characterization and molecular cloning of a heterodimeric β-galactosidase from the probiotic strain...

Characterizationandmolecular cloning ofa heterodimericb-galactosidase fromtheprobiotic strainLactobacillusacidophilus R22Thu-Ha Nguyen1,2, Barbara Splechtna1,2, Stanimira Krasteva1,2, Wolfgang Kneifel3, Klaus D. Kulbe2,Christina Divne4 & Dietmar Haltrich2

1Research Centre Applied Biocatalysis, Petersgasse, Graz, Austria; 2Division of Food-Biotechnology, Department of Food Sciences and Technology,

University of Natural Resources and Applied Life Sciences, Vienna, Austria; 3Division of Food Quality Assurance, Department of Food Sciences and

Technology, University of Natural Resources and Applied Life Sciences, Vienna, Austria; and 4Department of Biotechnology, Royal Institute of

Technology, Albanova University Centre, Stockholm, Sweden

Correspondence: Dietmar Haltrich, Division

of Food-Biotechnology, Department of Food

Sciences and Technology, University of

Natural Resources and Applied Life Sciences,

Muthgasse 18, A-1190 Vienna, Austria. Tel.:

143 1 36006 6275; fax: 143 1 36006 6251;

e-mail: [email protected]

Received 15 September 2006; revised 3

November 2006; accepted 14 December 2006.

First published online 15 January 2007.

DOI:10.1111/j.1574-6968.2006.00614.x

Editor: Atsushi Yokota

Keywords

b-galactosidase; galacto-oligosaccharides;

transgalactosylation; Lactobacillus acidophilus .

Abstract

b-Galactosidase from the probiotic strain Lactobacillus acidophilus R22 was

purified to apparent homogeneity by ammonium sulphate fractionation, hydro-

phobic interaction, and affinity chromatography. The enzyme is a heterodimer

consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis.

The optimum temperature of b-galactosidase activity was 55 1C (10-min assay)

and the range of pH 6.5–8, respectively, for both o-nitrophenyl-b-D-galactopyr-

anoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and

oNPG were 4.04� 0.26 mM, 28.8� 0.2 mmol D-glucose released per min per mg

protein, and 0.73� 0.07 mM, 361� 12 mmol o-nitrophenol released per min per

mg protein, respectively. The enzyme was inhibited by high concentrations of

oNPG with Ki,s = 31.7� 3.5 mM. The enzyme showed no specific requirements for

metal ions, with the exception of Mg21, which enhanced both activity and stability.

The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and

compared with other b-galactosidases from lactobacilli. b-Galactosidase from L.

acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS)

from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75%

lactose conversion.

Introduction

Lactobacillus acidophilus is a frequently occurring inhabitant

of the lower end of the small intestine where it both occupies

the lumen of the gut and adheres to the surface of the

intestinal walls (Itsaranuwat et al., 2003). Among others, it

was shown that L. acidophilus metabolizes any residual

lactose in the gut content, thus supporting lactose digestion

in consumers with low levels of indigenous lactase (b-

galactosidase) activity. Lactobacillus acidophilus is probably

among the best-known probiotic lactobacilli. A ‘probiotic’,

by general definition, is a live microbial feed supplement,

which beneficially affects the host by improving its intestinal

microbial balance (Fuller, 1989; Holzapfel & Schillinger,

2001). The first probiotic species introduced into research

was L. acidophilus by Hull and others in 1984 (Caglar et al.,

2005). An extensive number of studies have described the

potential benefits of probiotics including production of

important digestive enzymes, alleviation of symptoms of

lactose intolerance, cholesterol-lowering effects, reduction

of the risk of colon cancer, and stimulation of the immune

system (McDonough et al., 1987; Holzapfel et al., 1998;

Sanders, 1998; Klaenhammer & Kullen, 1999).

Certain oligosaccharides are considered to be beneficial

for human and animal hosts due to their ability to stimulate

selectively growth and/or activity of one or a limited number

of bacteria in the colon (Gibson & Roberfroid, 1995; Gibson,

1998), and are subsequently classified as ‘prebiotics’, new

functional food ingredients that are of considerable interest.

