J. Biol. Macromol., 16 (2), 43-53 (2016)

ArticleCharacterization of Two α-Glucosidases Purified 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 October 15, 2016; Accepted November 22, 2016
Two α-glucosidases, which hydrolyzed 4-nitrophenyl (4NP)-α-D-glucopyranoside, were purified andcharacterized from Bifidobacterium longum subsp. longum JCM 7052 grown on gum arabic. These
enzymes, named BlAglA1 and BlAglA2, had apparent molecular masses of 72.0 and 72.8 kDa estimatedby sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and 70.1 and 92.7 kDa by
PAGE without SDS, respectively. The amino acid sequences analyzed by MALDI-TOF-MS were foundin the genomes of several strains of B. longum subsp. longum. BlAglA1 and BlAglA2 showed hydrolyzing
activities toward isomaltose, trehalose, palatinose, isomaltotriose and panose, but did not toward sucrose,
maltose, amylose, amylopectin, and oyster glycogen, indicating that each enzyme belongs to oligo-1,6glucosidase (EC 3.2.1.10). Both enzymes showed optimal activities with 4NP-α-D-glucopyranoside at pH
5.5-6.0 and at 45-50°C. Values of Km and Vmax for 4NP-α-D-glucopyranoside, trehalose, palatinose, panose,
isomaltose, and isomaltotriose were also determined. The two oligo-1,6-glucosidases were shown to havetransglycosylation activities to synthesize oligosaccharides from trehalose, palatinose, isomaltose,
panose, and isomaltotriose to their selves. Both enzymes also catalyzed transglucosylation from palatinose to ethanol, 1-propanol, 2-propanol, 1-butanol, and 1-hexanol.
Keywords: α-glucosidase, oligo-1,6-glucosidase, transglycosylation, ethyl-α-D-glucoside,

Bifidobacterium longum

Introduction
Bifidobacteria, which are strictly anaerobic
Gram-positive bacteria, have many kinds of genes
to be adapted to grow on host produced glycan
such as mucins and milk oligosaccharides anddietary plant-derived oligo- and polysaccharides.
Many novel enzymes involved in utilization ofcarbohydrates, therefore, have been discovered,isolated and characterized [1-3].
This article is dedicated to Dr. Lars Ljungdahl
on the occasion of his 90th birthday.
*E-mail: iyamamoto@suma.kobe-wu.ac.jp

Among carbohydrate-hydrolyzing enzymes αglucosidases have been purified and characterized from only two species of Bifidobacteria,
Bifidobacterium adolescentis [4, 5] and Bifidobacterium breve [6-8]. α-Glucosidase is a generalterm for the enzymes able to hydrolyze α-glucoside bonds as shown in Fig. 1. Maltitol-hydrolyzing enzyme of B. adolescentis type a, E194a is amaltase acting as α-1,4-glucosidase [4]. AglA of B.
adolescentis DSM20083 (BaAglA) has substratespecificity toward α-1,6-linkage of glucosides,
AglB of this strain (BaAglB) prefers to α-1,4-glucoside bond [5]. B. breve UCC2003 contains Agl1

44

α-Glucosidases from Bifidobacterium longum

Other chemicals were of reagent grade and were
products of Wako Pure Chemical Industries, Ltd.

Bacterial strain and growth conditions
Bifidobacterium longum subsp. longum JCM

Fig. 1. Hydrolytic reactions catalyzed by
α-1,6-glucosidase and α-1,4-glucosidase.

(BbAgl1) and Agl2 (BbAgl2), these enzymes showing substrate specificity toward α-1,6-glucoside
linkage [6], Agl3 which can hydrolyze α-1,1-, α1,3-, α-1,4- and α-1,5-glucosidic linkages [7], andMelD being able to hydrolyze α-1,3-glucosidebond of melezitose [8].
Bifidobacterium longum subsp. longum JCM
7052 has been shown that enzyme activities ofseveral glycoside hydrolases, α- and β-galactosidases, α-L-arabinofuranosidase, α-L-arabinopyranosidase, α-L-fucosidase, and α-glucosidase,are increased during growth on gum arabic [9].
Two α-galactosidases and one β-galactosidase
have been purified and characterized from thisstrain [9-11]. The β-galactosidase encoded in thelacA gene appears to have multiple functions withactivities of α-L-fucosidase and α-L-arabinopyranosidase. In this study we focused on α-glucosidase, especially its transglycosylation activity to
produce oligosaccharides and alkyl glucosides
which are promised to exert beneficial effects on
maintenance of human health.
Materials and Methods
Chemicals Trehalose, palatinose, isomaltotriose, melibiose and gum arabic (from Acacia senegal) were obtained from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). Panose and isomaltose were obtained from Hayashibara Co. Ltd(Okayama, Japan). Isomaltotriose was obtained
from Tokyo Chemical Industry Co. Ltd. (Tokyo,
Japan). 4-Nitrophenyl (4NP) glycosides were purchased from Sigma-Aldrich Co. (St. Louis, USA).

