Characterization of a heterodimeric GH2 β-galactosidase from lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides

Article abstract of DOI:10.1021/jf103832q

The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli. Subsequently, the recombinant β-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity because o-nitrophenyl-β-d-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K_m and k_cat values for oNPG and lactose were 0.6 mM and 180 s^-1 and 20 mM and 43 s^-1, respectively. The enzyme is inhibited competitively by its two end-products d-galactose and d-glucose (K_i values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K _i/K_m, this inhibition is only very moderate and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of β-(1→6)-linked transgalactosylation products, whereas β-(1→3)-linked compounds were formed to a lesser extent and β-(1→4)-linked reaction products could not be detected.

Full text of DOI:10.1021/jf103832q

ARTICLE
pubs.acs.org/JAFC
Characterization of a Heterodimeric GH2 β-Galactosidase from
Lactobacillus sakei Lb790 and Formation of Prebiotic
Galacto-oligosaccharides
Sanaullah Iqbal,† Thu-Ha Nguyen,†,‡ Hoang Anh Nguyen,†,§ Tien Thanh Nguyen,|| Thomas Maischberger,†
Roman Kittl,† and Dietmar Haltrich*,†
†Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of Natural Resources and Life
Sciences, Vienna, Austria
‡ACIB GmbH, Graz, Austria
§Department of Biochemistry and Food Biotechnology, Hanoi University of Agriculture, Hanoi, Vietnam
School of Biotechnology and Food Technology, Hanoi University of Science and Technology, Hanoi, Vietnam
ABSTRACT: The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside
hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli. Subsequently, the recombinant β-galactosidase
LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity
because o-nitrophenyl-β-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP
analogues were not. The Km and kcat values for oNPG and lactose were 0.6 mM and 180 sꢀ1 and 20 mM and 43 sꢀ1, respectively.
The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (Ki values of 180 and 475 mM,
respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, Ki/Km, this inhibition is only very moderate
and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosyla-
tion activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L.
The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose
disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The
enzyme showed a strong preference for the formation of β-(1f6)-linked transgalactosylation products, whereas β-(1f3)-linked
compounds were formed to a lesser extent and β-(1f4)-linked reaction products could not be detected.
KEYWORDS: β-galactosidase, lactase, Lactobacillus sakei, transgalactosylation, galacto-oligosaccharides
’ INTRODUCTION
with modular organization, has been studied in considerable
detail.5,7,8 The β-galactosidase (EC 3.2.1.23) of L. sakei is
encoded by two genes, lacL and lacM, which are partly over-
lapping and out of frame, which suggests a coordinate regulation
at the level of translation to produce an equal amount of both
subunits that together form the active heterodimeric β-
galactosidase.8 Despite this previous study on the genes encoding
β-galactosidase in L. sakei, this lactose-hydrolyzing enzyme from
a technologically important organism has not been studied in
detail, and interestingly only very few lactobacillal β-galactosi-
dases have been characterized in depth with regard to their
biochemical properties.9ꢀ13 This is quite surprising considering
the importance of lactobacilli in food technology and especially
in dairy applications. The complete genome sequences of a
number of fermentative and commensal LAB species including
Lactic acid bacteria (LAB) have been used extensively for the
preservation and processing of foods such as milk, meat, and
vegetables. Lactobacillus sakei, an important member of LAB,
belongs to the main bacterial flora of fresh meat and fish and is
best known for its use in the fermentation of various meat
products, improving properties that allow better preservation
and storage of fresh meat. L. sakei has furthermore been isolated
from several other raw fermented food products of plant and
animal origin, including sauerkraut, sourdough, smoked fish, or
silage.1ꢀ4 To speed and improve the ripening process of fer-
mented meat products, glucose and sometimes lactose are added
as exogenous substrates. The utilization of the milk sugar lactose
is a primary function of lactobacilli and other LAB. The mechan-
ism by which lactose is transported into the cell determines
largely the subsequent pathway for the hydrolysis of this di-
saccharide. In several Lactobacillus spp. lactose is transported via
phosphotransferase systems, which results in phosphorylation of
lactose concurrent with its uptake. The resulting lactose-60-
phosphate is then hydrolyzed by phospho-β-galactosidase. Alter-
natively, lactose is taken up by secondary transport systems, and
lactose is further metabolized by β-galactosidase within the
cell.5,6 The organization of these genes involved in lactose
metabolism, which often form operons or operon-like structures
1
L. sakei have been published by now, which opens new
possibilities for comprehensive studies of a variety of biological
processes.
β-Galactosidases catalyze the hydrolysis of the β-1,4-D-glyco-
sidic linkage of lactose and structurally related substrates.