Galacto-oligosaccharides (GOS) are formed via the transga-

lactosylation reaction from lactose, which is catalysed by

b-galactosidases in addition to their hydrolytic activity.

These oligosaccharides are of great interest because of their

potentially prebiotic characteristics. A plethora of GOS is

also found in human milk, and these differently substituted

oligosaccharides are associated with a number of beneficial

FEMS Microbiol Lett 269 (2007) 136–144c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

effects for the breast-fed infant (Kunz et al., 2000). Using

b-galactosidases from probiotic strains for the formation of

prebiotic GOS is an interesting approach for the production

of new carbohydrate-based functional food ingredients.

Furthermore, the combination of these prebiotic GOS with

probiotic lactobacilli for the development of synbiotic

functional foods could introduce new dimensions of appli-

cations (Rastall & Maitin, 2002).

The objective of this study was to study the properties of

b-galactosidase from a probiotic strain, L. acidophilus R22,

and its transgalactosylation activity. The composition

of GOS formed by transgalactosylation of lactose using

b-galactosidase from L. acidophilus R22 was also compared

with the GOS mixtures formed using b-galactosidases from

other Lactobacillus species, namely Lactobacillus reuteri,

which were reported in a previous study (Splechtna et al.,

2006).

Materials and methods

Chemicals

All chemicals were purchased from Sigma (St Louis, MO)

unless otherwise stated, and were of the highest quality

available. MRS broth powder (for Lactobacillus broth ac-

cording to De Man, Rogosa and Sharpe) was obtained from

Merck (Darmstadt, Germany), and 1,4-dithiothreitol (DTT)

was from Roth (Karlsruhe, Germany). Glucose oxidase

(GOD) from Aspergillus niger (lyophilized, 205 U mg�1 en-

zyme preparation) was from Fluka (Buchs, Switzerland).

Horseradish peroxidase (POD) (lyophilized, 210 U mg�1)

and the test kit for the determination of D-glucose were

from Boehringer (Mannheim, Germany).

Strain and culture conditions

Lactobacillus acidophilus R22, originating from probiotic

yoghurt, was obtained from the culture collection of the

Division of Food Microbiology, BOKU University of Natur-

al Resources and Applied Life Sciences, Vienna. It was grown

anaerobically in MRS broth medium (peptone, 10 g L�1;

di-potassium hydrogen phosphate, 2 g L�1; meat extract,

8 g L�1; di-ammonium hydrogen citrate, 2 g L�1; yeast ex-

tract, 4 g L�1; sodium acetate, 5 g L�1; magnesium sulphate,

0.2 g L�1; Tween 80, 1 g L�1; manganese sulphate, 0.04 g L�1)

in which lactose served as the C-source (15 g L�1) at 37 1C.

Cells were harvested by centrifugation (8000 g, 15 min, 4 1C)

when lactose was almost depleted from the medium.

Purification of b-galactosidase fromL. acidophilus

Cells were resuspended in 50 mM sodium phosphate buffer,

pH 6.8, containing 20% w/v glycerol and 1 mM dithiothrei-

tol (buffer P) (Gopal et al., 2001; Nguyen et al., 2006),

disrupted using a French press (AMINCO, Maryland), and

debris was removed by centrifugation (25 000 g, 15 min,

4 1C) to obtain the crude cell extract. b-Galactosidase was

isolated from the cell extract using a purification protocol

based on ammonium sulphate precipitation, hydrophobic

interaction chromatography, and affinity chromatography

on p-aminobenzyl thiogalactoside agarose as described pre-

viously (Nguyen et al., 2006). The purified enzyme was

stored in 50 mM sodium phosphate buffer, pH 6.5, contain-

ing 20 mM MgCl2 at 4 1C.

Protein determination

Protein concentration was determined by the method of

Bradford (1976) using bovine serum albumin as the

standard.

