Critical Reviews in Biotechnology, 2010; 30(1): 41–62

REVIEW ARTICLE

β Galactosidases and their potential applications: a review

Qayyum Husain
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India
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Abstract
β Galactosidases have been obtained from microorganisms such as fungi, bacteria and yeasts; plants, animals
cells, and from recombinant sources. The enzyme has two main applications; the removal of lactose from milk
products for lactose intolerant people and the production of galactosylated products. In order to increase their
stability, reusability, and use in continuous reactors, these enzymes have been immobilized on both organic and
inorganic support via adsorption, covalent attachment, chemical aggregation, microencapsulation, and entrap-
ment. Free and immobilized preparations of β galactosidases have been exploited in various applications such as
industrial, biotechnological, medical, analytical, and in different other applications. β galactosidase is widely used
in food industry to improve sweetness, solubility, flavor, and digestibility of dairy products. Immobilized β galac-
tosidases are employed for the continuous hydrolysis of lactose from whey and milk in a number of reactors such
as hollow fiber reactors, tapered column reactors, packed bed reactors, fluidized bed reactors etc.
Keywords:  Immobilization; stabilization; β galactosidase; application; lactose
Abbreviations:  Aaβ-gal, Alicyclobacillus acidocaldarius β galactosidase; Con A, concanavalin; CBM, continuous
For personal use only.

batch mode; CMRR, continuous membrane recycle reactor; CBMR, cyclic batch membrane reactor; 3-D, three-
dimensional; ELISA, enzyme linked immunosorbent assay; FB, fluidized bed; FBRs, fluidized-bed reactors;
FBM, fluidized bed modes; FT-IR, fourier transform infra red; ESR, GH, glycoside hydrolase; GRAS, generally
regarded as safe; HFRs, hollow-fiber reactors; IMAC, immobilized metal affinity chromatography; LAB, lactic
acid bacteria; LB, Langmuir–Blodgett; MBR, membrane bioreactor; Mr, molecular weight; ONPG, o-nitrophenyl
galactoside; PBRs, packed-bed reactors; PFC, perfluorocarbon; PAGE, polyacrylamide gel electrophoresis;
PEG, polyethylene glycol; PsBGAL, Pisum sativum β galactosidase; PVA, polyvinyl chloride; STRs, stirred-tank
reactors; TCR, tapered column reactor; TBG, tomato β galactosidase; THP, tris(hydroxymethyl)phosphine.

Introduction milk and related dairy products for consumption by lactose

β Galactosidase (β-D-galactohydrolase, EC 3.2.1.23) is an
enzyme that hydrolyzes D-galactosyl residues from polymers,
oligosaccharides or secondary metabolites. Polysaccharide
specific β-galactosidases include β-galactanases, which
attack pectic polymers and β-galactosidases that attack
xyloglucans. These enzymes have two main applications;
the removal of lactose from milk products for lactose intol-
erant people and production of galactosylated products
(Hsu, Yu, and Chou, 2005; Heyman, 2006; Neri et al., 2008).
β Galactosidase is widely used in food industry to improve
sweetness, solubility, flavor and digestibility of dairy prod-
ucts (Richmond, Gray, and Stine, 1981; Grosova et  al.,
2008a). This enzyme has been used as a model for studying
its activity in amorphous matrices (Burin and Buera, 2002).
Enzymatic hydrolysis of lactose by β galactosidase is one of
the most popular technologies to produce lactose reduced
intolerant people (Ladero, Santos, and García-Ochoa, 2000;
Ladero et al., 2001; 2002; Jurado et al., 2002; Sener, Apar, and
Ozbek, 2006; Haider and Husain, 2008). Lactose is a hygro-
scopic sugar and has a strong tendency to absorb flavors
and odors and causes many defects in refrigerated products
such as crystallization in dairy foods, development of sandy
or gritty texture and deposit formation (Panesar et al., 2006;
2007). By hydrolyzing lactose with β galactosidase, the prob-
lems associated with whey disposal, lactose crystallization
in frozen concentrated deserts and milk consumption by
lactose-intolerant individuals can be eliminated (Kim and
Rajagopal, 2000; Bayramoglu et al., 2007).
β Galactosidases work in a relatively broad pH range:
enzymes from fungi act between pH 2.5–5.4, yeast and bac-
terial enzymes act between pH 6.0–7.0. Depending on the
natural source where lactose is present, pH values range

Address for Correspondence:  Qayyum Husain, Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh-202002, India; E-mail:
qayyumhusain@yahoo.co.in

(Accepted 20 August 2009)

ISSN 0738-8551 print/ISSN 1549-7801 online © 2010 Informa UK Ltd

DOI: 10.3109/07388550903330497 http://www.informahealthcare.com/bty
 42   Q. Husain
between ~ 3.5 or 5.6 of acid whey to 6.5 of milk. These enzymes
are classified in the family of glycoside hydrolase 2 (GH2) and
glycoside hydrolase 35 (GH35). The enzymes of GH2 family are
predominantly found in microorganisms whereas nearly 70%
of GH35 are present in plants (Arribas et al., 2000; Jacob et al.,
2000; Jacob, Peters, and Naim, 2002; Alcantara et al., 2006).
Sources of β galactosidases
β Galactosidases are found in microorganisms (bacteria,
fungi, yeasts), plants especially in almonds, peaches, apri-
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cots, apples and animal organs (Nagy et  al., 2001; Flood
and Kondo, 2004; Haider and Husain, 2007a). The major
industrial enzymes are obtained from Aspergillus sp. and
Kluyveromyces sp. β Galactosidase from Kluyveromyces lac­
tis is one of the most widely used enzymes (Zhou and Chen,
2001a; Jurado et al., 2002; Lee et al., 2003; Klewicki, 2007).
β Galactosidases from microorganisms
Fungi
Fungal β galactosidases generally have acidic pH-optima
in the range of 2.5–5.4 thus they are most effective for the
hydrolysis of lactose present in acidic products such as
whey. Fungal β galactosidases are thermostable enzymes;
however, they are more sensitive to product inhibition
For personal use only.
mainly by galactose (Boon, Janssen, and van’t Riet, 2000).
Filamentous fungi often utilize lactose only at very low rates,
for example, for the industrial production of penicillin by
Penicillium chrysogenum. In fungi two principal strategies
for the catabolism of lactose are realized: (1) extracellular
hydrolysis and subsequent uptake of resulting monomers
and (2) uptake of disaccharides (Pakula et al., 2005).
β Galactosidase purified from a culture of Aspergillus
oryzae by 2-propanol fractionation column chromatogra-
phy on DEAE-Sephadex A-50 and Sephadex G-200 showed
pH-optima of 4.5 with o-nitrophenyl β-D galactopyranoside
(ONPG) and 4.8 with lactose. The stable pH range was from
4.0 to 9.0 and the optimum-temperature was 46°C. The
apparent molecular weight (Mr) calculated by Sephadex
gel filtration and sucrose density gradient centrifugation
was about 105 kDa. The Michaelis constants were 7.2 × 10−4
M with ONPG and 1.8 × 10−2 M with lactose. Hg2+, Cu2+,
N-bromosuccinimide, and sodium lauryl sulfate caused
marked inhibition (Tanaka et al., 1975).
The three dimensional model of the active site of Asperg­
ill­us oryzae β galactosidase consists of two glutamic acid resi-
dues in a groove quite accessible to the solvent. Three exposed
tyrosine residues Tyr 138, Tyr 201, and Tyr 260 were found
within 1.0 nm from the active glutamic acid whereas within
the same distance no exposed arginine residue was seen. It
has been reported that binding through ­diazotization of tyro-
sine residues, protected the active site more than through
binding due to condensation (El-Masry et al., 2001a).
Bacteria
β Galactosidases from bacterial sources has been widely
used for the hydrolysis of lactose because of the ease of
fermentation, high activity of the enzyme and good stability
(Picard et al., 2005). Lactic acid bacteria (LAB) which con-
stitute a diverse group of lactococci, streptococci and lacto-
bacilli have become a focus of scientific studies for three
particular reasons: (1) lactose maldigesters may consume
some fermented dairy products with little or no adverse
effects, (2) LAB are generally regarded as safe (GRAS) so the
enzyme derived from them might be used without extensive
purification, and (3) some strains have probiotic activity
such as improved digestion of lactose (Vasiljevic and Jelen,
2002; Vinderola and Reinheimer, 2003).
Bifidobacterium is a probiotic organism and its β galac-
tosidase preparations are used in foods and food systems.
β Galactosidase production by Bifidobacterium longum CCRC
15708, Bifidobacterium longum B6 and Bifidobacterium
infantis CCRC 14633 were examined with Bifidobacterium
longum CCRC 15708, which showed highest production of
β galactosidase with highest specific activity (Hsu, Yu, and
Chou, 2005). Bifidobacterium sp. together with Lactobacillus
sp. are the bacteria most applied as probiotics because of
their potential health benefits. Bifidobacterium has been
chosen as a model bacterium for studying fermentation of
lactose by the colonic microbiota (Arunachalam, 2004).
Lactobacillus reuteri is a dominant strain of the hetero
fermentative Lactobacilli in the gastrointestinal tract of
humans and animals. Lactobacilli isolated from the stom-
ach contents of piglets were used for the production of
improved fermented milk products. Such Lactobacilli were
found in large numbers on the stratified, squamous epi-
thelium of the oesophagea. Porcine strains of Lactobacilli
would probably be capable of fermenting lactose in bovine
milk (Sung et al., 2003). β galactosidase activity is also abun-
dantly present in the colon of human beings. It catalyzes the
first step of lactose fermentation in colon and is often meas-
ured as an indication of the capacity of colonic microbiota
to ferment lactose present in the intestine (Jain, Gupta, and
Jain, 2007).
Yeasts
The yeast Kluyveromyces lactis is an important commercial
source of β galactosidase because its natural habitat is the
dairy environment (Kim, Ji, and Oh, 2004; Tello-Solis et al.,
2005). The production of β galactosidase by yeast could be
of interest since this enzyme is used by the food industry
for the production of reduced lactose milk, an outstanding
industrial product used by a large number of lactose-intol-
erant people (Pivarnik, Senecal, and Rand, 1995; Rech and
Ayub, 2007).
Lactose fermenting yeasts produce intracellular β
galactosidase and these are good sources of this enzyme
(Numanoglu and Sungur, 2004). This enzyme due to its
huge hydrolytic activity has been used to produce lactose-
free milk products. Yeast lactases are most active in the
buffers of pH 6.0–7.0 (Genari, Passos, and Passos, 2003).
Kluyveromyces marxianus presents the possibility of pro-
ducing homologous enzymes, such as β galactosidase, as
well as heterologous proteins and the capacity of growing