Received: October 1, 2010
Revised:
March 15, 2011
Accepted: March 15, 2011
Published: March 15, 2011
r
2011 American Chemical Society
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β-Galactosidases have two main biotechnological uses in the
food industry—the removal of lactose from milk and dairy
products14,15 as well as the production of galacto-oligosacchar-
ides (GalOS) exploiting the transglycosylation potential of these
enzymes.16ꢀ18 GalOS belong to the prebiotics, which are defined
as a “selectively fermented ingredient that allows specific
changes, both in the composition and/or activity in the gastro-
intestinal microbiota that confers benefits upon host well-being
and health”.19 GalOS have been classified as one of the few
proven prebiotics fulfilling the following three criteria: (i)
resistance to gastric acidity, hydrolysis by mammalian enzymes,
and gastrointestinal absorption; (ii) fermentation by intestinal
microflora; and (iii) selective stimulation of the growth and/or
activity of intestinal bacteria associated with health and well-
being.19
GalOS are complex mixtures of different oligosaccharides and
are attracting increasing attention as prebiotic ingredients in
various food applications including infant milk formula, as is
evident from a number of reviews on GalOS that have been
published recently.16ꢀ18 The spectrum of the oligosaccharides
making up these mixtures strongly depends on the source of the
β-galactosidase used for the biocatalytic reaction and on the
conversion conditions used in their production. Because these
differences in GalOS spectrum and yield are a result of structural
and/or mechanistic differences between β-galactosidases from
different sources, a detailed knowledge of novel, yet-unexplored
β-galactosidases from various strains can be of significant
interest.16,18 Rabiu et al.20 and Tzortzis et al.21 produced various
GalOS mixtures using lactose as substrate and β-galactosidases
from different probiotic bifidobacteria. Subsequently, they
showed that these different mixtures typically resulted in better
growth of that strain that had served as the source of the enzyme
for GalOS production. This concept can serve as the basis for a
new generation of functionally enhanced, targeted oligosacchar-
ides and has increased the interest in β-galactosidases from
beneficial probiotic organisms.22
genes encoding the large and small subunits (lacLM) of β-galactosidase
from L. sakei Lb790. E. coli cells were grown on LuriaꢀBertani (LB) agar
plates (10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl, and 15 g/L
agar) containing the appropriate antibiotics (100 μg/mL ampicillin or
50 μg/mL kanamycin) required for maintaining the plasmids or in
Terrific-Broth (TB) medium (12 g/L peptone, 24 g/L yeast extract,
0.4% (v/v) glycerol, and 13.6 g/L KH2PO4) supplemented with 100 μg/
mL ampicillin. E. coli DH5R electrocompetent cells were from
Invitrogen.
DNA Amplification and Subcloning of the β-Galactosi-
dase Genes. Plasmid pEH9S, which contains the complete genes
lacLM encoding heterodimeric β-galactosidase from L. sakei Lb790, the
construction of which was described previously,25 was used as template
for the amplification of these lacLM genes. The oligonucleotides
used for PCR amplification, LacLMSak_Forward (50-ACCATGGA-
ACCTAATATTCAATGGTTAG-30) and LacLMSak_Reverse (50-
TTTTCTCGAGTGAAAACGAAATTTCAA-30), were designed on
the basis of the published sequence of the β-galactosidase gene from
L. sakei subsp. sakei (GenBank accession no. X82287) and were obtained
from VBC Biotech (Vienna, Austria). These primers created a restriction
site (underlined above), NcoI and XhoI, at the 50 and 30 ends of the gene
fragment, respectively. The amplification was performed in a total
volume of 25 μL containing 0.2 mM of each deoxynucleotide triphos-
phate, 0.5 μM of each primer, 12.5 μL of 5ꢁ Phusion HF buffer (final
concentration of MgCl2 was 1.5 mM), 1 U of Phusion DNA Polymerase,
and 1ꢀ40 ng of plasmid DNA. The initial denaturation step at 98 °C for
30 s was followed by 30 cycles of denaturation at 98 °C for 10 s,
annealing at 60 °C for 20 s, and extension at 72 °C for 1 min. The final
cycle was followed by an additional 5 min elongation step at 72 °C. The
amplified PCR product was visualized by gel electrophoresis in an 0.8%
agarose gel (containing 0.2 μg/mL ethidium bromide) in 1ꢁ TAE
(TrisꢀacetateꢀEDTA) electrophoresis buffer (4.8 g/L Tris base, 1.2 g/
L acetic acid, 1 mM EDTA, pH 8.0) under UV light. The amplified
product was purified from the agarose gel using the Wizard SV gel and
PCR Clean-up System (Promega, Madison, WI). The Zero Blunt
TOPO PCR Cloning Kit containing the vector pCR-Blunt II-TOPO
(Invitrogen) was used for subcloning of PCR-amplified blunt end-
products. The resulting plasmid pBTlacLMSak contained the complete
genes (lacL and lacM) of β-galactosidase from L. sakei Lb790 as was
confirmed by sequencing performed by a commercial provider.
Construction of the lacLM Expression Vector. A DNA
fragment containing the β-galactosidase genes was obtained from the
pBTlacLMSak plasmid after digestion with NcoI and XhoI restriction
enzymes. The resulting fragments were then ligated into the NcoIꢀXhoI
fragment of expression vector pET21d (Novagen, Darmstadt,
Germany) so that a C-terminal His6-tag was added. The resulting
plasmid was transformed into E. coli DH5R electrocompetent cells,
and the correct sequence was verified by DNA sequencing. The
expression vector pSIlacLMSak thus obtained was then transformed
into E. coli BL21 Star (DE3) for the heterologous overexpression of the
β-galactosidase LacLM.
The objective of this work was to characterize in detail the β-
galactosidase from L. sakei Lb790 and to evaluate the transga-
lactosylation activity of this enzyme. Recent data are indicating
that L. sakei might be of interest as a beneficial LAB, making it
attractive as a probiotic strain,23 and hence prebiotic oligosac-
charides produced by its enzyme might be of functional interest.
’ MATERIALS AND METHODS
Chemicals and Enzymes. All chemicals were of the highest
quality available and were purchased from Sigma (St. Louis, MO) unless
otherwise stated. Phenylmethanesulfonyl fluoride (PMSF) and glucose
oxidase (GOD) from Aspergillus niger (lyophilized, 205 U/mg of enzyme
preparation) were obtained from Fluka (Buchs, Switzerland), whereas
isopropyl-β-D-thiogalactoside (IPTG) and 1,4-dithiothreitol (DTT)
were from Roth (Karlsruhe, Germany). The test kit for the determina-
tion of D-galactose was purchased from Megazyme (Wicklow, Ireland).
