J. Biol. Macromol., 9(2), 73-81 (2009)

Articleα-Galactosidase purified from Bifidobacterium longum
JCM 7052 grown on gum arabic
Naoko Saishin1 and Isamu Yamamoto2*

1Department of Food Nutrition, Kobe Women’s Junior College, 4-7-2 Minatojima-Nakamachi, Chuo-ku,

Kobe 605-0046 Japan and 2Laboratory of Molecular Microbiology, Graduate School of Life Science, Kobe
Women’s University, 2-1 Higashisuma Aoyama, Suma-ku, Kobe 654-8585, Japan
Received April 23, 2009; Accepted May 22, 2009.
Among some strains, Bifidobacterium longum JCM 7052 grew well anaerobically in a medium
with gum arabic, and showed high activities of α- and β-galactosidases. α-Galactosidase was purified
66-fold from B. longum JCM 7052 grown on gum arabic by ammonium sulfate fractionation, andchromatographies on Sepharose 4B, Q-Sepharose, Butyl-S Sepharose, and hydroxyapatite. The
enzyme had an apparent molecular mass of 79 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis (PAGE) and 150 kDa by PAGE without SDS, suggesting the dimeric nature of the
enzyme. This α-galactosidase showed optimal activity at pH 8.0 and at 40–45°C. Hydrolysis of
α-galactosides showed normal saturation kinetics: Km values for 4-nitrophenyl-α-D-galactopyranoside,
raffinose, and stachyose were 0.15, 88.3, and 126 mM, respectively. The activity of α-galactosidase wasinhibited competitively by tris (hydroxymethyl) aminomethane: its Ki was 32 mM. Transgalactosylation
activity was also observed from 4-nitrophenyl-α-D-galactopyranoside to galactose and melibiose.
Keywords: α-galactosidase, purification, Bifidobacterium longum, gum arabic
Introduction
Bifidobacteria are Gram-positive saccharolytic anaerobes that are natural commensals of
the gastrointestinal tract of humans andmammals and are thought to have healthpromoting effects, like the inhibition ofpathogenic microorganisms, antimutagenic andanticarcinogenic
activities,
preventionofdiarrhea, immune modulation, and reduction ofserum cholesterol levels [1-3]. Bifidobacterium
species can ferment many kinds of complex
carbohydrates including gum arabic andprobably can use them as important sources ofcarbon and energy [4-7].
Corresponding author: Isamu Yamamoto.
Phone: +81-78-737-2312, Fax: +81-78-732-5161
E-mail: iyamamoto@suma.kobe-wu.ac.jp
Gum arabic is a highly heterogeneouscomplex polysaccharide which consists of threemain fractions, arabinogalactan protein complex(AGP, 10%), arabinogalactan (AG, 90%), andglycoprotein (1%) [8]. Compositions of AG andcarbohydrate block of AGP are similar, galactose(40%), arabinose (30%), rhamnose (12%), andglucuronic acid (15%) [8-10]. It has a branchedstructure with a β-1,3 linked galactan backbone[9]. Gum arabic is highly water soluble and isused as a food additive. It has been known thatamong Bifidobacterium species some strains ofBifidobacterium longum ferment gum arabic[4-6]. The genome sequence of B. longum NCC
2705 revealed that this bacterium is able toproduce many kinds of glycoside hydrolase
including arabinosidases, α- and β-galactosidases,
α- and β-glucosidases, α-mannosidase and so on[11]. Recently, novel enzymes relating tocarbohydrate utilization were found in B. longum

