J.Biol. Macromol., 10 (1), 13-22 (2010)

ArticlePurification and characterization of α-galactosidase I from

Bifidobacterium longum subsp. longum JCM 7052
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 November 18, 2009; Accepted January 18, 2010.
α-Galactosidase I was purified and characterized from Bifidobacterium longum subsp. longum
JCM 7052 grown on glucose. It had a molecular mass of 162 kDa and consisted of two same subunits.
The molecular mass of the subunit was 85 kDa. MALDI-TOF-MS showed that this enzyme is ahomologue of BLD1483 and BL1518 found in the genomic sequences of B. longum subsp. longum
strains DJO10A and NCC 2705, respectively. α-Galactosidase I showed optimal activity at pH 7.5 and
45°C. Thermal stability was lost during 5 h incubation at 50°C, and was partly protected in thepresence of 0.15 M NaCl. The Km values for 4-nitrophenyl-α-D-galactopyranoside, raffinose, andstachyose were 0.34, 15.5, and 39.3 mM, respectively. The enzyme activity was inhibited competitively
by Tris (hydroxymethyl) aminomethane (Ki = 24 mM). Transglycosylation activity was also observed
from 4-nitrophenyl-α-D-galactopyranoside to galactose, arabinose, glucose, mannose, sucrose, melibiose,
and galactinol.
Keywords: α-galactosidase, purification, Bifidobacterium longum subsp. longum
Introduction
α-Galactosidases (EC 3.2.1.22) catalyze thehydrolysis of the terminal α-linked galactosideresidues from α-galactosides including melibiose,raffinose, stachyose, galactomannanes, andgalactanes [1]. α-Galactosidases are widelydistributed in microorganisms, plants, animals,
and humans [2-7]. The enzymes are known tohave transglycosylation activities, which are
expected to be powerful tools for oligosaccharide
synthesis [8].
Corresponding author: Isamu Yamamoto.
Phone: +81-78-737-2312, Fax: +81-78-732-5161
E-mail: iyamamoto@suma.kobe-wu.ac.jp

Bifidobacteria, predominant members ofintestinal microbiota in humans [9], have
α-galactosidase activities to use the variety of
α-galactosides as energy source. α-Galactosidases have been isolated either as native or
recombinant enzymes from Bifidobacterium
adolescentis DMS 20083 [8, 10, 11], Bifidobacterium breve 203 [12], Bifidobacteriumbifidum NCIBM 41171 [13], and Bifidobacterium
longum subsp. longum JCM 7052 [14]. These
enzymes evidently showed transgalactosylation
activities able to synthesize galacto-oligosaccharides.
B. longum subsp. longum JCM 7052 hasbeen found to grow on gum arabic [14]. A highactivity of α-galactosidase is observed in cells

14α-Galactosidase I from Bifidobacterium longum subsp. longum

grown on gum arabic, and the purified enzyme,
α-galactosidase X, shows optimal activity at pH
8.0. The cells grown on glucose also containα-galactosidase but its activity is one tenth ofthat obtained in the cells grown on gum arabic.
To learn whether these activities could be
attributed to different enzymes, we partiallypurified α-galactosidase from the cells grown on
glucose. This enzyme, α-galactosidase I, showed
weak binding affinity for Q-Sepharose comparedto α-galactosidase X, suggesting the two enzymes
are different proteins. In the present study
α-galactosidase I was purified and characterizedfrom B. longum subsp. longum JCM 7052 grownon glucose. Identification of the gene encoding foreach enzyme was attempted by means of
MALDI-TOF-MS.
Materials and Methods
Chemicals 4-Nitro-phenyl (4-NP) glycosides,
melibiose, stachyose, and 3,5-dinitrosalicylic acid
were purchased from Sigma-Aldrich Co. (St.
Louis, USA). Other chemicals were reagent
grade products of Wako Pure Chemical
Industries (Osaka, Japan).