All chromatographic materials were from Amersham Biosciences
(Uppsala, Sweden), and restriction enzymes and corresponding buffers
were from Fermentas (Vilnius, Lithuania).
Bacterial Strains and Culture Conditions. L. sakei Lb790, the
original source of the β-galactosidase genes, was isolated from meat as
described in Schillinger and L€ucke24 and was received from the culture
collection of the Norwegian University of Life Sciences, Ås, Norway.
Escherichia coli BL21 Star (DE3) (Invitrogen, Carlsbad, CA) was used as
expression host for the expression vector carrying the two overlapping
Expression of β-Galactosidase. E. coli BL21 Star (DE3) carrying
pSIlacLMSak was grown at 37 °C in 30 mL of TB medium containing
100 μg/mL ampicillin until an optical density at 600 nm (OD600) of 0.3
was reached. IPTG or lactose, added to the culture medium in final
concentrations of 0.1, 1, or 10 mM for IPTG and 5 or 10 g/L for lactose,
was then used as inducer, and the cultures were incubated further at
18 °C for 24 h until OD600 of 8.5 ( 1. Cells were then harvested by
centrifugation (3300g, 10 min, 4 °C), washed, resuspended in sodium
phosphate buffer (50 mM, pH 6.5), and disrupted by using a French
press (Aminco, Silver Spring, MD). Cell debris was removed by
centrifugation (16000g, 15 min, 4 °C) to obtain the crude cell extract.
Protein Purification. The crude cell extract was loaded onto a
10 mL Ni2þ-Sepharose fast flow column that was pre-equilibrated with
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buffer A (20 mM sodium phosphate, 0.5 M NaCl, and 20 mM imidazole,
pH 6.5). Proteins were eluted at a rate of 1.5 mL/min by using a linear
100 mL gradient of 20ꢀ500 mM imidazole. The active fractions were
pooled, desalted, and concentrated (Amicon Ultra Centrifugation filter
tubes, 10 kDa cutoff; Millipore, Billerica, MA). The purified β-galacto-
sidase was stored in 50 mM sodium phosphate buffer (pH 6.5) at 4 °C
for further analysis. Protein concentrations were determined according
to the method of Bradford using bovine serum albumin as standard.
Gel Electrophoresis and Activity Staining. Native polyacry-
lamide gel electrophoresis (PAGE), denaturing sodium dodecyl sulfateꢀ
polyacrylamide gel electrophoresis (SDS-PAGE), and activity staining
using 4-methylumbelliferyl β-D-galactoside (MUG) as the substrate, as
well as isoelectric focusing in the range of pH 3ꢀ10, were carried out as
previously described.26
Enzyme Assays. β-Galactosidase (β-gal) activity was determined
using o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose as the
substrate, as described previously.26,27 In brief, the standard assay of β-
gal activity was performed at 30 °C using oNPG as the substrate. The
reaction was started by adding 20 μL of enzyme sample to 480 μL of
22 mM oNPG in buffer (50 mM sodium phosphate buffer, pH 6.5) and
stopped after exactly 10 min by adding 750 μL of 0.4 M Na2CO3. The
release of o-nitrophenol (oNP) was assessed by measuring the absor-
bance at 420 nm. One unit of β-galactosidase (UoNPG) is defined as the
amount of enzyme releasing 1 μmol of oNP per minute under the
reaction conditions given above. When β-gal activity was determined
with its natural substrate lactose, 20 μL of enzyme sample was added to
480 μL of substrate solution (600 mM lactose in 50 mM sodium
phosphate buffer, pH 6.5) and incubated at 30 °C for 10 min. The
reaction was stopped by boiling the sample for 5 min. Glucose released
was measured with an enzymatic assay based on glucose oxidase (GOD)
and peroxidase (POD).28 One unit of lactase activity (ULac) refers to the
amount of enzyme necessary for the formation of 1 μmol of D-glucose
per minute under the described conditions. All measurements and
experiments were performed at least in duplicate, and the experimental
error was always below 5%.
Figure 1. SDS-PAGE analysis of recombinant β-galactosidase from L.
sakei Lb790. Lane 1 shows active staining of the purified β-galactosidase
with MUG (marked on the gel); lanes 2, 3, and 4 show Coomassie blue
staining of purified β-galactosidase, crude enzyme extract, and molecular
mass marker (Amersham), respectively.
o-nitrophenyl-R-D-glucopyranoside, p-nitrophenyl-β-D-glucopyranoside,
p-nitrophenyl-β-D-mannopyranoside, and p-nitrophenyl-β-D-xylopyrano-
side, were tested as potential substrates. Enzyme activities were measured
using 22 mM of each of the substrates under otherwise standard β-gal
assay conditions.
