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4990 J. Agric. Food Chem., Vol. 54, No. 14, 2006
Nguyen et al.
by centrifugation as described above. The supernatant was discarded;
the pellet was dissolved in 50 mM sodium phosphate buffer containing
1 M ammonium sulfate (buffer A).
potential of these enzymes which is based on the catalytic
mechanism of â-galactosidases (12, 14). The products of
transgalactosylation, galacto-oligosaccharides, are nondigestible
carbohydrates which meet the criteria of “prebiotics” and
therefore have attracted an increasing amount of attention.
â-Galactosidases have been isolated and characterized from
many different sources (12), yet the information about these
important enzymes from probiotic strains, such as the lactobacilli
or bifidobacteria, is still sparse.
L. reuteri is a dominant strain of the hetero-fermentative
lactobacilli in the gastrointestinal tract (GIT) of humans and
animals (15-17); in fact, it is the only Lactobacillus species
thought to inhabit the GIT of all vertebrates and mammals (18).
In this paper, we describe the purification and characterization
of two novel â-galactosidases extracted from L. reuteri strains
L103, originating from calf, and L461, isolated from suckling
pigs.
Hydrophobic Interaction Chromatography (HIC). The dissolved
pellet (200-400 mg of protein) was applied to a 400 mL phenyl-
Sepharose fast flow column (50 mm × 200 mm; Amersham, Uppsala,
Sweden) that was pre-equilibrated with buffer A. â-Galactosidase was
eluted at a rate of 15 mL/min with a 4000 mL linear gradient from 0
to 100% buffer B (50 mM sodium phosphate). The active fractions
were pooled and concentrated by ultrafiltration with a membrane with
a 10 kDa cutoff (Amicon, Beverly, MA).
Affinity Chromatography. The concentrated enzyme solution (≈40
mg of protein) was loaded onto the affinity column (10 mL of
p-aminobenzyl 1-thio-â-D-galactopyranoside immobilized on cross-
linked 4% beaded agarose; Sigma) that was pre-equilibrated with 50
mM sodium phosphate buffer (buffer B). The enzyme was eluted at a
rate of 0.5 mL/min by using a linear 0 to 1 M NaCl gradient in 10
column volumes. The active fractions were pooled, desalted, and
concentrated.
The pH values of the buffers used during purification of the
â-galactosidases from L103 and L461 (buffer A and buffer B) were
6.0 and 6.5, respectively. The purified enzymes were stored in 50 mM
sodium phosphate buffer (pH 6.5) (L461) and 50 mM sodium phosphate
buffer (pH 6.0) containing 1 mM DTT (L103) at 4 °C.
Protein Determination. The protein concentration was determined
by the method of Bradford (20) using bovine serum albumin as a
standard.
MATERIALS AND METHODS
Chemicals. All chemicals were purchased from Sigma (St. Louis,
MO) and were of the highest quality available unless otherwise stated.
MRS broth powder (for Lactobacillus broth according to De Man,
Rogosa, and Sharpe) was obtained from Merck (Darmstadt, Germany),
and 1,4-dithiothreitol (DTT) was from Roth (Karlsruhe, Germany).
Transgalacto-oligosaccharide powder (TOS) was from Yakult Honsha
(Tokyo, Japan), and galacto-oligosaccharides Elix’or (different brand
name Vivinal) were from Borculo Domo Ingredients (Zwolle, The
Netherlands). Glucose oxidase (GOD) from Aspergillus niger (lyoph-
ilized, 205 units/mg enzyme preparation) was from Fluka (Buchs,
Switzerland). Horseradish peroxidase (POD) (lyophilized, 210 units/
mg) and the test kit for the determination of the amount of lactose
were from Boehringer (Mannheim, Germany).
Strains and Culture Conditions for Screening. Fourteen strains
of Lactobacillus spp. were obtained from Lactosan (Starterkulturen
GmbH & Co. KG). The selected strains, with which most of the work
was performed, were isolated from calf (isolate L103) and suckling
pig (isolate L461). The strains were stored in sterile vials at -70 °C in
MRS broth medium containing glycerol (15%, v/v) and activated by
three successive transfers every 24 h in MRS broth medium. They were
grown on MRS broth medium (10 g/L peptone, 2 g/L dipotassium
hydrogen phosphate, 8 g/L meat extract, 2 g/L diammonium hydrogen
citrate, 4 g/L yeast extract, 5 g/L sodium acetate, 0.2 g/L magnesium
sulfate, 1 g/L Tween 80, and 0.04 g/L manganese sulfate), in which
glucose, lactose, or galacto-oligosaccharides served as the carbon source
(1 or 2%, w/v), and incubated anaerobically at 37 °C for 24 h. The
optical densities (OD600) of the cultures were measured with a
microplate reader (GENious, Tecan, Maennedorf, Switzerland) with
the readings being taken every 20 min for 24 h. To screen for
â-galactosidase activity, cells were harvested from a liquid culture by
centrifugation (10 000 rpm for 10 min at 4 °C) using an Eppendorf
centrifuge and resuspended in 50 mM sodium phosphate buffer (pH
6.8) containing 20% (w/v) glycerol and 1 mM dithiothreitol (buffer P)
(19). The cells were disrupted by ultrasonication, and debris was
removed by centrifugation (10 000 rpm for 15 min at 4 °C) to obtain
the cell-free extract.