7052 was used in this study. Bacterial cells weregrown in a medium of slightly modified Bifidobacterium medium, which contained (per liter)
Bacto-peptone (Difco Laboratories, USA), 10 g;yeast extract (Oriental Yeast Co., Ltd., Japan), 5
g; meat extract (Wako Pure Chemical Industries,
Ltd., Osaka, Japan), 5 g; K2HPO4, 3 g; sodium
ascorbate, 10 g; cysteine hydrochloride, 0.5 g; andTween 80, 1 ml. Gum arabic (10 g/l) was added ascarbon source instead of glucose. pH was adjusted
to 6.8. Cells were anaerobically grown at 37°C for
20 h. Anaerobic conditions were obtained by filling the neck of screw-capped bottles with themedium.
Assay of enzyme activities Standard assayof α-glucosidase activity was done with 4NP-α-Dglucopyranoside as substrate. The reaction mixture (1.0 ml) contained an enzyme preparation, 80
mM K-phosphate (pH 6.0) and 1.0 mM 4NP-α-Dglucopyranoside. The reactions were carried out
at 40°C for 10 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 wasdefined as the amount of enzyme that released
1 μmol of 4-nitrophenol per min from 4NP-α-Dglucopyranoside.
Glucose released by glycoside hydrolyzing
activity was assayed by using a glucose assay kit(LabAssayTM Glucose, Wako Pure Chemical
Industries, Ltd., Osaka, Japan). The reaction
mixture (50 µl) for glycoside hydrolyzation consisted of 20 mM saccharide, 80 mM K-phosphate(pH 6.0), and a purified enzyme. The enzyme
reaction was carried out at 40°C and stopped byheating in boiling water for 5 min. When trehalose, isomaltose, palatinose, isomaltotriose, or
panose were used as substrate, glucose production was observed proportionally during 5 min
incubation, while in the incubation for 10 min
products of transglycosylation were detected bythin layer chromatography (TLC) described below.
Therefore, the incubation for 5 min was chosen toSaishin et al.

determine Km and Vmax values. The reaction mixtures with the other saccharides (shown in Table2) as substrate were incubated for 30 min.
Determination of transglycosylation activity
was performed by TLC on HPTLC Silica gel 60
plates (Merck Ltd., Germany). Glucoside, 0.4 M
concentration of isomaltose, trehalose, palatinose,
isomaltotriose, or panose in 80 mM K-phosphate(pH 6.0), was incubated with 0.14-0.22 U ml-1 ofthe purified enzyme at 35°C for 0.5, 1, 3, 6, 12, 24,
48 and 72 h. Transglycosylation activities from 0.5
M palatinose to 1.0 M alcohols, ethanol, 1-propanol, 2-propanol, 1-butanol, 1-hexanol, or 1-octanol,
were also determined in 80 mM K-phosphate (pH6.0), with 0.2 U ml-1 of each enzyme at 35°C at
intervals indicated above. When 1-butanol, 1-hexanol, and 1-octanol were used as acceptor
molecules, 0.1% Tween 80 was added in the reaction mixtures. The reactions were stopped byheating in boiling water for 5 min. After centrifugation supernatants (0.5 or 1.0 µl) were spotted onasilica gel plate and saccharides were developed twice in a 2-propanol:1-butanol:water (2:2:1)
solvent system, and detected with spray of 10%
sulfuric acid in ethanol and heating for 15 min at
130°C.
Thermal stability of enzyme was assayed byincubating a purified enzyme (about 0.02 mg ofprotein/ml) at 20-60°C and drawing aliquots at intervals. The enzyme activity with 4NP-α-D-glucopyranoside was then determined according to thestandard assay procedure.
Activity staining of α-glucosidase in polyacrylamide gel after electrophoresis without SDS
was performed by putting the gel on a plastic
sheet with a few drops of 2 mM 4NP-α-D-glucopyranoside as substrate. Incubation for color
development was done at room temperature.
The protein concentrations were determinedby using Coomassie brilliant blue (Bio-Rad
Laboratories, USA). Bovine serum albumin wasused as the standard. All reactions were carriedout in triplicate.
Purification of α-glucosidase Wet cells (40 g)
were suspended in four volumes of 50 mM TrisHCl buffer (pH 7.2). The cells were sonicated(TOMY Ultrasonic Disruptor UD-200, Japan) in
an ice bath at 20 kHz, 140 W for 10 min and45