74

α-Galactosidase from Bifidobacterium longum[12-14].
α-Galactosidases (EC 3.2.1.22) hydrolyze the
α-1,6 linkages which are present in galactosaccharides such as melibiose, raffinose andstachyose. The enzymes are known to be widelydistributed in mammals, plants and microorganisms [15-19]. Degradation of oligosaccharides in plants is known to involve two distinct
types of α-galactosidase, acid α-galactosidase
active in acidic environments and alkaline
α-galactosidase active under neutral or alkalineconditions[19-21].
Some
speciesofBifidobacterium including B. longum also have
α-galactosidases whose activities are optimal at
pH 5.5-6.0 [22-25]. Glycolytic enzymes such an
α-galactosidase are often described as glycosidehydrolases, but some of them also showtransglycosylation activity, which is expected to
produce new oligosaccharides as dietary fibers.
In this study, we examined glycosidehydrolase activities in B. longum JCM 7052,
which was identified to be able to grow on gum
arabic, and α-galactosidase activity among
glycoside hydrolase activities tested wasobserved to be highest in gum arabic-grown cells.
We described purification and characterization of
α-galactosidase from B. longum JCM 7052 grownon gum arabic.
Materials and Methods
Chemicals Gum arabic (from Acacia senegal)
and raffinose were obtained from Wako Pure
Chemical Industries (Osaka, Japan). 4-Nitrophenyl (4-NP) glycosides, melibiose, stachyose,and 3,5-dinitrosalycylic acid were purchasedfrom Sigma-Aldrich Co. (St. Louis, USA). Otherchemicals were of reagent grade and were
products of Wako Pure Chemical Industries.

Bacterial strains and growth conditions
Bifidobacterium longum strains used in thisstudy were JCM 7052, JCM 1217, JCM 7053,
JCM 7055, JCM 7056, and 105-A. These strains
were anaerobically grown at 37°C on following
media. A medium modified from Bifidobacterium
medium described in the catalogue of JCM wasused as a basal medium. The basal medium
contained (g/l) polypepton (Nihon Seiyaku,
Japan), 10; extract from Bonito (Wako Pure

Chemical Industries), 5; yeast extract (Oriental
Yeast, Japan), 5; K2HPO4, 3; sodium ascorbate,
10; L-cysteine HCl, 0.5; Tween 80, 1 ml/l. pH wasadjusted to 6.8 with 2 N NaOH. Where indicated,glucose, gum arabic, lactose, or sucrose (10 g/l)
were added as carbon source. Anaerobic growth
conditions were attained by flashing CO2 for 3-5
min to butyl rubber-stoppered tubes or by fillingthe neck of screw-capped bottles with medium.
The basal medium with and without each carbon
source was inoculated with the overnight culturein the corresponding medium and incubated atintervals. The inoculum size was 1% volume of
media for each culture.
Cell growth was followed by measuring theabsorbance at 660 nm in a 10 mm-cuvette with aspectrophotometer (Hitachi UV-1900). Whenthe absorbance was over 0.5, cultures werediluted with 0.85% NaCl to give an absorbance
below 0.5. Cells were harvested by a centrifuge(18,000 x g for 15 min at 4°C) from cultures,
washed three times with 0.85% NaCl, and stored
at -20°C until use.
Assay of enzyme activities
Glycosidehydrolase activities were determined with
4-nitrophenyl (4-NP) glycosides as substrate.
Reaction mixture (1 ml) contained an enzyme
preparation, 80 mM K-phosphate (pH 7.0) and 1
mM 4-NP-glycoside. The reactions were carriedout at 30°C for 5 min and were terminated by theaddition of 0.5 ml of 0.2 M Na2CO3. The amountof 4-nitrophenol released was determined at
400 nm with an extinction coefficient of
18.3 mM−1 cm−1. One unit of enzyme activity isdefined as the amount of enzyme that released
1 μmol of 4-nitrophenol per min from
4-NP-glycoside.
Enzyme activities which hydrolyze oligosaccharides were determined by measuring theconcentration of reducing sugar formed inreaction mixtures at 540 nm using 3,5-dinitrosalitylic acid. Reaction mixtures containing 80
mM K-phosphate (pH 7.0), enzyme preparation,and substrate 100 mM raffinose, stachyose, or
0.5% gum arabic were incubated at 30°C for 60
min. One unit of enzyme activity was defined as
the amount of enzyme that produced 1 μmol of
reducing sugar per min from the oligosaccharide.