Bacterial strain and growth conditions
Bifidobacterium longum subsp. longum JCM

7052 was used in this study. The strain wasanaerobically grown at 37°C on Bifidobacterium
medium, which contained (g/l) Polypepton(Nihon Seiyaku, Japan), 10; extract from bonito(Wako Pure Chemical Industries), 5; yeast
extract (Oriental Yeast, Japan), 5; glucose, 10;
K2HPO4, 3; sodium ascorbate, 10; L-cysteine
HCl, 0.5; and Tween 80, 1 ml/l. pH was adjusted
to 6.8 with 2 N NaOH. 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 themedium. The medium was inoculated with 2%
volume of an overnight culture in the same
medium.
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 Standard assayof α-galactosidase activity was performed with
4-nitrophenyl (4-NP)- α-galactopyranoside as
substrate. The reaction mixture (1 ml) contained
an enzyme preparation, 80 mM K-phosphate (pH7.0) and 1 mM 4-NP-α-galactopyranoside. The
reactions were started by adding an enzyme
preparation. After incubation at 30°C for 10 min
the reactions were 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 unit of enzyme activity isdefined as the amount of enzyme that releases
1 μmol of 4-nitrophenol per min from 4-NP-αgalactopyranoside.
For assay of enzyme activities whichhydrolyze oligosaccharides, reducing sugar
produced from oligosaccharide was determinedin the reaction mixtures containing 80 mM
K-phosphate (pH 7.0), enzyme preparation, and100 mM raffinose or stachyose. After incubation
at 30°C for 60 min, 3,5-dinitrosalitylic acid wasadded to stop the reaction. The absorbance wasmeasured at 540 nm. Galactose was used as thestandard for calibration. One unit of enzyme
activity is defined as the amount of enzyme that
produces 1 μmol of reducing sugar per min fromthe oligosaccharide.
Transglycosylation activity was assayed bydetection of product in TLC with Silica gel 60
HPTLC plates (Merck Ltd., Germany). The
reaction mixtures (1 ml) contained 80 mM Kphosphate buffer (pH 7.0), 15 mM 4-NP-α-Dgal actopyranoside, 80 mM saccharide, and 0.2
units of purified α-galactosidase I, and wereincubated for 24 h. Saccharides were separated
in n-butanol:acetic acid:water (5:4:1), and weredetected with spray of 5% sulfuric acid in
ethanol.
Thermal stability was assayed by incubating
purified α-galactosidase I at 40, 45, 50 and 60°C
and drawing aliquots at intervals. The enzyme
activity was then determined by the standard
assay method described above.

N. Saishin et al.

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 α-galactosidase I Wet cells(30 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, 120 ml).
The precipitate
obtained between 30 and 70% saturation ofammonium sulfate was dissolved in 50 mM
Tris-HCl, pH 7.2 (Step 2. Ammonium sulfate
fraction, 7 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, 98 ml). The pooled
fractions were loaded on a column (1.5 x 7 cm) of
Q-Sepharose(GE
Healthcare,
Sweden)equilibrated in 50 mM Tris-HCl (pH 7.2)
containing 0.2 M NaCl. Proteins were eluted ata flow rate of 0.4 ml/min with a linear
concentration gradient of NaCl (0.2-0.7 M).
Fractions containing α-galactosidase activity
were pooled (Step 4. Q-Sepharose, 23 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 (1.84–0
M) at a flow rate of 1 ml/min. Fractions activewith α-galactosidase were pooled (Step 5. Butyl-S
Sepharose, 4.9 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 α-galactosidase activity was cut out

15of 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, 1.3 ml).

Molecular

massdeterminationThe
molecular mass of the native enzyme wasdetermined by concentration gradient PAGE
without SDS. The concentration gradient ofpolyacrylamide gel was 5-15% (w/v). A molecularweight marker set (NativeMark UnstainedProtein Standard, Invitrogen, USA) was used forcalibration. SDS-PAGE was carried out with
12.5% (w/v) polyacrylamide gel according toLaemmli [15]. Molecular weight marker proteinsfor SDS-PAGE (GE Healthcare, UK) were used
for calibration. Protein bands in gels were
stained with Coomassie blue R-250 (Bio-Rad
Laboratories, USA).