45

centrifuged at 20,000 x g for 20 min at 4°C. The
supernatant was used as crude extract (Step 1.
Crude extract, 165 ml). The precipitate obtained
between 30 and 60% saturation of (NH4)2SO4 wasdissolved in 50 mM Tris-HCl, pH 7.2 (Step 2.
Ammonium sulfate fraction, 13 ml). This solution
was 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 andeluted at a flow rate of 0.3 ml/min. Fractions
containing α-glucosidase activity were pooled(Step 3. Sepharose 4B, 76 ml). The pooled fractions were diluted with 10 volumes of cold
distilled water and loaded on a column (1.5 x 10cm) of Q-Sepharose (GE Healthcare, Sweden)
equilibrated in 50 mM Tris-HCl (pH 7.2). Proteins
were eluted at a flow rate of 0.3 ml/min with alinear concentration gradient of NaCl (0.1-0.32 M).
In this step two peaks of α-glucosidase activity
were found. One of the enzymes was named
BlAglA1 and another BlAglA2. Fractions containing each enzyme were pooled separately (Step 4A.
1st Q-Sepharose, 40 ml, and Step 4B, 30 ml). This
process was repeated twice for each enzyme (Step
5A. 2nd Q-Sepharose, 44 ml, and Step 5B, 25 ml).
The solution pooled was mixed with (NH4)2SO4 at
a concentration of 1.84 M, then was loaded on acolumn (1.5 x 4 cm) of Butyl-S Sepharose 6 Fast
Flow (GE, Sweden) equilibrated with 50 mM TrisHCl (pH 7.2) containing 1.84 M (NH4)2SO4.
Proteins were eluted using a decreasing stepwise
gradient of (NH4)2SO4 (1.1-0.7 M for both
enzymes) at a flow rate of 0.5 ml/min. Fractions
containing α-glucosidase activity were pooled(Step 6A. Butyl-S Sepharose, 50 ml; and Step 6B,
42 ml). Each enzyme pooled was desalted andconcentrated by centrifugation with a filtration
tube Vivaspin 4 (Sartorius, Germany). Concentrated preparations were subjected to 12.5% polyacrylamide gel electrophoresis (PAGE) without
sodium dodecyl sulfate (SDS). An area stainedwith α-glucosidase activity was cut out of the polyacrylamide gel and was soaked in 50 mM TrisHCl (pH 7.2). The enzyme was filtrated using agel extraction spin column (Bio-Rad, USA) andwas concentrated by centrifugation with a filtration tube Vivaspin 4 (Step 7A. Native PAGE, 0.61
ml; and Step 7B, 0.46 ml).

α-Glucosidases from Bifidobacterium longum

46

Table 1. Purification of α-glucosidases BlAglA1 and BlAglA2 from B. longum subsp. longum JCM 7052
grown on 1% gum arabic. 4NP-α-D-glucopyranoside was used as substrate.
Specific
Purification step

Protein

Total activity

activity

Yield

(mg)