Formation of Galacto-oligosaccharides. The synthesis of
galacto-oligosaccharides (GalOS) was carried out in discontinuous
mode at 37 °C using purified recombinant β-galactosidase from L. sakei
Lb790 (1.2 lactase U per mL of reaction mixture). Reaction conditions
were 215 g/L initial lactose concentration in sodium phosphate buffer
(50 mM, pH 6.5) containing 1 mM MgCl2. Continuous agitation was
applied at 300 rpm. Samples were withdrawn periodically, and the
composition of the GalOS mixture was analyzed by thin layer chroma-
tography (TLC), capillary electrophoresis (CE), and high-performance
anion-exchange chromatography with pulsed amperometric detection
(HPAEC-PAD), following methods described previously.29 Individual
GOS compounds were identified and quantified by using authentic
standards and the standard addition technique.29,30
Characterization of Recombinant β-Galactosidase. Steady-
state kinetic data were obtained at 30 °C using either oNPG or lactose as
the substrate in 50 mM sodium phosphate buffer (pH 6.5) with
concentrations ranging from 0 to 25 mM for oNPG and from 0 to
600 mM for lactose. Furthermore, the inhibition of oNPG hydrolysis by
D-glucose as well as the inhibition of lactose hydrolysis by D-galactose
was investigated, and the respective inhibition constants were deter-
mined. The kinetic parameters and the inhibition constants were
calculated using nonlinear regression, fitting the observed data to the
HenriꢀMichaelisꢀMenten equation using SigmaPlot (SPSS, Chicago,
IL).
The pH dependence of the enzymatic release of oNP from oNPG as
well as that of D-glucose from lactose was assessed in the range of pH
4ꢀ9 using BrittonꢀRobinson buffer (containing 20 mM each of
phosphoric, acetic, and boric acid adjusted to the required pH with
NaOH). The temperature dependence of β-gal activity (both oNPG and
lactase activity) was determined by measuring activity in the range
of 20ꢀ70 °C for 10 min. To determine the pH stability of L. sakei
β-galactosidase, enzyme samples were incubated at 30 °C and at pH
values ranging from 4 to 9 for up to 24 h, and the remaining β-gal activity
was measured for different time points using the standard oNPG assay.
The temperature stability of the enzyme was studied by incubating
enzyme samples in sodium phosphate buffer (50 mM, pH 6.5) at various
temperatures (4, 22, 30, 37, and 42 °C). At certain time intervals,
samples were withdrawn and the residual β-gal activity was measured
with oNPG as the substrate under standard assay conditions.
’ RESULTS
Overexpression in E. coli and Purification of β-Galactosi-
dase from L. sakei. A T7 RNA polymerase-based expression
system was used for the overexpression of the lacLM genes of L.
sakei in E. coli. The coding region of these two overlapping genes,
lacL and lacM,8 was cloned into the NcoI- and XhoI-cloning sites
of pET21d, resulting in the expression plasmid pSIlacLMSak.
Gene expression in E. coli BL21 Star (DE3) was induced after
OD600 reached 0.3, using different concentrations of IPTG
(0ꢀ10 mM) and lactose (5 and 10 g/L), and the cultivation
was continued at 18 °C for 20 h. The highest levels of β-gal
activity were obtained when 0.1 mM IPTG was used for
induction. Approximately 2800 U of β-gal activity/L of fermen-
tation medium was produced under these conditions with a
specific activity of 17 U/mg of protein. The His-tagged enzyme
was subsequently purified directly from the crude cell extract by a
single-step purification procedure using a Ni2þ-Sepharose fast
flow column, which gave an overall yield of ∼70% and an
apparently homogeneous β-galactosidase preparation with a
specific activity of 83 U/mg of protein.
Gel Electrophoresis Analysis. β-Galactosidase from L. sakei
is a heterodimer of ∼110 kDa, consisting of a large subunit of
∼72 kDa and a small subunit of ∼35 kDa as analyzed by SDS-
PAGE (Figure 1). This compares well to the calculated molecular
masses of the two subunits (72 457 and 35 254 Da, respectively)
deduced from their sequences. Activity staining of purified
Substrate Specificity. To determine the specificity of recombi-
nant β-galactosidase from L. sakei Lb790, several structurally related
chromogenic substrates, that is, p-nitrophenyl-R-D-galactopyranoside,
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Table 1. Kinetic Parameters for Recombinant, His-Tagged β-
Galactosidase from L. sakei Lb790 Overexpressed in E. coli for
the Hydrolysis of oNPG and Lactose
substrates
kinetic parameter
lactose
oNPG
Km (mM)
vmax (μmol minꢀ1 mgꢀ1
kcat (sꢀ1
20 ( 2
24 ( 1
43 ( 1
2100
0.60 ( 0.19
98 ( 8
)
)
180 ( 14
290000
kcat/Km (Mꢀ1 sꢀ1
)
Ki,Gal (mM)
180 ( 17
9.0
Ki,Gal/Km,Lac
Ki,Glc (mM)
475 ( 200
Ki,Glc/Km,oNPG
790
β-galactosidase (after preincubation with SDS buffer at 60 °C for
5 min to separate the subunits) directly on the SDS-PAGE gel
using 4-methylumbelliferyl β-D-galactoside as the substrate
showed that only the larger subunit exhibited activity with this
substrate (Figure 1), whereas the smaller subunit did not. In
addition, nondissociated β-galactosidase of Mr ∼110 000
showed activity on the SDS-PAGE gel under these conditions
(not shown). The treatment of the enzyme preparation with
denaturing SDS buffer at 99 °C for 5 min apparently resulted in
complete denaturation of both subunits, as is evident from the
total loss of activity on the gel. The isoelectric point (pI) of
recombinant β-galactosidase from L. sakei was determined by
isoelectric focusing, and it was found to be in the range of 5.2
(data not shown), which is in agreement with the calculated
theoretical pI of 5.05.
Figure 2. Kinetic analysis of β-galactosidase activity from L. sakei with
(A) chromogenic oNPG as the substrate and (B) lactose as the substrate.