N. Saishin and I. Yamamoto

Transglycosylation activity was assayed bydetection of product in HPTLC (Silica gel 60;
Merk Ltd., Germany). Reaction mixtures (1 ml)
contained 80 mM NaOH-glycine buffer (pH 8.0),
15 mM 4-NP-α-D-galactopyranoside, 80 mM
saccharides, and 0.2 units of purified
α-galactosidase, and were incubated for 5 h.
Saccharides were separated in n-butanol:acetic
acid:water (5:4:3), and detected with spray of 5%
sulfuric acid in ethanol.
Thermal stability assay was performed byincubating a purified enzyme at 40, 45, and 50°C
and drawing aliquots at intervals. The enzyme
activity was then determined with 4-NP-α-Dgalactopyranoside as substrate described above.
The protein concentrations were determinedaccording to the Lowry method for cells andcrude extracts, and by using coomassie brilliant
blue (Bio-Rad Laboratories, USA) in enzyme
purification. Bovine serum albumin was used as
the standard. All reactions were carried out in
triplicate.
Purification of α-galactosidase Wet cells (7 g)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, 37.5 ml).
The precipitate
obtained between 30 and 70% saturation ofammoniun sulfate was dissolved in 50 mM
Tris-HCl, pH 7.2 (Step 2. Ammonium sulfate
fraction, 9.1 ml). Proteins dissolved were loaded
on a column (2.5 x 90 cm) of Sepharose 4B (GEHealthcare, Sweden) equilibrated in 50 mM
Tris-HCl (pH 7.2) containing 0.1 M NaCl andeluted at a flow rate of 0.4 ml/min. Fractions
containing α-galactosidase activity were pooled(Step 3. Sepharose 4B, 54 ml). The pooled
fractions were loaded on a column (1.5 x 5 cm) of
Q-Sepharose(GE
Healthcare,
Sweden)equilibrated in 50 mM Tris-HCl(pH 7.2)
containing 0.1 M NaCl. Proteins were eluted ata flow rate of 0.5 ml/min with a linear
concentration gradient of NaCl (0.1-0.7 M).
Fractions containing α-galactosidase activity
were pooled (Step 4. Q-Sepharose, 14 ml). The

75

solution pooled was mixed with ammonium
sulfate at a concentration of 1.84 M, then was
loaded on a column (1.5 x 5 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 (1.84–0
M) at a flow rate of 1 ml/min. Fractions activewith α-galactosidase were pooled (Step 5. Butyl-S
Sepharose, 19.5 ml). The enzyme factions pooled
were desalted and concentrated by centrifugation
with a filtration tube Vivaspin 4 (Sartorius,
Germany). Concentrated proteins were loaded ona column (5.2 ml) of CHT Ceramic Hydroxyapatite Type I 40 μm (Bio-Rad Laboratories,
USA) equilibrated in 0.01 M K-phosphate (pH7.2). Proteins were eluted
by a stepwise
gradient of the phosphate buffer (0.01–0.5 M) ata flow rate of 1 ml/min (Step 6. Hydroxyapatite,
9.9 ml).
Molecular mass determinationThe
molecular mass of the native enzyme wasdetermined by polyacrylamide gel electrophoresis (PAGE) without sodium dodecyl sulfate(SDS). The concentration gradient of polyacrylamide gel was 5-15% (w/v). Molecularweight marker proteins for native PAGE (HMW
calibration kit, GE Healthcare, UK) were used
for calibration. SDS-PAGE was carried out with
12.5% (w/v) polyacrylamide gel according toLaemmli [26]. Molecular weight marker proteinsfor SDS-PAGE (GE Healthcare, UK) were used
for calibration. Protein bands in gels weredetected by staining with Coomassie blue R-250(Bio-Rad Laboratories, USA).
Results and Discussion
Cell growth of B. longum strains on gum arabic
Cell growth of some strains of B. longum was
examined on gum arabic under anaerobic
conditions. The basal medium without carbon
source gave no growth of any strains of B.
longum. All the tested strains grew well withglucose as carbon source and sedimentation ofcells occurred after 8 h incubation. Gum arabic3α-Galactosidase from Bifidobacterium longum

76

Table 1. Enzyme activities with 4-NP-glycoside in extracts from B. longum JCM 7052 grown on 1% carbohydrate
Gum arabic