(units)(units/mg of(%)

protein)
BlAglA1, BlAglA2

BlAglA1

BlAglA2

1. Crude extract

679

614

0.90

100

2. Ammonium sulfate fraction

243

283

1.16

46

3. Sepharose 4B

207

264

1.27

43

4A. 1st Q-Sepharose

54.0

155

2.87

25

5A. 2nd Q-Sepharose

41.3

151

3.66

25

6A. Butyl-S Sepharose

18.5

136

7.34

22

7A. Native PAGE

0.29

130

451.0

21

4B. 1st Q-Sepharose

32.4

100

3.09

16

5B. 2nd Q-Sepharose

21.5

87

4.03

14

6B. Butyl-S Sepharose

9.9

83

8.45

14

7B. Native PAGE

0.14

39

284.2

6

Molecular mass determination

SDS-PAGE
was carried out with a 5-20% (w/v) linear gradientpolyacrylamide slab gel (Wako Pure Chemical Industries, Ltd.) according to Laemmli [12]. Molecular weight marker proteins for SDS-PAGE(WAKO PLUS-VIEW™ Protein Size Marker)
were used for calibration. Native PAGE was doneusing the 5-20% (w/v) polyacrylamide gel and molecular mass was estimated with a molecularweight marker kit (NativeMarkTM, Invitrogen,
Carlsbad, CA, USA).

Protein identification by mass spectrometry

The Coomassie-stained protein bands were
excised from the SDS-PAGE gel, and subjected totryptic digestion as previously described [13].
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. BLAST [14,15] was also used to analyze distribution of theobtained peptides in proteins on the DDBJ
database.

Results
Purification of α-glucosidases from B. longum
subsp. longum JCM 7052
For purification of α-glucosidase B. longum
subsp. longum JCM 7052 grown on 1% gum
arabic was used to obtain a much amount of the
enzyme. The purification of α-glucosidase is summarized in Table 1. Two peaks of α-glucosidase
activity measured with 4NP-α-D-glucopyranosidewere separated by the 1st Q-Sepharose ion
exchange chromatography (Fig. 2). The enzymes
released at 0.3 and 0.2 M concentrations of NaCl
were named BlAglA1 and BlAglA2, respectively.
In hydrophobic chromatography with Butyl-S
Sepharose 6 Fast Flow, BlAglA1 and BlAglA2
were released at 0.9 and 1.0 M concentrations of(NH4)2SO4 in Tris-HCl (pH 7.2), respectively.
Finally, each α-glucosidase was purified as asingle protein band, which was evidently
separated by native PAGE. In consequence, αglucosidases BlAglA1 and BlAglA2 were purified
450- and 285-folds, respectively.
Molecular masses of α-glucosidases
SDS-PAGE showed that BlAglA1 andBlAglA2 were purified homogeneously and had

Saishin et al.

Identification of two α-glucosidases by mass spectrometry
The purified BlAglA1 was analyzed by MALDITOF-MS, MS/MS, and PMF search to identify its
putative gene. The PMF result (Accession number, gi|494117734; sequence coverage, 42.2%;score, 147) and two MS/MS results (1631.760(285-301), EGFLTVGEAPGVTAQR; score, 85)
and (2301.925 (398-417), GTPYIYQGEELGMTDAHFTR; score, 75) were obtained. For the purified
BlAglA2, the PMF result (Accession number,gi|658455158; sequence coverage, 62.2%; score,230) and two MS/MS results (2823.350 (515-540),
HGNATVATGEWNLVAADSEQVYSFTR; score,106) and (1919.841 (353-368), NAGWASLFFCNHDQPR; score, 131) were obtained. The two
couples of peptide sequences were also searchedagainst the genomic sequence of B. longum subsp.
longum JCM7052, which is still unpublished. The
couple of peptides obtained from BlAglA1 were
found in an α-amylase, and the couple of peptides
from BlAglA2 were also found in another αamylase.
BLAST search showed that the amino acidsequences of BlAglA1 and BlAglA2 werecompletely identical with those of the products ofgenes (locus tags of BLNIAS 02948 and BLNIAS
02851, respectively) in the genome of B. longum
subsp. longum KACC 91563 [16]. BlAglA1
consisted of 607 amino acids and had themolecular mass of 68256.70 Da. This molecularmass is approximately consistent with 72.0 kDa
determined by SDS-PAGE (Fig. 3A). BlAglA2 wascomposed of 606 amino acids and its molecularmass was 68547.12 Da. This value of molecularmass was approximately consistent with 72.8 kDa
estimated by SDS-PAGE (Fig. 3A).
In addition, similar amino acid sequences ofthe α-glucosidases were found in the characterized enzymes of two Bifidobacterium species. The
amino acid sequence of BlAglA1 is highly identical to that of BaAglA (85%) of B. adolescentis
47

2.5

4

3

2.0

0.4

2

0.3

1.5
A280

NaCl (M)Enzyme activity (Units）

apparent molecular masses of 72.0 and 72.8 kDa,
respectively (Fig. 3A). Molecular masses of
BlAglA1 and BlAglA2 were also estimated to be
70.1 and 92.7 kDa by PAGE without SDS,
respectively (Fig. 3B).