on different substrates including lactose as the sole carbon
and energy source (Furlan et al., 2000; Martins et al., 2002;
Ribeiro et al., 2007).
Cold-active acid β galactosidase from psychrophilic-ba-
sidiomycetous yeast Guehomyces pullulans can be applied
to the food industries and the enzyme may help in the uti-
lization of industrial β galactosidases for the hydrolysis of
lactose in milk and whey (Nakagawa et al., 2006).
β Galactosidases from plants
β Galactosidases are widely distributed in plant tissues.
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These enzymes have been shown to be involved in a number
of biological processes including plant growth, fruit ripen-
ing and in the hydrolysis of lactose. Molecular approaches
were also used to unravel the role of β galactosidases in fruit
development and ripening (Li et al., 2001; Lopez et al., 2002).
β galactosidase/exogalactanase activity has been reported
during tomato (Lycopersicon esculentum Mill.) fruit ripen-
ing, a family of seven tomato β galactosidase (TBG) cDNAs
was recognized. The shared amino acid sequence identity
among the seven TBG clones ranged from 33% to 79% and
these clones contained the putative active site-containing
consensus sequence pattern G-G-P-[LIVM]-x-Q-x-E-N-E-
[FY], which showed that they belonged to glycosyl hydro-
lase family 35 (Smith and Gross, 2000). This enzyme has an
For personal use only.
important role in ripening of persimmons because of signifi-
cant decrease in cell wall galactosyl content and associated
pectin degradation that helps in the ripening of fruit (Kang
et al., 1994). Lazan et al. (2004) isolated β galactosidase from
papaya which was responsible for the hydrolysis of its cell
wall and softening of the fruit during ripening.
β galactosidase activity was also found in cell wall pro-
teins of strawberry (Fragaria ananassa) fruits in different
developmental stages. Three full-length cDNAs (Faβgal1,
Faβgal2, and Faβgal3) encoding different β galactosidases
were isolated from a library representing red fruit tran-
scripts. Strawberry fruits soften markedly during ripening
and the process is accompanied by the release of free sugars
(Hadfield et al., 2000; Trainotti et al., 2001). β Galactosidase
activity has also been reported from the cotyledons of ger-
minated nasturtium (Tropaeolum majus L.) seeds and it was
described that the enzyme was involved in vivo hydrolysis
of storage xyloglucan (Edwards et al., 1988). β Galactosidase
isolated and purified from Cicer arietinum exhibited high
enzyme activity and this enzyme preparation was used for
the hydrolysis of milk in a convenient and unexpensive
way (Tu et  al., 1999). The isolation and characterization of
β galactosidases from cowpea, radish, mung bean, barley,
carrot, rice shoots, lupins, and kidney beans have also been
investigated (Konno and Katoh, 1992; Konno and Tsumki,
1993; Buckeridge and Reid, 1994; Li et  al., 2001; Biswas,
Kayastha, and Seckler, 2003). Multiple isozymes of β galac-
tosidases have been isolated from apple (Malus domestica),
muskmelon (Cucumis melo), Japanese pear (Pyrus pyri­
folia) and avocado (Persea americana) and their different
galactosyl-hydrolyzing abilities were characterized using
native cell wall polysaccharides and synthetic substrates
β galactosidases and their potential applications   43
(Ranwala, Suematsu, and Masuda, 1992; DeVeau et al., 1993;
Ross et  al., 1994; Kitagawa, Kanayama, and Yamaki, 1995;
Tateishi, Inoue, and Yamaki, 2001).
In higher plants, β galactosidase is the only enzyme that is
able to hydrolyze galactosyl residues from cell wall polysac-
charides and no enzyme capable of cleaving β-1,4-galactan
in an endo fashion has been identified (Smith, Starrett, and
Gross, 1998). Plant β galactosidases exhibited significant
differences from those isolated from bacteria. Bacterial
enzymes are generally tetrameric or monomeric and much
larger than the plant enzymes, which are generally dimeric
and much smaller (Flickinger and Drew, 1999; Biswas,
Kayastha, and Seckler, 2003). It has also been reported that
the pH-optima of the plant enzymes lie in the acidic range
while from bacteria lie near to the neutral range (McGee
and Muray, 1986). β galactosidase was isolated from almond
(Amygdalus communis) extract by ammonium sulfate pre-
cipitation. The partially purified β galactosidase showed pH
and temperature optima at pH 5.5 and 50°C. The enzyme
was significantly stable against heat, pH, Ca2+ ions, Mg2+
ions, and D-galactose. The almond β galactosidase retained
nearly 89% activity over 2 months storage at 4°C. Hydrolysis
of lactose in milk and whey was performed in a stirred batch
process by using this enzyme and it was observed that the
rate of hydrolysis of lactose increased continuously with
time. The enzyme could hydrolyze 94% lactose in buffer
solution and whey, whereas 90% lactose was hydrolyzed in
milk (Haider and Husain, 2007a). Dwevedi and Kayastha
(2009a) have purified a basic glycosylated β galactosidase
(PsBGAL) from pea seeds by 910-fold with a specific activ-
ity of 77.33 μmoL/min/mg protein. PsBGAL was efficiently
compartmentalized during seed maturation inside vacuoles
around pH 5.0. The enzyme was capable of hydrolyzing pea
seed xyloglucan, and it might be involved in altering the cell
wall architecture during seedling growth and development.
The presence of a protonated carboxyl group at the active
site of this β galactosidase has been confirmed by ioniza-
tion constant, thermodynamics and chemical modification
studies.
Recombinant β galactosidases
Recombinant β galactosidase was expressed in Pichia pas­
toris in a defined medium containing metals where magne-
sium and zinc were both required to support cell growth but
at significantly reduced levels compared to the control. The
cells grew to 12–15 generations when the medium was refor-
mulated with only zinc and magnesium. The product yields
of recombinant β galactosidase were markedly enhanced by
the concentrations of trace metals. By using response surface
and full factorial designs, maximum protein yield occurred
when the concentration of zinc salt was limited to the level
necessary only to support cell mass while protein yield posi-
tively correlated to increasing levels of the remaining trace
metal salts (Plantz et al., 2007). A thermostable β galactosi-
dase gene bgaB from bacterium, Bacillus stearothermophilus
was cloned and expressed in B. subtilis WB600. The optimum
pH and temperature for the cloned β galactosidase were pH
 44   Q. Husain
7.0 and 70°C. Kinetics of thermal inactivation and half-life
for this thermostable enzyme at 65°C and 70°C were 50 h and
9 h, respectively, and Km and Vmax values were 2.96 mM and
6.62 µmoL/min/mg. This enzyme exhibited a high level of
transgalactosylation activity in hydrolysis of lactose in milk.
The findings of the work suggested that this recombinant
thermostable β galactosidase might be suitable for both the
hydrolysis of lactose and the production of galactooligosac-
charides in milk processing (Chen et al., 2008).
Several recombinant proteins have been successfully
expressed using the δ-integrative systems. A flocculent
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Saccharomyces cerevisiae strain was constructed to secrete
stable Aspergillus niger β galactosidase in a continuous
high-cell-density bioreactor. The δ-sequences from the
yeast retrotransposon Ty1 were used as target sites for the
integration of the β galactosidase expression cassette. High-
copy-number transformants were successfully obtained
using the δ-integration system together with the dominant
selection antibiotic, G418 (Oliveira et  al., 2007). The yeast
oestrogen assay makes use of recombinant yeast cells
that have an oestrogen receptor expression cassette and a
reporter construct, coding for β galactosidase. The induc-
tion mechanism starts with the binding of oestrogenic com-
pounds to the oestrogen receptor. This complex activates
the production of β galactosidase. Thus the β galactosidase
For personal use only.
activity was a measure to the oestrogenic activity of chemical
compounds (Slepak et al., 2005).
The Kluyveromyces lactis LAC4 gene encoding β galac-
tosidase as a reporter gene and Candida famata mutant lac4
unable for lactose utilization as a recipient strain were used
in the recombinant system for the hydrolysis of lactose. The
Candida famata mutant lac4 was transformed with the plas-
mid containing analyzable promoters fused with the pro-
moter less LAC4 gene. Resulting transformants, unlike the
mutant lac4, were able to hydrolyze lactose as sole carbon
source (Ishchuk et al., 2007). Di Lauro et al. (2008) reported
the purification and characterization of a bacterial β galac-
tosidase from thermoacidophilic bacterium Alicyclobacillus
acidocaldarius, which is a rich source of glycoside hydrolases.
The enzyme was purified to 46-fold from A. acidocaldar­
ius extracts; the gene for Aaβ-gal encoded a new member
of the glycoside hydrolase family 42 (GH42). The gene for
this enzyme was cloned and expressed and recombinant
enzyme (Aaβ-gal) was characterized. The recombinant Aaβ-
gal was found to be optically active and stable at 65°C. The
cloning of β galactosidase from Lactobacillus reuteri L103
and its expression in Escherichia coli has been investigated.
Heterodimeric β galactosidase from L. reuteri is encoded by
two overlapping genes, lacL and lacM (Nguyen et al., 2006;
2007).
Methods of immobilization of β galactosidases
Immobilization is defined as an attachment or the entrap-
ment of a biocatalyst in a distinct phase that allows exchange
with, but is separated from the bulk phase in which substrate
effector or inhibitor molecules are dispersed and monitored
(Mateo et  al., 2007a; 2007b). Enzyme immobilization has
been studied for a number of years which indicates a contin-
ued interest in this area (Deng et al., 2004; Sanjay et al., 2005;
Delgado et al., 2006). This is likely due to the known benefits
of enzyme immobilization and the desire to improve immo-
bilization matrices and methods (Shibatane et  al., 2000;
Girelli and Mattei, 2005). The exact requirements for the
immobilizing matrix are dictated by the type of enzyme and
the intended application, the material being nondegradable
and compatible with the enzymes. The process for immobi-
lization should also be mild enough; during the immobili-
zation procedure enzyme should not denature and bind as
much enzyme as possible to the support (Khan, Akhtar, and
Husain, 2005; Kulshrestha, 2006a).
The immobilization of enzymes is a widely used
approach for obtaining reusable enzyme derivatives which
reduces the high enzyme cost associated with their pro-
duction and purification (Tanaka and Kawamoto, 1999).
Immobilization very often led to an enhancement in the
resistance of the enzymes against various denaturing fac-
tors such as extreme pH, temperature, high ionic strength,
chemical denaturants, proteases etc. High immobilization
yields, expression of high activity of the bound enzyme and
stabilization against inactivation mediated by several dena-
turing parameters were some of the important points which
determine the success of any immobilization procedure
(Cammarota and Freire, 2006). Immobilization of whole
cells eliminates the succeeding purification steps and in
some cases the enzymes were more stable if immobilized
within their natural environment, the cell (Genari, Passos,
and Passos, 2003; Krajewska, 2004).
The morphology of the support plays an important
role in continuous bioprocesses using immobilized bio-
catalysts (Taqieddin and Amiji, 2004; Milosavic et al., 2005).
Biocatalysts have been immobilized by using a variety of
supports and techniques (Palomo et al., 2007). Some poly-
meric supports have been used in the development of the
immobilized β galactosidases for industrial uses. They
offered the advantages of high mechanical strength, high
density, thermal stability, ease of handling, high flow rates in
continuous reactors and ease of regeneration. The matrices
used for enzyme immobilization coupling may be roughly
divided into two groups; natural and artificial polymers.
Natural polymers include alginate, hydrogels, chitosan etc.
Artificial polymers are acrylamide, polyacrylate, polysty-
rene, etc. (Caprio et al., 2000). Figure 1 demonstrates various
methods of enzyme immobilization.
Adsorption
This method is based on the physical adsorption of enzyme
on the surface of water-insoluble carriers. There is little or
no conformational change of the enzyme nor does destruc-
tion on its active site take place. Usually no reagents and
only a minimum of activation steps are required for the
adsorption process. The procedure is simple and unexpen-
sive (Khan, Akhtar, and Husain, 2006; Cunha et al., 2008). A
variety of support materials have been used for adsorption