Enzyme assays

b-Galactosidase activity was determined using o-nitrophe-

nyl-b-D-galactopyranoside (oNPG) and lactose as the sub-

strates as described previously (Nguyen et al., 2006). When

chromogenic oNPG was used as the substrate, the reaction

was initiated by adding 20 mL of enzyme solution to 480 mL

of 22 mM oNPG in 50 mM sodium phosphate buffer (pH

6.5) and stopped after 10 min of incubation at 30 1C by add-

ing 750mL of 0.4 M Na2CO3. The release of o-nitrophenol

was measured by determining the absorbance at 420 nm.

One unit of oNPG activity was defined as the amount of

enzyme releasing 1 mmol o-nitrophenol min�1 under the

described conditions.

When lactose was used as the substrate, 20 mL of enzyme

solution was added to 480 mL of 600 mM lactose solution in

50 mM sodium phosphate buffer, pH 6.5. After 10 min of

incubation at 30 1C, the reaction was stopped by heating the

reaction mixture at 99 1C for 5 min. The reaction mixture

was cooled to room temperature, and the release of D-

glucose was determined colourimetrically using the glucose

oxidase/peroxidase (GOD/POD) assay (Kunst et al., 1988).

One unit of lactase activity (ULac) was defined as the amount

of enzyme releasing 1 mmol of D-glucose per minute under

the given conditions.

Gel electrophoresis and active staining

Native polyacrylamide gel electrophoresis (PAGE) and de-

naturing sodium dodecyl sulphate polyacrylamide gel elec-

trophoresis (SDS-PAGE) were performed on a PhastSystem

unit (Amersham, Uppsala, Sweden) using precast polyacry-

lamide gels (PhastGel 8–25, Amersham). For SDS-PAGE,

the enzyme was preincubated with SDS buffer (47 mM Tris-

HCl, pH 6.8 containing 34 mg mL�1 SDS, 0.1 mg mL�1

bromophenol blue, 5% v/v mercaptoethanol, and 15% v/v

FEMS Microbiol Lett 269 (2007) 136–144 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

137b-Galactosidase from L. acidophilus

glycerol) at 60 1C for 5 min. Coomassie blue staining was

used for the visualization of the protein bands. Active

staining for the visualization of the bands with b-galactosi-

dase activity was carried out using 4-methylumbelliferyl-

b-D-galactoside (MUG) as the substrate as described pre-

viously (Nguyen et al., 2006).

Isoelectric focusing in the range of pH 3–10 was per-

formed using the Multiphor System (Amersham) and pre-

cast, dry polyacrylamide gels (CleanGel IEF, Amersham)

rehydrated with carrier ampholytes as recommended by the

supplier. The broad pI marker protein kit, pH 3–10,

(Amersham) was used to determine the pI value. Protein

bands were Coomassie stained following the instructions of

the manufacturer.

Steady-state kinetic measurements

All steady-state kinetic measurements were obtained at

30 1C using oNPG and lactose as the substrates in 50 mM

sodium phosphate buffer, pH 6.5, with concentrations

ranging from 0.5 to 22 mM for oNPG and from 1 to

600 mM for lactose, respectively. The kinetic parameters

were calculated by nonlinear regression, and the observed

data were fit to the Henri–Michaelis–Menten equation

(SigmaPlot, SPSS Inc., IL).

Effects of pH and temperature

Three buffer systems, sodium citrate (50 mM, pH 4.0–5.5),

sodium phosphate (50 mM, pH 6.0–7.5), and borate

(50 mM, pH 8.0–9.0), were used for measuring the pH

optimum of enzyme activity. To determine the pH stability,

the enzyme samples were incubated at various pH values

and 37 1C for up to 50 h, and the remaining enzyme activity

was measured at time intervals using oNPG as the substrate.

The temperature optimum of enzyme activity was measured

by assaying the enzyme samples over the range of 20–70 1C

for 10 min. The thermal stability of the enzyme was studied

by incubating the enzyme samples in 50 mM sodium

phosphate buffer, pH 6.5, at the desired temperatures; at

certain time intervals, samples were withdrawn and the

residual activity was measured with oNPG as the substrate

under standard assay conditions.

