J. Biol. Macromol., 10 (1), 23-31 (2010)

ArticleProperties of β-galactosidase purified from Bifidobacterium

longum subsp. longum JCM 7052 grown on gum arabic
Naoko Saishin1, Masami Ueta2, Akira Wada2, and Isamu Yamamoto3*
1Department

of Food Nutrition, Kobe Women’s Junior College, Chuo-ku, Kobe 605-0046 Japan,

2Yoshida Biological Laboratory, Yamashina-ku, Kyoto 607-8081, Japan, and 3Laboratory of Molecular

Microbiology, Graduate School of Life Science, Kobe Women’s University, Suma-ku, Kobe 654-8585,
Japan
Received December 18, 2009; Accepted January 25, 2010.
β-Galactosidase was purified and characterized from Bifidobacterium longum subsp. longum JCM
7052 grown on gum arabic. The molecular masses of the enzyme were estimated to be 77 and 110 kDa
by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate,respectively. MALDI-TOF-MS showed that the purified β-galactosidase was a homologue of the lacA1
gene product found in the genome of B. longum subsp. longum DJO10A. This β-galactosidase couldhydrolyze 4-nitrophenyl (NP)-β-D-galactopyranoside but neither lactose nor gum arabic. The Km value
for 4-NP-β-D-galactopyranoside was 0.42 ± 0.015 mM. Transglycosylation was observed from
4-NP-β-D-galactopyranoside to galactose and melibiose, but not from lactose. Maximal activities of
β-galactosidase were obtained at pH 7.0 and 50~55°C. The enzyme was stable at 35°C for 5 h, but was
gradually inactivated at higher than 40°C. Galactose was an effective competitive inhibitor: its
inhibition constant Ki was 5.8 ±0.38 mM.
Keywords: β-galactosidase, purification, Bifidobacterium longum subsp. longum
Introduction
Bifidobacteria are strictly anaerobic, Grampositive, and saccharolytic bacteria which
predominantly live in the intestinal tract ofhumans and animals. Some strains of this genus
are known to have many health-promoting or
probiotic activities [1, 2]. The growth ofbifidobacteria has been shown to be selectively
stimulated by various dietary carbohydrates
including gum arabic [3-6]. The genome sequenceof Bifidobacterium longum subsp. longum is now
available [7, 8], and it has revealed many genes
Corresponding author: Isamu Yamamoto.
Phone: +81-78-737-2312, Fax: +81-78-732-5161
E-mail: iyamamoto@suma.kobe-wu.ac.jp

for carbohydrate utilization. More than 30 genes
coding for glycoside hydrolases are found there
and some of them are predicted to be involved indegradation of complex carbohydrates.
Gum arabic is a soluble dietary fiber
obtained from the stems and branches of Acaciasenegal and Acacia seyal. It is composed of threemain fractions, arabinogalactan protein complex(AGP, 10%), arabinogalactan (AG, 90%), andglycoprotein (1%) [9]. AG and carbohydrate blockof AGP consist of a branched β-1,3-linked
galactose backbone with branches linked
through the 1,6-positions, and with arabinose,
rhamnose, and glucuronic acid in ramified side
chains [10, 11]. It is known that some strains ofBifidobacterium adolescentis and B. longum

