Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology

A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

Current trends of ß-galactosidase research and application

S. Sheik Asraf and P. Gunasekaran
Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj
University, Madurai – 625 021, India

Beta-galactosidase (ß-D-galactoside galactohydrolase, EC 3.2.1.23), has tremendous potential in research and application
in various fields like food, bioremediation, biosensor, diagnosis and treatment of disorders. The sources of the beta-
galactosidase are microorganisms, plants and animals. Microbial beta-galactosidase is of much interest because of their
thermostability, thermoacidophilic and thermoresistant properties. Whey, a by-product of cheese industry possess a
challenge in terms of down-stream processing and has been utilised for ethanol, exopolysaccharide and single cell protein
production by employing microbial beta-galactosidases. Transglycosylation and transgalactosylation properties of
microbial beta-galactosidases have been utilized for production of glucose, galactose, heteropolysaccharide, galacto-
oligosaccharides. Beta-galactosidase has wide range of medical and industrial applications. Immobilization of beta-
galactosidase improves its stability and reusage. Single molecule analysis of beta-galactosidase of Escherichia coli give in-
sights into its kinetic properties. Thus, research and development processes in beta-galactosidase have significant
applications.

Keywords beta-galactosidase; cold-adaptation; galactooligosaccharide; immobilization; lactose; single molecule; thermos-

table; transgalactosylation; whey

1. Introduction
Beta-galactosidases (EC 3.2.1.23) are present in a wide variety of organisms including plants, animals and
microorganisms, and are known to catalyze both hydrolytic and transglycosylation reactions. The thermostable beta-
galactosidase from Aspergillus niger, Bacillus stearothermophilus, Pyrococcus woesei, Thermus sp are relatively stable
from 35–80 °C. Cold-active and cold-adapted beta-galactosidase from psychrophilic microorganisms like Arthrobacter
psychrolactophilus, Pseudoalteromonas haloplanktis are in general quite efficient in compensating the reduction of
reaction rates by induced low temperatures through improvement of the turnover number (kcat) or of the physiological
efficiency (kcat/Km) and are relatively stable from 0–25 °C. Whey has been utilised for the production of ethanol,
exopolysaccharide and single cell protein by employing beta-galactosidase from microorganisms like Aspergillus
oryzae, Kluveromyces lactis, Kluveromyces marxianus, Lactobacillus delbrueckii subsp. bulgaricus, Saccharomyces
cerevisiae. Transglycosylation and transgalactosylation properties of beta-galactosidase from A. niger, Bacillus
megaterium, Beijerinckia indica, Bifidobacterium infantis, Bifidobacterium longum, Enterobacter cloacae, Geobacillus
stearothermophilus, K. marxianus, Lactobacillus sp, Lactobacillus reuteri, Penicillium expansum have been utilized for
production of glucose, galactose, heteropolysaccharide, galacto-oligosaccharides. Beta-galactosidase based medical and
industrial applications include cleavage of blood group A and B glycotopes, biosensor for specific lactose determination
in milk and disease diagnosis, treatment of lactose malsorption, production of lactose hydrolysed milk. Immobilization
of beta-galactosidase on anion exchange resin, cellulose-gelatin carrier system, DEAE agarose, glyoxyl / epoxy / BrCN
groups, glutaraldehyde, polyelectrolyte surfaces, silicon surface, sepabeads-epoxy supports partially modified with
boronate, iminodiacetic, metal chelates, and ethylenediamine improves its stability and reusage. Co-production of beta-
galactosidase with other enzymes like amylase, beta-glucosidase has been demonstrated in G. stearothermophilus.
Thus, the various beneficial properties of beta-galactosidase and its application across fields are discussed in this
chapter.

2. Cold-active and thermostable beta-galactosidases

The production of cold-stable β-D-galactosidases and microorganisms that resourcefully ferment lactose is of high
biotechnological interest, particularly for removal of lactose in milk and dairy products at low temperatures, cheese
whey bioremediation and bio-ethanol production (Table:1). Recently, a gene encoding β-D-galactosidase was isolated
from the genomic library of Antarctic bacterium Arthrobacter sp. 32c. Although, the highest activity of this purified
enzyme was found at 50 °C, 60 % of the highest activity of this enzyme was determined at 25 °C and 15 % of the
highest activity was detected at 0 °C. The cold-stable properties of Arthrobacter sp. 32c β-D-galactosidase could be
useful for commercial, industrial conversion of lactose into galactose and glucose in milk products and could be an
exciting substitute for the production of bioethanol from lactose-based feedstock [1].
Nakagawa et al. (2007) have overexpressed a cold-stable beta-galactosidase from A. psychrolactophilus strain F2 in
E. coli using the cold expression system. The purified recombinant enzyme, rBglAp exhibited similar enzymatic
properties to the native enzyme, it had high activity at 0 ºC, its most favourable temperature was 10 ºC, and it was

880 ©FORMATEX 2010

 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

achievable to swiftly inactivate the rBglAp at 50 ºC in 5 min. rBglAp was capable to hydrolyze both ONPG and lactose
with Km values of 2.7 and 42.1 mM, respectively, at 10 ºC. rBglAp is a cold-active and extremely heat labile enzyme
and has major possible application to the food industry [2]. Hoyoux et al. (2001) have purified beta-galactosidase from
the Antarctic Gram-negative bacterium P. haloplanktis TAE 79. The purified enzyme is characterized for optimum
activity at low temperature. Heat-induced unfolding examined by intrinsic fluorescence spectroscopy shown lower
melting point values for both P. haloplanktis wild-type and recombinant beta-galactosidase compared to the mesophilic
enzymes. Assays of hydrolysis of lactose in milk showed that P. haloplanktis beta-galactosidase is comparatively better
than current commercial beta-galactosidase, suggesting that the cold-active beta-galactosidase could be used in lactose
hydrolysis in dairy products processed in refrigerated plants [3].
Chen et al. (2008) have cloned and expressed a thermostable beta-galactosidase gene bgaB from B.
stearothermophilus in B. subtilis WB600. The optimum temperature for this beta-galactosidase activity was 70 °C.
Kinetics of thermal inactivation and half-life times for this thermostable enzyme at 65 and 70 °C were 50 and 9 h,
respectively. This enzyme exhibited a high level of transgalactosylation activity in hydrolysis of milk lactose. The
results suggest that this recombinant thermostable enzyme may be suitable for the processes such as hydrolysis of
lactose and production of galacto-oligosaccharides [4]. Lauro et al. (2008) have cloned, expressed, purified and
characterized a beta-galactosidase (Aaβ-gal) from thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The
recombinant Aaβ-gal is optimally active and stable at 65 °C [5].

Table 1 Cold-active, thermostable beta-galactosidases and their properties

Beta-galactosidase producers Thermostability (ºC) Reference

Arthrobacter sp. 32c 0-60 [1]
A. psychrolactophilus F2 0-50 [2]
P. haloplanktis TAE 79 0-40 [3]
B. stearothermophilus 45-80 [4]
A. acidocaldarius 40-90 [5]
T. ethanolicus 40-80 [6]

Volkov et al. (2005) have determined the nucleotide sequence of a 4936-bp genomic DNA fragment from the
thermophilic bacterium Thermoanaerobacter ethanolicus. The fragment contained three open reading frames (ORFs).
One of the ORF corresponded to the Lac A gene for a thermostable ß-galactosidase. Native recombinant LacA showed
the highest activity at 75–80 °C. Immobilized on aldehyde silochrome, LacA was even more thermostable and retained
its high activity [6]. Thus, the cold-active and thermostable beta-galactosidases play a vital role in hydrolysis of lactose,
bioethanol and galacto-oligosaccharide production because of their thermostable property.