LactoseGlucoseEnzyme activity (μmol/min/mg of protein) ± SE (3)
0.444

±

0.095

0.420

±

0.041

0.091

±

0.012

4-NP α-L-Arabinopyranoside

0.489

±

0.083

0.195

±

0.047

0.117

±

0.003

4-NP α-D-Galactopyranoside

1.96

±

0.059

0.404

±

0.055

0.176

±

0.019

4-NP β-D-Galactopyranoside

1.85

±

0.075

0.797

±

0.197

0.491

±

0.046

4-NP α-D-Glucopyranoside

1.12

±

0.116

0.864

±

0.125

0.500

±

0.052

4-NP β-D-fucopyranoside

0.319

±

0.062

0.084

±

0.008

0.148

±

0.009

caused cell growth of strains JCM 7052 and JCM
7053, whereas there were no growth on gum
arabic of the other strains JCM 1217, JCM 7055,
JCM 7056, and 105-A. High turbidities of theJCM 7052 and JCM 7053 cultures were observed
after 12 h and 24 h of incubation, respectively.
All the tested strains failed to grow on guar gum.
Glycoside hydrolase activities in B. longum JCM
7052
Some glycoside hydrolases presumed to be
involved in the degradation of gum arabic were
determined in B. longum JCM 7052. Hydrolytic
activities for gum arabic were observed in wholecells harvested at an early stationary phase of
growth on gum arabic and in crude extracts
prepared from them, to be 1.02 ± 0.060 and 1.65 ±0.21 μmol/60 min/mg of protein, respectively.
However, no activity was detected in supernatantof the culture. When 4-NP-glycosides were usedas substrate, activities of α- and β-galactosidases,
α-glucosidase, α-arabinofuranosidase, and α-arabinopyranosidase, were observed in crude
extracts (Table 1). Among these enzymes α- andβ-galactosidase activities were at highest levels
when cells were grown with gum arabic, about
10- and 4-times compared with those inglucose-grown cells, respectively. Hydrolytic
activities for 4-NP-glycosides were determined in
the extracts from cells harvested at several
stages of cell growth on gum arabic (Fig. 1).
α-Galactosidase activity was high in thestationary phase of growth, and activity levels
were maintained at least for 12 h in the growthphase. The highest level of β-galactosidase
activity was observed in the stationary growth

Enzyme activity（μmol/min/mg of protein)

4-NP α-L-Arabinofuranoside

3.0
1.5

2.5
2.0

1
1.5
1.0

0.5

Cell growth (A 6 6 0)

Substrate

0.5
0.0

0
0

4

8
12
16
Incubation time （h）

20

Fig 1. Enzyme activities during growth of B. longum
JCM 7052. ○, Cell growth; ▲, 4-NP-α-D-galactosidase; and ■, 4-NP-β-D-galactosidase.

phase. Activities of the other enzymes were low
during cell growth (data not shown). Thesefindings suggested that gum arabic washydrolyzed by unidentified enzymes on the cellsurface and was further degraded by at least αand β-galactosidases in the cytoplasm of B.
longum JCM 7052. Some properties of crude
α-galactosidase were reported in B. longum [22],while β-galactosidase was purified andcharacterized from B. longum CCRC 15708 [27].
Purification of α-galactosidase from B. longum
JCM 7052
For purification of α-galactosidase (EC
3.2.1.22) from B. longum, the strain JCM 7052
grown on 1% gum arabic was used.
The
purification of α-galactosidase from B. longum
JCM 7052 is summarized in Table 2. QSepharose ion exchange chromatography showeda peak of α-galactosidase activity at 0.53 M

N. Saishin and I. Yamamoto

77

Table 2. Purification of α-galactosidase from B. longum grown on 1% gum arabicSpecific activity

Protein

Total activity(mg)(units)

1. Crude extract

119

103

0.86

100

2. Ammonium sulfate fraction

26.4

74

2.80

72

3. Sepharose 4B

8.13

63

7.75

61

4. Q Sepharose

1.36

45

33.1

44

5. Butyl-S Sepharose

0.66

24

36.4

23

6. Hydroxyapatite

0.14

8

57.1

8

Purification step

concentration of NaCl. In hydrophobic chromatography with Butyl-S Sepharose 6 Fast Flow the
enzyme activity was released at 20% saturated
concentration of (NH4)2SO4 in Tris-HCl (pH 7.2).
Finally, α-galactosidase was desorbed fromhydroxyapatite by 0.1 M phosphate (pH 7.2).
α-Galactosidase was purified 66-fold and showeda single protein as shown in Fig. 2.