Enzyme Kinetics. The steady-state kinetic constants deter-
mined for the hydrolysis of the substrates lactose and oNPG as
well as the inhibition constants for both end-products, D-galac-
tose and D-glucose, for β-galactosidase from L. sakei are summar-
ized in Table 1; the kcat values were calculated on the basis of the
theoretical vmax values obtained from the experimental data by
nonlinear regression and using a molecular mass of 108.5 kDa for
the enzyme (LacLM plus His-tag). The catalytic efficiencies
(kcat/Km) for these two substrates, lactose and oNPG, indicate
that oNPG is the preferred substrate, because of the more
favorable Km and kcat values. As is evident from the Michaelisꢀ
Menten plots (Figure 2), β-galactosidase from L. sakei is inhibited by
elevated concentrations of the substrate oNPG, whereas no compar-
able substrate inhibition could be observed for lactose when present
in concentrations of up to 600 mM.
The hydrolysis end-product D-galactose was found to compe-
titively inhibit the hydrolysis of lactose. This inhibition, however,
is not pronounced as is obvious from the ratio of the Michaelis
constant for lactose and the inhibition constant for D-galactose
(Ki,Gal/Km,Lac = 9.0). oNPG was used as the substrate for
investigation of the inhibition by the second end-product,
D-glucose. The inhibitory effect of D-glucose is found to be even
less pronounced (Ki,Glc/Km,oNPG = 790), and D-glucose is also a
competitive inhibitor of β-gal activity.
The effects of D-galactose and D-glucose on β-gal activity
during the hydrolysis of oNPG were investigated in more detail
(Figure 3). Again, it is obvious that D-galactose is the more
efficient inhibitor. When added at a concentration of 200 mM to
the reaction mixture, β-galactosidase activity was reduced by
approximately 50%, whereas D-glucose in similar concentrations
Figure 3. Inhibition of β-galactosidase activity from L. sakei Lb790 by
its reaction products D-galactose (O) and D-glucose (b). oNPG (2 mM)
was used as substrate for assaying β-gal activity.
reduced the activity by only 14% compared to reactions without
the addition of the monosaccharides.
Effect of Temperature and pH on Enzyme Activity and
Stability. The optimum temperature of the activity of β-galac-
tosidase from L. sakei is 55 °C for both oNPG and lactose
hydrolysis (Figure 4A) for the 10 min assay. The Arrhenius plot
of the temperature dependence of lactase activity was found to be
linear in the range of 25ꢀ55 °C (data not shown), and the
activation energy Ea for β-galactosidase was calculated as 41 kJ/
mol. The pH optimum of β-gal activity is pH 6.5 when using
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Figure 6. Composition of the sugar mixture during lactose conversion
by recombinant β-galactosidase from L. sakei. The reaction was carried
out at 37 °C using 215 g/L initial lactose concentration in 50 mM
sodium phosphate buffer (pH 6.5), 1 mM MgCl2, and 1.2 ULac/mL of
homogeneous enzyme: ([) lactose; (b) glucose; (O) galactose; (2)
GalOS. Monosaccharides were measured enzymatically, and lactose and
GalOS were quantified by HPAEC-PAD and CE.
Figure 4. Temperature optimum (A) and pH optimum (B) of recom-
binant β-galactosidase from L. sakei: (O) oNPG as the substrate; (b)
lactose as the substrate.
The temperature stability of recombinant β-galactosidase
from L. sakei was analyzed at different temperatures under
otherwise standard assay conditions (pH 6.5). The enzyme has
a half-life time of activity (t1/2) of approximately 20 days at 4 °C,
and after a 1 h incubation at 37 and 42 °C without the addition of
any stabilizing reagents, the enzyme retained 56 and 34% of its
activity, respectively. Furthermore, the effect of various concen-
trations of Mg2þ on the thermal stability of the enzyme was
investigated. In the presence of 10 mM Mg2þ, the enzyme
retained 90% of its activity after a 5 h incubation at 42 °C. A
lower concentration of Mg2þ of only 1 mM proved to be less
effective with respect to stabilization, and >60% of the activity
was lost after 5 h at 42 °C, whereas the enzyme completely
inactivated within 5 h at that temperature without the addition of
Mg2þ (Figure 5A). These experiments not only showed a
significant stabilization but also an activation of β-galactosidase
activity in the presence of Mg2þ (Figure 5A). β-Galactosidase
from L. sakei is most stable in the pH range of 6.0ꢀ7.5, retaining
more than 80 and 60% of its activity after incubations of 2 and 10
h, respectively, in this pH range and at 30 °C (Figure 5B). The
enzyme is not stable at pH values below 5 and loses >85% of its
activity when kept at this pH and 30 °C within 1 h (data not
shown).
Substrate Specificity. To study the substrate specificity of β-
galactosidase from L. sakei Lb790, various structurally related
chromogenic substrates were used under standard assay condi-
tions. No activity (<1% relative to the standard substrate oNPG)
was detected when using p-nitrophenyl-R-D-galactopyranoside,
o-nitrophenyl-R-D-glucopyranoside, p-nitrophenyl-β-D-gluco-
pyranoside, p-nitrophenyl-β-D-mannopyranoside, and p-nitro-
phenyl-β-D-xylopyranoside as substrates, indicating that the
enzyme is a β-galactosidase with narrow substrate specificity.