Effects of various cations

To study the effect of various cations on b-galactosidase

activity (release of o-nitrophenol from oNPG), the enzyme

samples were assayed with 22 mM oNPG (in 10 mM Bis-

Tris, pH 6.5) in the presence of various cations with final

concentrations of 1.0, 10, or 100 mM (chloride or sulphate

form) at 30 1C for 10 min. The measured activities were

compared with the activity of the enzyme solution without

added cations under the same conditions.

Formation of GOS

The synthesis of GOS was carried out in the discontinuous

mode using purified b-galactosidase from L. acidophilus R22

(one lactase unit per mL of reaction mixture). Reaction

conditions were 205 g L�1 initial lactose concentration in

50 mM phosphate buffer (pH 6.5), varying process tem-

peratures (25, 30, 37, 42 1C), and constant agitation

(300 r.p.m.). Samples were withdrawn at intervals and the

composition of the GOS mixtures was analysed by capillary

electrophoresis (CE) and high-performance anion exchange

chromatography with pulsed amperometric detection

(HPAEC-PAD) following the methods described previously

(Splechtna et al., 2006).

DNA preparation

Chromosomal DNA was extracted from L. acidophilus R22

as described by Germond et al. (2003) with modifications.

The strain was grown anaerobically at 37 1C in MRS broth to

the mid-log phase. Cells were harvested by centrifugation

(8000 g, 10 min, 41), washed twice with 0.8% w/v NaCl, and

once with 60 mM EDTA. The cells were then subjected to

one freeze-and-thaw cycle, resuspended in TE buffer

(10 mM Tris-HCl pH 7.5, 1 mM EDTA) containing lyso-

zyme (8 mg mL�1) and mutanolysin (40 U mL�1), and in-

cubated at 37 1C for 1 h. One volume of 0.5% w/v SDS was

added to lyse the cells, and proteinase K was added to a final

concentration of 200 mg mL�1. After incubating the mixture

at 65 1C for 10 min, the DNA was extracted with phenol,

precipitated with isopropanol, and washed with 70% cold

ethanol. The DNA was then dissolved in TE buffer. After

DNA was dissolved, RNase A was added to a final concentra-

tion of 200 mg mL�1, and the solution was incubated at 35 1C

for 30 min. The final yield of DNA obtained was c.

0.5 mg mL�1.

DNA amplification procedure and subcloning ofb-galactosidase

The degenerated oligonucleotides used for PCR amplifica-

tion of the R22 b-galactosidase genes (lacLM) were designed

based on the published sequences of the L. acidophilus genes

for b-galactosidase (large subunit: GenBank accession no.

AB004867, small subunit: GenBank accession no.

AB004868) (Suzuki et al., 1997). The vector pCR-Blunt II-

TOPO (Invitrogen, Carlsbad, CA) was used for subcloning

the PCR-amplified product. Plasmid DNA was purified

using the Wizard Plus Miniprep DNA Purification System

(Promega, Madison, WI), and the resulting plasmid

pHAR22 contained the complete genes (lacL and lacM) of

b-galactosidase from L. acidophilus, which was confirmed by

sequencing.


138 T.-H. Nguyen et al.

Nucleotide sequencing and sequence analysis

The nucleotide sequence was determined by Bio S&T Inc.

(Montreal, Canada). Assembly and analysis of DNA se-

quences were performed using CHROMASPRO (version 1.33)

(Technelysium, Australia). The basis local alignment tool

(BLAST) from the National Center for Biotechnology Infor-

mation BLAST website was used for database searches.

Nucleotide sequence accession number

The GenBank accession number is EF053367 (nucleotide

sequence of both genes lacL and lacM).

Results

Purification of b-galactosidase fromL. acidophilus R22

b-Galactosidase was purified to apparent homogeneity from

lactose-grown cells of L. acidophilus R22 by a two-step

procedure (Table 1). The enzyme was purified c. 28-fold

from the crude cell extract to a specific activity of

230 U mg�1 of protein, using standard assay conditions with

oNPG as the substrate.