1 2
E
E
1. Adsorption
E
E
2. Covalent attachment
E E 3. Entrapment
4. Microencapsulation
3 4
E Enzyme molecule
E E
E Solid or porous supports
E
E E Porous polymeric matrix
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Semipermeable membrane
Figure 1.  Methods of enzyme immobilization.
and immobilization of biocatalysts. Some of the adsorbents
often used for adsorption includes magnetic particles, alu-
mina, amberlite CG-50, bentonite, calcium phosphate gels,
carbon, carboxy methyl cellulose, carboxy methyl sephadex,
collagen, DEAE-cellulose, DEAE-sephadex, glass, silica gel
titania (ceramics). Magnetic particles have wide range of
applications for the immobilization of cells and enzymes
(Tong, Xue, and Sun, 2000; Akgol et al., 2001; Liu et al., 2005;
Mandal et al., 2005; Amaral et al., 2006).
Chitosan is an ideal support for the adsorption of enzymes
For personal use only.
because of its many features such as hydrophilicity, biocom-
patibility, biodegradability and anti-bacterial properties.
In addition to this, chitosan appears to be an economically
attractive material because it is the second most abundant
biopolymer present in nature after cellulose (Juang, Wu, and
Tseng, 2001; Wu, Chen, and Ran, 2002). Thus chitosan-algi-
nate microcapsules were designed to meet the criteria speci-
fied for an immobilized system. Such a hybrid system pro-
vides many advantages over the use of chitosan or alginate
alone for adsorption of horseradish peroxidase (Taqieddin
et al., 2002).
β galactosidase from Aspergillus oryzae was immobi-
lized on colloidal liquid aphrons (surfactant-stabilized
solvent droplets) via hydrophobic interactions and this
procedure of immobilization greatly favored the stability
of the free enzyme. The activity of immobilized β galactosi-
dase increased 6-fold after immobilization and there was a
shift in the pH-optimum of the enzyme. The immobilized
enzyme showed a broader range and a maximal activity at
a higher pH (Lamb and Stuckey, 2000). β galactosidase in
Kluyveromyces lactis cells adsorbed on cellulose-gelatin
carrier was significantly more stable than without immobi-
lization. Immobilization eliminates the succeeding purifica-
tion steps and also decreased the cost of the β galactosidase
catalyst in the food industry (Numanoglu and Sungur, 2004).
Adsorption of β galactosidase on surfaces such as glass
beads, nylon-6, chitosan and bone powder has been used
for lactose hydrolysis in food industries (Portaccio et  al.,
1998; Sheu et al., 1998; Caprio et al., 2000).
The adsorption of enzymes onto composites based
on covalent coating of porous rigid supports with flexible
polymers containing a very high density of ion exchange
β galactosidases and their potential applications   45
moieties has been proposed as a very suitable method for
reversible but very strong protein immobilization (Mateo
et  al., 2000). β  galactosidase adsorption onto coated
membranes depends on various factors such as pH, ionic
strength, surface and protein properties such as isoelectric
point of the protein and history of dependence of protein-
adsorption kinetics (Calonder, Tie, and Van Tassel, 2001).
Since the activity of β galactosidase may be significantly
reduced or lost during adsorption and subsequent release,
the adsorbents should be chosen such that enzyme is
bound firmly with minimum inactivation. However, the
main drawback of this procedure is that desorption of the
protein occurs resulting from changes in temperature, pH
and ionic strength. This method is nonspecific (Noinville,
Revault, and Baron, 2002).
Adsorption of proteins on ionic exchangers is one of the
simplest used techniques for protein purification and immo-
bilization. Adsorption of proteins on these matrices requires
a multipoint attachment, with several groups of the protein,
the higher the number of groups in the protein interact-
ing with the support, the higher the adsorption strength
(Pessela et al., 2004a; Staby et al., 2005). Ionic adsorption of
proteins on ion exchangers is very rapid, simple and permits
the reuse of the support after the enzyme removal (Torres
et al., 2004).
Immobilization of thermophilic β galactosidase from
Thermus sp. has been performed via ionic adsorption onto
two different supports: a new anionic exchanger resin,
based on the coating of sepabeads (an epoxide-containing
commercial polymethacrylic carrier) with polyethylenimine
polymers or DEAE-agarose. Immobilization proceeded
very rapidly in both the cases and sepabeads supports
decreased product inhibition and thus complete hydrolysis
of lactose takes place in dairy products (Pessela et al., 2003).
β Galactosidases from Escherichia coli and Thermus sp. were
purified and reversibly immobilized on anionic exchangers
by controlling the support and the immobilization condi-
tions. Adsorption on very highly activated supports (e.g.,
containing 40 μmol of ionic groups per wet gram of 4 BCL
agarose) promotes a significant thermal stabilization of β
galactosidases obtained from both bacterial sources (Pessela
et al., 2006).
The adsorption of large proteins on highly activated sup-
ports involved multi interactions and yields a very strong
adsorption. β galactosidase from Thermus sp. T2 hardly
may be desorbed from highly activated immobilized metal
ion affinity chromatography (IMAC) supports may involve
many multi-interactions yielding a very strong adsorption,
in fact, multimeric α and β galactosidases from Thermus
sp. T2 hardly may be desorbed from highly activated IMAC
supports, even in the presence of 1 M imidazole. In the pres-
ence of 50 mM of imidazole, these very large proteins can be
adsorbed on such types of supports while the medium-small
proteins hardly adsorb even on highly activated supports.
Thus β galactosidase becomes 10-fold more stable than the
native enzyme. The involvement of large areas of these mul-
timeric enzymes in the adsorption process may promote a
 46   Q. Husain
multisubunit adsorption with stabilizing effects (Pessela
et al., 2007).
In a recent study, novel magnetic beads were prepared
from glycidylmethacrylate (GMA) and methylmethacrylate
(MMA) via suspension polymerization in the presence of
a cross-linker (i.e., ethylenedimethylmethacrylate). The
magnetic poly(GMA–MMA) beads were characterized with
a scanning electron microscope, FT-IR and ESR spectropho-
tometers. β galactosidase immobilized onto magnetic poly
(GMA-MMA) beads was used for the hydrolysis of lactose
in a bed reactor. The enzyme reactor operated continuously
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
at 35°C for 60 h and the immobilized enzyme lost nearly
12% of its original activity after this period (Bayramoglu
et al., 2007).
Covalent attachment
Covalent method is usually a permanent method for the
immobilization of enzymes. Covalent binding is a stable
method by the enzyme carrier bond, which prevents elution
of protein into the solution. The wide range of choices is pos-
sible by selecting carrier materials and binding method. This
allows a great deal of flexibility in designing an immobilized
enzyme with specific physical and chemical properties,
such as charge distribution, hydrophobic/hydrophilic ratio,
spacer arm separation, partition effect, etc. (Mozhaev et al.,
For personal use only.
1990). However, covalent methods are relatively expensive
and complicated. The activity yields may be low due to the
exposure of the enzyme to harsh environmental conditions.
Sometimes the active site of the enzymes may be modified
through the chemical reactions used to create covalent
bonding (Serio et al., 2003). The characteristics of the sup-
port, reactive groups and immobilization conditions need to
be carefully selected for the covalent attachment of a maxi-
mum number of enzyme groups. In fact, it is possible to cor-
relate the enzyme stabilization reached with the number of
enzyme-support linkages (Pedroche et al., 2007). Supports
containing epoxy groups seems to be useful to generate a
very intense multipoint covalent attachment with different
nucleophiles placed on the surface of enzyme molecules
(e.g., amino, thiol, hydroxyl groups) (Mateo et  al., 2007b).
The selection of suitable immobilization conditions is
important to maximize the multipoint covalent attachment.
Immobilization conditions should favor the enzyme-sup-
port reaction. Some of these critical variables are: reaction
time, pH of the used buffer, temperature, inhibitors or other
protein protectors (Mateo et al., 2002).
β galactosidase from Aspergillus oryzae was immobilized
via diazotization or condensation to nylon membranes
grafted with glycidyl methacrylate. Immobilization via dia-
zotization occurred through tyrosine residues, while immo-
bilization via condensation involved multipoint attachment
of the enzyme to the membrane through arginine residues.
It was found that the immobilization via condensation
strengthens the enzyme structure in contrast to the immobi-
lization by diazotization (El-Masry et al., 2001a). The immo-
bilization of β galactosidase from Kluyveromyces lactis on
graphite surface has received much attention as graphite has
been widely used as an enzyme carrier for covalent attach-
ment which involves Schiff’s base formation between the
active groups of graphite and the enzyme molecules. This
immobilized enzyme preparation was used for the hydroly-
sis of lactose (5%, w/v). The degree of lactose hydrolysis was
about 70% at 37°C over a period of 3 h. The immobilized β
galactosidase exhibited a good storage and operational sta-
bility (Zhou and Chen, 2001b).
β galactosidase from Aspergillus oryzae was covalently
bound to cotton cloth activated by tosyl chloride. This
immobilized β galactosidase preparation retained 85% of
the activity and had a half-life of 50 days at 50°C (Albayrak
and Yang, 2002). Immobilization of β galactosidase from
Kluyveromyces lactis has been performed by covalently
binding it on thiopropylagarose (Ovsejevi et  al., 2004).
Immobilization of LactozymTM, which is a commercially
available preparation of β galactosidase from Kluyveromyces
fragilis was immobilized on cellulose beads by covalent
attachment via epichlorohydrin coupling chemistry. The
immobilized preparation could be reused thrice and it can
hydrolyze milk lactose upto 60% within 5 h. Thus it was use-
ful for the hydrolysis of lactose in whey and in the produc-
tion of low lactose milk (Roy and Gupta, 2003). The kinetic
behaviour of β galactosidase from Kluyveromyces marxianus
(Saccharomyces lactis), covalently immobilized on different
oxides supports, such as alumina, silica, and silicated alu-
mina has been studied. The activity of immobilized enzyme
was strongly dependant on the chemical nature and physical
structure of the supports. As the particle size of the supports
was increased, the enzymatic activity decreased strongly
(Serio et al., 2003).
Talaromyces thermophilus CBS β-galactosidase was
immobilized by covalent attachment onto the insoluble
carrier Eupergit C with a high binding efficiency of 95%.
Immobilization increased both activity and stability at
higher pHs and temperatures when compared to the free
enzyme. The half-life of the β-galactosidase activity at 50°C
was determined to be 8 and 27 h for the free and immobi-
lized enzymes, respectively. The maximum yield of galac-
tooligosaccharides (GalOS) of 34% was obtained when the
degree of lactose conversion was roughly 80%. Hence, this
immobilized enzyme can be useful both for the cleavage of
lactose at higher temperatures and the formation of GalOS,
prebiotic sugars that have a number of interesting properties
for food applications (Nakkharat and Haltrich, 2006).
Goddard, Talbert, and Hotchkiss (2007) have reported
covalent attachment of β galactosidase to the surface-
functionalized low-density polyethylene films. A two-step
wet chemical functionalization introduced 15.7 nmoL/cm2
primary amines to the films surface. Glutaraldehyde was
employed to covalently attach β galactosidase to the surface
at a density of 6.0 μg protein per cm2 via reductive amina-
tion. The bond between the covalently attached enzyme and
the functionalized polyethylene withstood heat treatment
in the presence of an ionic denaturant with 74% enzyme
retention, thus it suggested that enzyme would not migrate
into the food product. β galactosidase from Kluyveromyces