24β-Galactosidase from Bifidobacterium longum subsp. longum

subsp. longum can ferment gum arabic [3]. For
the fermentation some glycoside hydrolytic
enzymes would be required to degrade gum
arabic: β-galactosidase, α-arabinopyranosidase,
α-arabinofuranosidase, arabinogalactan-endo-βgalactosidase and so on.
β-Galactosidase (EC 3.2.1.23) catalyzeshydrolysis of β-D-galactoside linkage, and someβ-galactosidases may have an activity oftransferring one or more D-galactosyl units onto
lactose [12]. Various β-galactosidases have beencharacterized in Bifidobacterium species, B.
adolescentis [13, 14], Bifidobacterium bifidum[15-17], Bifidobacterium longum subsp. infantis[16, 18, 19], and B. longum subsp. longum [20-22].
Glycoside hydrolases (GHs) including β-galactosidase constitute 113 protein families [23].
Lactose-degrading β-galactosidases are classified
as GH2 family and others classified as GH42
family which show activity toward β-galactosyl
linkages in oligosaccharides [14, 24].
We have previously shown that B. longum
subsp. longum JCM 7052 is able to grow on gum
arabic as carbon source and contains a highactivity of β-galactosidase as well as α-galactosidase [25]. These enzymes are considered
probably to contribute to degradation of thepolysaccharide. In this paper, some properties of
β-galactosidase purified from B. longum subsp.
longum JCM 7052 grown on gum arabic aredescribed.
Materials and Methods
Chemicals
Nitrophenyl (NP)-glycosides,and 3,5-dinitrosalitylic acid were purchased from
Sigma-Aldrich (St. Louis, Mo, USA). Gum arabic(Acacia senegal) was obtained from Wako Pure
Chemical Industries (Osaka, Japan). Otherchemicals of reagent grade were products of
Wako Pure Chemical Industries.

Bacterial strain and growth conditions
Bifidobacterium longum subsp. longum JCM

7052 was used in this study. Bacterial cells weregrown in a medium of slightly modified
Bifidobacterium medium, which contained (g/l)
Polypepton (Nihon Seiyaku, Japan), 10; yeast
extract (Oriental Yeast Co. Japan), 5; meat
extract (Wako Pure Chemical Industries, Japan),5; K2HPO4, 3; sodium ascorbate, 10; cysteinehydrochloride, 0.5; and Tween 80, 1 ml/l. Gum
arabic (10 g/l) was added as carbon source
instead of glucose. pH was adjusted to 6.8. Cells
were anaerobically grown at 37°C for 16 h.
Anaerobic conditions were obtained by filling theneck of screw-capped bottles with the medium.
Assay of enzyme activities Standard assayof β-galactosidase activity was performed with
4-nitrophenyl (4-NP)- β-D-galactopyranoside as
substrate. The reaction mixture (1 ml) contained
an enzyme preparation, 80 mM K-phosphate (pH7.0) and 1 mM 4-NP-β-D-galactopyranoside. The
reactions were carried out at 30°C for 10 min andwere terminated by the addition of 0.5 ml of
0.2 M Na2CO3. The amount of 4-nitrophenol
released was determined at 400 nm with an
extinction coefficient of 18.3 mM−1 cm−1. One unitof enzyme activity is defined as the amount of
enzyme that releases 1 μmol of 4-nitrophenol per
min from 4-NP-β-D- galactopyranoside.
For assay of activities which hydrolyze
oligosaccharides, reducing sugar produced fromoligosaccharide was determined in the reaction
mixtures containing 80 mM K-phosphate (pH7.0), enzyme preparation, and 100 mM lactose or
0.1% gum arabic. After incubation at 30°C for 60
min, 3,5-dinitrosalitylic acid was added to stop
the reaction. The absorbance was measured at
540 nm. Galactose was used as the standard forcalibration. One unit of enzyme activity isdefined as the amount of enzyme that produces
1 μmol of reducing sugar per min from theoligosaccharide.
Transglycosylation activity was assayed bydetection of product in HPTLC (Silica gel 60;
Merck Ltd., Germany). The Reaction mixtures(1 ml) contained 80 mM K-phosphate buffer (pH7.0), 15 mM 4-NP-β-D-galactopyranoside, 80 mM
saccharide, and 0.2 units of purified
β-galactosidase, and were incubated at 30°C for 3,
6, and 12 h. Saccharides were separated in
n-butanol:acetic acid:water (5:4:1), and detectedwith spray of 5% sulfuric acid in 20% ethanol.
Thermal stability was assayed by incubatinga partially purified enzyme at 35, 40, 45, 50 and
55°C and drawing aliquots at intervals. The
enzyme activity was then determined according