Properties of b-galactosidase from L.acidophilus R22

b-Galactosidase from L. acidophilus R22 is a heterodimer

(Mr�105 kDa) consisting of a large subunit of 72 kDa and a

smaller one of 35 kDa as judged by native PAGE (Fig. 1a)

and SDS-PAGE (Fig. 1b). Active staining on the native PAGE

gel using MUG as the substrate showed a single band of c.

105 kDa. Active staining on the SDS-PAGE gel after pre-

incubating the enzyme with denaturing SDS buffer at 60 1C

for 5 min showed that one band corresponding to the larger

subunit exhibited activity with MUG while the smaller

one did not. The isoelectric point of b-galactosidase from

L. acidophilus R22 was determined by isoelectric focusing,

and it was found to be c. 4.8 (data not shown).

Enzyme kinetics

The steady-state kinetic constants were determined for the

hydrolysis of lactose and oNPG. Kinetic analysis with

increasing concentrations of lactose as the substrate

(Fig. 2a) showed Michaelis–Menten kinetics with the

following parameters obtained by nonlinear regression:

Vmax = 28.8� 0.2 [mmol D-glucose released min�1

(mg protein)�1] and Km = 4.04� 0.26 (mM). Figure 2b

shows that the enzyme activity was inhibited by high

concentrations of oNPG and the following kinetic para-

meters were obtained: Vmax = 361� 12 [mmol o-nitrophenol

released min�1 (mg protein)�1], Km = 0.73� 0.07 (mM),

and Ki,s = 31.7� 3.5 (mM). The kcat values calculated were

50.4 and 632 s�1 when lactose and oNPG were used as the

Table 1. Purification of b-galactosidase from Lactobacillus acidophilus R22

Purification steps

Total

activity (U)

Total

protein (mg)

Specific

activity (U mg�1)

Purification

factor

Recovery

(%)

Crude cell extract 5120 616 8.3 1.0 100

HIC (Phenyl-Sepharose) 1040 70 14.8 1.8 20.2

Affinity chromatography

(p-aminobenzyl thiogalactoside-agarose)

405 1.76 230 27.7 7.9

1 2 3

1 2 3

669 kDa

440 kDa

232 kDa

250 kDa

75 kDa

140 kDa

67 kDa

37 kDa

10 kDa

(a)

(b)

Fig. 1. Native PAGE (a) and SDS-PAGE (b) of purified b-galactosidase

from Lactobacillus acidophilus R22. (a) Lane 1, active staining with MUG;

lane 2, molecular weight markers; lane 3, Coomassie blue staining of b-

galactosidase. (b) Lane 1, active staining with MUG; lane 2, recombinant

molecular weight markers (Bio-Rad); lane 3, Coomassie blue staining of

b-galactosidase.



substrates, respectively. The catalytic efficiencies kcat/Km of

12.5 and 865 (mM�1 s�1) for the two substrates, lactose and

oNPG, respectively, indicate that oNPG is the preferred

substrate of b-galactosidase from L. acidophilus R22.

Effects of pH and temperature

The optimal pH of b-galactosidase activity from L. acido-

philus R22 is in the range between pH 6.5 and 8.0 for both

lactose and oNPG hydrolysis. The enzyme is most stable at

pH 6.5, retaining more than 80% of its activity when

incubated at pH 6.5 and 37 1C for 48 h. The optimum

temperature of both lactose and oNPG hydrolysis was 55 1C

under standard assay conditions (10 min). The effect of

temperature on the stability of b-galactosidases from L.

acidophilus R22 was investigated in the presence of 20 mM

MgCl2. In the absence of Mg21, the enzyme lost its activity

very rapidly (data not shown). b-Galactosidase from L.

acidophilus R22 is most stable at 4 1C; the enzyme comple-

tely retained its activity after 30 days, and more than 90% of

its activity was observed after 45 days at 4 1C. The half-life

times (t1/2) of activity at 22, 37, and 45 1C were 180, 120, and

24 h, respectively.