lactis was covalently immobilized on cotton fabric, which
used glutaraldehyde as a crosslinking reagent. The cotton
fiber has been used as a carrier for the immobilization of β
galactosidase, due to its low cost and large surface area per
unit mass. This procedure does not involve complex opera-
tion and expensive equipment. Thus a pilot scale model was
set up to hydrolyze lactose at an industrial level (Zhou et al.,
2003; Li et  al., 2007). β galactosidase from Kluyveromyces
lactis was covalently attached to a polysiloxane-polyvinyl
alcohol magnetic (mPOS-PVA) composite, using glutar-
aldehyde as an activating agent. The mPOS–PVA bound
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
β galactosidase preparation showed higher operational
and thermal stability as compared to the free enzyme. The
immobilized β galactosidase was more effective in hydro-
lyzing lactose from milk than the soluble enzyme (Neri
et al., 2008).
Elnashar and Yassin (2008) investigated the hydrolysis of
lactose by covalently immobilized β galactosidase on ther-
mally stable carrageenan coated with chitosan (hydrogel).
The modification of carrageenan improved the gel’s thermal
stability in solutions from 35°C to 95°C. The hydrogel was
effective for the immobilization β galactosidase where 11 U/g
wet gel was immobilized with 50% enzyme loading capacity.
Immobilized β galactosidase showed a minor shift in their
optimum pH from 4.5–5 to 5–5.5 and temperature from 50°C
For personal use only.
to 45–55°C, which exhibited higher catalytic activity and sta-
bility at broad spectrum pH and temperature ranges as com-
pared to soluble enzyme. The free and immobilized enzyme
preparations exhibited lactose conversion of 87% and 70%
at 7 h, respectively. The operational stability showed 97%
retention of the enzyme activity after 15 repeated uses, which
demonstrated that the covalently immobilized enzyme was
not leaching from the surface of the gel.
Immobilization carriers; macro-, micro-, and nanosized
chitosan particles were prepared by precipitation, emul-
sion cross-linking and ionic gelation methods, respectively.
Effects of particle preparation parameters on particle size
were investigated. The activity of β galactosidase covalently
immobilized to different size particles were compared with
each other and free enzyme. The enzyme immobilized on
nanoparticles, by means of an ionotropic gelation method
by sodium sulphate as gelation agent, exhibited high-
est activity. β galactosidase immobilized on macro- and
microspheres exhibited remarkably high storage stability
in aqueous solution, with a minor loss of 5% activity after
3 weeks storage at 4°C and pH 7.0 (Biró et al., 2008). Zhang
et al. (2009) prepared magnetic beads from artemisia seed
gum, chitosan, and magnetic fluid in the presence of glu-
taraldehyde. The reactive aldehyde groups of the magnetic
beads were allowed to react with the amino groups of the
enzymes. The activated magnetic beads were used for the
covalent immobilization of β galactosidase. β galactosidase
immobilized on the magnetic beads resulted in an increase
in enzyme stability. Optimum operational temperature for
immobilized enzyme was increased to 10°C than that of the
free enzyme.
β galactosidases and their potential applications   47
It has been described that β galactosidase, PsβGAL (from
Pisum sativum) was found extremely unstable with loss of
over 80% enzyme activity within 24 h at 4°C when the protein
concentration was lower than 0.1 mg/mL. Ps βGAL (21.9 μg)
was immobilized by 62.56% onto activated 100 mg of
amberlite MB-150 beads using 4% glutaraldehyde in 50 mM
sodium phosphate buffer, pH 6.0. Enzyme immobilization
onto amberlite MB-150 beads had significantly stabilized
the enzyme structure because it retained 100% activity over a
period of 12 months at 27°C. Amberlite−PsβGAL hydrolyzed
64.57% and 69.18% lactose from milk and milk whey within
10 h at 27°C, respectively. Immobilized enzyme was repeat-
edly used for over 10 uses without any loss in activity. Due to
easy accessibility of enzyme source, ease of its immobiliza-
tion on amberlite, lower cost of amberlite, enhanced stabil-
ity of amberlite−PsBGAL and comparable lactose hydrolysis
in milk and milk whey, this immobilized preparation can
be exploited at an industrial scale (Dwevedi and Kayastha,
2009b).
Chemical aggregation
Crosslinking is based on the formation of covalent bonds
between enzyme molecules, by means of bi- or multifunc-
tional reagents, leading to three dimensional crosslinked
aggregates. Due to crosslinking very little desorption occurs
as the enzyme is strongly bound. One additional advan-
tage of crosslinked enzyme preparations is that there is no
requirement for support for the immobilization of enzymes.
Crosslinking is best used in conjunction with one of the
other methods. It is used mostly as a means of stabilizing
adsorbed enzymes and also for preventing leakage (Lopez-
Gallego et al., 2005a). However, crosslinking may cause sig-
nificant changes in the active site of enzymes and also severe
diffusion limitations may lead to significant loss of activity.
Loss of enzyme activity also occurs during crosslinking of
enzyme by using bifunctional or multifunctional reagents.
The most commonly used reagent for crosslinking include
glutaraldehyde. Other reagents used for crosslinking are
bis-diazobenzidine 2,2,-disulphonic acid, diazobenzidine,
N,N-hexamethylene bis-isodiacetamide, hexamethylene
diisocyanate (Lopez-Gallego et al., 2005b).
Entrapment
Entrapment of enzymes is defined as the trapping of
enzymes into the lattices of a semipermeable gel or in a
polymeric network of matrices. Entrapment in natural gels
is one of the commonly used methods for biocatalyst immo-
bilization (Desai, Dave, and Devi, 2004; Won et al., 2005; Wu
et al., 2007).
Calcium alginate-mediated entrapment has attracted
much attention for the immobilization of enzymes. Alginate
is one of the frequently used polymers for the immobiliza-
tion and entrapment of β galactosidases. An alginate-chi-
tosan hybrid gel was prepared to enhance the stability of
alginate beads and to avoid the leakage of entrapped mate-
rial (Gåserød, Sannes, and Skjåk-Braek, 1999; Won et  al.,
2005). Alginate as an immobilization matrix is also used
 48   Q. Husain
in combination with gelatin to entrap Aspergillus oryzae
β galactosidase. The entrapped enzyme showed good opera-
tional stability (Tanriseven and Dogan, 2002).
Some workers have reported that pectin beads were
significantly more stable than alginate beads and therefore
these workers have prepared hybrid beads of alginate and
pectin (Matto and Husain, 2006; Satar, Matto, and Husain,
2008). In order to minimize the cost of the immobiliza-
tion, our group has developed another hybrid alginate and
starch gel for the entrapment of peroxidase and this matrix
has proved its potential for enzyme immobilization (Matto
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
and Husain, 2009). Mammarella and Rubiolo (2005) dem-
onstrated the entrapment of Kluyveromyces fragilis β galac-
tosidase in alginate-carrageenan gels beads. The presence
of carrageenan had a favorable influence on the enzyme
catalyzed reaction because the gel is formed within K+ ions,
which increased the activity of the enzyme.
Hydrogels are a class of three-dimensional, highly
hydrated polymeric networks whose macromolecular prop-
erties are similar to the natural extracellular matrix (Peppas
et al., 2000; Montembault, Viton, and Domard, 2005). One
of the recently used polymeric hydrogels, polyvinyl alco-
hol (PVA) has offered several advantages as compared to
other known matrices. The advantages are low toxicity,
mechanical and good long-term stability, low biodegrad-
For personal use only.
ability and no side effects on enzyme catalyzed reactions
(Parascandola, Branduardi, and De Alteriis, 2006; Rebros
et al., 2006). Synthetic polymeric PVA gel was used for the
entrapment of beta galactosidase and has shown its poten-
tial including mild conditions of preparation stability, bio-
compatibility, structural strength and diffusive properties
(Rossi et al., 1999). β galactosidase from Aspergillus oryzae
was entrapped in lens-shaped PVA hydrogel capsules, called
LentiKats (diameter 3–4 mm, thickness 200–400 μm) for the
hydrolysis of lactose (Grosova et al., 2008b).
The nanocomposites of silica gel were prepared by the
sol-gel technique in the aqueous solutions which has drawn
attention to entrap enzymes without their covalent bond-
ing to the matrix. The porous silica matrix provides enzyme
accessibility to external reagents and removal of the reaction
products through pore diffusion (Shchipunov et al., 2004).
Entrapment of enzymes in polymeric membranes is one
of the most advantageous methods because it is rapid and
simple. It retained very high enzyme activity and requires
no chemical reaction which leads to the inactivation of the
enzyme (Duran et al., 2002). The major limitation is that the
entrapped enzyme is prohibited from interaction with high
molecular weight (Mr) substrates that are unable to diffuse
into the matrix. Consequently, only reactions involving
relatively small reactants and products may be carried out
successfully (Tischer and Wedekind, 1999; Kierstan and
Bucke, 2000; Liang, Li, and Yang, 2000). There are several
reports about the leakage of enzymes from porous poly-
meric matrices, calcium alginate gel, poly(vinyl) hydrogel
and sol gel (Blandino, Macías, and Cantero, 2000; Veronese
et al., 2001; Musthapa et al., 2004). In order to prevent the
leaching of enzymes from entrapped gels, a number of
attempts have been made to increase the molecular dimen-
sions of the enzymes prior to their final entrapment such as
the crosslinking of the enzymes by using bi- or multifunc-
tional reagents, preparation of insoluble lectin or antibody
enzyme complexes or the adsorption of enzyme on the
fine particles (Musthapa et al., 2004; Betancor et al., 2005;
Satar, Matto, and Husain, 2008; Matto and Husain, 2009).
There should be sufficient cavities inside the gel to accom-
modate the trapped species, connected to channels which
allow substrates and product diffusion (Wilson et al., 2004;
Betancor et al., 2005).
Microencapsulation
Immobilization of biomolecules within mineral hosts such
as silica gel has become a challenging approach to design
new materials with biotechnological applications. The
microcapsule type involves an entrapment within a semi-
permeable membrane. The preparation of enzyme micro-
capsules requires extremely well controlled conditions.
The biopolymer should provide a suitable environment for
the microencapsulation of enzymes (Uludag, De Vos, and
Tresco, 2000; Coradin and Livage 2003).
The liposome based microencapsulation within an
amphiphatic liquid-surfactant membrane prepared from
liquid, usually phospholipids has attracted attention. In
microencapsulation, lipid vesicles are carriers for the
enzymes, protecting the enzymes from getting in direct con-
tact with the substrate. The choice of an encapsulation helps
in the efficiency and preservation of catalytic activity (Walde
and Ichikawa, 2001; Monnard, 2003). In such a lipid vesicle
assisted lactose hydrolysis process, the entrapped enzyme is
added to milk and is assumed to be released into the intes-
tine by the presence of bile salts, allowing the degradation of
lactose (Kim et al., 1999).
Liposomes are composed of only amphiphilic layers of
lipids which are usually not rigid enough for the microen-
capsulation of enzymes, therefore cholesterol layers are also
incorporated into lipid bilayers to change liposome prop-
erties. Cholesterol layers improve retention of enzymes in
liposomes by decreasing the bilayer permeability but reduce
the concentration of enzymes that can be incorporated by
reducing polypeptide affinity. The entrapment of β galac-
tosidase in liposomes may be useful in order to overcome
the shortcoming of the taste of hydrolyzed-lactose milk and
protecting the biocatalysts from inactivation by proteolytic
enzymes (Betageri and Kulkarni, 1999; Laridi et al., 2003).
β galactosidase immobilized in liposomes prepared by
the dehydration–rehydration method showed high resist-
ance to proteolysis, retaining about 93% and 75% of initial
activity after 6 and 24 h exposure to protease. Moreover,
liposome microencapsulated β galactosidase exhibited
a remarkable increase in thermal stability (Rodriguez-
Nogales and Delgadillo, 2005; Rodriguez-Nogales, Garcia,
and Marina, 2006). β galactosidase has been encapsulated
within reversed micelles, which were formed by mixing the
surfactant with an organic solvent, for example, aerosol OT/
isooctane reverse micelles and in the membrane type, the