N. Saishin et al.

25
to the standard assay procedure.
The protein concentrations were determinedby using Coomassie brilliant blue (Bio-Rad
Laboratories, USA). Bovine serum albumin
was used as the standard. All reactions werecarried out in triplicate.
Purification of β-galactosidase Wet cells (10g) were suspended in four volumes of 50 mM
Tris-HCl buffer (pH 7.2). The cells were sonicated(TOMY Ultrasonic Disruptor UD-200, Japan) in
an ice bath at 20 kHz, 140 W for 5 min andcentrifuged at 20,000 x g for 30 min at 4°C. The
supernatant was used as crude extract (Step 1.
Crude extract, 40 ml). The precipitate obtained
between 30 and 70% saturation of ammonium
sulfate was dissolved in 50 mM Tris-HCl, pH 7.2(Step 2. Ammonium sulfate fraction, 4.4 ml).
Proteins dissolved were loaded on a column (2.5 x
90 cm) of Sepharose 4B (GE Healthcare, Sweden)
equilibrated in 50 mM Tris-HCl (pH 7.2)
containing 0.1 M NaCl and eluted at a flow rateof 0.4 ml/min. Fractions containing β-galactosidase activity were pooled (Step 3. Sepharose 4B,
77 ml). The pooled fractions were loaded on acolumn (1.5 x 7 cm) of Q-Sepharose (GEHealthcare, Sweden) equilibrated in 50 mM
Tris-HCl (pH 7.2) containing 0.1 M NaCl.
Proteins were eluted at a flow rate of 0.4 ml/min
with a linear concentration gradient of NaCl
(0.1~0.7 M). Fractions containing β-galactosidase
activity were pooled (Step 4. Q-Sepharose, 28 ml).
The solution pooled was mixed with ammonium
sulfate at a concentration of 1.84 M, then was
loaded on a column (1.5 x 7 cm) of Butyl-S
Sepharose 6 Fast Flow (Amersham Biosciences,
Sweden) equilibrated with 50 mM Tris-HCl (pH7.2) containing 1.84 M ammonium sulfate.
Proteins were eluted
using a decreasing
stepwise gradient of ammonium sulfate (1.84~0M) at a flow rate of 1 ml/min. Fractions activewith β-galactosidase were pooled (Step 5. Butyl-S
Sepharose, 6.0 ml). The enzyme pooled wasdesalted and concentrated by centrifugation witha filtration tube Vivaspin 4 (Sartorius, Germany).
Concentrated proteins were loaded on 6%
polyacrylamide gel electrophoresis (PAGE)
without sodium dodecyl sulfate (SDS). An area
stained with β-galactosidase activity was cut outof the polyacrylamide gel and was soaked in 50
mM Tris-HCl (pH 7.2).
The enzyme wasfiltrated using a gel extraction spin column(Bio-Rad, USA) and was concentrated bycentrifugation with a filtration tube Vivaspin 4(Step 6. Native PAGE, 4.0 ml).

Molecular mass determination SDS-PAGE

was carried out with 12.5% (w/v) polyacrylamide
gel according to Laemmli [26]. Molecular weightmarker proteins for SDS-PAGE (GE Healthcare,UK) were used for calibration. Native PAGE wasdone using a 5~15% (w/v) linear gradient slab gel(Wako Pure Chemical Industries). Molecularweight marker proteins for native PAGE (HMW
calibration kit, GE Healthcare, UK) were used
for calibration. Activity staining of β-galactosidase on the gel was done using 4-NP- β-D-galactopyranoside as substrate. Protein bands ingels were stained with Coomassie blue R-250(Bio-Rad Laboratories, USA).

Protein identification by mass spectrometry

The Coomassie-stained protein bands were
excised from the SDS-PAGE gel, and subjected
to tryptic digestion as previously described [27].
Matrix-assisted laser desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry (MS)and tandem mass spectrometry (MS/MS) were
performed on an Ultraflex MS system (Bruker
Daltonik GmbH, Bremen, Germany). Peptide
mass fingerprinting (PMF) searches were
performed by using the Mascot Search engine
against the NCBI protein database.
Results
Purification of β-galactosidase and its molecularmass
A summary of a purification of β-galactosidase from B. longum subsp. longum JCM
7052 grown on gum arabic is given in Table 1.
When the enzyme was separated by means ofchromatography, β-galactosidase activity was
eluted by 0.55 M NaCl and by 1.1 M (NH4)2SO4
in ion exchange chromatography with QSepharose and in hydrophobic chromatography
with Butyl-S Sepharose, respectively. At final