47

0.2

1.0

0.1
0.0

1

0.5

0

0.0
0

20

40

60

80

Fraction number (4ml/Tube)

Fig. 2. Ion exchange chromatography on
Q-Sepharose of α-glucosidases from B.
longum subsp. longum JCM 7052. Enzyme
activities were determined with 4NP-α-Dglucopyranoside as substrate. Symbols: ●,enzyme activity; ▲, NaCl (0.10-0.32 M); and, A280.
●Ａ

kDa 1 M 2

Ｂ

kDa 1 M 2
1048→
720→
480→

200→
140→
100→
73→
57→

242→
146→
66→

41→
29→

Fig. 3. Molecular mass determination of
α-glucosidases by SDS-PAGE [A] and native
PAGE [B]. Lane M, marker proteins; lane 1,
purified BlAglA1; and lane 2, purified
BlAglA2. Proteins were stained by Coomassie
blue R-250.
DSM20083, BbAgl1 (91%) and BbAgl2 (88%) of B.
breve UCC2003. The amino acid sequence of
BlAglA2, which was homologous with that ofα-Glucosidases from Bifidobacterium longum

48

Table 2. Hydrolytic activities of BlAglA1 andBlAglA2 toward some saccharides.
Substrate
Disaccharides
Trehalose
SucroseTuranose
MaltoseCellobiose
LactosePalatinose
Isomaltose
Melibiose
Trisaccharides
Melezitose
Maltotriose
PanoseIsomaltotriose

Hydrolytic activity

BlAglA1

BlAglA2

＋

＋

－

－

－

－

－

－

－

－

－

－

＋

＋

＋＋

＋＋

－

－

－

－

－

－

＋＋＋

＋＋

＋＋＋

＋＋＋

Other substrates－－
Glycogen－－
Amylose－－Amylopectin
Glucose released after 30 min incubation wasdetermined as described in Materials andMethods. The enzyme used was 63 mU in eachreaction. Scales were: －, 0-0.01 µmol glucose; +,0.01-0.05 µmol glucose; ++, 0.05-0.10 µmol
glucose; and +++, 0.10-1.0 µmol glucose.

BlAglA1 (74% identical), was identical to those of
BaAglA (86%), BbAgl1 (74%), and BbAgl2 (72%).
Substrate specificities of α-glucosidases
The enzymes BlAglA1 and BlAglA2 showedhydrolytic activities against 4NP-α-D-glucopyranoside but not against other 4NP-glycosides,
4NP-α-D-galactopyranoside, 4NP-α-D-mannopyranoside, 4NP-α-L-rhamnopyranoside, 4NP-α-Lfucopyranoside,
4NP-α-L-arabinopyranoside,
4NP-α-L-arabinofuranoside, 4NP-β-D-glucopyranoside, 4NP-β-D-galactopyranoside, 2-nitrophenyl-β-D-galactopyranoside, 4NP-β-D-fucopyranoside and 4NP-β-L-fucopyranoside.
Hydrolytic activities toward some saccharides
were tested for BlAglA1 and BlAglA2 by measuring glucose released after 30 min incubation at pH
6.0 and 40°C in the K-phosphate buffer (Table 2).
Both BlAglA1 and BlAglA2 were capable ofreleasing glucose from some saccharides having
α-1,6-linkage, isomaltose, palatinose, isomaltotriose, and panose. Trehalose, which has α-1,1-linkage between two glucoses, was alsohydrolyzed by both enzymes at lesser extent.
However, no hydrolysis products were found fromthe other saccharides listed in Table 2, indicatingthat both enzymes BlAglA1 and BlAglA2 cannot
catalyze hydrolysis of α-1,2-β-linkage in sucrose,
α-1,3-linkage in turanose and melezitose, and α1,4-likage in maltose, maltotriose, and amylose.
These findings including the amino acid sequence

