxian

sSup

plem

entedwheywith

cauliflow

erwaste

Batch

[24]

Yeast

Kluyverom

yces

marxian

sCBS78

94Lactose-based

chem

ically

definedmedium

Batch

[25]

Yeast

Kluyverom

yces

marxian

sNCIM

3551

Whey

Batch

[26]

Yeast

Kluyverom

yces

marxian

sCCT78

02Lactose-based

chem

ically

definedmedium

Batch

[27]

Yeast

Kluyverom

yces

marxian

sMTCC13

88Whey

Batch

[28]

Yeast

Kluyverom

yces

frag

ilisNRRL-Y-110

9Lactose-based

chem

ically

definedmedium

Batch

andfedbatch

[29]

Yeast

Recom

binant

Saccharomyces

cerevisiae

W303

Lactose-based

yeast-definedmineral

medium

Batch

[30]

Yeast

Saccha

romyces

cerevisiae

Lactose-based

chem

icallydefinedmedium,de-proteinatedwhey

Fed

batch

[31]

Yeast

Can

dida

pseudo

trop

icallis

Whey

Batch

[32]

Bacteria

ClonedEscherichia

coliXL-1

Blue,XL-1

BlueFF,T

B-1

andN48

30Basal

medium

with

IPTG

Batch

[33]

Bacteria

Lactoba

cillu

ssp.B

ulga

ricusCHRHan

sen

Lb-12

Com

binatio

nof

skim

milk

powder,glucose,andyeastextract

Batch

[34]

Bacteria

Bacillus

sp.M

PTK12

1Lactose-based

chem

ically

definedmedium

Batch

[35]

Bacteria

Bacillus

sp.

Luriamedium

whereindividu

ally

galactose,glucose,mannitol,

lactose,andxy

lose

wereadded

Batch

[36]

Bacteria

Bacillus

licheniform

isATCC12

759

Luria

medium

Batch

[37]

Bacteria

Lactoba

cillu

sacidop

hilusNRRL44

95Acidwhey

Batch

[16]

Bacteria

Bifido

bacterium

animalisBd1

2,Lactoba

cillu

sdelbrueckiissp.

bulgaricus

ATCC11

842

Chemically

definedmedium

where

individu

ally

glucose,

lactose,andgalactosewereused.Moreover,individu

ally

pepton

e,yeastextract,casein

hydrolysate,tryp

tone,or

ammon

ium

sulfatewereused

Batch

[17]

Bacteria

Bifido

bacterium

animalisBd1

2,Lactoba

cillu

sdelbrueckiissp.

bulgaricus

ATCC11

842

De-proteinatedwheywhere

proteinwas

supp

lementedby

individu

ally

pepton

e,yeastextract,casein

hydrolysate,

tryp

tone,andam

mon

ium

sulfate

Batch

[18]

Bacteria

Lactoba

cillu

splan

tarum

Pi06

Lactose-based

chem

ically

definedmedium

Batch

[19]

Bacteria

Lactoba

cillu

sacidop

hilus

de-M

annRog

osaandSharpemedium

Batch

[20]

Bacteria

Streptococcusthermop

hilus

Whey-basedmedium

Batch

[38]

Bacteria

Lactoba

cillu

sdelbrueckiissp.

bulgaricus

ATCC11

842

Skim

milk

,mod

ified

de-M

annRog

osaSharpemedium,w

hey,

andwheyperm

eate-based

brothwereused.A

ddition

ally,y

east

extractwas

added

Batch

[21]

Bacteria

Bacillus

coag

ulan

sRCS3

Lactose-based

chem

ically

definedmedium

Batch

[39]

Fun

gus

Aspergillu

sflavus

Lactose-based

chem

ically

definedmedium

Batch

[40]

Fun

gus

Penicillium

chrysogenu

mNCAIM

0023

7Lactose-based

chem

ically

definedmedium

Batch

[41]

Fun

gus

Aspergillu

soryzae

PTCC51

63Wheyandchem

ically

definedmedium

with

different

carboh

ydrate,i.e.,lactose,wheat

barn,and

soyb

eanmeal

Batch

[42]

Fun

gus

LacA

from

Aspergillu

soryzae,exp

ressingin

Saccha

romyces

cerevisiae

NCYC86

9-A3/

pVK1.1

Wheat

bran

andrice

husk

Batch

[43]

Fun

gus

Rhizomucor

sp.

Lactose-based

chem

ically

definedmedium

Batch

[44]

Fun

gus

Aspergillu

sniger

Lactose-based

yeastnitrog

enbase

medium

andsemi-synthetic

lactosemedium

Con

tinuo

us[45]



Table

2.Differentmethodologyforβ-galactosidasepurification.

Sou

rce

Purificatio

nprocedure

Characteristicsof

purified

enzyme

Reference

Lactoba

cillu

smurinus

Purified

292-fold

bychromatog

raph

yon

Ultrog

elACA

34,D

EAE–S

ephadexA-50

columns

andby

affinity

chromatog

raph

yin

agarose-p-am

inop

heny

l-β-

D-thiog

alactoside

Subunitsaresamemolecular

weight.M.W

.is170kg

mol

�1.

Maxim

umenzymaticactiv

ityat318KandpH

7inpresence

of50

mM

phosphatebuffer.T

heKmforONPG

andONPG

+20

×10

�3M

oflactoseare480×10

�6and870×10

�6M,respectively.

Inhibitors

are10

×10

�3M

CaC

l 2,g

lutathione,and

cysteine,and

stim

ulator

is10

×10

�3M

MgC

l 2.A

ctivatorsaremercaptoethanolanddithiotreitol.

Enzym

aticactiv

ityisnotshownin

presence

ofp-β-galactosidase.

[46]

Bacillus

sp.3

088

ZnC

l 2precipitatio

n,ionexchange

mem

brane

chromatography,

hydrophobicinteraction

chromatographyandgelfi

ltration

chromatography

M.W

.is48

4kg

mol

�1,associatewith

subu

nits11

5,86

.5,72.5,45

.7,

and41

.2kg

mol

�1.O

ptim

umpH

andtemperature

are8and

333.15

K,respectiv

ely.

Isoelectricpo

intis6.2.

Kmis6.34

×10

�3

and6.18

×10

�3M

forONPG

andlactose,respectiv

ely.

Inhibitors

aregalactosedivalent

Hg,

Cu,

andAg.

Metalactiv

ator

isdivalent

Mg.

EDTA

dono

taffect

theenzymeactiv

ity.

[47]

Lactoba

cillu

sreuteri

Ammon

ium

sulfateprecipitatio

n,hy

drop

hobic

interactionchromatog

raph

y,andaffinity

chromatog

raph

y

Heterod

imer

enzyme,M.W

.is10

5kg

mol

�1(m

onom

ersare72

and

35kg

mol

�1).Isoelectricpointsare4.6–4.8and3.8–4.0forL

.reuteri

L103andL.reuteriL461,respectiv

ely.Optim

umpH

is6–8.Inhibitor

isD-glucose

andactiv

atorsareNa(+),Mn(+2),and

K(+).