enzyme was separated from the reaction solution by an
ultrafiltration membrane, a micro-filtration membrane or a
hollow fibre (Chen and Ou-Yang, 2004).
A novel alginate chitosan core-shell microcapsule tech-
nology has been developed for the microencapsulation of β
galactosidase. The enzyme was immobilized in either a cal-
cium alginate or barium alginate core surrounded by a perm-
selective chitosan shell. The microcapsule core was formed
by crosslinking sodium alginate either by calcium or barium
ions. The crosslinked alginate core was uniformly coated by
a chitosan layer and crosslinked with sodium tripolyphos-
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
phate. However, the phosphate ions of sodium tripolyphos-
phate penetrate the calcium alginate and released calcium
ions from alginate and liquefy the core. Thus, β galactosidase
was immobilized either in a liquid core (Ca2+ alginate) or
solid core (Ba2+ alginate) microcapsules. The enzyme was
immobilized and protected in the inner biocompatible algi-
nate core while the outer chitosan shell dictated the trans-
port properties. Ca2+ ions used as crosslinking agent for the
alginate microcapsules, produced 60% loading efficiency,
while Ba2+ ions crosslinked alginate microcapsules showed
100% loading efficiency (Taqieddin and Amiji, 2004).
Hydrogels have been used as matrices for the encapsula-
tion of living cells and for the controlled release of pharma-
ceutically active proteins. For these applications, it is often
For personal use only.
required that the gels degrade under physiological condi-
tions. Many crosslinking methods have been developed
and are presently available for the preparation of hydrogels.
However, the crosslinking agents used are often toxic com-
pounds, which have to be extracted from the gels before
they can be applied. Moreover, crosslinking agents can give
unwanted reactions with the bioactive substances present
in the hydrogel matrix (Stenekes et al., 2000; Hennink and
Nostrum, 2002).
Bioaffinity immobilization
The basis of the bioaffinity technique is the biospecific inter-
action between two affinity groups. This technique is not
only an efficient method for the determination, isolation and
applications of antibodies, antigens, cells, enzymes, proteins,
hormones etc. but it is also useful for the study of supramo-
lecular structures in relation to their microenvironment. The
advantages of oriented immobilization of biologically active
proteins are good steric accessibility of active binding sites
and increased stability. Oriented immobilization of various
affinity ligands on the porous or nonporous solid supports,
can provide us with efficient biospecific adsorption of bio-
molecules suitable for a variety of applications. Bioaffinity
immobilized biomolecules can be studied as a model for
the study of chemical processes in the living cells (Turkova,
1999; Khan, Akhtar, and Husain, 2005).
Bioaffinity based methods have several advantages over
the other known methods used for the immobilization of
enzymes due to their reversibility, lack of chemical modi-
fication and usually an accompanying an enhancement in
stability. This method is also useful for the immobilization
of enzymes directly from the crude homogenate, and thus
β galactosidases and their potential applications   49
reducing the high cost of enzyme purification (Akhtar et al.,
2005; Kulshrestha Husain, 2006b). Thus, higher enzyme
activity is preserved since no modification/distortion of the
enzyme active site occurs. Also, as the active site is blocked
less by the matrix, the steric accessibility allows more free
access for the incoming substrate and outgoing products
(Dalal and Gupta, 2007).
Fraguas, Bolon, and Viera (1999) reported the bioaffinity
adsorption of Aspergillus oryzae β galactosidase on concana-
valin A (Con A)-Sepharose. The low cost of Con A-Sepharose
and the fact that the carrier can be reused makes it a more
convenient, less expensive and a more practical solid phase
than other supports. This preparation was successfully
hydrolyzed about 60% lactose in a batch process after 5 h.
Bioaffinity based adsorption of enzymes is simple, reversible
and retains very high enzyme activity for the immobilized
protein The support can be reused after its regeneration by
removing the inactivated enzyme.
Haider and Husain (2007b) prepared insoluble Con
A-β galactosidase complex by using jack bean extract and
Aspergillus oryzae enzyme and the complex was crosslinked
by glutaraldehyde. Soluble enzyme, Con A-β galactosidase
complex and crosslinked Con A-β galactosidase complex
preparations were entrapped into calcium alginate beads
which provided suitability to use these preparations in
the continuous reactors. Entrapped crosslinked Con A-β
galactosidase complex preparation was superior in stability
against various physical and chemical denaturants as com-
pared to the other entrapped preparations. The entrapped
crosslinked Con A-β galactosidase retained 95% activity
after its 7th repeated use and showed 93% activity over two
months storage at 4°C. A novel therapeutic agent, in the
form of β galactosidase, by immobilizing this enzyme on the
surface of Con A layered calcium alginate-starch beads, was
developed. Immobilized β galactosidase showed remarkably
high stability against conditions of the digestive system such
as pH, salivary amylase, pepsin and trypsin. Immobilized β
galactosidase exhibited significant broadening in pH and
temperature activity profiles as compared to free enzyme.
This immobilized β galactosidase retained 65% activity even
after its sixth repeated use. This immobilized β galactosidase
preparation was superior in stability to denaturation induced
by some chemical and physical agents and in hydrolyzing
lactose from whey and milk in a batch process and it hydro-
lyzed 89% and 79% lactose from whey in 3 h and milk in 4 h,
respectively. Immobilized β galactosidase retained 61% of its
original activity after 2 months storage at 4°C while the free
enzyme retained only 37% activity under similar incubation
conditions (Haider and Husain, 2008; Haider and Husain,
2009b).
Polyclonal antibody bound cellulose was employed
for the immobilization of β galactosidase from Aspergillus
oryzae. Immobilized β galactosidase had a broad-spectrum
pH optima between pH 4.0 to pH 6.0 while the free enzyme
exhibited an activity peak at pH 4.6. IgG-cellulose bound
β galactosidase showed a shifting in temperature optima
from 50°C to 60°C. Immunoaffinity immobilized enzyme
 50   Q. Husain
was significantly more stable against the inactivation medi-
ated by pH, heat and product inhibition as compared to
free enzyme. IgG-cellulose adsorbed β galactosidase and
exhibited 80% activity even after two months storage at
4°C whereas the free enzyme showed only 35% of the ini-
tial activity. Immunoaffinity bound β galactosidase had
shown 46% activity after its 10th repeated use (Haider and
Husain, 2009c).
Immobilized metal affinity chromatography (IMAC) is
a separation technique that uses covalently bound chelat-
ing compounds on solid chromatographic supports to bind
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
metal ions, which serve as affinity ligands for various pro-
teins, making use of coordinative binding of some amino
acid residues exposed on the surface of the protein. Although
compared to other affinity separation technologies it cannot
be classified as highly specific, several amino acids especially
histidine, lysine, cysteine, proline, arginine and methionine
are susceptible to metal-catalyzed oxidation reactions that
produce highly reactive radical intermediates which can
damage a variety of proteins (Porekar and Menart, 2001).
A strategy to selectively adsorb large proteins on IMAC
supports has been reported for purifying thermophilic β
galactosidase containing a polyhistidine tag overexpressed
in Escherichia coli by using a single chromatographic step.
New immobilized IMAC matrices containing a high con-
For personal use only.
centration of metal-chelate moieties, completely coated
with inert flexible and hydrophilic dextrans are reported to
improve the purification of polyhistidine tagged proteins
(Pessela et  al., 2004b). The purification of a recombinant
multimeric enzyme, β galactosidase from Thermus sp. strain
T2 has been employed to investigate the performance of
such types of chromatographic media. IMAC supports with
a high concentration of metal chelate groups promoted a
rapid adsorption of poly-His tagged proteins during IMAC.
These supports also favored the promotion of undesirable
multipunctual adsorptions and problems may arise for the
simple and effective purification of poly-His tagged proteins:
(a) more than 30% of the natural proteins contained in crude
extracts from E. coli get adsorbed, beside target recom-
binant protein, on these IMAC supports via multipoint weak
adsorptions (b) the multimeric poly-His tagged enzyme
may become adsorbed via several poly-His tags belonging
to different subunits. Thus, the desorption of pure enzyme
from the support may become quite difficult, for example, it
is not fully desorbed from the support even in the presence
of 200 mM of imidazole. The coating of these IMAC supports
with dextrans significantly minimized undesired multipoint
adsorptions. The dextran coating of chromatographic matri-
ces appeared to allow the formation of strong one-point
adsorptions that involved small areas of protein and support
surface (Mateo et al., 2001).
Thus, affinity binding offers very mild, controlled adsorp-
tion of biocatalysts onto the supports and is likely to be of
continuing value for the immobilization of sensitive biocata-
lysts. It is also gaining increasing acceptance in construction
of sensitive enzyme based devices, separation techniques as
well as for other applications. The oriented immobilization
of antibodies on solid-phase materials has been used as an
affinity matrix in many areas such as purification, diagnos-
tic immunoassays and immunosensors (Lu, Smyth, and
O’Kennedy, 1996; Nisnevitch and Firer, 2001).
Applications of immobilized β galactosidases
Enzymes are biological catalysts that serve different functions
in the body and have attracted a wide range of interest from
fundamental academic research to many different indus-
trial applications (Pierre, 2004; Hartmann, 2005; Kallenberg,
Rantwijk, and Sheldon, 2005). The immobilized enzyme
retains the basic biochemical activity of the free catalyst
while taking on the physical characteristics of the support.
Applications of free enzyme were limited in industrial proc-
esses as regard to the problem of instability and rapid loss
of catalytic activity during the operation and storage periods
which may be due to an autolysis effect/protein unfolding
or aggregation. The removal of free enzyme from the reac-
tion mixture was also difficult and caused contamination of
product (Szczodrak, 2000; Xi et al., 2005).
Immobilized enzymes have been mostly used in the
production of food, pharmaceuticals and other biologically
important fine products (Bayramoglu et  al., 2004; 2007).
Enzyme reuse provides a number of cost advantages that
are often an essential prerequisite for various applications in
industries (Tischer and Kasche, 1999; Norouzian, 2003).
Industrial applications of β galactosidases
Enzymatic hydrolysis of lactose either from milk/whey or
pure lactose has been accomplished in various reactors of
different configurations, under varying operating condi-
tions of temperature and pH (Table 1). Since the cost of the
enzyme is the most important factor that determines proc-
ess economy, only continuous systems that involve the reuse
of a single batch of enzyme can be considered (Albayrak
and Yang, 2002). Technically feasible processes include the
direct addition of soluble enzyme with subsequent recy-
cling of the soluble enzyme by membrane processes; or
use of immobilized enzymes. An inexpensive batch process
using in-house production of crude enzyme preparations
could make manufacturing of lactose hydrolyzed products
feasible even for smaller dairy plants (Vasiljevic and Jelen,
2001; Haider and Husain, 2007a). But immobilization of β
galactosidase offers numerous advantages over free form
in choice of batch or continuous process, rapid termination
of reactions, controlled product formation, ease of enzyme
removal from the reaction mixture and adaptability to vari-
ous engineering designs (Matioli, Mores, and Zanin, 2003; Li
et al., 2007; Haider and Husain, 2009a).
Reactors containing immobilized β galactosidase have
been extensively studied because this is the critical point in
the industrial production of lactose free milk and whey. The
enzyme reactors mainly used in lactose hydrolysis include
enzyme-membrane systems, fluidized-bed reactors (FBRs),
hollow-fiber reactors (HFRs), packed-bed reactors (PBRs),
and stirred-tank reactors, STRs (Ganesh, Joshi, and Sawant,
 β galactosidases and their potential applications   51