Synthesis of GalOS. Lactose conversion and product forma-
tion of a typical discontinuous conversion reaction, using 215 g/
L initial lactose in 50 mM sodium phosphate buffer þ 1 mM
MgCl2, pH 6.5, and 1.2 ULac/mL of β-gal activity at 37 °C, are
shown in Figure 6. As the reaction proceeded, D-glucose and
D-galactose, the primary hydrolysis products, were formed but
Figure 5. (A) Stability of recombinant β-galactosidase from L. sakei at
42 °C without the presence of MgCl2 (O) and with the presence of
1 mM (b) and 10 mM (ꢁ) MgCl2. (B) pH stability of recombinant β-
galactosidase from L. sakei Lb790 at 30 °C in BrittonꢀRobinson buffer
(pH 5ꢀ9): residual activity after 2 h (ꢁ), 5 h (b), and 10 h (O).
oNPG as substrate, whereas an optimum pH of 7.5 was found for
lactose (Figure 4B). Overall, the pH curve is rather narrow for
this β-galactosidase, showing 80% of its maximal activity in a pH
range of only 1ꢀ1.5 units.
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Table 2. Individual GalOS Components Produced by
β-Galactosidase from Lactobacillus sakei Lb790a
degree of lactose conversion
15%
35%
60%
77%
85%
90%
GalOS component (g/L)
D-Galp-(1f3)-D-Glc
D-Galp-(1f6)-D-Glc
(allolactose)
1
2
nd
3.31
0.44
8.00
0.44
15.1
0.94
19.3
1.82
21.9
2.20
20.0
3
4
5
6
D-Galp-(1f3)-D-Gal
D-Galp-(1f6)-D-Gal
D-Galp-(1f6)-D-Lac
D-Galp-(1f3)-D-Lac
GalOS component (mM)
D-Galp-(1f3)-D-Glc
D-Galp-(1f6)-D-Glc
(allolactose)
nd
0.41
5.02
27.1
0.90
1.15
7.36
26.6
2.59
1.70
12.4
33.2
5.08
3.01
13.5
26.4
2.41
2.21
15.1
21.7
2.10
1.40
17.5
0.69
1
2
nd
9.68
1.29
23.4
1.29
43.9
2.75
56.4
5.32
64.0
6.43
58.5
3
4
5
6
D-Galp-(1f3)-D-Gal
D-Galp-(1f6)-D-Gal
D-Galp-(1f6)-D-Lac
D-Galp-(1f3)-D-Lac
GalOS (% mass of
total GOS)
nd
1.19
14.7
53.8
1.79
3.36
21.5
52.8
5.14
4.97
36.3
65.9
10.1
8.80
39.5
52.4
4.78
6.46
44.3
43.1
4.17
4.09
34.6
1.37
1
2
D-Galp-(1f3)-D-Glc
D-Galp-(1f6)-D-Glc
(allolactose)
nd
14.1
0.93
16.9
0.71
24.4
1.05
21.6
2.19
26.3
2.87
26.1
3
4
5
6
D-Galp-(1f3)-D-Gal
D-Galp-(1f6)-D-Gal
D-Galp-(1f6)-D-Lac
D-Galp-(1f3)-D-Lac
nd
0.86
10.7
57.6
1.90
1.86
12.0
43.2
4.20
1.90
13.9
37.2
5.69
3.61
16.2
31.7
2.89
2.87
19.7
28.3
2.74
5.97
74.4
2.95
a The reaction was carried out at 37 °C using 215 g/L initial lactose
concentration in 50 mM sodium phosphate buffer (pH 6.5), 1 mM
MgCl2, and 1.2 ULac/mL of homogenous enzyme.
Figure 7. Formation and degradation of GalOS during lactose conver-
sion by β-galactosidase form L. sakei Lb790 with 215 g/L initial lactose
concentration in 50 mM sodium phosphate buffer (pH 6.5) at 37 °C and
1 mM MgCl2: (O) disaccharides; (9) trisaccharides; (b) tetrasacchar-
ides; (2) total GalOS.
As the conversion of lactose proceeded further, the concentra-
tions of the main hydrolysis products, D-galactose and D-
glucose, increased, and these in turn became prominent
acceptors for the galactosyl moiety. Hence, disaccharides
other than lactose were also formed to a significant extent at
higher lactose conversion. Furthermore, disaccharides can
result from β-gal-catalyzed hydrolysis of trisaccharides, which
are present in higher concentrations at latter phases of the
conversion. Non-lactose disaccharides were dominant by
weight at above 85ꢀ90% lactose conversion.
also GalOS as a result of the transgalactosylation reaction
catalyzed by the enzyme. Between 3 and 4 h of reaction, the
concentration of total GalOS reached at maximum of 89 g/L,
corresponding to approximately 41% of the total sugars. There-
after, the concentration of GalOS decreased because these
oligosaccharides are not stable end-products of a thermodyna-
mically controlled reaction; they are only transiently formed and
are subject to hydrolysis, which became more pronounced when
the substrate lactose was depleted or their concentration reached
a higher, critical concentration for hydrolysis (Figure 6). In
addition to hydrolysis of these newly formed oligosaccharides,
GalOS can also serve as acceptor for galactosyl transfer as can the
primary hydrolysis products D-glucose and D-galactose, and
therefore the amount and composition of GalOS change drama-
tically during the course of the reaction, as is illustrated in more
detail in Figure 7. Up to ∼80% lactose conversion, the amount of
GalOS, expressed by their relative concentration (percentage of
GalOS of total sugars), was constantly rising. The maximal
concentration of GalOS was found at 77% lactose conversion
(Figure 7A), and the GalOS mixture at this reaction point was
composed of mainly non-lactose disaccharides, trisaccharides,
and tetrasaccharides with approximately 38, 57, and 5% of total
GalOS formed, respectively. At the beginning of the reaction,
trisaccharides were predominantly formed, constituting up to
∼80% of total GalOS during this initial reaction phase
(Figure 7B). This is not unexpected because lactose is the most
abundant carbohydrate species in the reaction mixture acting as
the main galactosyl acceptor during the first phase of the reaction.