3. Immobilization of beta-galactosidase
Immobilization has shown to improve beta-galactosidase’s stability and reusage (Table: 2).The immobilization of the ß-
galactosidase of Thermus sp. T2 was performed using ionic adsorption onto two different supports: a new anionic
exchanger resin, based on the coating of Sepabeads internal surfaces with polyethylenimine (PEI) polymers, and
conventional DEAE-agarose. Immobilization proceeded unusually rapid in both cases, but the adsorption strength was
much greater in the case of PEI-Sepabeads than in DEAE-supports at both pH 5 and 7. Interestingly, the PEI-
derivatives remained wholly active at pH 5 and 7 after several weeks of incubation at 50 ºC, conditions that permit the
lactose hydrolysis in milk [7].
Pessela et al. (2004) have used a battery of new heterofunctional epoxy supports to immobilize beta-galactosidase.
The capability of a standard Sepabeads-epoxy support to immobilize beta-galactosidase from Thermus sp. strain T2 can
be equal with other Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and
ethylenediamine. Immobilization yields depended on the support, ranging from 95 % using Sepabeadsepoxy-chelate,
Sepabeads-epoxy-amino, or Sepabeads-epoxy-boronic to 5 % using Sepabeads-epoxy-IDA. In count, rate of
immobilization differed when using different supports. Amazingly, the immobilized beta-galactosidase derivatives
showed outstandingly improved but different stabilities after favoring multipoint covalent attachment by long-term
alkaline incubation. The enzyme immobilized on Sepabeadsepoxy-boronic was found to be the most steady. The
crosslinking with aldehyde-dextran allowed the stabilization of the quaternary structure of the enzyme. The optimal
derivative was extremely active in lactose hydrolysis even at 70 °C (over 1000 IU/g), maintaining its activity after
extended incubation times under these conditions and with no risk of product contamination with enzyme subunits [8].
An immobilized preparation of the beta-galactosidase of E. coli using diverse supports and immobilization strategies
(bearing glyoxyl, epoxy, BrCN groups or by glutaraldehyde crosslinking on matrices containing primary amino groups)
have been obtained. In each and every one cases, the immobilization yield was 100 % with activity recoveries between
50 % and 100 % (using o-NPG as substrate). The enzyme immobilized on Eupergit 250 L exhibited an increase in the

©FORMATEX 2010 881

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

enzyme activity by a factor of 2. Synthetic activity / hydrolytic activity ratio (Vs/Vh) was lower than 0.1 with the
enzyme immobilized on BrCN at 4 ºC and pH 7, while the soluble enzyme gave a ratio of 0.46 and the immobilized
enzyme on Eupergit 250 L gave a ratio of 0.8. Eupergit C immobilized enzyme and soluble enzyme showed enhanced
Vs/Vh ratio when temperature was decreased [9].
Immobilization of ß-galactosidase-producing permeabilized dead cells of K. lactis ATCC 8583 into gelatin using
glutaraldehyde as cross-linker has been performed. Thirty percent activity was obtained by immobilized cells relative to
free disrupted cells [10]. Hamlin et al. (2007) have constructed an electrostatic self-assembly (ESA) for the
immobilization of beta-galactosidase onto polyelectrolyte multilayer assemblies of the polyanion poly [1-[4-(3-carboxy-
4 hydroxyphenylazo) benzenesulfonamido]-1, 2-ethanediyl, sodium salt] (PAZO) and the polycation poly
(ethylenimine) (PEI) [11].

Table 2 Immobilization agents of beta-galactosidases

Beta-galactosidase producers Immobilization agents Reference

Thermus sp. T2 Sepabeads-epoxy supports, [7, 8]
Sepabeads-epoxy-chelate,
Sepabeads-epoxy-amino,
Sepabeads-epoxy-boronic,
Sepabeads-epoxy-IDA,
PEI-sepabeads
E. coli Glyoxyl, epoxy, BrCN groups [9]
K. lactis ATCC 8583 Gelatine [10]
A. oryzae Silica [12]
Streptococcus thermophilus Calcium alginate (CA), [13]
Κ-carrageenan,
gellan-xanthan (GX)
K. lactis , A. oryzae Poly (vinylalcohol) hydrogel [14]

Mariotti et al. (2008) have considered the hydrolysis of whey lactose by immobilized beta-galactosidase of A. oryzae
on silica. The most excellent immobilization results were attained by using glutaraldehyde as support activator and
enzyme stabilizer. The optimized enzyme concentration for immobilization was 15-20 mg g-1 of support [12]. The
usage of calcium alginate (CA), Κ-carrageenan and gellan-xanthan (GX) gel beads for entrapment of cells of
Streptococcus thermophilus containing ß-galactosidase enhanced the stability of enzyme at higher temperatures (>55
ºC) [13]. Grosová et al. (2009) have immobilized ß-galactosidases of K. lactis and A. oryzae and yeasts in poly
vinylalcohol hydrogel lens-shaped capsules. In the process of SSF with co-immobilized enzyme of K. lactis and S.
cerevisiae, the galactose output increased from 3 g l-1 h-1 to 4.1 g l-1 h-1, thus condensed the time of preparation of D-
galactose [14]. Thus, immobilization has shown to improve the stability of beta-galactosidase and reduces the
processing time in food and other industries.

4. Notable industrial applications of beta-galactosidase

Microbial beta-galactosidases have a prominent position in terms of their role in production of various industrially-
relevant products like biosensor, lactose hydrolyzed milk, ethanol. Marrakchi et al. (2008) have developed a biosensor
associating two distinct enzymatic activities, that of the beta-galactosidase and that of the glucose oxidase, in order to
apply it for the quantitative detection of lactose in commercial milk samples. To eliminate interferences with glucose, a
differential mode of measurement was used in this biosensor [15]. A putative beta-galactosidase from Streptococcus
mitis with a choline-binding domain was recently identified. This peculiar property makes it useful for biotechnological
applications [16]. Panesar et al. (2007) have carried out trialling to overcome the problem of enzyme extraction and
poor permeability of cell membrane to lactose. Permeabilized K. marxianus NCIM 3465 cells were used for the
production of lactose-hydrolyzed milk. The ethanol-permeabilized yeast cells gave 89 % hydrolysis of milk lactose
under optimized conditions [17].
Domingues et al. (2005) have investigated the constant production of extracellular heterologous beta-galactosidase
and ethanol by a recombinant flocculating S. cerevisiae. Jointly with extracellular beta-galactosidase production, an
ethanol productivity of 9 g/1 h was obtained for the bioreactor fed with 50 g/l initial lactose concentration at 0.45 h−1
dilution rate. In adding together to beta-galactosidase and ethanol production, this system allowed for complete lactose
metabolism [18]. In 2005, a kinetic analysis of alcoholic fermentation of lactose using strain NCYC869-A3/T1, a
recombinant S. cerevisiae flocculent strain expressing both the LAC4 (coding for ß-galactosidase) and LAC12 (lactose
permease) genes of K. Lactis was carried out. The lactose was wholly utilized in all the fermentations. The increase in
ethanol production improved linearly when the initial lactose concentration was increased between 5 and 200 g L-1.