Enzyme Assays. â-Galactosidase activity was determined using
o-nitrophenyl â-D-galactopyranoside (oNPG) and lactose as the sub-
strates.
Assay with Chromogenic Glycoside. When chromogenic oNPG was
used as the substrate, the determination of â-galactosidase activity was
carried out at 30 °C with 22 mM oNPG in 50 mM sodium phosphate
buffer (pH 6.5). The reaction was initiated by adding 20 µL of enzyme
solution to 480 µL of the substrate solution, and then the mixture was
incubated for 10 min using an Eppendorf (Hamburg, Germany)
thermomixer compact. Agitation was carried out at 600 rpm. After the
incubation time, the reaction was stopped by adding 750 µL of 0.4 M
Na2CO3. The release of o-nitrophenol (oNP) was assessed by determin-
ing the absorbance at 420 nm. One unit of oNPG activity was defined
as the amount of enzyme releasing 1 µmol of oNP per minute under
the described conditions.
Assay with Lactose. When lactose was used as the substrate, 20 µL
of an enzyme solution was added to 480 µL of a 600 mM lactose
solution in 50 mM sodium phosphate buffer (pH 6.5). The reaction
mixture was incubated at 30 °C using an Eppendorf thermomixer
compact (600 rpm). After 10 min, the reaction was stopped by heating
the reaction mixture at 99 °C for 5 min. After the sample had been
cooled to room temperature, the release of D-glucose was assessed
colorimetrically using the GOD/POD assay (21) by adding 60 µL of
this reaction mixture to 600 µL of a solution containing GOD (94 µg/
mL), POD (6.1 µg/mL), 4-aminoantipyrine (157 µg/mL), and phenol
(1.95%, v/v) in 0.1 potassium phosphate buffer (pH 7.0). This assay
mixture (660 µL) was incubated in the dark at room temperature for
40 min, and the absorbance at 546 nm was measured. One unit of lactase
activity was defined as the amount of enzyme releasing 1 µmol of
D-glucose per minute under the given conditions.
All measurements and experiments were performed at least in
duplicate, and the experimental error never exceeded 5%.
Fermentation of the Selected Lactobacillus Strains. The strains
were grown anaerobically in MRS broth medium containing 1% (w/v)
lactose in a 50 L laboratory fermentor (strain L. reuteri L461) and in
a 220 L industrial fermentor (strain L. reuteri L103) at 37 °C and with
an initial pH of 6.5. Precultures were grown overnight in MRS broth
medium [1% (w/v) lactose] and inoculated into the fermentors to a
final concentration of 0.5% inoculum. The pH was allowed to drop to
5.5 and was then regulated at this value.
Enzyme Purification. Approximately 50 g of biomass (wet weight)
was suspended in 100 mL of buffer P. The cells were disrupted by
using a French press (Aminco), and debris was removed by centrifuga-
tion (25000g for 15 min at 4 °C) to obtain the crude extract.
Ammonium Sulfate Precipitation. Ammonium sulfate was slowly
added to crude extract to 60% saturation. The precipitate was obtained
Determination of Molecular Masses. Electrophoretic Analysis and
ActiVe Staining. Native polyacrylamide gel electrophoresis (PAGE) and
denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) were performed on a PhastSystem unit (Amersham)
using precast polyacrylamide gels (PhastGel 8-25). For SDS-PAGE,
the enzyme was preincubated with SDS buffer [47 mM Tris-HCl (pH
6.8) containing 34 mg/mL SDS, 0.1 mg/mL bromophenol blue, 5%
(v/v) mercaptoethanol, and 15% (v/v) glycerol] at 60 °C for 5 min.
Coomassie blue staining was used for the visualization of the protein
bands. Active staining for the visualization of the bands with â-galac-
tosidase activity was carried out by applying filter paper soaked with
the staining solution [50 mM sodium phosphate buffer (pH 6.5) and