Protein identification by mass spectrometry

The proteins in the SDS-PAGE gel were digested
using sequence-grade modified trypsin (Promega,
Madison, USA) using the procedures of Tanaka
et al. [16], with slight modifications. The
identification of each protein was carried out by
peptide mass fingerprinting (PMF) analysis
using matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) mass spectrometry(MS) and tandem mass spectrometry (MS/MS),
using an Ultraflex MS system (Bruker Daltonik
GmbH, Bremen, Germany). The PMF results
were used to search the NCBI protein databaseusing the Mascot Search engine. The PMF resultand one MS/MS result were combined to makethe results of the search reliable.
Results
Purification of α-galactosidase I
The cells grown on glucose of Bifidobacterium longum subsp. longum JCM 7052
possess α-galactosidase activity [14]. Q-Sepharose ion exchange chromatography showed two
peaks of α-galactosidase activity in extracts ofthe cells which were grown in a stationary
phase of 12 h incubation (Fig. 1A).

α-Galactosidase I from Bifidobacterium longum subsp. longum

16

Table 1. Purification of α-galactosidase I from B. longum subsp. longum JCM 7052 grown on 1% glucose
Specific activity

Protein

Total activity(mg)(units)

1. Crude extract

310

40.2

0.130

100

2. Ammonium sulfate fraction

198

30.5

0.154

76

3. Sepharose 4B

150

26.9

0.180

67

4. Q Sepharose

41.9

18.5

0.442

46

5. Butyl-S Sepharose

3.90

4.8

1.22

12

6. Native PAGE

0.18

3.1

17.8

8

Purification step

The major and minor peaks were eluted at
0.43 and 0.53 M NaCl, respectively. The latterpeak of the activity seemed to be the same as the
enzyme of α-galactosidase which was inducible
by gum arabic (Fig. 1B). The inducible α-galactosidase has been purified and characterizedfrom the same strain grown on gum arabic [14].
We, therefore, attempted to purify anotherα-galactosidase from the cells grown on 1%
glucose. We call the enzyme purified in thisstudy α-galactosidase I, and the previously
purified enzyme α-galactosidase X.
α-Galactosidase I was purified to an electrophoretically single band, and an overall yield of
8% was obtained (Table 1). The enzyme, which
was eluted at 0.43 M NaCl from Q-Sepharose,
was released by 0.5 M ammonium sulfate inhydrophobic chromatography with Butyl-S
Sepharose 6 Fast Flow. In the last step we used
native PAGE for separation of the enzyme, andthe obtained preparation contained only a single
protein, which was confirmed by SDS-PAGE.
Determination of the molecular mass of
α-galactosidase I
Molecular mass of α-galactosidase I wasdetermined by PAGE with and without SDS.
SDS-PAGE showed the molecular mass of
α-galactosidase I to be 85 kDa (Fig. 2A). The
molecular mass was also determined to be 162
kDa by native PAGE (Fig. 2B). These results
indicate that α-galactosidase I consists of two
same subunits. These values of molecular mass
are obviously different from those of the(units/mg of

Yield
(%)

protein)(A)(B)

Fig. 1. Ion exchange chromatography on Q Sepharose of
α-galactosidases from B. longum subsps. longum JCM
7052. Enzyme preparations of α-galactosidase obtainedby gel filtration with Sepharose 4B were applied oncolumns (1.5×5 cm) of Q Sepharose and eluted with an
NaCl gradient in 50 mM Tris-HCl, pH 7.2. (A), an
enzyme preparation obtained from cells grown on 1%
glucose; (B), from cells grown on 1% gum arabic. ●,α-galactosidase activity; ○, absorbance at 280 nm; △,concentrations of NaCl. Each fraction contained 2.4 ml.

N. Saishin et al.

(A)
kDa

M

(B)
kDa

1

M

1

242 →
97 →
66 →

←85 kDa

45 →

66 →

30 →

20 →

20.1→
Fig.

2.