Table 1.  Hydrolysis of lactose under different operating conditions by immobilized β galactosidase.
Source of β Support used for Source of
galactosidase immobilization lactose Operating conditions Percent conversion Reference
Recombinant Chitosan using Milk 2 h in a reactor 80% Chen et al., 2009
β-galactosidase Tris(hydroxymethyl)
from Bacillus phosphine (THP) &
stearothermophilus glutaralde-hyde
Aspergillus niger Concanavalin A layered Whey pH 4.8 at 37°C in 3 h 89% Haider and Husain,
calcium alginate-starch Milk pH 6.6 at 37°C in 4 h 79% 2009b
hybrid beads
Kluyveromyces lactis Polysiloxane-polyvinyl Skimmed Milk pH 6.5, 25°C, at the end 90% hydrolysis of lactose. Neri et al., 2008.
alcohol magnetic of 120 min, batch process, Enzyme conc. 10 mg/mL
stirred conditions.
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10

Escherichia coli Magnetic poly (GMA- Pure lactose pH 7.0, 35°C hydrolysis of Enzyme conc. 3150 U. Flow Bayramoglu et al.,
MMA) 200 mM lactose in bed reactor. rate of 20 mL/h for 60 h, 2007.
88% hydrolysis of lactose
Aspergillus oryzae Lens-shaped polyvinyl Lactose solu- pH 4.6 at 45°C 35 100% Grosova et al., 2008b
alcohol capsules tion (10%, w/v) repeated batch runs
and 530 h of ­continuous
hydrolysis of lactose.
Kluyveromyces fragilis Cellulose beads via Milk and whey pH 6.6, 30°C, at the end Whey lactose: 94% in FBM, Roy and Gupta, 2003.
epichlorohydrin of 30 min in CBM and 80% in CBM. Milk: 7% in
coupling FBM. FBM
Thermophilic Sepa beads Novo buffer pH 6.5, 50°C, at the end Lactose hydrolysis 99% Pessela et al., 2003.
(Thermus sp. T2) (50 gL−1) of 30 min in batch reactor
Kluyveromyces fragilis Silica-alumina Milk buffer pH 7.0, 40°C, 300 rpm, 37% and 17% at enzyme Ladero, Santos, and
(50 gL−1) at the end of 30 min in conc. of 9.45 and 4.75 mg/ García-Ochoa, 2000.
batch reactor. mL
For personal use only.

Aspergillus oryzae Cross-linked poly(vinyl Lactose and at 50°C and pH 5.0 in 95% Rejikumar and
alcohol) or natural milk whey ­citrate-phosphate buffer Surekha, 2001
polysaccharide chitosan
Kluyveromyces fragilis Silanized porous Whey permeate 50°C at pH 6.0 (86–90%) both in batch Szczodrak, 2000
glass modified by process and in a recycling
glutaraldehyde packed-bed bioreactor

2000; Bodalo et al., 2001; Albayrak and Yang, 2002; Carrara,
Mammarella, and Rubiolo 2003; Ozdural et al., 2003; Jurado
et al., 2005; Novalin, Neuhaus, and Kulbe, 2005). Presently
in commercial applications lactose hydrolysis is commonly
carried out in STRs using free and immobilized β galactosi-
dase with a batch operation mode (Li et al., 2007; Haider and
Husain, 2009b). Batch hydrolysis of lactose in milk and whey
is the traditional procedure and is simple and easy to con-
trol. However, there are several disadvantages with respect
to batch hydrolysis mainly related to high enzyme and labor
costs (Rios et al., 2004). β Galactosidase, immobilized onto
highly activated supports, has allowed the continuous reuse
of the enzyme in order to improve its global yield as com-
pared to a batch procedure. Since there was loss of enzyme
activity and constraints for diffusion into the support, the
use of this technique is limited (Lamas et  al., 2001; Sousa
et  al., 2004; Tardioli et  al., 2005). The biocatalyst prepara-
tion and the bioreactor configuration offer some advantages
compared to the conventional methods because they allow
a higher concentration of biomass to be immobilized and a
larger surface area per unit mass. Such a system was shown
to be useful for the hydrolysis of whey/milk lactose to give
a more fermentable substrate (Genari, Passos, and Passos,
2003).
In a membrane bioreactor (MBR), the biocatalyst is
confined in a well-defined region of space by means of
a selective membrane, or immobilized by adsorption or
entrapment within the membrane itself (Diano et al., 2004;
Curcio, Calabro, and Iorio, 2006). MBR for lactose or whey
hydrolysis, practically involve the use of a thermostatic stir-
ring vessel that provides the time for hydrolysis, the content
of which is continuously circulated towards an ultrafiltra-
tion module. The use of MBR as continuous systems for the
hydrolysis of lactose (whole milk/cheese whey) is an effec-
tive technique. Both lactose conversion and recovery of high
Mr proteins can be accomplished in a single step (Tragardh,
1991; Giorno and Drioli, 2000). The main advantage of this
configuration is the possibility to work with free enzyme in
homogeneous solutions with its substrate, thus fully exploit-
ing its native kinetic properties. The major disadvantages of
this configuration are the increased risk of microbial con-
tamination, especially during prolonged operation times
at ambient temperatures and the clogging of ultra-filtration
membranes with milk proteins. These drawbacks can at least
partially be eliminated by operating the system at relatively
high temperatures and by using deproteinated substrates
such as whey permeate (Hatzinikolaou et al., 2005).
Continuous reaction and simultaneous separation of
products from the reaction mixture can be achieved with a
continuous membrane recycle reactor (CMRR) (Prata-Vidal
et al., 2001; Guadix, Camacho, and Guadix, 2006). In spite
of these important advantages, a number of drawbacks of
 52   Q. Husain
CMRR must be pointed out such as permeate flux decline
due to membrane fouling and the frequent purges required
to eliminate nonreacting substrates (Giorno and Drioli,
2000; Rios et  al., 2004). The production of reduced-anti-
genicity whey protein hydrolysates in a cyclic batch mem-
brane reactor (CBMR) by subtilisin at 60°C and pH 8.5 has
been described here. The operation of CBMR comprises
three steps, namely, hydrolysis, ultrafiltration and enzyme
recycling, which were repeated a number of times. The
antigenicity was reduced to 1000 times. Polyethersulphone
membrane (8000 Da) was used in order to retain the
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
enzyme and allow the permeation of the product, peptides.
A theoretical model was developed, which was validated
by experiments. The optimization of the CBMR was per-
formed, taking the total amount of enzyme as the objective
function. The optimum number of enzyme reuses were cal-
culated and resulted to be up to 5. Compared to the tradi-
tional single batch operation mode, a save of enzyme up to
59% can be achieved. In order to profit from the advantages
of both batch and membrane recycle reactor, a membrane
reactor operating in a cyclic batch mode for the production
of a reduced-antigenicity whey protein hydrolyzates with
subtilisin at 60°C and pH 8.5 has been demonstrated (Prieto
et al., 2007).
A Fluidized-bed reactor (FBR) was used to hydrolyze lac-
For personal use only.
tose present in milk whey and whole milk by β galactosidase
immobilized on cellulose beads (Roy and Gupta, 2003). But
lactose conversion could not proceed beyond the point of
60% in whole milk, due to presence of fat that impairs the
performance of FBR. FBRs are suitable when substrates are
viscous or particulate as these reactors produce less pressure
loss and a more uniform distribution of the flow through the
axial section of the reactor. Furthermore, mechanical stirring
is not necessary and the support is not damaged by impact
with the impeller (Bodalo et al., 1995).
The use of hollow-fibre (HF) modules as a catalytic biore-
actor is growing due to the advantages that these HF reactors
present, such as the high membrane surface to volume ratio
and the high density of catalyst that these reactor systems
allow in a small reactor volume (Calabro,Curcio, and Iorio,
2002). For the reuse of the β galactosidase in the HF bioreac-
tor, the enzyme is usually immobilized by physical or chemi-
cal binding to the membrane or by physical retention in the
lumen or in the extra capillary space (Salzman et al., 1999;
Knezevic et al., 2004; Jurado et al., 2006).
Packed-bed reactors (PBRs) have been widely used for the
hydrolysis of lactose by immobilized β galactosidase. Cotton
fibre was applied as the carrier that was unexpensive and
has a large surface area per unit mass; the immobilization
procedure does not involve a complex operation or expen-
sive equipment. Thus this method could be easily employed
at an industrial scale for large scale hydrolysis of lactose
(Zhou et  al., 2003). PBRs have the advantage of a higher
average reaction rate compared to the continuous stirred
tank reactors because (i) the inhibition effect decreases due
to the small difference between substrate and product con-
centrations in the reactor and (ii) the enzyme loss is reduced
due to the absence of collisions between biocatalyst parti-
cles and impeller and liquid shearing. β galactosidase from
Aspergillus oryzae was immobilized onto either ­cross-linked
PVC or natural polysaccharide chitosan and these immo-
bilized enzyme preparations were employed for the con-
tinuous hydrolysis of lactose and milk whey in a fixed-bed
reactor. Several experimental conditions such as reactor
temperature, substrate concentration, pH and flow rates
were optimized for the effective operation of the reactor. The
optimum pH and temperature for the immobilized enzyme
in the reactor was observed as pH 5.0 and 50–60°C, respec-
tively. Almost 95% lactose was hydrolyzed under optimal
conditions. The immobilized enzyme systems were seen to
retain their activity even after 12–20 cycles (Rejikumar and
Surekha, 2001).
A mathematical model for a number of working con-
ditions was numerically solved to investigate a fixed bed
reactor with a biocatalyst. The effect of some parameters,
such as bed swelling, mass transfer limitations, axial disper-
sion, residence times and inlet lactose concentration was
investigated. Michaelis–Menten kinetics with competitive
product inhibition was examined in order to demonstrate
lactose hydrolysis by β galactosidase. Lactose conversion
was used to determine the appropriate conditions for the
immobilized enzyme reactor. The model predicted the
experimental errors as less than 2.5% (Mammarella and
Rubiolo, 2006). Immobilization of β galactosidase from
Bacillus circulans offered several advantages compared to
the free enzyme such as improvement of enzyme stability
and the continuous operation in a PBR (Hernaiz and Crout,
2000). Al-Mufta and Abu-Reesh (2005) have examined the
effect of internal mass transfer and product inhibition on
a simulated immobilized β galactosidase catalyzed reactor
for the hydrolysis of lactose. A general mathematical model
was developed to predict the performance and simulation
of a packed-bed immobilized enzyme reactor catalyzing
lactose hydrolysis, which follows Michaelis–Menten kinet-
ics with competitive galactose inhibition. The perform-
ance of a packed-bed immobilized enzyme reactor was
evaluated by considering the effects of various diffusional
phenomena such as axial dispersion, internal and external
mass transfer limitations. It was demonstrated that these
effects reduced the internal effectiveness factor. In a recent
study, β galactosidase from Kluyveromyces lactis has been
coupled by using glutaraldehyde as the cross-linking rea-
gent. A pilot-scale module with a 10-L packed-bed reactor
was set up to hydrolyze lactose from whole milk. The extent
of the lactose hydrolysis and the productivity of the reactor
changed with the residence time in the continuous opera-
tion mode (Li et al., 2007).
Haider and Husain (2009a) have demonstrated that
entrapped crosslinked Con A-β galactosidase was more
superior in hydrolysis of lactose present in whey and milk
in batch processes as compared to the entrapped soluble β
galactosidase. The efficiency of columns, containing algi-
nate entrapped soluble and crosslinked Con A complex of
β galactosidase was monitored at various flow rates at room