A detailed analysis of the main GalOS components, which
were identified and quantitated by HPAEC-PAD using authen-
tic standards and the standard addition technique, over the
time course of the reaction is given in Table 2. The L. sakei
β-galactosidase shows a strong preference for the formation of
β-(1f6) linkages in its transgalactosylation mode, whereas
β-(1f3)-containing compounds are formed to a lesser extent,
and β-(1f4)-containing GalOS compounds could not be
identified among the major reaction products. The main com-
ponents of this GalOS mixture were identified as D-Galp-(1f6)-
D-Glc (allolactose), D-Galp-(1f3)-D-Glc, D-Galp-(1f6)-D-Gal,
D-Galp-(1f3)-D-Gal, D-Galp-(1f6)-Lac, and D-Galp-(1f3)-
Lac.
’ DISCUSSION
L. sakei is an important member of the LAB because it is widely
used as a starter for the manufacture of fermented sausages and
other meat products. Meat has a low carbohydrate content,
with glucose and ribose, originating from glycogen and ATP,
respectively, being the main sugars,1,2,31 and exogenous glucose
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Journal of Agricultural and Food Chemistry
ARTICLE
or lactose is usually added to accelerate the ripening process of
these fermented meat products. β-Galactosidases represent one
possible way of introducing lactose into the metabolism of
lactobacilli through intracellular cleavage of this disaccharide to
galactose and glucose. Lactobacillus spp. can carry β-galactosi-
dases belonging to two different glycoside hydrolase (GH)
families, 2 and 42.13,16 Recent genome projects revealed that
certain species such as L. acidophilus or L. plantarum can carry at
least two genes encoding β-galactosidases belonging to GH2
(lacLM) and GH42 (lacA), whereas other strains such as L. sakei
with its modestly sized genome possess only the genes encoding
the β-galactosidase of the LacLM type.
Arguably a better way to compare this inhibition, however, is by
considering the ratio of the inhibition constant Ki,Gal to the
Michaelis constant Km,Lac, which can be interpreted as a speci-
ficity constant that determines preferential binding of the sub-
strate lactose versus that of the competitive inhibitor D-
galactose.17,38 Therefore, a high value for this ratio is important
for an efficient hydrolysis, with only moderate product inhibition.
This ratio, Ki,Gal/Km,Lac, was calculated to be 9.0 for β-gal from L.
sakei, which is higher than the respective ratio for most other β-
galactosidases (e.g., 0.0055 for β-gal from B. licheniformis,36 0.4
for Caldicellulosiruptor saccharolyticus,38 4.0 for β-gal from S.
solfataricus 37). This ratio is also higher than that previously
β-Galactosidase from L. sakei was produced heterologously
because the yields obtained with the wild-type strain were very
low (data not shown). Recently, we compared the expression of
four different lactobacillal β-galactosidases, all of the LacLM
type, in L. plantarum and L. sakei25 as well as in Lactococcus
lactis.32 Whereas the expression levels of β-gal from L. plantarum
and L. reuteri were generally high with these different hosts,
expression of L. acidophilus β-gal was high only in Lactococcus
lactis, reaching expression levels of ∼85 mg of recombinant
protein/L without further optimization. Interestingly, L. sakei
β-gal expressed relatively poorly in these three hosts, even
when homologously overexpressed in L. sakei with typical
expression levels found in the range of 10ꢀ15 mg/L. Whereas
overexpression in E. coli yielded sufficient enzyme for the
subsequent characterization and transgylcosylation experi-
ments, the yields of 33 mg/L are also only rather modest for
E. coli as expression host. When using the identical expression
system, E. coli BL21 Star (DE3) and an expression plasmid
based on pET21d carrying lacLM, we obtained expression
levels of ∼690 mg/L for the overexpression of L. reuteri
β-gal.27 We previously proposed that rarely used and unfavor-
able codons in the start of the lacLM genes from L. sakei could
be responsible for the inefficient expression in the two Lactobacillus
species and Lactococcus lactis,25,32 and this assumption also seems to
hold for E. coli.
We recently characterized several β-galactosidases of the
LacLM type isolated from different lactobacilli.26,33ꢀ35 The
enzyme from L. sakei shows properties that are in general quite
similar to those found for the other lactobacillal β-galactosidases,
for example, with respect to pH and temperature optima or
kinetic properties. One noteworthy aspect of L. sakei β-gal is its
Km value of 20 mM (corresponding to approximately 6.8 g/L
lactose), which is relatively low when compared to those of other
microbial β-galactosidases (see, e.g., the BRENDA Enzyme
Database; http://www.brenda-enzymes.org). This low Michaelis
constant ensures efficient utilization of lactose by L. sakei even
when this disaccharide is present in low concentrations as, for
example, in sausage production. An additional attractive property
that is of interest for a potential application of L. sakei β-gal is its
very slight inhibition by the end-products D-galactose and D-
glucose. This end-product inhibition of β-gal activity can be a
severe limitation in technical processes, especially when lactose-
free or lactose-reduced products are the goal, because complete
lactose hydrolysis is very difficult to achieve when this inhibition
is pronounced. We determined an inhibition constant Ki,Gal of
180 mM for L. sakei β-gal and D-galactose, which is considerably
higher (and hence indicates less inhibition) than reported for
most other microbial β-galactosidases, with Ki,Gal values typically
found in the range of 0.93 mM for β-gal from Bacillus licheni-
formis 36 to 366 mM for the enzyme from Sulfolobus solfataricus.37
reported by us for β-gal from L. reuteri (Km,Lac = 31 mM; Ki,Gal =
89 mM; Ki,Gal/Km,Lac = 3.0),26 indicating that β-gal from L. sakei
could be advantageous for complete lactose hydrolysis because of
this very slight product inhibition. This weak inhibition is also
obvious in the comparison of the inhibition constant Ki,Gal and
the Michaelis constants for the artificial substrates oNPG or
pNPG Km,NPG, which can be retrieved more frequently from the
scientific literature. The ratio Ki,Gal/Km,NPG was calculated to be
300 for β-gal from L. sakei, whereas the corresponding ratio for
the commercially important enzymes from Kluyveromyces lactis
and Aspergillus niger are 12.539 and 0.36,40 indicating again the
much less pronounced inhibition by D-galactose of the L. sakei
enzyme.