882 ©FORMATEX 2010

 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

Ethanol productivity improved with increasing initial lactose concentration up to 150 g L-1 (1.23 g L-1 h-1) [19].
Rodríguez et al. (2006) have constructed and analyzed two hybrid proteins from the beta-galactosidase of K. lactis,
intracellular, and its A. niger homologue that is extracellular. One of the hybrid proteins obtained has interesting
properties for its biotechnological utilization that increases the yield of the protein released to the growth medium.
Changes introduced in the construction, besides to improve secretion, conferred to the protein biochemical
characteristics of biotechnological interest [20]. Thus, beta-galactosidase plays a key role in food and other allied
industries.

4.1 Galacto-oligosaccharides production by beta-galactosidase

In recent years, much investigation has been carried out in the field of pro- and prebiotics as functional foods. Galacto-
oligosaccharides (GOS) are used as nondigestible, carbohydrate-based food ingredients in human and animal nutrition.
Much of the research is focussed upon microorganisms that produce beta-galactosidases with improved quality for
production of galacto-oligosaccharides (Table: 3). The synthesis of GOS with a high yield of 55 % from 275 g/L lactose
at 50 °C for 12 h was performed using transglycosylating beta-galactosidase producing E. cloacae. The enzyme showed
a extensive range of acceptor specificity for transglycosylation and catalyzed glycosyl transfer from ONPGal to various
chemicals resulting in novel saccharide yields from 0.8% to 23.5 % [21]. Wu et al. (2006) have screened a mutant strain
of B. indica L3 for the production of heteropolysaccharide-7 (PS-7). The highest amount of PS-7 formed by the mutant
was 2.88 g/L with a viscosity of 4530 cP in lactose-based MSM medium. The PS-7 manufacture was enhanced by the
addition of 4 g/L glucose into lactose-based MSM medium, reaching 5.52 g/L with a viscosity of 39531 cP. PS-7 of
6.18 g/L with a viscosity of 45772 cP was produced from the mutant grown in whey medium. The PS-7 production
from the mutant reached 7.04 g/L when 4 g/L glucose was added to the whey medium [22].
Li et al. (2009) have cloned a novel gene encoding transglycosylating beta-galactosidase (BGase) from P. expansum
F3 and subsequently expressed on the cell surface of S. cerevisiae EBY-100 by galactose induction. The BGase-
anchored yeast could directly utilize lactose to produce GOS, as well as the by-products glucose and a small quantity of
galactose. The glucose was consumed by the yeast, and the galactose was used for enzyme expression, thus to a great
extent facilitating GOS synthesis. The GOS yield reached 43.64 % when the recombinant yeast was cultivated in yeast
nitrogen base-Casamino Acids medium containing 100 g/liter initial lactose at 25 °C for 5 days [23]. In 2007, the
process by which GOS formed from lactose was optimized using beta-galactosidase from Lactobacillus sp. It proved to
be beneficial to directly apply the crude cell-free enzyme extract for the conversion, since similar GOS yields and
composition were obtained as when using the pure enzyme preparation, but expensive purification could be avoided
[24]. Hsu et al. (2007) have studied the production of GOSs by transgalactosylation using ß-galactosidase of B. longum
BCRC 15708. Two types of GOSs, tri- and tetrasaccharides, were formed after ß-galactosidase action on 40 % lactose.
Trisaccharides were the major type of GOS formed. In general, an increase of the initial lactose concentration in the
reaction mixture resulted in a higher GOS production. A highest yield of 32.5 % (w/w) GOSs could be achieved from
40 % lactose solution at 45 °C, pH 6.8, when the lactose conversion was 59.4 %. The corresponding productivity of
GOSs was 13.0 g / L-1 h-1 [25].
A mutagenesis advance was applied to the beta-galactosidase (BgaB) of G. stearothermophilus KVE39 to improve
its enzymatic transglycosylation of lactose into oligosaccharides. A straightforward screening strategy based on the
reduction of the hydrolysis of a potential transglycosylation product (lactosucrose), provided mutant enzymes
possessing enhanced synthetic properties for the autocondensation product from nitrophenyl-galactoside and GOS from
lactose. Alter of one arginine residue to lysine (R109K) augmented the oligosaccharide yield compared to that of the
wild-type BgaB. Consequently, Site-saturation mutagenesis at this position demonstrated that valine and tryptophan
further enlarged the transglycosylation performance of BgaB. During the transglycosylation reaction with lactose of the
evolved ß-galactosidases, a key trisaccharide [ß-D-galactopyranosyl-(1→3)-ß-D-galactopyranosyl-(1→4)-D-
glucopyranoside (3'-galactosyl-lactose)] was formed [26]. Spletchna et al. (2007) have investigated GOS formation
from lactose in discontinuous and constant modes of conversion using beta-galactosidase (ß-gal) from L. reuteri. In the
continuous stirred tank reactor, ß-gal from L. reuteri showed up to 2-fold higher specificity toward the formation of ß-
(1→6)-linked GOS, with ß-D-Galp-(1→6)-D-Glc and ß-D-Galp-(1→6)-D-Gal being the main GOS components
formed under these conditions [27].
Cheng et al. (2006) have used Bacillus sp for the production of low-content GOS from lactose that resulted in the
highest yield of trisaccharides and tetrasaccharides. GOS production was improved by mixing beta-galactosidase with
glucose oxidase. The low content GOS syrups, produced by beta-galactosidase was subjected to the fermentation by K.
marxianus, whereby glucose, galactose, lactose and other disaccharides were at a low level, resulting in up to 97 % and
98 % on a dry weight basis of high-content GOS with the yields of 31 % and 32 %, respectively [28].
In 2008, a procedure was proposed for producing non-monosaccharide and high-purity GOS from lactose by P.
expansum F3 β-galactosidase immobilized in calcium alginate. A purity of 28.7 % (w/w) GOS was obtained from 380
g/L lactose solution at pH 5.4 and 50 ºC. The immobilized enzyme was used for repeated GOS synthesis and showed
good working stability. Digestible sugars in the GOS were dwindling after fermentation with S. cerevisiae L1 or K.
lactis L3 entrapped in the calcium alginate. Purity greater than 37 % with yields greater than 27 % of non-
monosaccharide GOS were obtained by S. cerevisiae L1 for 19 batches and purity greater than 97 % with a yield greater

©FORMATEX 2010 883

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

than 20 % of high-purity GOS was produced using K. lactis L3 for two batches [29]. Layer and Fischer (2006) have
performed in vitro glycosylation of peptides and proteins by trans-galactosylation of protected serine and threonine by
β-D-galactosidase. The trans-mono-galactosylation of serine with a surplus of lactose produced 28 % of N-tert-
butoxycarbonyl-1-O-β-D-galactopyranosyl-L-serinemethylester. The same transformational conditions, when applied to
threonine, produced N-tert-butoxycarbonyl-1-O-β-D-galactopyranosyl-L-threonine-methylester in lower quantities.
Mono-galactosylated serine and threonine are further galactosylated in the examined experimental setup to yield bi-
galactosylated products also, especially at 50 °C with completely dissolved lactose [30].