temperature, 30–32°C for the continuous hydrolysis of 0.1 M
lactose for over 2 months. A recombinant thermostable β
galactosidase from Bacillus stearothermophilus was immo-
bilized onto chitosan using Tris(hydroxymethyl)phosphine
(THP) and glutaraldehyde and a PBR was employed to
hydrolyze lactose in milk. The thermal and storage stability
of THP-immobilized β galactosidase was superior to that
of free and glutaraldehyde-immobilized enzyme prepara-
tions. The THP-immobilized β galactosidase retained higher
activity in the presence of Ca2+ than the free enzyme and was
stable during the storage at 4°C for 6 weeks, whereas the free
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
enzyme lost nearly 31% of its original activity under identi-
cal storage conditions. In the reactor more than 80% lactose
from milk was hydrolyzed after 2 h of operation. Therefore,
THP-immobilized recombinant thermostable β galactosi-
dase from Bacillus stearothermophilus has the potential for
application in the production of lactose free milk (Chen
et al., 2009).
A tapered column reactor (TCR) narrower at the bottom
and wider at the top, with continuous up flow has been
developed as an effective bioreactor for a wide variety of
applications. In this reactor, the superficial liquid velocity
decreases with the height. This type of reactor was success-
fully used in affinity purification, ethanol fermentation and
waste water treatment. β galactosidase was immobilized
For personal use only.
on the surface of perfluorocarbon (PFC) droplets by per-
fluorobenzyl chloride as a modifier. The optimal conditions
for introducing the enzyme to the PFC were determined
in order to find the maximal activity for the hydrolysis of
o-nitrophenyl-β-D -galactoside. There were no problems
with nonuniform distribution of enzyme and internal dif-
fusion resistance of the substrate. This immobilized β galac-
tosidase was successfully employed in a TCR. The results
showed that PFC droplets stabilized the operation in a TCR
with upflow and allowed more wide operating conditions
than in a conventional cylindrical column reactor (Yang,
Shinoda, and Goto, 1998).
Biotechnological applications of β galactosidases
β Galactosidases from thermophile microorganisms have
natural built-in stability to temperature and other inactiva-
tion agents and could be useful both in solution and immobi-
lized form in food industries, allowing for a simultaneous soft
thermal treatment and the low hydrolysis of lactose (Ladero
et al., 2003; 2006). Thermophilic β galactosidases represent
a very useful alternative to the mesophilic enzymes and
these enzymes have been used in the industrial processing
of dairy products along with heat treatment to sterilize the
product. The enzymatic treatment of these kinds of materials
at high temperatures has a number of generally recognized
advantages in the industry, such as the lowering of microbial
contamination (Pessela et al., 2003).
Cold-active β galactosidase, which hydrolyzes lactose to
its basic components, is one of the important food-­industrial
enzymes. Cold active β galactosidase was isolated from
Psychrophilic yeasts, which grow on lactose as a sole carbon
source at low temperature and under acidic conditions.
β galactosidases and their potential applications   53
The Gram-positive Antarctic bacterium Arthrobacter sp.
C2-2 contains two, possibly three cold-active isoenzymes
of β galactosidase. The C2-2-1 isoenzyme was cloned,
purified and characterized. This β galactosidase was clas-
sified as being a member of the family 2 of glycosidases.
(­Karasova-Lipovova et  al., 2003; Nakagawa et  al., 2003;
Turkiewicz et al., 2003; Cieslinski et al., 2005).
In biotechnological processes, an ultrasonication
method is widely used for laboratory scale work and it does
not require sophisticated equipment or extensive technical
training. The structure and function of biological molecules
can be changed by ultrasound irradiation. Therefore, the
level of intensities of ultrasound play the major part in the
activity or inactivity of many enzymes after ultrasonication.
Sonication resulted in release of higher β galactosidase activ-
ity from lactic acid bacterial cells to the culture medium.
However, lactose hydrolysis was enhanced only when β
galactosidase was effectively released. The degree of lactose
hydrolysis obtained was nearly 75% which was much higher
than those in conventional fermentation, below 40%. The
findings of this work exhibited that a high viable cell count
and a high degree of lactose hydrolysis could be simultane-
ously obtained by a suitable sonication method (Wang and
Sakakibara, 1997; Ozbek and Ulgen, 2000).
The use of whole cells as a source of β galactosidase has
been found to be an interesting alternative for the lactose
hydrolysis but a major drawback in the use of whole cells is
the poor permeability of the cell membrane to lactose (Joshi
et  al., 1987). The use of permeabilization technology can
overcome this problem and be helpful in the development
of a low-cost technology for the hydrolysis of lactose. Thus
permeabilization technology was applied for the production
of lactose-hydrolyzed milk using yeast cells. The ethanol-
permeabilized yeast cells gave 89% hydrolysis of milk lac-
tose under optimized conditions (Lee, Kin, and Oh, 2004;
Panesar et al., 2006).
Dairy whey is a contaminant product with a high organic
chemistry demand and after hydrolysis it may be used as
cattle food resources and in the food industry for develop-
ment of new products with no lactose content (Ladero,
Santos, and García-Ochoa, 2000). Cheese whey is a highly
polluting product, consisting of 0.7% (w/v) protein, 5% (w/v)
lactose, 93% (w/v) water and salts. This organic waste could
be used as a cheap, readily available substrate for microbial
cell cultivation after the hydrolysis of lactose by β galactosi-
dase (Szczodrak, 2000; Rech and Ayub, 2007). Whey proteins
(such as α lactalbumin) have excellent functional proper-
ties, which can be recovered by ultrafiltration and hydro-
lyzed to produce many useful pharmaceutical intermedi-
ates. β galactosidase can be employed directly to the cheese
whey/permeate stream resulting in high sweetness syrups
that can be used as an additive in ice-creams, desserts, etc
(Dominques, Lima, and Teixeira, 2005).
Medical applications of β galactosidases
The interest of the dairy industry in lactose hydrolysis has
been driven mainly by the fact that more than 70% of the
 54   Q. Husain
world’s population suffer from the inability to digest lactose
or lactose containing products due to the lactose intolerance
symptoms caused by the lack of β galactosidase activity in
the mucosa of the small intestine (Lee and Krasinski, 1998;
Nogales and Lopez, 2006).
The prevalence of lactose intolerance varies from <5%
to almost 100% between different populations in the world
(Figure 2). It is estimated that 75% of adults worldwide exhib-
ited some decrease in lactase activity during adulthood. The
frequency of decreased lactase activity ranges from 5% in
northern Europe, up to 71% for Southern Europe, to more
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
than 90% in some African and Asian countries (Bulhoese
et al., 2008). In general, the frequency is high outside Europe,
with the highest prevalence in far-east Asia. However,
exceptions occur, for example, milk-dependent nomads in
the Afro-Arabian desert zone (Swallow, 2003; Ridefelt and
Hakansson, 2005).
Lactose is the dominant carbohydrate in milk, which is
the only significant natural source of lactose. The inabil-
ity to completely digest lactose by the human population
is termed as lactose intolerance (Montalto et  al., 2006;
Thompkinson and Kharb, 2007). Intestinal digestion of lac-
tose involves breakdown of lactose into glucose and galac-
tose by membrane bound lactase located on the brush bor-
der of the small intestine. The resulting monosaccharides
For personal use only.
are absorbed into the portal circulation. In people with
lactose maldigestion, a portion of lactose is not digested in
the small intestine; it passes into the large intestine where it
is fermented by colonic microflora. The fermentation pro-
duces short chain fatty acids and gases, including H2, CO2,
and sometimes methane (Wang et  al., 1998; Pribila et  al.,
2000; Wilson, 2005).
African Americans
American Indians
Figure 2.  Distribution of lactose intolerance in different populations of the World (Wikipedia). From Wikipedia, the free encyclopedia, File:LacIntol-
World2.png, Date/Time 02:10, 25 January 2007 Dimensions 1427 × 628 (52 KB) User Kmusse.
The symptoms of lactose intolerance are abdominal pain
and distention, abdominal colic, diarrhoea and nausea
(Cappello and Marzio, 2005). Lactose intolerance is diag-
nosed by some of the common tests such as lactose intol-
erance test where the blood sugar levels before and after
a drink containing lactose is measured, if the blood sugar
rises above a critical threshold then the person is not lactose
intolerant. The breath test, which is performed after drinking
a lactose concentrated drink; is where the breath is analyzed
for H2 gas, which is only present when lactose is fermented
(Rizkalla et al., 2000; Vonk et al., 2000; Kerber et al., 2007). The
third test is endoscopy, which involves inserting a tube into
a person’s small intestine via the stomach. The tube enables
doctors to inspect the lining of the stomach and to obtain a
tissue sample for biopsy (Suarez, Savaiano, and Levitt, 1995;
Nilsson et al., 2004; Rasinpera et al., 2004).
A suitable, economically feasible process for lactose
hydrolysis may eliminate the problems related to lactose
intolerance mentioned earlier. Lactose hydrolysis in milk
and other dairy products is achieved by an acidic or enzy-
matic process. The use of enzymes allows milder conditions
of temperature and pH. However, the acidic method causes
several problems such as protein denaturation and the for-
mation of a brown colored product and yield of undesirable
by-products such as toxic substances like lysino-alanine
(Sinha et  al., 2007). The enzymatic process of hydrolysis
of lactose to glucose and galactose is catalyzed by β galac-
tosidases, which are widely found in nature, appearing in
microorganisms, plants and animal tissues (Sener, Apar, and
Ozbek, 2006).
Low β galactosidase activity has been demonstrated
in HIV-infected patients. It is possible that lactose
Lactose Intolerance
91–100%
81–90%
71–80% Australian Aborigines
61–70%
51–60%
41–50%
31–40%
21–30%
11–20%
1–10%
0%
No Data