Effects of various cations

Various cations were tested with respect to a possible

stimulating or inhibitory effect on enzyme activity. The

purified enzyme showed no specific requirements for metal

ions, with the exception of Mg21. The enzyme was activated

by 21% and 28% in the presence of 1 and 10 mM Mg21,

respectively. Complete inhibition of the enzyme activity was

observed in the presence of 10 mM Mn21, Cu21, or Zn21,

while the inhibitory effect of 10 mM Ca21 was c. 45%.

Synthesis of oligosaccharides

Figure 3 shows the course of formation of GOS by b-

galactosidase from L. acidophilus R22 in a discontinuous

[Lactose] (mM)

0 100 200 300 400 500 600

Vel

ocity

(µm

ol g

luco

se r

elea

sed

min

−1 m

g pr

otei

n−1 )

0

10

20

30

Velocity / [Lactose] 0 1 2 3 4 5 6

Vel

ocity

0

10

20

30

[oNPG] (mM)0 5 10 15 20 25

Rat

e (µ

mol

oN

P r

elea

sed

min

−1 m

g pr

otei

n−1 )

0

100

200

300

(a)

(b)

Fig. 2. Kinetic analysis of b-galactosidase from Lactobacillus acidophilus

R22 with (a) lactose as the substrate: the inset diagram shows the

Eadie–Hofstee plot; and (b) chromogenic oNPG as the substrate.

Time (h)

0 10 20 30 40

Car

bohy

drat

es (

g L−1

)

0

50

100

150

200

250

Lactose conversion (%)

0 20 60 8040 100

% G

OS

of t

otal

sug

ars

0

10

20

30

40

(a)

(b)

Fig. 3. Course of formation of GOS by b-galactosidase from Lactobacillus

acidophilus R22 in the discontinuous lactose conversion process at 30 1C,

using 205 g L�1 initial lactose in 50 mM phosphate buffer 11 mM MgCl2,

pH 6.5, and 1 ULac mL�1 of b-galactosidase. (a) Time course of reaction for

lactose conversion; symbols: lactose ( ), glucose (�), galactose (&), GOS

(�); (b) Formation of GOS during lactose conversion; symbols: total GOS

(�), disaccharides (�), trisaccharides (&), tetrasaccharides (D).



lactose conversion process at 30 1C, using 205 g L�1 initial

lactose in 50 mM phosphate buffer 11 mM MgCl2, pH 6.5,

and 1 ULac mL�1 of b-galactosidases. The maximum GOS

yield of about 38.5% of total sugars was obtained at around

75% lactose conversion, of which 15.5% were nonlactose

disaccharides, 19% were trisaccharides, and 4% were tetra-

saccharides. Process parameters were chosen in the range in

which the enzyme is most stable; however, varying the

process temperature had no effect on the yield and compo-

sition of GOS.

Comparison of amino acid sequences

The comparison on the amino acid level between pairs of

b-galactosidases from L. acidophilus R22, L. acidophilus

NCFM, L. acidophilus JCM1229, and some other Lactoba-

cillus spp. is presented in Table 2. The large subunit (lacL) of

b-galactosidase from L. acidophilus R22 shows 100% iden-

tity to the reported sequence of lacL from L. acidophilus

NCFM, and 99% identity to the sequence of lacL from

L. acidophilus JCM1229. The only difference between R22

lacL and the latter protein is found at position His302,

which is replaced by Asn in JCM1229 lacL. The small

subunit (lacM) of b-galactosidase from L. acidophilus R22

shows 100% identity to both reported sequences of lacM

from these L. acidophilus strains.

Discussion

b-Galactosidases of lactobacilli play an important role in a

number of commercial processes, e.g. milk processing or

cheese making (Hebert et al., 2000; Adam et al., 2004).