Table 3 Galactooligosaccharides production by beta-galactosidase

Beta-galactosidase producers Galactooligosaccharides (GOS) Reference

and by-products
E. cloacae GOS, glucose, galactose [21]
B. indica L3 Heteropolysaccharide-7 [22]
P. expansum F3 GOS, glucose, galactose [23,29]
Lactobacillus sp ß-D-Galp-(1→6)-D-Glc, [24]
ß-D-Galp-(1→6)-D-Lac,
ß-D-Galp-(1→6)-D-Gal,
β-D-Galp-(1→3)-D-Lac,
β -D-Galp-(1→3)-D-Gal
B. longum BCRC 15708 tri-, tetrasaccharides, lactose, [25]
galactose, glucose
G. stearothermophilus KVE39 Lactosucrose, ß-D galactopyranosyl-(1→3)-ß-D- [26]
galactopyranosyl-(1→4)-D-glucopyranoside (3'-
galactosyl-lactose)]
L. reuteri ß-D-Galp-(1→6)-D-Glc, [27]
ß-D-Galp-(1→6)-D-Gal,
β -D-Galp-(1→3)-D-Gal
β-D-Galp-(1→6)-D-Lac
β-D-Galp-(1→3)-D-Lac
L. bulgaricus Sialyllactose, 14 other oligosaccharides [31]
L. delbrueckii subsp. bulgaricus Galactose, lactic acid , acetic acid, ethanol [35]
B. infantis GOS, lactose, monosaccharides [37]
Lactobacillus plantarum ß-D-Galp-(1→6)-D-Lac, [38]
ß-D-Galp-(1→6)-D-Glc
Bacillus circulans N-acetylactosamine, N-acetylglucosamine [39]

In 2009, oligosaccharides in bovine cheese whey permeate was characterized by a combination of nanoelectrospray
Fourier transform ion cyclotron resonance mass spectrometry and matrix-assisted laser desorption/ionization Fourier
transform ion cyclotron resonance mass spectrometry. In adding together to sialyllactose (the most abundant
oligosaccharide in bovine colostrum), 14 other oligosaccharides were identified, half of which have the same
composition of human milk oligosaccharides. These oligosaccharides could potentially be used as additives in infant
formula and products for the pharmaceutical industry [31]. Spletchna et al. (2006) have formed prebiotic GOS from
lactose using the β-D-galactosidases (β -Gals) of L. reuteri L103 and L461. Greatest GOS yields were 38 % when using
an initial lactose concentration of 205 g/L and at 80 % lactose conversion. Disaccharides other than lactose and
trisaccharides made up the enormous majority of GOS formed. The main products were identified as β-D-Galp-(1→6)-
D-Glc (allolactose), β-D-Galp-(1→6)-D-Gal, β-D-Galp-(1→3)-D-Gal, β-D-Galp-(1→6)-Lac, and β-D-Galp-(1→3)-
Lac. There were no key products with β1→4 linkages. Both intermolecular and intramolecular transgalactosylation
were observed. D-Galactose proved to be a very competent galactosyl acceptor; thus, a relatively large amount of
galactobioses was formed [32].
Li et al. (2008) have analyzed a novel beta-galactosidase BgaBM from B. megaterium that displayed wide acceptor
specificity for transglycosylation with a series of acceptors, including pentose, hexose, hydroxyl, and alkyl alcohol
using o-nitrophenyl-β-D-galactoside (ONPG) as a donor [33]. Kwon et al. (2007) have established that β-D-
galactosidase displayed on Bacillus spores by fusion to the spore coat proteins may be used as a whole-cell immobilized
biocatalyst for transgalactosylation in water-solvent biphasic reaction systems resulting in the synthesis of octyl-β-D-
galactopyranoside at concentrations up to 27.7 mM (8.1 g/liter) with a conversion yield of 27.7 % (wt/wt) after 24 h
from 100 mM lactose and 100 mM octanol dissolved in phosphate buffer and ethyl ether, respectively [34]. In 2007, an
exopolysaccharide producing strain of L. delbrueckii subsp. bulgaricus was isolated from yogurts grown at dilution

884 ©FORMATEX 2010

 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

rates between 0.06 and 0.8 h−1. A major fraction of the galactose moiety from lactose from deproteinized whey was
metabolized; molar yield of galactose from lactose varied between 0.173 and 0.791 with increasing dilution rates. The
process was heterofermentative with maximum concentrations of lactic acid (30.7 g L−1), acetic acid (11.7 g L−1) and
ethanol (0.96 g L−1) obtained at dilution rates of 0.12, 0.36 and 0.12 h−1, respectively. Greatest EPS concentration (830
mg L−1) and maximum specific EPS production rate [188 mg (g biomass h)−1] were obtained at a dilution rates of 0.36
h−1 and 0.67 h−1 respectively [35]. Nguyen et al (2007) have established the synthesis of prebiotic GOS from lactose,
with the maximum GOS yield of 38.5 % of total sugars at about 75 % lactose conversion using heterodimeric beta-
galactosidase from the probiotic strain Lactobacillus acidophilus R22 [36].
In 2008, synthesis of GOS from 36 % lactose using a recombinant beta-galactosidase of B. infantis in Pichia pastoris
was investigated. The trangalactosylation ratio reached up to 25.2 % with 83.1 % conversion of initial lactose and the
highest yield of GOS was 40.6 %. The GOS syrup was possessed of a 13.43 % GOS, 5.06 % lactose, and 8.76 %
monosaccharides. The prebiotic effect of GOS promoted the growth of B. breve ATCC 15700 and L. acidophilus ATCC
33323 [37]. Iqbal et al. (2010) have observed that a recombinant beta-galactosidase from Lactobacillus plantarum has a
high transgalactosylation activity and was used for the synthesis of prebiotic GOS. The maximal GOS yield was 41 %
(w/w) of total sugars at 85 % lactose conversion (600 mM initial lactose concentration). The main individual products
formed were ß-D-Galp-(1→6)-D-Lac, accounting for 34 % of total GOS, and ß-D-Galp-(1→6)-D-Glc, totalling up 29
% of total GOS [38]. In 2002, the use of ionic liquids as substitute solvents for enzyme catalysis was investigated. Beta-
galactosidase from Bacillus circulans catalysed the synthesis of N-acetylactosamine starting from lactose and N-
acetylglucosamine in a transglycoslyation reaction. The adding up of 25 % v/v of 1, 3-di-methyl-imidazolmethyl sulfate
as a water-miscible ionic liquid suppresses the secondary hydrolysis of the formed product, resulting in doubling-up the
yield to almost 60 %. The enzyme can be reused a number of times after ultrafiltration of the reaction mixture without
loss of activity [39]. Thus, beta-galactosidase plays a significant role in production of galacto-oligosaccharides that can
be used as food and feed for human-beings and animals respectively.