[48]

Bacillus

circulan

sGel

perm

eatio

nchromatog

raph

y,affinity

chromatog

raph

y,andam

mon

ium

sulfate

precipitatio

n

M.W

.is21

2kg

mol

�1,com

posedby

145and86

kgmol

�1.K

mfor

threesubunitsare3.6×10

�3,5.0×10

�3 ,and3.3×10

�3M

forO

NPG

,respectiv

elyandthoseare3.7×10

�3,2.94×10

�3 ,and2.71

×10

�3M,

respectiv

elyconsideringlactoseas

asubstrate.

[49]

(Exp

ressed

inEscherichia

coli)

Colum

nchromatog

raph

ieson

resource

Qand

seph

acrylS-200

HR

Potentialof

GOSsynthesis

[50]

Streptococcusthermop

hilus

Ammon

ium

sulfateprecipitatio

nandgel

perm

eatio

nchromatog

raph

yIntracellular,M.W

.is53

0kg

mol

�1.O

ptim

umpH

is7.1.

Galactose

iscompetitiveinhibitor(K

i60

×10

�3M),KmforO

NPGandlactose

are0.98

×10

�3and6.9×10

�3M,respectiv

ely.

[51]

Streptococcuslactis

Protaminesulfateprecipitatio

n,am

mon

ium

sulfateprecipitatio

n,andionexchange

chromatog

raph

y

Enzym

eismostlabile

whenitissuspendedat

cold

(278

.15K)

phosph

atebu

ffer.In

presence

ofhigh

concentrationam

mon

ium

sulfate(0.85M),enzymeishigh

lyactiv

eandstabilizes.

[52]

Bacillus

stearothermop

hilus

(exp

ressed

inBacillus

subtilis)

Heattreatment,am

monium

sulfateprecipitatio

n,ionexchange,and

gelfi

ltrationchromatography

Intracellular,M.W

.is70

kgmol�1 .Isoelectricpointis5.1.Optim

umtemperatureandpH

are343.15

Kand7,respectively.Kineticsof

thermalinactivationandhalflifeat338.15

and343.15

Kare18

×10

4

and324×10

2s.KmandVmaxare2.96

×10

�3M

and0.11

×10

�3

kmols�

1kg

ofprotein,respectively.InhibitorsareFe(2+),Z

n(2+

),Cu(2+

),Pb

(2+),S

n(2+),and

thiolagent.

[53]

Bacillus

megaterium

Son

ication,

ammon

ium

sulfateprecipitatio

n,desaltatio

n,columnchromatog

raph

yusing

DEAE–sepharose

fastflow

column,

andaffinity

chromatog

raph

y

M.W

.is118kg

mol

�1.O

ptim

umpH

7.5–8.0andtemperature328K.

Enzym

eisstableatpH

6.0–9.0andbelow313K.T

heKmandVmax

values

forONPG

andlactoseare9.5mM,16.6mM.m

in�1and

12.6mM,5

4.4mM.m

in�1 ,respectiv

ely.

[54]

Bacillus

licheniform

is(cloned

andexpressedin

Escherichia

coli)

Ionexchange

chromatog

raph

yon

NiS

epharose

6fastflow

columnandultrafiltration

Hom

odim

eric,op

timum

temperature,andpH

are32

3.15

Kand

6.5,

respectiv

ely.

KmforlactoseandONPG

are16

9×10

�3and

13.7×10

�3M,respectively.

Inhibitors

areglucoseandgalactose.

Metal

activ

atorsareNa+,K

+,M

g+2,M

n+2,and

Ca+

2.

[55]



Table

2.(Continued

)

Sou

rce

Purificatio

nprocedure

Characteristicsof

purified

enzyme

Reference

Arthrob

actersp.

Ionexchange

chromatographyon

DEAE–

sepharose,

affinity

chromatographyon

PABTG-sepharose,

andgelfi

ltrationon

SephacrylS

-300

Intracellular,ho

mod

imeric,each

subu

nitM.W

.is11

6kg

mol

�1.

Optim

umpH

andtemperature

are6–8and29

8.15

K,respectiv

ely.

Activatorsarethiolcompo

unds,Na(+),andK

(+).Inactiv

ated

by4-chloromercuribenzoicacid,Pb(2+),Zn(2+),andCu(2+)

[56]

Bifido

bacterium

long

umCCRC15

708

Ultrason

icationandsubsequently

purified

byQ

Fast-Flow

chromatog

raph

yandgel

chromatog

raph

yon

aSup

erose6HRcolumn

Finally

15.7-fold,

ayieldof

29.3%,andaspecificactiv

ityof

168.6*

106Ukg

-1protein(β-galactosidase)hasbeen

achh

ived.T

heMW

was

357kg

.mol-1

asdeterm

ined

from

Native-PAGE.The

pHandtemperature

optim

aof

thepu

rified

β-galactosidasewere7.0and

323K,respectivelyconsideringONPGas

asubstrate,.T

heenzyme

was

stableatatemperature

upto

313KandatpH

values

of6.5-7.0.

Valuesof

Km

andVmax

forthispu

rified

enzymewerefoun

dto

be0.85

mM

and70

.67*1

06U

kg-1,respectiv

ely.

Itwas

foun

dthat

Na+

andK+stim

ulated

theenzymeup

to10

-fold,

while

Fe3

+,F

e2+,

Co2

+,C

u2+,C

a2+,Z

n2+,M

n2+andMg2

+inhibitedtheactiv

ityof

β-galactosidase.

Furthermoreitwas

foun

dthat

glucose,galactose,

maltose,orraffino

seexertedlittle

orno

effecton

theβ-galactosidase

activ

ity,lactoseandfructose

inhibitedtheenzymeactiv

ity.The

effectof

lactoseon

theenzymeactiv

ityforONPGisprob

ably

acase

ofcompetitiveinhibitio

n.

[57]

Bifido

bacterium

bifidu

mUltrason

ictreatm

entin

thepresence

ofTriton

X-100

Enzym

ehasdifferentsubu

nits,such

as16

3,17

0,17

8,and

190kg

mol

�1.T

heM.W

.is36

2kg

mol

�1andisoelectricpo

intis

5.25

.The

purified

enzymeisstableattemperaturesbelow31

8Kand

over

thepH

rang

esfrom

6.5–

8.Lactose

hydrolysisby

thepu

rified

enzymetake

places

atpH

6.5andtemperature

310K.

[58]

Bifido

bacterium

infantis

(exp

ressed

inEscherichia

coli)

Ammon

ium

sulfateprecipitatio

n,dialysis,

anionexchange

column(M

onoQ

HR5/5,

Pharm

acia)usingaFPLCsystem

(Pharm

acia).

Activeβ-galactosidasefractio

nshasbeen

collected

from

severalchromatog

raph

icruns

andconcentrated

usingtheCentriplussystem

(Millipore).The

activ

efractio

nof

β-galactosidasehasbeen

obtained

from

ion

exchange

chromatog

raph

y.