β-Galactosidase from Bifidobacterium longum subsp. longum

26

Table 1. Purification of β-galactosidase from B. longum subsp. longum JCM 7052 grown on 1% gum arabic
Purification step

Protein
(mg)

Total

Specific

activity

activity(units)(units/mg of protein)

Yield
(%)

1. Crude extract

283

128

0.45

100

2. Ammonium sulfate fraction

104

72.5

0.70

57

3. Sepharose 4B

73.4

65.2

0.89

51

4. Q Sepharose

26.6

38.1

1.43

30

5. Butyl-S Sepharose

1.76

13.1

7.41

10

6. Native PAGE

0.52

6.3

12.1

5

step of purification, the enzyme obtained was
obviously homogenous in SDS-PAGE (Fig. 1A).
The molecular mass of the enzyme wasdetermined by SDS-PAGE and native gradient
PAGE. SDS-PAGE showed a single band ofprotein and its molecular mass to be 77 kDa (Fig.
1A). Native gradient PAGE also showed a single
protein with a molecular mass of about 110 kDa(Fig. 1B). These results suggested that the
enzyme was a dimeric protein.
Identification of β-galactosidase by mass spectrometry
The purified enzyme was subjected to PMF
analysis in order to identify its putative gene.
The PMF result (Accession number, gi|
189439789; sequence coverage, 43.7%; score, 210)
and two MS/MS results (1795.875 (637-651),
ADETGENHFVFLFNR;
score,
150)
and(1882.879 (60-74), LEPEEGVYDFDWLDR; score,92) were obtained. These results indicate thatthe purified β-galactosidase is evidently identicalwith the product of the lacA1 gene (the locus tag
BLD 0926) in the genomic DNA of B. longum
subsp. longum DJO10A [8]. The same amino acidsequence is also found as BL 1168 in the genomeof B. longum subsp. longum NCC 2705 [7].
This gene product consists of 691 amino acids
and has the molecular mass of 77,414 Da. Thisvalue is consistent with the molecular mass of
77 kDa determined by SDS-PAGE. The BLD
0926 β-galactosidase belongs to GH42 family.
β-Galactosidase encoded in the lacZ gene isanother enzyme which has been found in B.
longum subsp. longum [7, 8].

(A)
kDa

(B)
kDa

M 1

M 1

669 →
97 →
66 →
45 →

440 →
232 →

←77 kDa140 →←110 kDa

30 →
20.1→
14.4→

Fig

1.

67 →

Molecular

massdeterminationof

β-galactosidase by SDS-PAGE (A) and native
gradient PAGE (B). Lanes: M, marker proteins;
1, purified enzyme. Proteins were stained by
Coomassie blue R-250.

Substrate specificity and transglycosylation
activity of β-galactosidase
The β-galactosidase purified from B. longum
subsp. longum JCM 7052 displayed a narrow
substrate range. The enzyme hydrolyzed 4-NP-βD-galactopyranoside (100% of activity), 2-NP-β-Dgalactopyranoside (31%), and 4-NP-β-D-fucopyranoside (8%), but not 4-NP-β-D-glucopyranoside and 4-NP-α-linked compounds with
L-arabinofuranoside, L-arabinopyranoside, D-galactopyranoside, D-glucopyranoside, D-mannopyranoside, L-rhamnopyranoside, and L-fucopyranoside. The Km values for 4-NP-β-D-galactopyranoside and 2-NP-β-D-galactopyranosidewere determined by double reciprocal plots to be
0.42 ±0.015 and 0.78 ±0.039 mM, respectively.