4.2 Role of beta-galactosidase in whey utilization

Whey has enormous therapeutic applications due to its composition in terms of proteins, lactose, minerals and valuable
milk nutrients. The disposal of whey remains a major problem for the dairy industry especially in developing countries
where a relatively insignificant part of whey is used for production of protein concentrates or permeates and a
significant part of it is disposed off into the water streams causing severe water pollution resulting in high BOD and 5–6
% dissolved solids. The major options for treatment or bioconversion of whey into commercially important products,
ethanol and β- galactosidase (Table: 4) which finds an increasing use because of growing lactose intolerant population.
Thus, microbial beta-galactosidases production is an important area in whey utilization.
Oberoi et al. (2008) have found that K. marxianus NCIM 3465 showed greatest beta-galactosidase activity of 1.62 IU
mg-1 dry weight using whey and cauliflower waste. Although a minor increase in enzyme production was seen by
incorporating 5 % to 10 % cauliflower waste in whey, nearly 15% increase in beta-galactosidase production was
observed when cauliflower waste level was increased to 20%. Supplementing whey with 20 % cauliflower waste also
lowered the production time. Lactose concentration in whey, mainly responsible for increasing the BOD of the effluent
water, decreased from 4.2 % to nearly 0 % at 24 h. Thus, this study established that both these by-products ⁄ residues
could be effectively used for beta-galactosidase production at commercial scale [40]. In 2004, a strain of K. lactis M2
obtained from whey samples has maximum enzyme activity (up to 8103 EU/ml). This yeast strain could be of valuable
application in bioconversion of whey [41]. Oda and Nakamura (2009) have found that NBRC 1963 of K. marxianus
converted lactose [in media containing 20 % (w/v) sugar cheese whey] most economically to ethanol [42].
Several mathematical models have been developed for ethanol production from whey using beta-galactosidase. In
2007, mathematical models for semi-continuous ethanolic fermentation in a whey medium employing coimmobilized S.
cerevisiae strain and β-D-galactosidase was developed. Kinetic parameters of biomass growth, experimentally
determined fluxes of ethanol and water, kinetic constants of ethanol separation, the degree of sugar utilization and
ethanol productivity, the time of ethanol separation were predicted using this model [43]. Hatzinikolaou et al. (2005)
have meticulously examined kinetics and stability of beta-galactosidase of a wild type strain of A. niger in the direction
of its potential use for the hydrolysis of acid whey permeate lactose. An incorporated process, concerning the
simultaneous hydrolysis–ultrafiltration of whey lactose that included the specific kinetic properties of the beta-
galactosidase was developed and modelled [44]. Ozmihci and Kargi (2007) have used lactose utilizing yeast strain, K.
marxianus DSMZ-7239 for ethanol production from cheese-whey powder (CWP) solution in batch experiments and
developed a kinetic model describing the rate of sugar utilization and substrate inhibition as function of the initial
substrate and the biomass concentrations [45]. In 2009, a mathematical model for ethanol fermentation with yeast S.
cerevisiae and beta-galactosidase on whey was developed [46].
Rech and Ayub (2007) have reported the consequence of simple feeding strategies to obtain high-cell-density
cultures of K. marxianus maximizing β–galactosidase productivity using cheese whey as basic medium. The best fed-
batch strategy was established to be the feeding of three-fold lactose concentration in the cheese whey-medium during
25 h, resulted in beta-galactosidase productivity of 291 U/L h, signifying an increase of more than 50 % compared to
batch cultivations [47]. Bansal et al. (2007) have carried out studies related to beta-galactosidase production using whey

©FORMATEX 2010 885

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

containing 4.4 % (w/v) lactose inoculated with K. marxianus MTCC 1389 and alleviated water pollution problems
caused due to its disposal into the water streams [48]. Saad (2004) has demonstrated that submerged culture of
Aspergillus japonicus produced β-D-galactosidase, with 2.95 U mg-1 protein specific activity, when developed on
cheese whey permeate fortified with 0.5 % yeast after 4 days incubation at 28 °C. Rates of lactose hydrolysis in whey
was about 55 %, after 4 h incubation at 45 °C. This enzyme was found suitable for obtaining fermentable sugars from
whey wastes [49].

Table 4 Beta-galactosidases used for ethanol production from whey

Beta-galactosidase producers Reference

K. marxianus NCIM 3465 [40]
K. lactis M2 [41]
K. marxianus NBRC 1963 [42]
S. cerevisiae [43]
A. niger [44]
K. marxianus DSMZ-7239 [45]
S. cerevisiae [46]
K. marxianus [47]
K. marxianus MTCC 1389 [48]
Aspergillus japonicus [49]
Streptococcus thermophilus 95/2, [51]
Lactobacillus delbrueckii subsp. bulgaricus 77

In 2009, appropriate conditions for the production of beta-galactosidase from whey permeate has been evaluated.
This enzyme is to be used in the production of lactose-hydrolyzed milk [50]. Tari et al. (2009) have investigated beta-
galactosidase production by Streptococcus thermophilus 95/2 (St 95/2) and Lactobacillus delbrueckii ssp. bulgaricus 77
(Lb 77) in a medium containing whey (5%), corn steep liquor (4%), potassium phosphate (2 %) and peptone (2 %) at
43 ºC for 8 h using RSM. The associative growth provided 6.4 % and 39 % more beta-galactosidase activity using pure
St 95/2 and Lb 77 strains, respectively [51]. Ozmihci and Kargi (2008) have studied ethanol fermentation of cheese
whey powder solution using the pure culture of K. marxianus (DSMZ 7239). Total Sugar of 50 gL−1 was fermented to
ethanol in a continuously operated packed column bioreactor (PCBR) using olive pits as support particles for cell
attachment [52]. Thus, whey utilization by beta-galactosidase reduces the burden of water pollution and provides
beneficial products like ethanol and protein concentrates.

4.3 Medical and veterinary applications of beta-galactosidase

Beta-galactosidase with its transgalactosylation property finds prominent medical applications such as treatment of
disorders and development of digestive supplements. Anderson et al. (2005) have isolated an endo beta-galactosidase
from Clostridium perfringens ATCC 10543 capable of liberating both the A trisaccharide and B trisaccharide from
glycoconjugates containing blood group A and B glycotopes, respectively. Recombinant EABase damaged the blood
group A and B antigenicity of human type A and B erythrocytes and also released A-Tri and B-Tri from blood group
A+- and B+-containing glycoconjugates. The exceptional specificity of this beta-galactosidase should make it useful for
studying the structure and function of blood group A- and B-containing glycoconjugates [53]. In 2009, a recombinant
endo-beta-galactosidase (ABase), which releases A/B antigen was developed. It removed 82 % of A antigen and 95 %
of B antigen in human A/B red blood cells, and concealed anti-A/B antibody binding and complement activation
effectively. It was also found to remain active at 4 °C. in vivo infusion into a blood type A demonstrated a marked
reduction of A antigen expression in the glomeruli of kidney (85 % at 1 h, 9 % at 4 h and 13 % at 24 h) and the
sinusoids of liver (47 % at 1 h, 1 % at 4 h and 3 % at 24 h) without grave adverse effects. This substitute approach
might be useful for minimizing antibody removal and anti-B cell immunosuppression as an adjuvant therapy in ABO-
incompatible kidney, liver and possibly heart transplantation [54]. Liu and Roffler (2006) have examined the expression
of E. coli beta-galactosidase in muscle fibers and concluded that repeated intramuscular injections of beta-galactosidase
can encourage strong immune responses in immune-competent animals and cause abolition of transduced muscle fibers
by inflammatory cells [55].
Recently, a beta-galactosidase from the mesoacidophilic fungus Bispora sp. MEY-1 under simulated gastric
conditions, has shown greater stability (100%) and hydrolysis ratio (>80 %) toward milk lactose than the commercially
available beta-galactosidase from A. oryzae ATCC 20423. Thus, this beta-galactosidase may be a superior digestive
supplement for alleviating symptoms associated with lactase deficiency [56]. Sanchez-Aparicio et al. (2009) have
developed recombinant β-D-galactosidases accommodating one or two different peptides from the foot-and-mouth
disease virus nonstructural protein 3B per enzyme monomer that allowed differentiation between sera of FMDV-
infected pigs, cattle, and sheep and those of naïve and conventionally vaccinated animals. These FMDV infection-

886 ©FORMATEX 2010

 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

specific biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected
animals [57].