Another aspect that makes this particular enzyme attractive is
its application in the synthesis of oligosaccharides. Using β-gal
from L. sakei and lactose at an initial concentration of 215 g/L, we
obtained GalOS in a total yield of 41%. This efficiency in
producing GalOS in high yields seems to be superior to that
found for other β-galactosidases from various LAB, which have
reported yields ranging from 28% for β-gal from L. plantarum to
∼38% for L. acidophilus, L. reuteri, and Streptococcus thermophilus
when using comparable initial concentrations of lactose.11,13,34,35
The lactose concentration of 215 g/L that was used here is
relatively low compared to other studies.16ꢀ18 However, this
concentration was selected deliberately because it corresponds to
the solubility limit of lactose under ambient conditions and
reflects the realistic technological situation. Lactose concentra-
tion is one of the most important parameters affecting oligosac-
charide yields,16ꢀ18 and hence supersaturated lactose solutions
as used by others in concentrations of up to 600 g/L could further
improve GalOS yields when using β-gal from L. sakei.
β-Galactosidase from L. sakei formed GalOS structurally
similar to those obtained with other β-galactosidases from
LAB,13,16,29,33,35 yet proportions of individual components varied
to some extent. The predominant oligosaccharide products were
identified as β-D-Galp-(1f6)-D-Glc (allolactose), β-D-
Galp-(1f6)-D-Gal, β-D-Galp-(1f3)-D-Glc, β-D-Galp-(1f3)-
D-Gal, β-D-Galp-(1f6)-Lac, and β-D-Galp-(1f3)-Lac, indicat-
ing that this β-galactosidase has a propensity to synthesize
β-(1f6)- and β-(1f3)-linked GalOS. At maximal GalOS
production (77% lactose conversion), β-D-Galp-(1f6)-D-Lac
was the most abundant oligosaccharide species formed, compris-
ing 37% of total GalOS, followed by β-D-Galp-(1f6)-D-Glc as
the second most important GalOS compound produced (23% of
total GOS). Especially, this yield of the trisaccharide is signifi-
cantly higher than that obtained with β-gal from, for example, L.
reuteri or L. plantarum.33 Hence, different enzymes can be used -
to tailor GalOS mixtures to contain certain desired oligosacchar-
ide structures. Current commercial GOS products contain struc-
tures with predominant β-(1f4)-linkages,18,29,41 whereas the
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Journal of Agricultural and Food Chemistry
ARTICLE
lactobacillal enzymes show a strong propensity to form
β-(1f6)-linked transgalactosylation products. In a study on
the structureꢀfunction relationship of various disaccharides with
respect to their prebiotic effect, it was shown that among a group
of galactose-containing disaccharides, those containing a (1f6)-
linkage supported growth of bifidobacteria best in mixed culture
populations.42 Because of this strong bifidogenic effect of these
compounds, a GalOS mixture produced with β-galactosidase
from L. sakei that is rich in these β-(1f6)-linked oligosacchar-
ides should therefore be of considerable interest as a prebiotic
oligosaccharide mixture.
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’ ABBREVIATIONS USED
β-gal, β-galactosidase; CE, capillary electrophoresis; DTT, 1,4-
dithiothreitol; GalOS, galacto-oligosaccharides; GOD, glucose
oxidase; HPAEC-PAD, high-performance anion exchange chroma-
tography with pulsed amperometric detection; IPTG, isopropyl-β-
D-thiogalactoside; Lac, lactose; MUG, 4-methylumbelliferyl β-D-ga-
lactoside; oNP, o-nitrophenol; oNPG, o-nitrophenyl-β-D-galacto-
pyranoside; pNPG, p-nitrophenyl-β-D-galactopyranoside; PMSF,
phenylmethanesulfonyl fluoride.
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’ AUTHOR INFORMATION
(16) Gosling, A.; Stevens, G. W.; Barber, A. R.; Kentish, S. E.; Gras,
S. L. Recent advances refining galactooligosaccharide production from
lactose. Food Chem. 2010, 121, 307–318.
Corresponding Author
*Postal address: Lebensmittel-Biotechnologie, Department f€ur
Lebensmittelwissenschaften und -technologie, BOKU Uni-
versit€at f€ur Bodenkultur Wien, Muthgasse 18, A-1190 Wien,
Austria. Phone: þ43 1 47654 6141. Fax: þ43 1 47654 6199.
E-mail: dietmar.haltrich@boku.ac.at.
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microbial β-galactosidase: current state and perspectives. Appl. Micro-
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Funding Sources
This research work was supported by the Higher Education
Commission, Islamabad, Pakistan, through a scholarship to S.I.
H.A.N. and T.T.N. thank ASEA-Uninet and the Austrian Academic
Exchange Service for a Technology Southeast Asia Grant.
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