N. Saishin et al.

Enzyme activity（％）

80
60
40
20
0
6

6

7

7

8

8

9

pH
Fig 2. Effects of pH on the activity of thepurified β-galactosidase. Buffers used were, ●,80 mM K-phosphate (pH 5.5–8.0); and ▲, 80
mM Tris-HCl (pH 6.5–8.5 ).
100
Enzyme activity (％)

Effects of pH, temperature, and chemicals on the
enzyme activity
In these experiments on characterization of
the catalytic properties, the enzyme preparation
obtained at Butyl-S Sepharose step of purification was used. To observe pH-dependency ofenzyme activity, β-galactosidase was incubatedin 80 mM K-phosphate buffer with 1 mM
4-NP-β-D-galactopyranoside at 30°C for 10 min.
The highest activity was obtained at pH 7.0 (Fig.
2). In the assay in 80 mM Tris-HCl buffer
enzyme activities obtained were slightly low.
The optimal temperature for β-galactosidase
activity was 50~55°C at pH 7.0 in a 5 minreaction (Fig. 3A). The enzyme was stable during
5 h incubation at 35°C, but very instable athigher than 40°C (Fig. 3B). NaCl of 0.15 M
concentration was slightly effective to stabilize
the enzyme.
Of metal chloride salts tested (Table 2) 1 mM
Cu2+ inhibited the β-galactosidase activity by
50%, while Ba2+, Ca2+, Co2+, Fe3+, Mg2+, Mn2+,
Ni2+, and Zn2+ showed almost no effects on the
enzyme activity. No effects of 10 mM NaCl and
10 mM KCl were observed on the enzyme
activity.
A metal chelator EDTA did not affect theactivity of β-galactosidase.
The enzyme activity was determined in thepresence of 20 mM galactose, glucose, or lactose(Table 2). Galactose showed about 60% inhibitionof the activity, and glucose and lactose 17% and

100

80
60
40
20
0

Enzyme activity (％)

Maximal velocities (Vmax ) for 4-NP- and 2-NP-βD-galactopyranosides were also shown to be 5.21
±0.38 and 2.16 ±0.062 µmol/min/mg of protein,respectively.
Lactose and gum arabic were not hydrolyzed
by β-galactosidase during 1 h incubation at 30°C
and pH 7.0.
Transgalactosylation assay with the purified
β-galactosidase was performed as described inMaterials and Methods. When 4-NP-β-D-galactopyranoside was used as a donor of galactosylgroup, an apparently single product was formed
with galactose and melibiose as acceptors (datanot shown). Transgalactosylation from lactose tothese acceptor sugars was not observed.

27

20

30

0

1

40
50
Temperature (℃)

60

100
80
60
40
20
0
2

3

4

5

Incubation time (h)Fig 3. Effects of temperature on the activity (A)and stability (B) of the purified β-galactosidase.
Symbols in (B), ●, 35℃; ■, 40℃; ○, 45℃; ▲,50 ℃ ; and ◇ , 55 ℃ .

Solid lines indicate

activities in the absence of NaCl, and dotted lines
in the presence of 0.15 M NaCl.

β-Galactosidase from Bifidobacterium longum subsp. longum

28

Table 2. Effects of various reagents on the
β-galactosidase activity
Relative
activity (%)

Reagents
None
BaCl2
CaCl2
CoCl2
CuCl2
FeCl3
MgCl2
MnCl2
NiCl2
ZnCl2
NaCl
KCl
EDTA
Galactose
GlucoseLactose
Tris*

100
100
99
90
50
100
98
100
100
100
98
96
100
43
83
81
73
54

1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
10 mM
10 mM
1 mM
20 mM
20 mM
20 mM
50 mM
100 mM

1/v(μmol/min/mg of protein)-1*Tris (hydroxymethyl) aminomethane

8
6
4
2
0
0

1

2

3

4

-1

1/s (mM )Fig 4.

Inhibitory effects of D-galactose on theactivity of the purified β-galactosidase. Double
reciprocal plots of β-galactosidase activity against
4-NP- β-D-galactopyranoside in the presence ofD-galactose. For the enzyme activity assay,

K-phosphate buffer (pH 7.0) was used. The
concentrations of galactose were 0 (●), 10 (■), 20(▲), and 30 mM (◆).