5. Individual molecule studies of beta-galactosidase

Individual molecule analysis of beta-galactosidase of E. coli give in-sights into its kinetic properties. Craig and Dovichi
(1998) have incubated E. coli beta-galactosidase molecules with fluorogenic substrate. Individual molecules were found
to differ with respect to their activities. Molecules showed a 23-fold distribution of activities with the bulk of molecules
within a 4-fold distribution [58]. In 2000, it was observed that when E. coli ß-galactosidase stored at 25 ºC in buffer
containing no added MgCl2, over half the enzyme molecules became inactive. However, those molecules which
retained activity remained dynamic for the duration of the experiment. This indicated that there exist two populations of
E. coli ß-galactosidase, one which requires storage in the presence of the higher concentration of Mg2+ in order to
remain active. [59].
Craig et al. (2003) have performed single enzyme molecule assays on E. coli beta-galactosidase from three different
sets of samples. The first set consisted of lysates of induced cells from 5 diverse strains of the bacteria, as well as 2
dissimilar commercial preparations of the enzyme. These samples were found to have considerably different
distributions of single molecule activities. For the second set of samples, beta-galactosidase expression was induced for
1.5 h, followed by further incubation where expression was subdued. Assays were performed on the lysates of the
preinduction and on the lysates from aliquots taken set times post induction. The freshly induced enzyme had a 25 %
higher average single molecule activity than the basally expressed enzyme. This average activity returned to the basal
value 3.5 h post induction and remained unaffected thereafter. Finally, beta-galactosidase was induced at 26 and 42°C.
The samples were found to be indistinguishable with respect to their average single molecule activities [60].
In 2003, it was found that individual molecules of beta-galactosidase molecules from the crystallized enzyme as well
as the original enzyme preparation displayed a range of activity of 20-fold or greater. Molecules obtained from two
diverse crystals had identical activity distributions of 31600 ± 1100 and 31800 ±1100 reactions min-1 (enzyme
molecule)-1. This activity was found to be drastically different from that of the enzyme used to grow the crystals, which
showed an activity distribution of 38500 ±900 reactions min-1 (enzyme molecule)-1[61].
Nichols et al. (2007) have induced beta-galactosidase in E. coli wild type strains ATCC 8677 and 35321 in the
presence of various protease inhibitors. The presence of the protease inhibitors had a least effect on the average and
distribution of single molecule activities. Assays performed on beta-galactosidase from strains 8677 and 35321
demonstrated that the relative activities of the enzyme for the diverse substrates differed between the strains. These two
strains differ in their primary sequence by a single amino acid substitution in position 280, which is in the region of the
active site [62]. Nichols and Craig (2008) have performed the single enzyme molecule assays of in vivo and in vitro
synthesized beta-galactosidase from wild-type E. coli strains ATCC 35321 and 8677. The average combined turnover
numbers of the enzyme from wild-type E. coli strains ATCC 35321 and 8677 synthesized in vivo and in vitro and with
the presence and absence of a His6 tag were found to differ considerably. This indicates that the in vivo and in vitro
produced enzymes are not alike and the presence of a C-terminal His6 tag alters the activity of ß-galactosidase [63].
Nichols and Craig (2008) have calculated the electrophoretic mobility and catalytic activity of individual molecules
of E. coli beta-galactosidase. Together the mobility and activity were reproducible for each molecule but differed
between individual molecules. There was no experimental relationship between the experimental activities and
electrophoretic mobilities. If the finding that single beta-galactosidase molecules have heterogeneous electrophoretic
mobility can be extended to other proteins, this may prevent the resolution possible for capillary zone electrophoresis
protein separations [64]. In 2008, CE-LIF detection-based assay for the study of individual molecules of E. coli ß-
galactosidase was developed. The assay allows for the real-time measurement of the electrophoretic mobility, catalytic
activity and the difference in activity over time of single enzyme molecules. In adding together to showing the
microheterogeneity of the enzyme molecules with respect to mobility and activity, it was established that at superior
temperatures the enzyme activity changed over time. Incubation at different temperatures showed that single beta-
galactosidase molecules exhibited differences in their change in activity upon a modification in incubation temperature.
In addition, thermal denaturation was found to cause a quick and absolute loss of activity [65]. Thus, study of individual
molecules of beta-galactosidase has shown the previously unstudied features of the enzyme.

6. Conclusion
Research and development in the beta-galactosidase will help to address the problems faced in the food and allied
industries that look for enzymes with novel properties like cold-stability and thermo-active. Immobilization of beta-
galactosidase will reduce the cost of production of food products and allow for reusage of the enzyme. Novel
galactooligosaccharides production by beta-galactosidase will pave the way for development of prebiotics that can be
used as food supplement. Whey utilization by beta-galactosidase will help to reduce the water pollution caused by lack
of downstream-processing and lead to production of products like bioethanol and lactose-hydrolysed milk. Beta-

©FORMATEX 2010 887

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

galactosidase’s capability has been realised by the pleothera of products like biosensors, digestive supplements.
Individual molecule study of beta-galactosidase has shown the various unknown kinetic properties of beta-
galactosidase. Thus, research and development of beta-galactosidase finds application in several industries.

Acknowledgements: Authors thank Department of Science and Technology, New Delhi (SR/SO/BB-50/2007) for the Junior
Research Fellowship to SSA through the Centre for excellence in Genomic Sciences. Authors thank University Grants Commission,
New Delhi for providing facility and financial support through Networking Resource Centre in Biological Sciences, School of
Biological Sciences, Madurai Kamaraj University. SSA thanks all the authors listed in references for their generous help by sending
their publication.