The

enzymeistetram

er,w

ithan

M.W

.ofabou

t47

0kg

mol

�1,and

itssubu

nitis11

5kg

mol

�1.T

heop

timum

temperature

andpH

for

ONPG

andlactoseare333K,pH7.5and323K,pH7.5,respectiv

ely.

The

enzymeisstableover

apH

rangeof

5.0–8.5andremains

activ

eformorethan

4800

satpH

7.0,323K.T

heenzymeactiv

ityhasbeen

significantly

increasedby

reducing

agents.M

axim

umactiv

ityhas

been

show

nin

presence

ofboth

Na+

andK+ataconcentrationof

10×10

�3M.T

heenzymeisstrongly

inhibitedby

p-chloromercuribenzoicacid,d

ivalentm

etalcatio

nsandCr3+,and

toalesser

extent

byEDTAandurea.T

hehydrolyticactiv

ityusing

lactoseas

asubstratehasbeen

significantly

inhibitedby

galactose.

The

KmandVmaxvalues

forONPG

andlactoseare2.6×10

�3M,

262×10

6Ukg

�1and73.8×10

�3M,1.28×10

6Ukg

�1 ,respectiv

ely.

The

enzymepossessesstrong

transgalactosylatio

nactiv

ity.T

heproductio

nrateof

GOSfrom

20%

lactoseat303and333Kis

120×10

�3kg

L�1,and

thisrateincreasesto190×10

�3kg

L�1when

30%

lactosehasbeen

used

asasubstrate.

[59]

Cryptococcuslaurentii

OKN-4

Colum

nchromatog

raph

iesby

DEAE–S

ephadex

A-50,TSK

gelToy

oPearlHW-55S

,andTSK

gelDEAE-5PW

M.W

.is200kg

mol

�1.hom

odim

eric,optim

umpH

is4.3,andenzyme

isstableatpH

between2.8and9.3.

The

optim

umtemperature

is333Kandenzymeisstableattemperature

below330.5Kfor600s

[60]

(Continues)



Table

2.(Continued

)

Sou

rce

Purificatio

nprocedure

Characteristicsof

purified

enzyme

Reference

incubatio

n.The

Kmvalues

oftheenzymeare18.2×10

�3M

and

11.4×10

�3M,and

thevalues

ofVmaxare1.28

×10

�3km

ols�

1kg

ofprotein,

and0.09

×10

�3km

ols�

1kg

ofproteinforONPG

and

lactose,respectiv

ely.The

enzymeisstrongly

inhibitedby

Hg2

+,A

g+,

2-mercaptoethanol,g

lucose,m

altose,and

maltotriose.E

nzym

eis

potentialo

fGOSsynthesis.

Aspergillu

soryzae

2-prop

anol

fractio

natio

n,column

chromatog

raph

yon

DEAE–S

ephadex

A-50,

andSephadexG-200

Extracellu

lar,op

timum

pHforONPG

andlactoseare4.5and4.8,

respectiv

ely.

Optim

umtemperature

is31

9.15

K.K

mare7.2×10

�4

and1.8×10

�2M

forONPG

andlactoserespectiv

ely.

Inhibitors

are

divalent

Hg,

Cu,

N-bromosuccinimide,

andsodium

laurylsulfate.

App

arentM.W

.is10

5kg

mol

�1.

[61]

Metal-ion

affinity

chromatog

raph

y(IMAC)

follo

wed

bysize-exclusion

separatio

nExtracellu

lar,M.W

.is11

3kg

mol

�1.M

utantenzym

ehasfive

times

high

ercatalytic

activ

ityon

thesynthetic

substrateONPG

compared

with

thewild

-typ

eenzyme.Moreover,themutantenzymeismore

thermoresistantcomparedwith

thewild

type.

[62]

Aspergillu

sniger

Gel

filtration,

anionexchange

chromatog

raph

yon

DEAE–S

epharose

CL-6B,hy

drop

hobic

chromatog

raph

yon

octyl-SepharoseCL-4Band

catio

nexchange

chromatog

raph

yon

CMSepharose

CL-6B

Glycoproteinin

nature.A

ssociateswith

threesubu

nits,and

each

M.

W.are12

4,15

0,and17

3kg

mol

�1.Isoelectric

pointis4.6.

Optim

umpH

liesbetweenin

2.5to

4.0.

Heatstableup

to33

3.15

K.

Kmvaluevaries

from

85×10

�3to

125×10

�3M

forlactose.Km

valuevaries

2.4×10

�3M

forONPG.V

maxvalues

are10

4×10

3un

itenzymekg

ofproteinand12

1×10

3un

itenzymekg

ofproteinat

303.15

KforONPG

andlactose,respectiv

ely.

[63]

Aspergillu

saculeatus

Proteinprecipitationby

Pectinex

UltraSP

followed

bydesalting

andcolumnchromatographyusing

DEAE–Sepharose

fastflow

.

The

M.W

.is12

0kg

mol

�1(app

roximately),isoelectricpo

intlies

between5.3and5.7andisop

timally

activ

eat

pH5.4and

temperature

328–

333K.Based

ontheN-terminal

aminoacid

sequ

ence,the

enzymeprob

ably

belong

sto

family

35of

theglycosyl

hydrolases.Enzym

eispo

tentialof

synthesizing

GOS.

Enzym

ehasactiv

itytowards

twoexo-po

ly-saccharide(EPSs),

having

lactosyl

side

chains

attached

todifferentbackbo

nestructures.The

enzymedegraded

O-deacetylatedEPSB89

1faster

than

EPSB39

.Moreover,thepresence

ofacetyl

grou

psin

EPSB89

1slow

sdo

wnthehy

drolyzingrate

buttheenzymeis

still

able

toreleaseallterm

inally

linkedgalactose.

[64]

Penicillium

chrysogenu

mPrecipitatio

nwith

ammonium

sulfate,ion

exchange

chromatographyon

DEAE–S

ephadex,

affinitychromatography,andchromatefocusing

Intracellular,Sp

ecificactiv

ityis5.84

×10

3Ukg

ofprotein.Optim

umtemperature

andpH

are303.15

Kand4,

respectiv

ely.Kmand

isoelectricpointare

1.81

×10

�3M

and4.6forONPG

.Multim

eric

enzyme,M.W

.is270kg

mol�1,and

M.W

.ofsinglemonom

eris

66kg

mol

�1

[41]

Kluyverom

yces

lactis

Gel

filtrationchromatog

raph

y,ionexchange

chromatog

raph

y,andaffinity

chromatog

raph

yAffinity

chromatog

raph

yisshow

nbetterresults

than

gelp

ermeatio

nchromatog

raph

y,andionexchange

chromatog

raph

y.Sim

ilar

molecular

weigh

tsub

unit.

Enzym

eisconfi

rmed

byspecificantig

en-

antib

odyreactio

n(ELISA

test).