Surprisingly, b-galactosidases from lactobacilli have received

very little scientific attention, and only a few enzymes have

been characterized to date (Cesca et al., 1984; Kim &

Rajagopal, 2000; Nguyen et al., 2006). Recently, an addi-

tional attractive application of b-galactosidases from pro-

biotic bacteria such as bifidobacteria or lactobacilli has been

proposed, namely their use in the production of tailor-made

prebiotics targeting specifically at advantageous and bene-

ficial intestinal microorganisms (Rabiu et al., 2001; Rastall &

Maitin, 2002). b-Galactosidases, which efficiently hydrolyse

GOS structures, can also form GOS when they act in their

transgalactosylation mode. It is conceivable that b-galacto-

sidases from probiotic microorganisms produce GOS struc-

tures that have a strong prebiotic potential.

b-Galactosidase from the probiotic strain L. acidophilus

R22 was studied in detail pertaining to some of its physical

and biochemical properties. This enzyme is a heterodimer

with a molecular mass of c. 105 kDa. In accordance with

reports in the literature on the sequences of b-galactosidases

from L. acidophilus (Suzuki et al., 1997) and Lactobacillus

helveticus (Fortina et al., 2003), and with a previous study on

b-galactosidases from L. reuteri (Nguyen et al., 2006), the

enzyme from L. acidophilus R22 consists of two subunits of

c. 35 and 72 kDa, which is in good agreement with the

calculated molecular masses of 35 817 and 73 253 Da (http://

au.expasy.org/tools/protparam.html) deduced from their

sequences. It was reported previously that the larger subunit

of L. reuteri b-galactosidases showed activity after subunit

dissociation and separation by SDS-PAGE while the smaller

subunit was inactive. It is noteworthy that this observation

was also made with b-galactosidases from L. acidophilus

R22.

The steady-state kinetic constants were determined for

the hydrolysis of lactose, the natural substrate, and oNPG,

an artificial chromogenic substrate. Substrate inhibition was

observed at a high concentration of oNPG, while it was not

the case for lactose. The Km value of 4 mM determined for

lactose is lower than the values for b-galactosidases from

Lactobacillus spp. reported hitherto, 14 mM for Lactobacillus

crispatus (Kim & Rajagopal, 2000), 13 mM for L. reuteri

L103 b-galactosidases, and 31 mM for L. reuteri L461 b-

galactosidases (Nguyen et al., 2006), and compares even

more favourably with the reported values for some com-

monly used commercial b-galactosidases (Aspergillus oryzae,

36–180 mM; A. niger, 54–100 mM; Kluyveromyces fragilis,

15–52 mM; Kluyveromyces lactis, 35 mM) (Jurado et al.,

2002; de Roos, 2004). This low Km value of b-galactosidase

from L. acidophilus R22 can be useful when a close to

complete conversion of lactose is desired, e.g. in lactose-free

dairy products.

b-Galactosidase from L. acidophilus R22 shows a specific

requirement for Mg21 for both its activity and stability. This

requirement for divalent ions for optimal stability and

activity is well described for several microbial b-galactosi-

dases, most prominently for the Escherichia coli lacZ b-

galactosidase (Nakayama & Amachi, 1999); yet, the exact

mechanisms of activation and stabilization by metal ions are

still debated. In a recent structural study, a magnesium ion

was identified in the active site of lacZ, close to the site where

various substrate analogues and inhibitors bind (Juers et al.,

2000). As b-galactosidase from L. acidophilus R22 shows a

comparable requirement for Mg21, it can be speculated that

this enzyme also has a binding site for Mg21 close to its

active site.

The main GOS structures formed from lactose by b-

galactosidases from L. acidophilus R22 are very similar to

those found for b-galactosidases from L. reuteri (Splechtna

et al., 2006). These predominant oligosaccharide pro-

ducts are b-D-Galp-(1 ! 6)-D-Glc (allolactose), b-D-

Galp-(1 ! 6)-D-Gal, b-D-Galp-(1 ! 3)-D-Gal, b-D-Galp-

(1 ! 3)-D-Glc, b-D-Galp-(1 ! 6)-Lac and b-D-Galp-

(1 ! 3)-Lac. Hence, b-galactosidase from L. acidophilus

R22 is very similar to b-galactosidase from L. reuteri in that

they show a high specificity for the formation of b1 ! 6

and b1 ! 3 linkages, and no major products with b1 ! 4



Tab

le2.