References
[1] Hildebrandt P, Wanarska M, Kur J. A new cold-adapted β-D-galactosidase from the Antarctic Arthrobacter sp. 32c – gene
cloning, overexpression, purification and properties. BMC Microbiology. 2009; 9(151):1-11.
[2] Nakagawa T, Ikehata R, Myoda T, Miyaji T, Tomizuka N. Overexpression and functional analysis of cold-active ß-
galactosidase from Arthrobacter psychrolactophilus strain F2. Protein Expression and Purification. 2007; 54:295–299.
[3] Hoyoux A, Jennes I, Dubois P, Genicot S, Dubail F, Francois JM, Baise E, Feller G, Gerday C. Cold-adapted ß-galactosidase
from the Antarctic psychrophile Pseudoalteromonas haloplanktis. Applied and Environmental Microbiology.2001; 67(4):1529–
1535.
[4] Chen W, Chen H, Xia Y, Zhao J, Tian F, Zhang H. Production, purification, and characterization of a potential thermostable
galactosidase for milk lactose hydrolysis from Bacillus stearothermophilus. J. Dairy Sci. 2008; 91:1751–1758.
[5] Lauro BD, Strazzulli A, Perugino G, Cara FL, Bedini E, Corsaro MM, Rossi M, Moracci M. Isolation and characterization of a
new family 42 β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: Identification of the
active site residues. Biochimica et Biophysica Acta.2008;1784:292–301.
[6] Volkov IY, Lunina NA, Berezina OV, Velikodvorskaya GA, Zverlov VV. Thermoanaerobacter ethanolicus gene cluster
containing the α– and ß-Galactosidase genes mel A and lac A and properties of recombinant LacA. Molecular Biology. 2005;
39(6):799–805.
[7] Pessela BCC, Fernández-Lafuente R, Fuentes M, Vián A, Garc´ıa JL, Carrascosa AV, Mateo C, Guisán JM. Reversible
immobilization of a thermophilic ß-galactosidase via ionic adsorption on PEI-coated sepabeads. Enzyme and Microbial
Technology. 2003; 32:369–374.
[8] Pessela BCC, Mateo C, Fuentes M, Vian A, Garcı´a JL, Carrascosa AV, Guisa´n JM, Ferna´ndez-Lafuente R. Stabilization of a
multimeric ß-Galactosidase from Thermus sp. Strain T2 by immobilization on novel heterofunctional epoxy supports plus
aldehyde-dextran cross-linking. Biotechnol. Prog. 2004;20,388-392.
[9] Pessela BCC, Dellamora-Ortiz G, Betancor L, Fuentes M, Guis´an JM, Fernandez-Lafuente R. Modulation of the catalytic
properties of multimeric ß-galactosidase from E. coli by using different immobilization protocols. Enzyme and Microbial
Technology. 2007; 40:310–315.
[10] Numano˘glu Y, Sungur S. ß-Galactosidase from Kluyveromyces lactis cell disruption and enzyme immobilization using a
cellulose–gelatin carrier system. Process Biochemistry. 2004; 39:703–709.
[11] Hamlin RE, Dayton TL, Johnson LE, Johal MS. A QCM study of the immobilization of ß-Galactosidase on polyelectrolyte
surfaces: effect of the terminal polyion on enzymatic surface activity. Langmuir.2007; 23: 4432-4437.
[12] Mariotti MP, Yamanaka H, Araujo AR, Trevisan HC. Hydrolysis of whey lactose by immobilized ß-Galactosidase. Braz. arch.
biol. technol. 2008; 51(6):1233-1240.
[13] Goel A, Sharma RK, Tandon HKL. A comparison of different polymeric gels for entrapment of cells of Streptococcus
thermophilus containing ß-galactosidase. J Food Sci Technol. 2006; 43(5):526-531.
[14] Grosová Z, Rosenberg M, Gdovin M, Sláviková L, Rebroš M. Production of D-galactose using ß-galactosidase and
Saccharomyces cerevisiae entrapped in poly (vinylalcohol) hydrogel. Food Chemistry. 2009; 116:96–100.
[15] Marrakchi M, Dzyadevych SV, Lagarde F, Martelet C, Jaffrezic-Renault N. Conductometric biosensor based on glucose
oxidase and beta-galactosidase for specific lactose determination in milk. Material Science and Engineering C. 2008; 28:872-
875.
[16] Campuzano S, Serra B, Llull D, García JL, García P. Cloning, expression, and characterization of a peculiar choline-binding ß-
Galactosidase from Streptococcus mitis. Applied and environmental microbiology. 2009; 75(18):5972–5980.
[17] Panesar R, Panesar PS, Singh RS, Kennedy JF, Bera MB. Production of lactose-hydrolyzed milk using ethanol permeabilized
yeast cells. Food Chemistry.2007; 101:786–790.
[18] Domingues L, Lima N, Teixeira JA. Aspergillus niger ß-galactosidase production by yeast in a continuous high cell density
reactor. Process Biochemistry .2005; 40:1151–1154.
[19] Guimaraes P, Klein J, Domingues L, Teixeira JA. Fermentation performance of a recombinant lactose-consuming flocculating
Saccharomyces cerevisiae Strain. Braz. J. Food Technol. 2005; 34-39.
[20] Rodríguez ÁP, Leiro RF, Trillo MC, Cerdán ME, Siso MIG, Becerra M. Secretion and properties of a hybrid Kluyveromyces
lactis-Aspergillus niger β-galactosidase. Microbial Cell Factories. 2006; 5(41):1-13.
[21] Lu L, Xiao M, Li Z, Li Y, Wang F. A novel transglycosylating ß-galactosidase from Enterobacter cloacae B5. Process
Biochemistry.2009; 44:232–236.
[22] Wu JR, Son JH, Kim KM, Lee JW, Kim SK. Beijerinckia indica L3 fermentation for the effective production of
heteropolysaccharide-7 using the dairy byproduct whey as medium. Process Biochemistry. 2006; 41:289–292.
[23] Li Y, Lu L, Wang H , Xu X, Xiao M. Cell surface engineering of a ß-Galactosidase for galactooligosaccharide synthesis.
Applied and environmental microbiology. 2009; 75(18):5938–5942.