[65]

Kluyverom

yces

lactis

Gelfiltrationon

Superose

12PC

3.2/30

column

andionexchange

Intracellular,M.W

.of

sing

lemon

omer

is12

4kg

mol

�1

[66]



Table

2.(Continued

)

Sou

rce

Purificatio

nprocedure

Characteristicsof

purified

enzyme

Reference

chromatographyon

MonoQPC

1.6/5column

usingaFP

LCsystem

Celld

isruption,

DEAE–S

ephadexionexchange

chromatography,

andchromatographyon

hydroxylapatite

M.W

.is13

5kg

mol

�1.O

ptim

umpH

is7.25

.Kmis12

×10

�3to

17×10

�3M

forlactose.Kmis1.6×10

�3M

forONPG.Metal

activ

atorsareNa(+)andMn(+2).

[67]

Kluyverom

yces

marxian

usExtractionwith

2%chloroform

,aceton

e,and

ammon

ium

sulfateprecipitatio

nM.W

.is28

0kg

mol

�1.O

ptim

umtemperature

andpH

are31

8.15

to32

5.15

Kand6.2,

respectiv

ely.

Inactiv

ates

in24

0sat32

9.15

K.K

mare3.1×10

�3and25

×10

�3M

forONPG.Inhibitio

nconstantsare

58×10

�3,1

10×10

�3,1

11×10

�3,and

52×10

�3M

forONPG,

galactose,ribo

se,andlactose.Inhibitors

arealso

P-chlorom

ercuribenzoate

anddithiothreito

l.

[68]

Saccha

romyces

lactis

Ammonium

sulfatefractio

natio

n,column

chromatographyon

Sephadex

G-100,and

DEAE–S

ephadexA-50,

78-foldpurificatio

n

Optim

umpH

is7.2.

Metal

activ

ator

divalent

magnesium

,Kmis

1.18

×10

�3M

forONPG.

[69]

Saccha

ropo

lysporarectivirgu

laFPDA

13columnchromatog

raph

y,am

mon

ium

sulfatefractio

natio

n,Ultrog

elAcA

34column

chromatog

raph

y,DEAE–S

epharose

CL-6B

columnchromatog

raph

y,TSK

gelDEAE-

Toy

opearlpak65

0SHigh-pressure

liquid

chromatog

raph

y(H

PLC)

Extracellu

larthermostable,relativ

eM.W

.of

14500

0ands°

20,w

of7.1s.Michaelisconstant

Kmis0.75

×10

�6kM

andmolecular

activ

ity(k

cat)is63

.1s�

1atpH

7.2and32

8KforO

NPGwhereas

Km

is0.04

×10

�6kM

andk c

atis55

.8s�

1forp

-NPG.E

nzym

ehasahigh

transgalactosylatio

nactiv

ity.The

enzymereacts

with

1.75

Mlactoseat333KandpH

7.0for7.92

×10

4sto

obtain

maxim

umyieldoligosaccharides

(41%

(w/w)).T

hegeneralstructure

forthemajor

transgalactosylic

productscanbe

expressedas

(Gal) c-

Glc,w

here

nis1,

2,3,

and4with

aglucoseatareducing

term

inal.

[70]

Sterigm

atom

yces

elviae

Centrifug

ation,

celldisrup

tion,

heat

treatm

ent,

DEAE–T

oyop

earlchromatog

raph

y,salting

out,Butyl-Toy

opearlchromatog

raph

y,Chrom

atofocusingchromatog

raph

y,andPATG

chromatog

raph

y

M.W

.is17

0and86

kgmol

�1fordenaturedenzyme,isoelectric

point4.1.

The

optim

altemperature

is35

8K,anditisstable

attemperaturesup

to35

3K

for360

0s.The

optim

alpH

rang

eforthe

enzymeis4.5to

5.0andtheenzymeisstable

atpH

2.5to

7.0.

Ezymeisinhibitedby

Hg2

+.T

heKmvalues

forONPG

andl

actose

are9.5×10

�3and2.4×10

�3M,respectively.

The

Vmax

for

thesesubstrates

are96

mols�

1kg

ofproteinand4mols�

1kg

ofprotein,

respectiv

ely.

The

enzymepo

ssessesahigh

levelof

transgalactosylatio

nactiv

ity(higheryield39

%from

200gL�1

lactose),which

prod

uces

GOSinclud

ingtri-andtetrasaccharides.

[71]

Therm

usaquaticus

Gel

filtrationchromatog

raph

y(U

ltrog

elAcA

34),anionexchange

(Mon

oQ),andgel

filtration(Sup

erose-12

)

M.W

.is70

0kg

mol

�1andsubu

nitis59

±1kg

mol

�1.Isoelectric

pointis4.9.

The

optim

umtemperature

andpH

forenzymeactiv

ityare35

3K

and5.5,

respectiv

ely.

The

enzymeisstable

over

awide

pHrang

e(pH3–12

),andthethermostabilityof

theenzymeis

enhanced

byCaC

l 2.T

heenzymeissign

ificantly

activ

ated

byalkali

andalkalin

e-earth-metal

salts.Inhibitors

arethiol-bind

ingagents,

glucose,andgalactose.The

enzymespecificforb-D

anom

eric

linkagesandtheidentityof

theaglycone

moietyalso

influenced

enzymeactiv

itydram

atically.Enzym

eispo

tentialof

GOS

synthesis.

[72]



CHARACTERIZATION OF β-GALACTOSIDASE

Apart from catalyzing the hydrolysis of β-galactosidesinto monosaccharides by lactase, the enzyme may alsocleave fucosides and arabinosides with much lowerefficiency. Lactase is often confused as an alternativename for β-galactosidase, but it is merely a sub-classof β-galactosidase. In other words, β-galactosidase isan exoglycosidase, which hydrolyzes the β-glycosidicbond formed between galactose and its organicmoiety.[73] Molecular weight, amino acids chain length,position of the active site, pH, and optimum thermalstability are significantly differed by the microbialsources.[74] The choice of suitable β-galactosidase sourcedepends on the condition of reaction. For example, dairyyeasts with optimum pH (6.5–7.0) are habitually used forthe hydrolysis of lactose in milk or sweet whey.[75] On theother hand, the fungal β-galactosidases with optimum pH(3.0–5.0) are more suitable for acidic whey hydrolysis.[76]

The activity of different β-galactosidases also dependson presence of ions. The fungal β-galactosidasesare active without presence of ions as cofactors; whereβ-galactosidase isolated from K. lactis shows its higheractivity in presence of Mn2+ and Na+. β-galactosidasesynthesized from K. fragilis are mostly active in presenceof Mn2+, Mg2+, and K+.[77] On the contrary, Ca2+

and heavy metals inhibit the enzyme activity of allβ-galactosidases.[78] Properties of β-galactosidasesynthesized by different microorganisms are describedin Table 3.In 1970, 1024 amino acids of β-galactosidase of E.