Table 3. Kinetic parameters of BlAglA1 and BlAglA2 purified from B. longum subsp. longum
JCM 7052.
Substrate

BlAglA1

Km

BlAglA2

Vmax

Km

Vmax

1.5 ± 0.2
122.0 ± 8.5
02.0 ± 0.1
64.2 ± 0.6
4NP-α-D-glucopyranoside
Trehalose
14.3 ± 1.9
01.5 ± 0.4
07.4 ± 0.5
1.3 ± 0.6
Palatinose
32.2 ± 1.5
15.3 ± 2.5
33.5 ± 3.0
16.4 ± 1.8
Isomaltose
35.0 ± 2.2
17.0 ± 1.0
38.6 ± 3.6
22.6 ± 3.7
Panose38.3 ± 0.7
47.7 ± 2.9
24.1 ± 2.3
31.6 ± 0.6
Isomaltotriose
41.7 ± 0.5
25.3 ± 2.2
43.3 ± 1.0
17.2 ± 1.1
68.6 ± 2.7
24.2 ± 2.8
37.3 ± 3.8
12.0 ± 2.3
Ethyl-α-D-glucoside
The standard assay of enzyme activity was used for 4NP-α-D-glucopyranoside as substrate. Glucose
released from the other substrate was determined as described in Materials and Methods.
Km values are expressed as mM and Vmax values are expressed as µmol min-1 mg-1 of protein. All
values are means of three experiments ± standard errors.

Saishin et al.

velocity (Vmax) for 4NP-α-D-glucopyranoside,trehalose, palatinose, isomaltose, panose,
isomaltotriose, and ethyl-α-D-glucoside wereshown in Table 3.

similarities of the two enzymes indicated BlAglA1
and BlAglA2 to be oligo-1,6-glucosidase (EC
3.2.1.10) belonging to glycoside hydrolase family
13 [17, 18].
Values of Michaelis constant Km and maximal

Ａ

Ｂ
100

Enzyme activity (％）

100

Enzyme activity (％）

49

80
60
40
20

80
60
40
20
0

0
3

4

5

6

pH

7

8

9

10

3

4

5

6

pH

7

8

9

10

Fig. 4. Effects of pH on the activity of the purified α-glucosidases BlAglA1 [A] and BlAglA2[B]. Buffers used were ■, 80 mM acetate (pH 3.5–6.5); ●, 80 mM K-phosphate (pH 5.58.0); ○, 80 mM glycine-NaOH (pH 7.5–10.0); and ▲, 80 mM Tris-HCl (pH 6.5–9.0 ).

Ｂ

Ａ

100

80

Enzyme activity (％）

Enzyme activity (％）

100

60
40
20
0

80
60
40
20
0

20

25

30

35

40

45

50

55

60

20

25

30

35

40

45

50

55

60

Temperature (℃ )Temperature (℃ )Fig. 5. Effects of temperature on the activity of the purified α-glucosidases BlAglA1 [A] andBlAglA2 [B].

Ａ

B
100

Enzyme activity (％）

Enzyme activity (％）

100
80
60
40
20
0

80
60
40
20
0

0

1

2

3

4

Incubation time (h)

5

0

1

2

3

4

Incubation time (h)5Fig. 6.
Effects of temperature on the stability of the purified BlAgl1 [A] and BlAgl2 [B].
Enzyme activities were assayed at intervals after incubation of enzyme solutions at 40°C (●),
45°C (▲), 50°C (○), and 55°C (■).
49