Yield(units/mg of(%)

protein)（Ａ）（Ｂ）

kDa M １

kDa M １

97→
66→
45→

669→
440→
←79kDa 232→140→

←150 kDa

67→

Molecular mass of α-galactosidase
SDS-PAGE showed a molecular mass of
α-galactosidase to be 79 kDa (Fig. 2A).
Molecular mass was also determined to be 150
kDa by native PAGE (Fig. 2B). These results
suggested that α-galactosidase of B. longum is
active in dimeric form. The genome sequence of
B. longum NCC 2705 revealed that this strainprobably contains two genes encoding for
α-galactosidase, BL0177 and BL1518 [11].
BL1518 consists of 768 amino acids and has
molecular mass of 83 kDa. Another possible
α-galactosidase BL0177 is composed of 469
amino acids and has molecular mass of 51 kDa.
As far as we know, this is the first report ofpurification and characterization of α-galactosidase from B. longum. The purified α-galactosidase appeared to be a homologue of BL1518
enzyme if B. longum JCM 7052 had two kinds of
α-galactosidase gene in its genome.

30→
20.1→
Fig

2.

Molecular

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

no activity with other 4-NP-α-glycosides, 4-NP4-NP-α-D-mannopyranα-D-glucopyranoside,
oside, 4-NP-α-L-rhamnopyranoside, 4-NP-α-Lfucopyranoside,
4-NP-α-L-arabinopyranoside,and 4-NP-α-L-arabinofuranoside. 4-NP-β-D-galactopyranoside and 4-NP-β-D-fucopyranosidegave no enzyme activity. Michaelis constant Km
was determined by double reciprocal plots to be
0.15 mM for 4-NP-α-D-galactopyranoside.
Maximal velocity (Vmax) was also shown to be 5.1
μmol/min/mg of protein.
Raffinose and stachyose were alsohydrolyzed by α-galactosidase. Km values were
determined to be 88.3 and 126 mM for raffinoseand stachyose, respectively. Values of Vmax were45.1 and 60.3 μmol/min/mg of protein in the

Substrate specificities of α-galactosidase
Substrate specificity of the purified
α-galactosidase was investigated with 4-NPglycoside compounds. Hydrolytic activity wasobserved with 4-NP-α-D-galactopyranoside, but
5α-Galactosidase from Bifidobacterium longum

Effects of pH, temperature, and chemicals on the
enzyme activity.
The optimal pH was assayed by incubating
the purified enzyme with 4-NP-α-galactopyranoside in 80 mM buffers ranging from pH
5.0 to pH 9.5. The highest activity of α-galactosidase was obtained at pH 8.0 in 80 mM
glycine-NaOH buffer (Fig. 3). pH dependency of
α-galactosidase activity was also investigated in
extracts prepared from the other species of B.
longum. All strains tested JCM 1217, JCM 7055,
and 105-A contained α-galactosidase activity andshowed maximal activity at pH 8.0 (data notshown). These findings indicated that B. longum
had an α-galactosidase operating optimally insemialkaline environments, and this enzyme isdistinct from the α-galactosidase active at pH 5.8[22]. α-Galactosidases have been purified fromthe other three species of Bifidobacterium [23-25],
and their optimal activities are found at pH
5.5-6.0.
When α-galactosidase activity was
assayed in Tris-HCl buffers, activities observedat pH 6.5–9.0 were lower than the activities
observed in the other buffers (Fig. 3). Double
reciprocal plots showed that the enzyme wasinhibited competitively by tris (hydroxymethyl)aminomethane (Fig. 4), and its inhibition
constant Ki was estimated to be 32 mM in Dixon
plot.
The optimal temperature for α-galactosidase
was 40–45°C at pH 7.5 in a 5 min reaction (Fig.
5A), but a remarkable decline in activity wasobserved during 5 h incubation at 40–45°C and
its instability was protected partly by 0.15 and
0.3 M NaCl (Fig. 5B). However, the enzyme
activity was decreased by 30% after 30 min of
incubation at 50°C and was not maintained evenin the presence of 0.15 M NaCl. The enzyme
activity in the presence of 0.15 M NaCl was100
Enzyme activity（％）

reactionwithraffinoseandstachyose,
respectively. This enzyme hydrolyzed melibiose
to galactose and glucose, those products weredetected by HPTLC (data not shown). No
reducing sugar was produced from 0.5% gum
arabic during 1 h incubation with the enzyme(7.2 mU of 4-NP-α-galactopyranoside hydrolytic
activity) at 37°C and pH 7.0.