coli were first sequenced.[93] After 24 years, four chainscomprising the protein were discovered to be 464 kgmol�1 tetramer with 222-point symmetry. Every unitof β-galactosidase contains five domains; whereas theactive site persists in the third domain. This enzymecan be split into two peptides, LacZα and LacZΩ, noneof them is active but both spontaneously reassemble afunctional enzyme. This characteristic is used for manycloning vectors to achieve α-complementation inspecific laboratory strains of E. coli, where the plasmidencodes the small LacZα while the large LacZΩ isencoded by the bacterial chromosome. Aftermath,when DNA fragments inserted in the vector,production of LacZα disrupted, the cells revealed noβ-galactosidase activity, were subjected to the blue/whitescreening of recombinant clones further.[94] The activesite of β-galactosidase catalyzes the hydrolysis ofdisaccharide substrate via ‘shallow’ and ‘deep’ binding.The beta-linkage of the substrate was cleaved by aterminal carboxyl group on the side chain of glutamicacid.[95] It has been determined that the DNA fragmentof thermophilic bacterium Thermoanaerobacterethanolicus contains three open reading frames. One ofthe open reading frames corresponded to the LacA genefor a thermostable s-galactosidase and the nativerecombinant LacA showed the highest activity at

348K–353K. Immobilized on aldehyde silochrome,LacA was even more thermo stable and retained highactivity.[96] It has been found that individual moleculesof β-galactosidase from the crystallize enzyme as wellas the original enzyme displayed a range of activity of20-fold or greater. Molecules obtained from two diversecrystals have identical activity distributions, i.e.,31 600 ± 1100 and 31 800 ± 1100 reactions per minuteper enzyme molecule. This activity of the enzyme wasfound to be drastically different from that of the enzyme,which was used to grow the crystals (showed an activitydistribution of 38 500±900 reactions per minute perenzyme molecule).[97] Induced β-galactosidase in E. coliwild-type strains ATCC 8677 and 35321 in the presenceof various protease inhibitors have been studied. Thepresence of the protease inhibitors had least effect onthe average distribution of single molecule activities,and the relative activities of the enzyme for the diversesubstrates differed between the strains.[98] Average-combined turnover numbers of the enzyme from wild-type E. coli strains ATCC 35321 and 8677 in vivo andin vitro conditions in the presence and absence of His6tag differed considerably. This indicated that synthesizedenzymes in both conditions (vivo and vitro) were notalike and presence of a C-terminal His6 tag altered theactivity of s-galactosidase.[99] Moreover, it was foundthat electrophoretic mobility and catalytic activity ofindividual molecules of β-galactosidase synthesized byE. coli were different, although they had potentiality toact on the same substrate molecule.[100]

IMMOBILIZATION OF β-GALACTOSIDASE

Immobilization has shown to improve the stability ofβ-galactosidase, reusages, and reduces the processingtime in food and other industries. For example, theimmobilization of β-galactosidase of Thermus sp. T2was performed using ionic adsorption by a new anionicexchanger resins (based on coating of Sepabeadsinternal surfaces with polyethylenimine) andconventional DEAE–agarose. Immobilization wascarried out in both cases, but the adsorption strengthshowed much greater in the case of PEI–Sepabeads thanin DEAE supported at both pH5.0 and 7.0. Also, thePEI–Sepabeads remained wholly active, and after severalweeks of incubation at 323K, it showed the lactosehydrolysis in milk.[101] Also a new hetero functionalepoxy supports were used for immobilization ofβ-galactosidase. The capability of a standardSepabeads-epoxy supports to immobilize β-galactosidasefrom Thermus sp. strain T2 that equalized with otherSepabeads-epoxy, which supports partially modificationusing boronate, iminodiacetic, metal chelates andethylenediamine. Here, immobilization was dependedon the support, ranging from 95% to 5% using



Table

3.Properties

ofβ-galactosidasesynthesized

bydifferentmicroorgan

isms.

Typ

eof

consortiu

mNam

eof

consortiu

mTem

perature

(K)

pHHalflife(s)

Specific

activ

ity(U

mg�

1)

Reference

Bacteria

Arthrob

acterpsychrolactoph

ilus

333

836

00(303

K)

20[79]

Bacillus

circulan

s32

35–

6—

5.1

[50]

Bacillus

megaterium

313

6–9

—60

[54]

Bacillus

stearothermop

hilus

343

73.24

×10

4(343

K)

125

[53]

Bifido

bacterium

adolescentis

323

660

0(323

K)

526

[80]

Bifido

bacterium

bifidu

m31

06.5

1.44

×10

4(318

K)

—[58]

Bifido

bacterium

infantis

323–

333

7.5

7200

(333

K)

569

[59]

Cryptococcuslaurentii

331

4.3

600(331

K)

12[60]

Enterob

acterag

glom

eran

s31

0–31

37.5–

8—

—[81]

Lactoba

cillu

sacidop

hilus

328

6.5–

81.72

8×10

5(310

K)

230

[82]

Lactoba

cillu

sreuteri

323

6–8

—18

0[48]

Streptococcuspn

eumon

ia30

35.5–

7.5

——

[83]

Alicycloba

cillu

sacidocalda

rius

338

5.5

360(353

K)

592

[84]

Caldicellu

losiruptor

saccha

rolyticus

353

61.72

8×10

5(343

K)

211

[85]

Geoba

cillu

sstearothermop

hilus

338

6.5

—0.5

[86]

Saccha

ropo

lysporarectivirgu

la33

36.5–

7.2

1.44

×10

4(333

K)

—[70]

Therm

otog

amaritima

353–

358

6.5

960(363

K)

70[87]

Thu

rmus

sp.

343

6.5

7.2×10

4(343

K)

—[88]

Thu

rmus

aqua

ticus

353

5.5

—5.7

[72]

Yeast

Kluyverom

yces

frag

ilis

303

6.5

1.8×10

4(313

K)

—[88]

Kluyverom

yces

lactis

310–

313

6.6–

7—

—[89]

Sporob

olom

yces

sing

ularis

303

4—

8.7

[90]

Sterigmatom

yces

elviae

358

4.5–

536

00(353

K)

20[71]

Bullera

sing

ularis

323

572

00(318

K)

56[91]

Fun

gus

Aspergillu

soryzae

303

4.8

600(343

K)

40[62]

Aspergillu

saculeatus

328–

333

5.4

—24

[64]

Archae

Sulfo

lobu

ssolfa

taricus

348

6.5

1.08

×10

4(358

K)

116

[92]



Sepabeads-epoxy-chelate, Sepabeads-epoxy-amino, orSepabeads-epoxy-boronic using Sepabeads-epoxy-IDA.Amazingly, the immobilized β-galactosidase derivativesshowed outstandingly good result but different stabilitieshad been notified after favoring multipoint covalentattachment by long-term alkaline incubation. Theenzyme immobilized on Sepabeads-epoxy-boronicwas found to be the steadiest. The crosslinking withaldehyde-dextran allowed the stabilization of thequaternary structure of the enzyme. The optimalderivative was extremely active in lactose hydrolysiseven at 343 K (over 1000 IU g�1), maintaining itsactivity after extended incubation times with no riskof product contamination with enzyme subunits.[102]