19%, respectively. The inhibitory effect ofgalactose was competitive and its inhibition
constant Ki was 5.8 ±0.38 mM (Fig. 4).
Effect of Tris (hydroxymethyl) aminomethane was also investigated because Tris has
been known to inhibit competitively someglycoside hydrolases [25, 28, 29]. β-Galactosidase
activity was inhibited 27% and 46% in thepresence of 50 and 100 mM Tris, respectively. A
possible inhibition type was shown to becompetitive by double reciprocal plots and an
inhibition constant Ki of Tris was about 98.0 ±1.16 mM in Dixon plots. These results suggested
weak interaction between β-galactosidase andTris.
Discussion
β-Galactosidase was purified from B. longum
subsp. longum JCM 7052 grown on gum arabic.
Its primary protein structure was partly
determined by mass spectrometry and showedthat the purified β-galactosidase was ahomologue of BLD 0926 and BL 1168, whichhave been found in the genomes of B. longum
subsp. longum strains DJO10A and NCC 2705,
respectively [7, 8]. This enzyme is encoded in thelacA1 gene, but not in the lacZ gene. The purified
β-galactosidase showed activities hydrolyzing
4-NP-β-D-fucopyranoside with 8% of reactivity to
4-NP-β-D-galactopyranoside, but did not hydrolyze lactose and gum arabic. Maximal activities
were obtained at pH 7.0 and at 55°C. However,
the enzyme was completely inactivated during 3
h incubation at 55°C. Neither activation nor
inhibition of β-galactosidase activity occurred in
the presence of monovalent and divalent cations
except for Cu2+, which inhibited by 50% at 1 mM
concentration. β-Galactosidase was competitively
inhibited by D-galactose with Ki of 5.8 mM.
Transglycosylation activity was also observed
from 4-NP-β-D-galactopyranoside, but not fromlactose.
So far, β-galactosidases have been purified
and characterized from two strains of B. longum
subsp. longum. However, the molecular mass andcatalytic properties of each enzyme are quite
different from those of the β-galactosidase
purified in this study. β-Galactosidase with an

N. Saishin et al.

29
activity of hydrolyzing 4-NP-β-D-fucoside has
been purified from B. longum subsp. longum 401[20]. Its molecular mass is 330 kDa. The enzyme
activity is not activated by any cations but
inhibited by 1 mM CuCl2, ZnCl2, and MgCl2
about 90%, 55%, and 43%, respectively. This
enzyme is thermo-instable: the enzyme loses 75%
of its initial activity during incubation at 45°C
for 10 min. The sodium-stimulated β-galactosidase has been isolated from B. longum subsp.
longum CCRC 15708 [22]. This enzyme has themolecular mass of 357 kDa as a native form, andits activity is inhibited by lactose in a competitive
manner but not by galactose. There are no
information about subunit structures in bothstudies.
The lacZ gene encoding β-galactosidase has
been characterized in B. longum subsp. longum
MB219 [21]. Transformation with a lacZ-carrying
plasmid can make some strains that do not growon lactose use this sugar as a carbon source. The
genome sequence of B. longum subsp. longum [7,8] clearly shows that there are three genes, lacZ,
lacA1, and lacA2, for β-galactosidases whose
deduced molecular masses are 114, 77.4, and78.9 kDa, respectively. It is recently revealed
that the lacZ gene is inducible in B. longum
subsp. longum NCC2705 by lactose, maltose, andfructo-oligosaccharide [29]. On the other hand,
the lacA2 gene and genes coding for an ABC
sugar permease transporter within the samegene cluster are induced when B. longum subsp.
longum LMG 13197 is grown in the presence ofgalacto-oligosaccharide [31]. β-Galactosidase
activity is increased about 4-fold when B. longum
subsp. longum JCM 7052 is grown on gum arabic[25]. In this study we identified the gum
arabic-inducible β-galactosidase was the productof the lacA1 gene, which was classified as GH42
family. This enzyme could not hydrolyze lactose
and gum arabic, though the whole cell of B.
longum subsp. longum JCM 7052 was capable ofhydrolyzing gum arabic [25]. Galacto-oligosaccharides produced by hydrolyzing gum arabic
would be imported into the cell. If one ofphysiological roles of the purified β-galactosidase
would be to hydrolyze the galacto-oligosaccharides inside the cell, the β-galactosidase may

have specificity for β-1,3- or β-1,6-linked galactosides because gum arabic consists of aβ-1,3-linked galactose backbone with branches of
β-1,6-linked galactose chains.
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Communicated by Ueno Hiroshi