malabsorption may be responsible for some of the intesti-
nal symptoms of AIDS. Lactose malabsorption has been
reported in adult patients with various stages of HIV infec-
tion by the hydrogen breath test (Heise et al., 1991; Corazza
et  al., 1997). A study has shown that short-term treatment
with rifaximin, an antibiotic reduces the curve of excretion
of H2 in patients suggesting a potential beneficial effect in
patients with lactose intolerance (Stefano et al., 2000). It was
shown that a 10  day therapy with rifaximin at 800 mg/day
reduces symptom score and normalizes the breath H2 curve
in lactose intolerant patients (Cappello and Marzio, 2005).
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
Analytical applications of β galactosidases
Biosensors are important tools in a variety of fields including
immunoassays, toxicology analysis, forensics, drug screen-
ing, gene expression analysis, gene identification, agro diag-
nostics and pharmacogenetics (Huang et al., 2002; Levicky
and Horgan, 2005). They play an important role in address-
ing the accurate characterization of emerging human health
challenges and in accomplishing environmental analysis
(Kumbhat et  al., 2007). It combines the selectivity of the
molecular recognition of the biomolecules and the sensitiv-
ity of the signal transducers (Cooper, 2002; Patel, 2002). A
wide variety of biosensors have been developed using dif-
ferent biorecognition elements such as enzymes, antibod-
For personal use only.
ies, peptides, whole cell and nucleic acids (Shankaran et al.,
2005; Yemini et  al., 2005; Rogers, 2006). Quality control of
milk and its derivatives is a very demanding field and the
need for sensitive, time-saving and accurate analytical meth-
ods has to be developed for the hydrolysis of lactose present
in milk and whey. The various low and high cost analytical
methods used for lactose analysis are mass spectrometry,
nuclear magnetic resonance spectroscopy and high per-
formance liquid chromatography (Cataldi, Angelotti, and
Bianco, 2003). Milk contains a lot of different components
with their own functional properties such as casein, lactose,
milk fat and whey. Lactose is the characteristic carbohydrate
of bovine milk, present at ~4.8% (w/v) (Audic, Chaufer, and
Daufin, 2003).
β galactosidase has been covalently immobilized onto
gold-coated magnetoelastic film via a self-assembled mon-
olayer of ω-carboxylic acid alkylthiol. The application of
magnetoelastic transduction allowed for wireless monitoring
of enzymatic activity through the associated changes in the
frequency and amplitude of magnetic fields. The methods
developed within this work considered for the fabrication
of wireless enzyme sensing systems, which could be used
as another means for screening of any biological processes
involving the precipitation of analyte species. An indolyl
galactopyranoside substrate was specifically used together
with an azo dye as the precipitating system. The immobilized
β galactosidase showed an apparent Michaelis−Menten
constant, KM of 1.2 mM for the indolyl galactopyranoside.
Calibration plots for both substrates and inhibitors were
obtained in order to establish the suitability of this sensing
system. Kinetic parameters for nonprecipitating substrates
were calculated in the presence of a precipitating substrate
β galactosidases and their potential applications   55
by way of a competitive inhibition study by β galactosi-
dase attached to magnetoelastic strips (Ball, Puckett, and
Bachas, 2003).
β Galactosidase immobilized by the solid-phase method
continues to be the focus of interest in both analytical and
process technology. To use enzyme electrodes as biosen-
sors the enzymes have to be immobilized on the surface of
the electrodes. The enzyme has many analytical uses, being
a favourite label in various affinity recognition techniques
such as enzyme linked immunosorbent assays (ELISA)
(Manta et  al., 2003). Lukacheva et  al. (2007) have devel-
oped a procedure for the determination of glucose and
lactose in food products with the use of biosensors based
on Berlin Blue. β Galactosidase catalyzed the hydrolysis of
β galactosides into monosaccharides. It acted on different
substrates including ganglioside GM1, lactosylceramides,
lactose and various glycoproteins. β Galactosidase was
immobilized into polyelectrolyte membranes by the use of
organic solvents and this immobilized enzyme was applied
to the development of biosensing elements for biosensors.
The following preparations were used: the enzymes glucose
oxidase and β galactosidase and a perfluorosulfonated pol-
ymer. The glucose and lactose biosensors based on Berlin
blue as a signal transducer and polyelectrolyte membranes
exhibited high sensitivity, low detection limits and fast
response.
A glucose oxidase biosensor based on Clark electrode
was used in order to monitor β galactosidase activity.
Immobilization of glucose oxidase was made by gelatin
and glutaraldehyde as cross-linking agent. Optimum tem-
perature, thermal stability, optimum pH, buffer system,
lactose concentration and their effects on the biosensor
system, repeatability, reproducibility, and storage and
operational stabilities of the biosensor were identified. A
linear detection range for β galactosidase was observed
between 9.4 × 10−5 and 3.2 × 10−2 U/mL. β galactosidase
activity in artificial intestinal juice was investigated by the
biosensor and the results obtained were compared with
a reference spectrophotometric method (Sezgintürk and
Dinçkaya, 2008).
Hybrid biosensors for the measurement of lactose have
been developed. S. cerevisiae was entrapped in agar. β
Galactosidase of Enterobacter agglomerans was adsorbed
onto diethylaminoethyl cellulose. Catalytic components
were confined between the surface of a gas-permeable
membrane of a CO2 electrode and a dialysis membrane.
β Galactosidase hydrolyzed lactose and S. cerevisiae fer-
mented glucose and galactose producing CO2. This gas
was measured by a CO2 electrode. The lifetime of these
biosensors was two weeks and two months, respectively.
The results were applied to lactose analysis in milk samples
of several dairy brands by comparison with a spectropho-
tometric method (Amarita, Fernandez, and Alkorta, 1997).
A manometric sensor previously developed to measure
urea was modified to measure glucose and lactose through
enzymatic oxidation. Change in pressure in an enclosed
cavity was correlated to the depletion of oxygen resulting
 56   Q. Husain
from the enzymatic oxidation of glucose or lactose. The
response of the sensor was linear and could be made
adjustable over a wide range by adjusting the amount of
sample loaded into the fixed volume reactor. Glucose was
measured after the hydrolysis of lactose by β galactosidase
for assaying the lactose. Milk lactose was also estimated
by lactose assay procedure after diluting the samples. The
30-min incubation time of lactose standards with β galac-
tosidase was shown to be adequate to hydrolyze all of the
lactose to glucose and galactose and the incubation period
could have been made as short as 5 min without any loss
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Quebec Montreal on 11/19/10
of sensitivity (Jenkins and Delwiche, 2003; Sezginturk and
Dinckaya, 2008).
A number of alternative strategies to immobilize enzymes
for their applications in biosensors have been developed in
recent years. Entrapment of enzymes within silica-based
nanospheres formed through silicification reactions pro-
vided high loading capacities for enzyme immobilization,
which resulted in high volumetric activity and enhanced
mechanical stability. Here, β galactosidase from E. coli was
used as a model enzyme due to its suitability as a biosensor
for lactose detection. The immobilization strategy resulted
in a three-dimensional network of silica attached directly
at the silicon surface, it provided a significant increase
in surface area and a corresponding 3.5-fold increase in
For personal use only.
enzyme loading compared to enzyme attached directly at
the surface. The maximum activity recovered for a silicon
square sample of 0.5 × 0.5 cm was 0.045 IU, using the direct
attachment of the enzyme through glutaraldehyde and
0.16 IU, when using silica nanospheres. Immobilized
β  galactosidase obtained by silica deposition was stable
and retained more than 80% of the original activity after
10 days at 24°C. The ability to generate three-dimensional
structures with enhanced loading capacity for biosensing
molecules has a promising future in biosensor technology
(Betancor al., 2008).
An amperometric lactose biosensor was developed by
immobilizing β galactosidase and galactose oxidase in
Langmuir–Blodgett (LB) films of poly (3-hexyl thiophene)/
stearic acid for estimation of lactose in milk and its prod-
ucts to prevent lactose intolerance. The enzyme immobi-
lized LB film was used as a working electrode and platinum
as a reference electrode (Sharma et  al., 2004). Another
amperometric biosensor prepared by immobilization of
β galactosidase and glucose oxidase onto a glassy carbon
electrode coated with mercury thin film for lactose meas-
urement has been developed. The enzyme was immobilized
by gelatin and glutaraldehyde onto a mercury thin film
electrode. The biosensor was applied to the determination
of lactose in milk samples and the results obtained with
the biosensor were validated by Somogyi–Nelson method
(Goktug, Sezginturk, and Dinckaya, 2005). Biosensors
offered advantages such as reliability, sensitivity, accuracy,
ease of handling and low cost compared with conventional
detection methods. These characteristics render an enzyme
based biosensor ideal for biomedical applications (Liang,
Li, and Yang, 2000).
Acknowledgements
The author is grateful to the Council of Science & Technology,
Uttar Pradesh Lucnow, India for funding a project entitled
“Immobilization of plant and fungal β galactosidases by
using bioaffinity supports: Its applications in the hydrolysis
of lactose in whey and milk.”
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
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