Protocol for immobilization of β-galactosidasesynthesized by E. coli using diverse supports (glyoxyl,epoxy, BrCN groups, or by glutaraldehyde crosslinkingon matrix, containing primary amino groups), andstrategies have been studied. In each case, theimmobilization yield showed 100%with active recoveriesbetween 50% and 100% (using ortho-nitrophenyl-β-galactoside as substrate). Ratio of synthetic activityto hydrolytic activity (Vs/Vh) was lower than 0.1 whensoluble enzyme and the Eupergit 250 L enzymewere immobilized on BrCN at 277 K and pH 7.0,resulting 0.46 and 0.8, respectively.[103] Immobilizationof β-galactosidase producing permeabilized dead cellsof K. lactis ATCC 8583 into gelatin usingglutaraldehyde as crosslinker was performed, where30% activity obtained by immobilized cells relative tofree disrupted cells.[104] The usage of calcium alginate,Κ-carrageenan, and gellan-xanthan gel beads forentrapment of β-galactosidase synthesized byStreptococcus thermophilus, enhanced the stability ofenzyme at higher temperatures (>298K).[105] Solid statefermentation with co-immobilized β-galactosidasessynthesized by K. lactis, Aspergillus oryzae, and yeastsin polyvinyl alcohol hydrogel lens-shaped capsules havebeen performed. In the process the enzyme, synthesizedfrom Kluyveromyces lactis and Saccharomycescerevisiae showed the highest activity (galactose outputincreased from 3 to 4.1 g l�1 h�1), which reduced thereduction of processing time.[106] Differentmethodologies for immobilization of β-galactosidaseshave been described in Table 4.

INDUSTRIALIZED APPLICATIONS OFβ-GALACTOSIDASE

Microbial β-galactosidase plays a tremendouslyessential role in the production of various industrialrelevant products such as biosensor, lactose-hydrolyzed milk, ethanol, and GOS, also it has beenused in the field of bioremediation, diagnosis, and intreatment of lactose digestion disorder etc.

Use of β-galactosidase in biosensor

A biosensor associated with two distinct enzymaticactivities (β-galactosidase and glucose peroxidase) hasbeen developed for quantitative detection of lactose incommercial samples of milk. To avoid interferenceswith glucose, a degree of different mode ofmeasurement was done using biosensor.[146]

Foresighting this technique, presumed β-galactosidasefrom Streptococcus mitis with a choline-bindingdomain was identified recently. This remarkableproperty makes it differentially functional property forbiotechnological applications.[147]

Intended as lactose-hydrolyzed milk production

The cold–stable properties of Arthrobacter sp. 32csynthesized β-galactosidase could be useful forcommercial, industrial conversion of lactose intogalactose and glucose in milk products.[56] It has beenreported that rudimentary β-galactosidase extractproduced by Lactobacillus ssp. bulgaricus CHRHansen Lb-12 was applied in sterile milk, which hasbeen processed through ultra-high temperature method(UHT milk) for hydrolyzing lactose. Optimum amountof crude β-galactosidase extract and Maxilact enzymefor producing lactose-hydrolyzed milk was observedto be 0.418 and 0.512UmL�1 respectively during 6 hof processing. Using more than 418 U L�1 of crudeβ-galactosidase extract showed undesirable acidity oflactose-hydrolyzed milk that significantly increased attemperature of between 288 and 290K, whileenrichment of acidity in lactose-hydrolyzed milkproduced through Maxilact enzyme was not significant.Total count of lactose-hydrolyzed milk by 418UL�1

of crude β-galactosidase extract, after 2.16 × 104 s ofprocessing was significant high (5 to 30 Colony FormingUnit). Sensory estimation of lactose-hydrolyzed milkand ordinary ultra-high temperature milk (controlled)did not show any major differences with respect toacceptability of sweetness, taste, and color.[33] Thedifficulty in enzyme extraction and poor permeabilityof cell membrane to lactose was solved whenpermeabilized Kluyveromyces marxianus NCIM 3465cells were used for the production of lactose-hydrolyzedmilk. The ethanol-permeabilized yeast cells showed 89%of hydrolysed lactose under optimized conditions.[148]

Role of β-galactosidase in whey utilization

The execution of profound environmental legislationfor recycling and reuse of waste materials is grabbinglots of headlines at present century. Since many years,dairy products have been used as valuable ingredientsby the confectionery industry, being a huge economicalsource of capital, especially in the tropical andsubtropical countries. The large volume of byproduct



of the dairy industry, namely, whey, creates severedisposal problem. Concerning about environmentalaspects, research has been emphasized on membraneseparation technology, providing remarkable newopportunities for large-scale protein and lactosefractionation. They intrude proteins that are multi-functional food ingredients of high nutritional valueand offer a wide range of functional properties allowingdevelopment of new products and optimization ofexisting products within considerable low cost, butpresence of high concentration of lactose in aqueousmedium after separation of proteins from whey createssevere disposal problem.[149–153] Different strategies

have been attempted for whey lactose conversion, thoseare related to the synthesis of nutraceuticals GOS, as wellas alcohol from whey permeates would ensure ultimateconsumption and utilization of casein whey.[154,155]

Function of β-galactosidase in ethanolfermentationThe idea of kinetic analysis of alcoholic fermentationof lactose using a recombinant flocculent strain ofSaccharomyces cerevisiae NCYC869-A3/T1,expressing both the LAC4 (coding for β-galactosidase)and LAC12 (lactose permease) genes ofKluyveromyces Lactis conceded. The lactose was

Table 4. Different techniques for immobilization of β-galactosidases.

Immobilization method Source of β-galactosidase Immobilization matrix References

Covalent binding Aspergillus oryzae Amino-epoxy Sepabead [107]

Covalent binding Aspergillus oryzae Chitosan bead and nylon membrane [108]

Entrapment Aspergillus oryzae Nylon-6 and zeolite [109]

Physical adsorption Aspergillus oryzae Phenol-formaldehyde resin [110]

Physical adsorption Aspergillus oryzae Polyvinyl chloride and silica gel membrane [111]

Covalent binding Aspergillus oryzae Polyvinyl alcohol hydrogel and magneticFe3O4-chitosan as supporting agent

[112]

Covalent binding Aspergillus oryzae Silica [113]

Covalent binding Aspergillus oryzae Silica gel activated with TiCl3 and FeCl3[114]

Entrapment Aspergillus oryzae Spongy polyvinyl alcohol Cryogel [115]

Physical adsorption Aspergillus oryzae Celite and chitosan [116]

Covalent binding Aspergillus oryzae Cotton cloth and activated with tosyl chloride [117]

Physical adsorption Aspergillus niger Porous ceramic monolith [118]

Covalent binding Kluyveromyces lactis Magnetic polysiloxane-polyvinyl alcohol [119]

Entrapment Kluyveromyces bulgaricus Alginate using BaCl3[120]