Am

ino

acid

iden

tity

bet

wee

npai

rsofb-

gal

acto

sidas

esfr

om

Lact

obac

illus

spp.w

ith

the

Gen

Ban

kac

cess

ion

num

ber

sgiv

enfo

rth

ere

spec

tive

sequen

ces

%A

min

oac

idid

entity

withb-

gal

acto

sidas

ela

rge

subunit

(lacL

)fr

om

:

L.ac

idophilu

s

R22

L.ac

idophilu

s

NC

FM

L.re

ute

ri

L103

L.hel

veticu

s

L.jo

hnso

nii

NC

C533

L.pla

nta

rum

WC

FS1

L.pla

nta

rum

WC

FS1

L.co

ryniform

isL.

sake

iE.

coli

lacz

(a)La

rge

subunit

(lacL

)

L.ac

idophilu

sR22

EF053367

100

L.ac

idophilu

sN

CFM

AA

V43287

100

100

L.ac

idophilu

sJC

M1229

BA

A20536

99

99

100

L.re

ute

riL1

03

ABF7

2116

74

74

74

100

L.hel

veticu

sC

AD

55499

77

77

77

73

100

L.jo

hnso

nii

NC

C533

AA

S08676

75

75

75

69

72

100

L.pla

nta

rum

WC

FS1

CA

D65569

64

64

64

64

64

62

100

L.co

ryniform

isA

BD

96610

63

63

63

64

64

62

99

100

L.sa

keiC

AA

57730

59

59

59

58

58

58

59

59

100

E.co

lila

czV

00296

33

33

33

33

33

34

35

35

33

100

%A

min

oac

idid

entity

withb-

gal

acto

sidas

esm

alls

ubunit

(lacM

)fr

om

:

L.ac

idophilu

s

R22

L.ac

idophilu

s

NC

FM

L.ac

idophilu

s

JCM

1229

L.re

ute

ri

L103

L.hel

veticu

s

L.jo

hnso

nii

NC

C533

L.pla

nta

rum

WC

FS1

L.co

ryniform

isL.

sake

i

(b)Sm

alls

ubunit

(lacM

)

L.ac

idophilu

sR22

EF053367

100

L .ac

idophilu

sN

CFM

AA

V43288

100

100

L.ac

idophilu

sJC

M1229

BA

A20537

100

100

100

L.re

ute

riL1

03

ABF7

2117

71

71

71

100

L.hel

veticu

sC

AD

55500

78

78

78

70

100

L.jo

hnso

nii

NC

C533

AA

S08675

71

71

71

65

71

100

L.pla

nta

rum

WC

FS1

CA

D65570

64

64

64

68

63

61

100

L.co

ryniform

isA

BD

96612

64

64

64

68

63

61

98

100

L.sa

keiC

AA

57731

57

57

57

58

55

54

58

58

100



linkages are formed. A strong prebiotic effect was attributed

to some of these sugars in a recent study comparing a

number of disaccharides with respect to in vitro prebiotic

selectivity (Sanz et al., 2005). Therefore, it is likely that the

GOS mixtures produced by b-galactosidase from L. acido-

philus have a significant potential as a prebiotic.

Comparison of the amino acid sequences of several

b-galactosidases showed high identity between both lacL

and lacM from L. acidophilus and other heterodimeric

b-galactosidases from lactobacilli, which were in the range

of 57–78%, with the highest identities found between lacLM

R22 and L. helveticus and Lactobacillus johnsonii b-galacto-

sidases. In fact, these two species are closely related to

L. acidophilus (Stiles & Holzapfel, 1997; Holzapfel et al.,

2001). Furthermore, the large subunit (lacL) of L. acidophi-

lus b-galactosidase shows 33% amino acid identity to lacZ of

E. coli and has thus been classified into glycosyl hydrolase

family 2 (Henrissat, 1991; Coutinho & Henrissat 1999;

Nakayama & Amachi, 1999).

Acknowledgement

This research study was supported by the Research Centre

Applied Biocatalysis (Graz, Austria).

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characterization and molecular cloning of a heterodimeric β-galactosidase from the probiotic strain...

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