888 ©FORMATEX 2010

 Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

[24] Splechtna B, Nguyen TH, Zehetner R, Lettner HP, Lorenz W, Haltrich D. Process development for the production of prebiotic
galacto-oligosaccharides from lactose using β-galactosidase from Lactobacillus sp. Biotechnol. J. 2007; 2:480–485.
[25] Hsu CA, Lee SL, Chou CC. Enzymatic production of galactooligosaccharides by ß-Galactosidase from Bifidobacterium longum
BCRC 15708. J. Agric. Food Chem. 2007; 55:2225-2230.
[26] Placier G, Watzlawick H, Rabiller C, Mattes R. Evolved ß-galactosidases from Geobacillus stearothermophilus with improved
transgalactosylation yield for galacto-oligosaccharide production. Applied And Environmental Microbiology. 2009;
75(19):6312–6321.
[27] Splechtna B, Nguyen TH, Haltrich D. Comparison between discontinuous and continuous lactose conversion processes for the
production of prebiotic galacto-oligosaccharides using ß-Galactosidase from Lactobacillus reuteri. J. Agric. Food Chem. 2007;
55:6772-6777.
[28] Cheng CC, Yu MC, Cheng TC, Sheu DC, Duan KJ, Tai WL. Production of high-content galacto-oligosaccharide by enzyme
catalysis and fermentation with Kluyveromyces marxianus. Biotechnol Lett. 2006; 28:793–797.
[29] Li Z, Xiao M, Lu L, Li Y. Production of non-monosaccharide and high-purity galactooligosaccharides by immobilized enzyme
catalysis and fermentation with immobilized yeast cells. Process Biochemistry. 2008; 43:896–899.
[30] Layer A, Fischer F. Enzymatic O-Galactosylation of protected Serine and Threonine by ß-Galactosidase employing high lactose
concentrations. Ind. Eng. Chem. Res. 2006; 45:6619-6621.
[31] Barile D, Tao N, Lebrilla CB, Coisson JD, Arlorio M, German JB. Permeate from cheese whey ultrafiltration is a source of milk
oligosaccharides. International Dairy Journal. 2009; 19:524–530.
[32] Splechtna B, Nguyen TH, Steinbock M, Kulbe KD, Lorenz W, Haltrich D. Production of prebiotic galacto-oligosaccharides
from lactose using ß-Galactosidases from Lactobacillus reuteri. J. Agric. Food Chem. 2006; 54:4999-5006.
[33] Li Y, Wang H, Lu L, Li Z, Xu X, Xiao M. Purification and characterization of a novel β-Galactosidase with
transglycosylation activity from Bacillus megaterium 2-37-4-1. Appl Biochem Biotechnol. 2008; DOI 10.1007/s12010-008-
8310-4.
[34] Kwon SJ, Jung HC, Pan JG. Transgalactosylation in a water-solvent biphasic reaction system with ß-Galactosidase displayed on
the surfaces of Bacillus subtilis spores. Applied and environmental microbiology. 2007; 73(7):2251–2256.
[35] Shene C, Bravo S. Whey fermentation by Lactobacillus delbrueckii subsp. bulgaricus for exopolysaccharide production in
continuous culture. Enzyme and Microbial Technology. 2007; 40:1578–1584.
[36] Nguyen TH, Splechtna B, Krasteva1 S, Kneife W, Kulbe KD, Divne C, Haltrich D. Characterization and molecular cloning of
a heterodimeric ß-galactosidase from the probiotic strain Lactobacillus acidophilus R22. FEMS Microbiol Lett. 2007;269:136–
144.
[37] Jung SJ, Lee BH. Production and application of galacto-oligosaccharides from lactose by a recombinant ß-galactosidase of
Bifidobacterium infantis overproduced by Pichia pastoris. Food. Sci. Biotechnol. 2008; 17(3):514-518.
[38] Iqbal S, Nguyen TH, Nguyen TT, Maischberger T, Haltrich D. ß-Galactosidase from Lactobacillus plantarum WCFS1:
biochemical characterization and formation of prebiotic galacto-oligosaccharides. Carbohydr.
Res.2010;doi:10.1016/j.carres.2010.03.028
[39] Kaftzik N, Wasserscheid P, Kragl U. Use of ionic liquids to increase the yield and enzyme stability in the ß-galactosidase
catalysed synthesis of N-Acetyllactosamine. Organic Process Research & Development. 2002; 6:553-557.
[40] Oberoi HS, Bansal S, Dhillon GS. Enhanced ß-galactosidase production by supplementing whey with cauliflower waste.
International Journal of Food Science and Technology. 2008; 43:1499–1504.
[41] Moeini H, Vallian S, Nahvi I. Isolation and identification of yeast strains capable of producing single cell protein from whey in
co-cultures with Saccharomyces cerevisiae. Iranian Journal of Biotechnology. 2004; 2(1):13-18.
[42] Oda Y, Nakamura K. Production of ethanol from the mixture of beet molasses and cheese whey by a 2-deoxyglucose-resistant
mutant of Kluyveromyces marxianus.FEMS Yeast Res. 2009; 1–7.
[43] Staniszewski M, Kujawski W, Lewandowska M. Ethanol production from whey in bioreactor with co-immobilized enzyme and
yeast cells followed by pervaporative recovery of product – kinetic model predictions. Journal of Food Engineering. 2007;
82:618–625.
[44] Hatzinikolaou DG, Katsifas E, Mamma D, Karagouni AD, Christakopoulos P, Kekos D. Modeling of the simultaneous
hydrolysis–ultrafiltration of whey permeate by a thermostable ß-galactosidase from Aspergillus niger. Biochemical Engineering
Journal. 2005; 24:161–172.
[45] Ozmihci S, Kargi F. Kinetics of batch ethanol fermentation of cheese-whey powder (CWP) solution as function of substrate and
yeast concentrations. Bioresource Technology. 2007; 98:2978–2984.
[46] Staniszewski M, Kujawski W, Lewandowska M. Semi-continuous ethanol production in bioreactor from whey with co-
immobilized enzyme and yeast cells followed by pervaporative recovery of product – kinetic model predictions considering
glucose repression. Journal of Food Engineering. 2009; 91:240–249.
[47] Rech R, Ayub MAZ. Simplified feeding strategies for fed-batch cultivation of Kluyveromyces marxianus in cheese whey.
Process Biochemistry.2007; 42:873–877.
[48] Bansal S, Oberoi HS, Dhillon GS, Patil RT. Production of β- galactosidase by Kluyveromyces marxianus MTCC 1388 using
whey and effect of four different methods of enzyme extraction on β- galactosidase activity. Indian J. Microbiol. 2008; 48:337-
341.
[49] Saad RR. Purification and some properties of β-galactosidase from Aspergillus japonicus. Annals of Microbiology. 2004;
54(3):299-306.
[50] Jokar A, Karbassi A. Determination of proper conditions for the production of crude beta-galactosidase using Lactobacillus
delbrueckii ssp bulgaricus. J. Agric. Sci. Technol. 2009; 11: 301-308.
[51] Tari C, Ustok FI, Harsa S. Optimization of the associative growth of novel yoghurt cultures in the production of biomass, ß-
galactosidase and lactic acid using response surface methodology. International Dairy Journal. 2009; 19:236–243.
[52] Ozmihci S, Kargi F. Ethanol production from cheese whey powder solution in a packed column bioreactor at different hydraulic
residence times. Biochemical Engineering Journal. 2008; 42:180–185.

©FORMATEX 2010 889

Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology
A. Méndez-Vilas (Ed.)
_______________________________________________________________________________________

[53] Anderson KM, Ashida H, Maskos K, Dell A, Li SC, Li YT. A Clostridial endo-ß-galactosidase that cleaves both blood group A
and B glycotopes:The first member of a new glycoside hydrolase family, GH98. The Journal of Biological Chemistry. 2005;
280(9):7720-7728.
[54] Kobayashi T, Liu D, Ogawa H, Miwa Y, Nagasaka T, Maruyama S, Li Y, Onishi A, Iwamoto M, Kuzuya T, Kadomatsu K,
Uchida K, Nakao A. Removal of blood group A/B antigen in organs by ex vivo and in vivo administration of endo-ß-
galactosidase (ABase) for ABO-incompatible transplantation. Transplant Immunology. 2009; 20: 132–138.
[55] Liu AB, Roffler SR. Anti-CTLA4 scFv, a single-chain antibody, protects the expression of E. coli. beta-galactosidase during
repeated intramuscular injections in immune-competent mice. Tzu Chi Med J. 2006; 18(4):259-265.
[56] Wang H, Luo H, Bai Y, Wang Y, Yang P, Shi P, Zhang W, Fan Y, Yao B. An acidophilic β-Galactosidase from Bispora sp.
MEY-1 with high lactose hydrolytic activity under simulated gastric conditions. J. Agric. Food Chem. 2009; 57:5535–5541
[57] Sa´nchez-Aparicio MT, Rosas MF, Ferraz RM, Delgui L, Veloso JJ, Blanco E, Villaverde A, Sobrino F. Discriminating foot-
and-mouth disease virus-infected and vaccinated animals by use of ß-Galactosidase allosteric biosensors. Clinical and vaccine
immunology. 2009, 16(8):1228–1235.
[58] Craig DB, Dovichi NJ. Escherichia coli ß-galactosidase is heterogeneous with respect to the activity of individual molecules.
Can. J. Chem. 1998; 76:623-626.
[59] Craig DB, Hall T, Goltz DM. Escherichia coli ß-galactosidase is heterogeneous with respect to a requirement for magnesium.
BioMetals. 2000; 13: 223–229.
[60] Craig DB, Nachtigall JT, Ash HL, Shoemaker GK, Dyck AC, Wawrykow TMJ, Gudbjartson HL. Differences in the average
single molecule activities of E. coli ß-Galactosidase: effect of source, enzyme molecule age and temperature of induction.
Journal of Protein Chemistry. 2003; 22(6):555-561.
[61] Shoemaker GK, Juers DH, Coombs JML, Matthews BW, Craig DB. Crystallization of ß-Galactosidase does not reduce the
range of activity of individual molecules. Biochemistry. 2003; 42: 1707-1710.
[62] Nichols ER, Gavina JMA, McLeod RG, Craig DB. Single molecule assays of ß-Galactosidase from two wild-type Strains of E.
coli: effects of protease inhibitors on microheterogeneity and different relative activities with differing substrates. The Protein
Journal. 2007; 26(2):95-105.
[63] Nichols ER, Craig DB. Single molecule assays reveal differences between in vitro and in vivo synthesized ß-galactosidase.
Protein J. 2008; 27:376–383.
[64] Nichols ER, Craig DB. Measurement of the differences in electrophoretic mobilities of individual molecules of E. coli ß-
galactosidase provides insight into structural differences which underlie enzyme microheterogeneity. Electrophoresis. 2008;
29:4257–4269.
[65] Craig DB, Nichols ER. Continuous flow assay for the simultaneous measurement of the electrophoretic mobility, catalytic
activity and its variation over time of individual molecules of Escherichia coli ß-galactosidase. Electrophoresis. 2008; 29:4298–
4303.

890 ©FORMATEX 2010