Physical adsorption Kluyveromyces fragilis Cellulose beads [121]

Physical adsorption Kluyveromyces fragilis andKluyveromyces lactis

Chitosan [3]

Physical adsorption Kluyveromyces fragilis Chitosan [122]

Physical adsorption Kluyveromyces fragilis Chitosan bead [123]

Covalent binding Kluyveromyces lactis Cotton fabric [124]

Physical adsorption Kluyveromyces lactis CPC-silica and agarose [125]

Entrapment Kluyveromyces lactis, Aspergillusoryzae and Saccharomycescerevisiae

Poly(vinylalcohol) hydrogel [126]

Covalent binding Kluyveromyces lactis Corn grits [127]

Covalent binding Kluyveromyces lactis Thiosulfinate/thiosulfonate [128]

Covalent binding Kluyveromyces lactis Graphite surface [129]

Covalent binding Kluyveromyces fragilis Silica-alumina [130]

Entrapment Thermus aquaticus YT-1 Agarose bead [131]

Physical adsorption Thermus sp. T2 PEI–Sepabeads, DEAE–agarose [132]

Entrapment Penicillium expansum F3 Calcium alginate [133]

Covalent binding Saccharomyces anamensis Calcium alginate [134]

Physical adsorption Pisum sativum Sephadex G-75 and chitosan beads [135]

Covalent binding Lactobacillus bulgaricus Egg shells [136]

Physical adsorption Bacillus stearothermophilus Chitosan [137]

Physical adsorption Bacillus circulans Polyvinyl chloride and silica [138]

Covalent binding Bacillus circulans Eupergit C (Spherical acrylic polymer) [139]

Entrapment Escherichia coli Polyacrylamide gel [140]

Covalent binding Escherichia coli Polyvinyl alcohol [141]

Physical adsorption Escherichia coli Chromosorb-W [142]

Covalent binding Escherichia coli (Recombinantβ-galactosidase)

Cyanuric chloride-activated cellulose [143]

Covalent binding Escherichia coli Hen egg white [144]

Covalent binding Escherichia coli Gelatine [145]



https://www.researchgate.net/publication/232327318_Separation_of_Proteins_by_Charged_Ultrafiltration_Membranes?el=1_x_8&enrichId=rgreq-a6017d31-708c-46b6-ac5a-b4b3e3b6a1e9&enrichSource=Y292ZXJQYWdlOzI2MjExNDQ5NTtBUzoyMzA3NjA3NzkwODc4ODBAMTQzMjAyOTA2NTcyMA==

completely utilized in all fermentative processes, and ithas been observed that with increase of initial lactoseconcentration (5 to 200 gL�1), the level of ethanolproduction increased linearly.[156] Meticulously, kineticmodel with respect to biomass growth, lactosehydrolysis, and ethanol production using β-galactosidasesynthesized by Kluyveromyces sp. was performedconsidering whey permeate and cheese whey powderas growth medium.[157–159] Moreover, Saccharomycescerevisiae has also been reported for production ofethanol considering concentrated deproteinized whey,cheese whey powder, and salted cheese whey as feedstock.[160–162] Ethanol fermentation of cheese wheypowder solution using the pure culture ofKluyveromycesmarxianus (DSMZ 7239) was studied in packed columnbioreactor using olive pits as sustaining particles forcell attachment.[163] Researches have also beenconducted for ethanol production by membranerecycle bioreactor and using co-immobilized S.cerevisiae strain and β-galactosidase in semi-continuous fermentation process considering wheypermeate and whey medium, respectively.[164,165]

Thus, whey utilization by β-galactosidase reduces theburden of water pollution and establishes the conceptof recycling and reuse of waste materials.

Galato-oligosaccharide production byβ-galactosidaseIn recent years, many investigations have been carriedout in the field of prebiotics, considered as functionalfood. Among them, one of the recognized functionalfood ingredients is oligosaccharides. They are

carbohydrates containing three to ten sugar units boundwith glycosidic bonds. It has been seen that there are anumber of classes of oligosaccharides, but amongthem, GOS has attracted particular attention becauseof their presence in human breast milk. GOS are non-digestible, carbohydrate-based food ingredient,responsible for human and animal nutrition. Productionof GOS by transgalactosylation activity results theformation of 4′- or 6′-galactosylactose, longeroligosaccharides, transgalactosylated disaccharides,and nonreducing oligosaccharides in presence ofβ-galactosidases. Depending on the source of enzymeand conditions of reaction, various glycosidic linkages,such as β(1,2), β(1,3), β (1,4), and β (1,6), are formedin the end product (GOS).[166] It has been found thatthe amount of GOS synthesis from lactose dependson the initial concentrations of lactose present inthe reaction mixture instead of the concentration ofβ-galactosidase. There are different phenomena thathave been reported to elucidate the synthesismechanism of GOS, which are depicted in Figs 4–6.It is believed that the presence of GOS in human

milk influences the growth of bifidobacteria in thegastrointestinal tract of newly born and breast-fedinfants. GOS fraction (referred to as bifidus factor) incow’s milk also provides several health benefits.[170]

GOS is stable under acidic conditions during foodprocessing as well as maintain excellent taste quality,which makes them popular as an active ingredient ina wide variety of food products. They pass throughthe small intestine without being digested for lowcaloric 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 fromBoon et al. (1999)).[167]



microorganisms in the oral cavity.[171] The detail workscarried out in this direction have been represented inTable 5.

Applications β-galactosidase in medical andimmunology research

Clostridium perfringens ATCC 10543 synthesizedendo β-galactosidase, which is capable of liberatingbeneficial A trisaccharide and B trisaccharide fromglycoconjugates containing blood group A and Bglycotopes, respectively, was isolated by Andersonet al., 2005. Recombinant EABase damages the bloodgroup A and B antigenicity of human type A and Berythrocytes with the release of A-Tri and B-Tri fromblood group A+ and B+ containing glycoconjugates.Here, the incomparable specificity of β-galactosidase wasuseful for studying the structure and function of bloodgroupA-containing and B-containing glycoconjugates.[193]

The recombinant endo-β-galactosidase (ABase), whichreleases A/B antigen was developed in 2009. Itremoved 82% of A antigen and 95% of B antigen fromhuman A/B red blood cells and concealed anti-A/Bantibody binding, also the complement activationeffectively. In vivo infusion into a blood type Ademonstrated the reduction of A antigen expressionin the glomeruli of kidney (85% at 3600 s, 9% at1.44 × 104 s, and 13% at 8.64 × 104 s) and the sinusoidsof liver (47% at 3600 s, 1% at 1.44 × 104 s, and 3% at8.64 × 104 s) without any grave adverse effects. Thissubstitute approach remains useful for minimizing

antibody removal and anti-B cell immunosuppressionas an adjuvant therapy of ABO incompatible kidney,liver, and possibly heart transplantation.[194]