←162 kDa
146 →Molecular

massdeterminationof

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

previously purified enzyme α-galactosidase X (79and 150 kDas [14]).
Identification of α-galactosidase I by mass
spectrometry
We have identified the purified enzyme bymeans of MALDI-TOF-MS as described in the
Materials and Methods. The PMF result wasused to search the NCBI protein database using
the Mascot Search engine. The PMF result(Accession number, gi|46190700; Sequence
coverage, 33.1%; score, 153) and two MS/MS
results (1714.891 (535-551), TAHPGLEIESCSSG
GGR; score, 111) and (1917.171 (747-763),
YGIRPPSLHPQQAVLLK; score, 97) werecombined and searched again. Both amino acidsequences were coincided with those in
α-galactosidase (Accession number, gi|46190700;
combined score, 303), which has been found andgiven the locus tag BLD 1483 in the genomic
sequence of B. longum subsp. longum DJO10A[17]. α-Galactosidase I was, therefore, identified
as a homologue of BLD 1483 found in B. longum
subsp. longum DJO10A. This α-galactosidase
consists of 763 amino acids and has themolecular mass of 83,309 Da. This value of themolecular mass is consistent with that of 85 kDa
determined by SDS-PAGE. The BLD 1483
α-galactosidase belongs to the glycosyl hydrolase(GH) 31 family.

17

Substrate specificities of α-galactosidase I
Substrate specificity of the purified
α-galactosidase I was investigated with 4-NPglycoside compounds. Hydrolytic activity wasobserved with 4-NP-α-D-galactopyranoside, but
no activity with other 4-NP-α-glycosides, 4-NP-αD-glucopyranoside, 4-NP-α-D-mannopyranoside,
4-NP-α-L-fuco4-NP-α-L-rhamnopyranoside,pyranoside, 4-NP-α-L-arabinopyranoside, and
4-NP-α-L-arabinofuranoside. 4-NP-β-D-galactopyranoside and 4-NP-β-D-fucopyranoside gave no
enzyme activity. The Km and Vmax values for
4-NP-α-D-galactopyranoside were determined bydouble reciprocal plots to be 0.34 mM and 75.8μmol/min/mg of protein, respectively. Raffinoseand stachyose were also hydrolyzed by
α-galactosidase I. The Km values for raffinoseand stachyose were determined to be 15.5 and39.3 mM, respectively. The Vmax values were
22.8 and 28.8 μmol/min/mg of protein in thereactionwithraffinoseandstachyose,
respectively. In previous study, α-galactosidase X
showed the Km values higher than 80 mM for
raffinose and stachyose [14]. The affinity of
α-galactosidase I for these α-galactosides
appeared to be higher than that of α-galactosidase X.
Effects of pH, temperature, and chemicals on
enzyme activity
The optimal pH was assayed by incubating
the purified α-galactosidase I with 4-NP-αgalactopyranoside in 80 mM buffers ranging
from pH 5.0 to pH 10.0. The highest activity of
α-galactosidase was obtained at pH 7.5 in 80 mM
glycine-NaOH buffer (Fig. 3). When α-galactosidase activity was determined in Tris-HCl
buffers (pH 6.5–9.0), the activities were lower
than those observed in the other buffers (Fig. 3).
The enzyme activities determined in 80 mM
K-phosphate, pH 7.5 were lowered by 60% in thepresence of 60 mM Tris-HCl, pH 7.5. Double
reciprocal plots showed that the enzyme wasinhibited competitively by Tris (hydroxymethyl)aminomethane (Fig. 4), and its inhibition
constant Ki was estimated to be 24 mM in Dixon
plot.

α-Galactosidase I from Bifidobacterium longum subsp. longum

18

100

Enzyme activity (％)

Enzyme activity（％）

100
80
60
40
20

(A)

80
60
40
20
0

0

20
5

6

7

8

9

pH

100

α-galactosidase I. Buffers used were at 80 mM. ■,Na-acetate (pH 5.0–5.5); ● , K-phosphate (pH
5.5–8.0); ○ , glycine-NaOH (pH 7.0–10.0); and ▲ ,(μmol/min/mg of protein)-1

Tris-HCl (pH 6.5–9.0).