80
60
40
20
0
5

6

7

8

9

10

ｐＨ
Fig 3.

Effects of pH on activity of α-galactosidase

purified from B. longum JCM 7052. ●, Buffers usedwere 80 mM acetate (pH 5.0–5.5), phosphate (pH

6.0–7.0), and glycine-NaOH (pH 7.5–9.5). ▲, 80mM
Tris-HCl （pH 6.5–9.0)

0.3

1/v
(μmol/min/mg of protein) -1

78

0.2

0.1

0
0

2

4
1/s (mM)

Fig 4.

6-1

Inhibitory effects of Tris on activity of thepurified α-galactosidase. Double reciprocal plots of
α-galactosidase activity against

4-NP-α-D-galacto-pyranoside in the presence of Tris-HCl. For the
enzyme activity assasy, K-phosphate buffer (pH7.5)
was used. The concentrations of Tris were 0 (●), 60（■) , and 120 mM (▲).

inhibited about 10% (data not shown). Though noattempt has been done to examine effects of NaClon thermostability of α-galactosidase as far as weknow, the stabilization of enzyme would endow itwith great industrial potential.
To determine the effects of chemicals, enzyme
activities were measured in the presence of 1 mM
metal chloride salts or EDTA. α-Galactosidase
activity was inhibited about 40% by Cu2+ andabout 10–15% by Fe2+, Mg2+, Mn2+, and Co2+.

N. Saishin and I. Yamamoto

79

Enzyme activity (％)

100

evaluation of the reaction conditions will be thesubject of further investigation.

（Ａ）

80
60

In this study B. longum JCM 7052 was foundto be able to grow on gum arabic as a carbon andenergy source. α- and β-Galactosidase activities
were observed at higher levels in extracts from
cells grown on gum arabic compared with thosein cells grown on glucose. This probably suggeststhat α- and β-galactosidases play some roles indegradation of gum arabic. The subunit of the
α-galactosidase had the molecular mass of 79
kDa, suggesting that the purified α-galactosidase appeared to be a homologue of BL1518
enzyme in B. longum NCC 2705 [11]. The
purified enzyme seemed to be a semialkaline
α-galactosidase which showed an optimal
activity at pH 8.0, though acid α-galactosidases
have been found in the other species of
Bifidobacterium [22-25]. It is necessary to finda gene encoding the semialkaline α-galactosidase
and to clarify the role of the enzyme in
metabolism of gum arabic in B. longum JCM
7052.

40
20
0
20

30
40
50
Temperature (℃)

Enzyme activity (％)

100

60

（B）

80
60
40
20
0
0

1

2
3
4
Incubation tｉｍｅ （ｈ）5

Fig 5. Effects of temperature on the activity （Ａ） andstability （ Ｂ ） of the purified α-galactosidase.

In

Acknowledgement
The authors would like to thank Marsha
Hayashi for her critical reading of this paper.
This research was supported partly by a grant(No. kr51421702) from Yukiyoshi Institute.

（Ｂ）, ■, 40℃; ○, 45℃; and ▲, 50℃. Solid lines
indicate activities in the absence of NaCl, dotted lines
in the presence of 0.15 M NaCl, broken line in thepresence of 0.3 M NaCl.

EDTA was less active as a chelating reagent for
α-galactosidase activity. Contrary to our results,
Garro et al. [22] reported that Mn2+ showed apositive effect on activity of the acidic α-galactosidase in crude extracts from B. longum CRL
849.

References
1.

2.

Transgalactosylation activity of the enzyme
α-Galactosidase is known to have transgalactosylation activity [24, 25, 28, 29]. The
purified α-galactosidase was briefly tested tohave galactosyl transferring activity. HPTLC
showed a new saccharide in the reaction mixture
containing 4-NP-α-D-galactopyranoside andgalactose (data not shown). A similar result wasalso obtained by mixing 4-NP-α-D-galctopyranoside and melibiose. However, we could not
identify products of transgalactosylation. The

3.

4.

7

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Br. J. Nutr. 55, 261-266.

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Communicated by Ito Susumu

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