Expression of β-galactosidase, synthesized by E. coliwithin muscle fibers has been demonstrated by Liuand Roffler, 2006. They conclude that repeatedintramuscular injections of β-galactosidase couldencourage strong immune responses amongimmuno-competent animals and cause eliminationof transduce muscle fibers by inflammatory cells.[195]

Recently, β-galactosidase from the mesoacidophilicfungus Bispora sp. MEY-1 under simulated gastricconditions has shown greater stability (100%) andhydrolysis ratio (>80%) toward milk lactose thancommercially available β-galactosidase from Aspergillusoryzae ATCC 20423. Thus, this β-galactosidase may bea better digestive supplement for alleviatingsymptoms associated with lactase deficiency.[196]

Recombinant β-galactosidases in cooperating one ortwo different peptides from the foot-and-mouthdisease virus (FMDV) nonstructural protein 3B perenzyme monomer, granted specified differentiationbetween sera of FMDV-infected pigs, cattle, andsheep, and those of native and conventionallyvaccinated animals. These FMDV infection-specificbiosensors can provide effectual and versatilealternatives for the serological differentiation ofFMDV-infected animals.[197]

SCOPE FOR FURTHER STUDY

β-galactosidase belongs to lactase group, whichhydrolyzes β-glycosidic bond formed betweengalactose and its organic moiety. Moreover, in somecases, it takes part in transgalactosidase reaction. Thedisadvantage of wild-type β-galactosidase is its loweractivity as well as it is inhibited by hydrolyzed product,glucose, and galactose. Therefore, research regardingtransgenic β-galactosidase synthesis will boost uphydrolytic activity as well as GOS synthesis. Although,synthesis mechanism of β-galactosidase is well establishedfor E. coli, still much more information is needed for othermicroorganism. Although, it is recommended thatprobiotic consortium (lactic acid bacteria) is much moreacceptable for food grade β-galactosidase synthesis, butbecause of product inhibition by lactic acid, researchon bioprocess design is in demand. More than 90-foldpurification of β-galactosidase is yet to achieved. In thiscircumstances, suitable purification device as well asdevelopment of purification protocol for high throughputis required. Research in the line of process intensificationof industrial β-galactosidase production and purificationshould be a great challenge. It is obvious, immobilizedβ-galactosidase provides several benefits for GOSsynthesis but due to lower activity at immobilizecondition; studies on the active site of enzyme and

Figure 6. Schematic diagram of galato-oligosaccharidesynthesis from lactose (Figure adapted from Palai et al.(2012)).[169]

Figure 5. Schematic diagram of galato-oligosaccharidesynthesis from lactose (Figure adapted from Neri et al.(2008)).[168]



choice of suitable matrix are required. Without anycontradiction, β-galactosidase plays several roles inbiopharmaceutical, food, and dairy industries. Researchfor more applications in several fields particularly cancerdetection and pharmaceutical research will boost upimmunological research and biotechnological economy.

CONCLUSION

In this review, authors have tried their best toaccumulate all the information regarding production,purification, characterization, immobilization, andapplication of β-galactosidase. β-galactosidase is oneof the important enzyme that not only offers nutri-tional applications but also used for waste treatment.β-galactosidase used in food, pharmaceuticals, anddairy industries for producing low lactose-containingfood product, sweeteners, GOS, and also used for dairywaste treatment. It is also used for ethanol production,which is a challenging step in present century. Severalmicrobes, especially probiotic consortia arerecommended for enzyme production using casein wheyas a growth medium. Purified β-galactosidase will ensure

the high activity as well as poses better catalysisproperties. Enzyme immobilization provides enzymereutilization, also the immobilized enzyme showed highlevel of hydrolysis and thus it can be applied successfullyfor hydrolyzing lactose in milk and whey. The isolation ofpyschrophilic bacteria with cold active β-galactosidasehas opened up the possibility of processing of GOS atlow temperatures. On the other hand, thermostableenzyme has the unique ability to retain their activity athigher temperature for prolonged periods, and the processis less prone to microbial contamination due to higheroperating temperature. Therefore, it may be concludedthat utilization of β-galactosidase is a twofold solution ofbiotechnology economy and waste treatment.Furthermore, this work will provide a ready referencefor future scientific research regarding β-galactosidase.

Acknowledgements

Arijit Nath acknowledges Council of Scientific andIndustrial Research (CSIR), New Delhi for financial

Table 5. Different microorganisms for galato-oligosaccharide synthesis and parameters of transgalactosylationreaction.

Nature of enzyme Enzyme sourceTemperature

(K) pHSynthesis rate(g L�1 h�1) Reference

Crude Aspergillus oryzae 313 4.5 24 [172]

Crude Bacillus circulans 313 6 2.2 [173]

Crude Bifidobacterium longum 318 6.8 13 [174]

Crude Geobacillus stearothermophilus 310 6.5 0.4 [86]

Recombinant Geobacillus stearothermophilus R109W 310 6.5 6.9 [86]

Crude Lactobacillus reuteri 303 6.5 3.9 [175]

Crude Talaromyces thermophilus 313 6.5 13 [176]

Purified Bullera singularis 323 5 3.9 [91]

Purified Enterobacter agglomerans 323 7.5 3.9 [81]

Purified Lactobacillus acidophilus 303 6.5 7.9 [82]

Purified Penicillium simplicissimum 323 6.5 11 [177]

Recombinant Bifidobacterium infantis 333 7.5 13 [59]

Recombinant Sulfolobus solfataricus 353 6 5.6 [178]

Recombinant Thermotoga maritima 353 6 18 [179]

Whole cells Bifidobacterium bifidum 312 6.8 65 [180]

Toluene-treated cells Rhodotorula minuta 333 6 3.2 [181]

Immobilized Aspergillus candidus 313 6.5 87 [182]

Immobilized Bacillus circulans 313 6 4.2 [183]

Immobilized Bullera singularis 318 3.7 4.8 [184]

Immobilized Kluyveromyces lactis 313 7 25 [185]

Immobilized cells Talaromyces thermophilus 313 6.5 18.75 [186]

Immobilized cell Cryptococcus laurentii 313 4.3 3.6 [187]

Crude Talaromyces thermophilus 313 6.5 — [186]

Crude Penicillium sp. 328 4 — [188]

Purified Saccharopolyspora rectivirgula 333 6.5 — [70]

Purified Sterigmatomyces elviae 333 5 3.2 [71]

Recombinant Pyrococcus furiosus 353 5 — [189]

Recombinant Thermus sp. 343 7 — [190]

Immobilized Sirobasidium magnum 323 — 3.2 [191]

Immobilized Sirobasidium magnum 323 — 5.8 [191]

Immobilized Aspergillus oryzae 313 4.5 106 [117]

Immobilized cell Sporobolomyces singularis 328 5.0 8.7 [192]



support as an SRF. The reported work is a part of aUniversity Grants Commission (UGC) Major project,entitled ‘Production and Purification of β-galactosidase from Milk Whey-based Lactic AcidBacteria using Fermentation and Membrane-basedSeparation Techniques”. The contribution of UGC isgratefully acknowledged.

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