25
20

Enzyme activity (％)

Fig. 3. Effects of pH on activity of the purified

1/v

30
40
50
Temperature (℃)

10

60

(B)

80
60
40
20
0

15

0
10

1

2
3
4
Incubation time (h)5Fig. 5. Effects of temperature on the activity (A) and5

stability (B) of the purified α-galactosidase I. In (B),

0
0

2

4

6
8
-1
1/s (mM )10

12

Fig. 4. Inhibitory effects of Tris (hydroxymethyl)■, 40℃; ○, 45℃; ▲, 50℃; and ◇, 60℃. Solid lines
indicate activities in the absence of NaCl, dotted lines
in the presence of 0.15 M NaCl, and a broken line in
the presence of 0.3 M NaCl.

aminomethane on activity of the purified α-galactosidase I. Double reciprocal plots of α-galactosidase
activity against 4-NP-α-D-galactopyranoside in thepresence of Tris-HCl (pH 7.5). For the enzyme activity
assay, K-phosphate buffer (pH7.5) was used. The
concentrations of Tris were 0 (●), 60 (■), and 120 mM(▲).

The optimal temperature for α-galactosidase
activity was 45°C at pH 7.5 in a 10 min reaction(Fig. 5A), but the enzyme activity was partly lost
during 5 h incubation at 45°C. The enzyme wasalmost completely inactivated during 5 h
incubation at 50°C and its stability was partly
protected by 0.15 NaCl (Fig. 5B). The enzyme

activity was decreased by 30% after 30 min of
incubation at 60°C and was not maintained evenin the presence of 0.15 M NaCl. Thermostabilizing effects of NaCl on α-galactosidase I
would endow it with great industrial potential.
The effects of chemicals on the enzyme
activity were also determined in the presence of 1
mM metal chloride salts or EDTA (Table 2).
α-Galactosidase I activity was inhibited about
20% by Cu2+. However, other metal ions Fe3+,
Mg2+, Mn2+, and Co2+ showed almost no effects on
the enzyme. EDTA also worked less actively as achelating reagent for α-galactosidase activity.
These results are different from those observed

N. Saishin et al.

Table 2.

Effects of some reagents on the activityof α-ｇalactosidase I from B. longum subsp. longum
JCM 7052.
Reagents

Relative activity(1 mM)

（％）

None

100

BaCl2

98

CaCl2

103

CoCl2

104

CuCl2

83

FeCl3

105

MgCl2

105

MnCl2

106

NiCl2

105

ZnCl2

109

EDTA

98

in α-galactosidase X, which was slightly
inhibited about 40% and 10-15% by Cu2+ and theother ions, respectively [14]. No activation by
Mn2+ was observed with our enzyme, although
Garro et al. [19] reported that Mn2+ showed apositive effect on activity of the acidic
α-galactosidase in crude extracts from B. longum
CRL 849.
Transgalactosylation activity of α-galactosidase I
Most α-galactosidases often have transgalactosylation activity [1, 8]. The purified α-galactosidase I was briefly tested to have galactosyltransferring activity.
TLC showed a newsaccharide in the reaction mixture containing
4-NP-α-D-galactopyranoside and galactose (datanot shown). Similar results were also obtained bywithmixing4-NP-α-D-galctopyranosidearabinose, glucose, mannose, sucrose, melibiose,
and galactinol. However, we could not identify
products of transgalactosylation.
Discussion
In this study B. longum subsp. longum JCM
7052 was found to possess at least two kinds of
α-galactoside hydrolase, α-galactosidases I and X.
MALDI-TOF-MS
analysis
showedthat
α-galactosidase I is a homologue of BLD1483,

19

which is found in the genome of B. longum subsp.
longum strains DJO10A [17]. Almost the sameamino acid sequence in BLD 1483 has beenfound in the deduced gene product BL1518 in the
genome of B. longum subsp. longum NCC2705[18]. α-Galactosidase I seemed to serve indegradation of galactosides having α-1,6-linkage
because this enzyme showed apparently highaffinity for raffinose and stachyose. Moreover,
BL1518 has been shown to be induced when B.
longum subsp. longum NCC 2705 was grown on
raffinose [19]. On the other hand, α-galactosidase
X was synthesized at high levels when B. longum
subsp. longum JCM 7052 was grown on gum
arabic as a carbon source [14]. However,physiological roles of α-galactosidase X are notclear because α-1,4- and α-1,6-linked galactoses
are not found in gum arabic. No homologue of
α-galactosidase X was found in genomic datacompiled so far (unpublished results). This may
suggest that B. longum subsp. longum JCM 7052
would have some additional genes specific fordegradation of gum arabic and α-galactosidase X
would be one of products of those genes. Isolationof genes encoding some enzymes and proteins
involved in degradation of gum arabic is now inprocess with probes designated on the basis ofamino acid sequences found in α-galactosidase X.
α-Galactosidase I showed the optimal
activity around neutral pH of 7.5 as
α-galactosidase X had done so at pH 8.0 [14].
Such a property of each enzyme is different fromthat of an α-galactosidase partially purified from
B. longum CRL 849 showing highest activity at
pH 5.8 [20]. α-Galactosidases so far purified fromthe other species of Bifidobacterium are mostactive at pH 5.5-6.0 [8, 10-13], though thehighest transgalactosylation activity of the B.
adolescentis enzyme is obtained at pH 7.0-8.0 [8,11]. In the CaZy database α-galactosidases
cloned from these Bifidobacterium species [11-13]
including BL1518/BLD1483 [17, 18] areclassified into the GH31 family (http://www.
cazy.org/fam/GH31.html). Alignment of theamino acid sequences of α-galactosidases fromfour species of Bifidobacterium shows that theamino acid sequences are homologous with
65-69% identity [12]. The catalytic mechanism of

20

α-Galactosidase I from Bifidobacterium longum subsp. longum

the GH31 family is less clear, though two
aspartic residues are confirmed to be involved in
the catalytic site of α-galactosidase from
Thermotoga maritima [21]. Catalytic andstructural studies are necessary to identify theamino acid residues which cause the alteration of
pH optima.
α-Galactosidase I activity was competitively
inhibited by Tris as was α-galactosidase X [14].
Actions of Tris have been observed in someenzymes. Sucrase activity in intestinal brushborder is also competitively inhibited by Tris [22].
Vesseur et al [22] have interpreted the inhibitory
effect in terms of a mechanistic model in which
Tris binds at two distinct sites in the enzyme’s
active center. Binding at the glucosyl sub-site
occurs through the free hydroxyl groups of Tris.
This positioning facilitates the interaction
between the amino group of Tris and a proton
donor in the enzyme’s active center. In this way
the competitive inhibition of sucrose is achieved.
Tris has been recently suggested to be acompetitive inhibitor of starch hydrolyzing
activity of Bacillus licheniformis α-amylase, andto have a high tendency to bind the enzyme
active site [23]. β-Galactosidase partially purified
from B. longum subsp. longum JCM 7052 also
showed low activities in Tris-HCl buffer
compared with those observed in phosphatebuffer (unpublished results). These findings
suggest that Tris (hydroxymethyl) aminomethane would be effective as a competitive
inhibitor in catalysis of some glycosidases.
α-Galactosidase I showed transgalactosylation activity from 4-NP-α-D-galctopyranoside toglucose, mannose, sucrose, melibiose, andgalactinol. α-Galactosidases have been known tohydrolyze α-1,6-linkage of galactosides, and to
produce new α-1,6-galactosides by means oftransgalactosylation activity [1, 8]. Recently,
Zhao et al [12] showed that α-galactosidase
purified from Bifidobacterium breve 203
synthesizes galactosyl-α-1,4-linked compound,galactosyl-α-1,4-galactosyl-α-1,6-glucose
frommelibiose
and4-NP-α-D-galctopyranoside.
However, we could not identify the products oftransgalactosylation. The evaluation of reaction
conditions and product identification will be thesubject of further investigation.
Due to enhanced solubility and reaction
rates, most industrial enzymatic processes arecarried out at elevated temperatures. Thermal
inactivation is, therefore, the most frequently
encountered cause of enzyme inactivation. We
showed that α-galactosidase I could be stabilizedagainst heat inactivation by adding 0.15-0.3 M
NaCl (Fig. 5B). NaCl is one of salts which haveeffects to stabilize proteins by reducing thesolubility of hydrophobic residues on the protein
molecule and by enhancing water clusters
around the protein [24]. However, suchproperties of NaCl appeared not to be effective on
stabilization of proteins at temperatures higher
than 50℃.
Acknowledgement
The authors would like to thank Ann B. Caryfor her critical reading of this paper. This
research was supported partly by a grant (No.
kr51421702) from Yukiyoshi Institute.
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Communicated by Ito Susumu