α-Glucosidases from Bifidobacterium longum

50

Effects of pH, temperature, and chemicals on the
enzyme activity.
The optimal pH was assayed by incubating
the purified enzyme with 4NP-α-glucopyranosidein 80 mM buffers ranging from pH 3.5 to pH 10.0.
For both enzymes BlAglA1 and BlAglA2, thehighest activity of α-glucosidases were obtainedat pH 5.5-6.0 in 80 mM K-phosphate buffer(Fig. 4A and B).
The optimal temperature of BlAglA1 wasobserved in hydrolyzation of 4NP-α-D-glucopyranoside at 40-45°C (Fig. 5A). The enzyme
activity gradually decreased during 5 h incubation at 45 and 50°C (Fig. 6A), and a remarkabledecline in the activity occurred during 2 h incubation at 55°C (Fig. 6A). The optimal temperaturefor BlAglA2 was observed at 45-50°C (Fig. 5B).
BlAglA2 was unstable rather than BlAglA1, sothat the activity of BlAgl2 was lost after 1 h
incubation at 55°C (Fig. 6B)
To determine the effects of chemicals,
hydrolytic activities of BlAglA1 and BlAglA2 were
measured using 4NP-α-D-glucopyranoside in thepresence of 1 mM metal chloride salts or EDTA
(Table 4). For both enzymes Cu2+ inhibited about
60% of their enzyme activities and Ca2+, Co2+,

Mg2+, Mn2+, Ni2+, and Zn2+ did about 10–20%.
EDTA was less active as a chelating reagent for
both enzymes.
Transglycosylation activity of the enzymes
α-Glucosidases are known to have transglycosylation activity [5, 6, 19, 20]. The purified
enzymes BlAglA1 and BlAglA2 were briefly
tested to have glucosyl transferring activity. TLC
showed that oligosaccharides were synthesized in
the reaction mixtures containing isomaltose,
palatinose, isomaltotriose, or panose. Especially,
tetra- and pentasaccharides were produced frompanose and isomaltotriose. When isomaltose wasused as a glucosyl donor, isomaltotriose was
obviously produced (Fig. 7, lanes 10 and 11),suggesting the formation of α-1,6-linkage in the
products of transglucosylation by BlAglA1 andBlAglA2.

－Glu

Table 4. Effects of metal ions and EDTA on thehydrolytic activities of purified BlAglA1 andBlAglA2. 4NP-α-D-glucoside was used as substrate.
Reagents
None
BaCl2
CaCl2
CoCl2
CuCl2
FeCl2
MgCl2
MnCl2
NiCl2
ZnCl2
NaCl （10 mM）
KCl （10 mM）
EDTA

－Mal
－IM
－IMT

Relative activity (%)

BlAglA1
100
95
90
86
43
94
90
82
87
89
97
103
99

BlAglA2
100
97
89
89
39
96
87
82
89
92
99
101
99

Divalent metal ions and EDTA were added at 1.0
mM to the reaction mixture.

1 2 3 4 5 6 7 8 9 10 11 12 13 14
Fig. 7. TLC analysis of the transglycosylation
activities of BlAglA1 and BlAglA2. Standard
carbohydrates used were trehalose (lane 3),
palatinose (lane 6), panose (lane 9), and a
mixture of glucose (Glu), maltose (Mal),
isomaltose (IM), and isomaltotriose (IMT)(lane 14). The reaction mixtures contained
trehalose (lanes 1 and 2), palatinose (lanes 4
and 5), panose (lanes 7 and 8), isomaltose(lanes 10 and 11), and isomaltotriose (lanes
12 and 13). In the reaction mixture BlAglA1
was added in lanes 1, 4, 7, 10 and 12, andBlAglA2 was in lanes 2, 5, 8, 11, and 13.

Saishin et al.

Transglucosylation to alcohols was also
examined with purified BlAglA1 and BlAglA2 by
TLC (Fig. 8). Under the used conditions thestandard ethyl-α-D-glucoside moved faster thanglucose, suggesting alkyl-glucosides formed in thereactions would be detected at upper region of thegel plate. In the reactions with palatinose as adonor and ethanol, 1-propanol, 2-propanol,
1-butanol, or 1-hexanol as acceptors, products of
the respective reactions were found at upper placethan the position of glucose or fructose developedin the silica gel plate, and ethyl-α-D-glucoside was
identified as the product of transglycosylation toethanol. This result suggests that both oligo-1,6glucosidases BlAglA1 and BlAglA2 transfers
glucose to alcohol to produce alkyl-α-D-glucoside.
In the reaction with 1-octanol, only a faint spot
was found at an upper place of TLC gel. Whenglycerol was used as an acceptor of glucose, no
products were detected by TLC (data not shown).
Discussion
In B. longum subsp. longum JCM 7052 theincrease in α-glucosidase activity by growing on
gum arabic seemed to be attributed to one or bothof two oligo-1,6-glucosidases. Both enzymes
BlAglA1 and BlAglA2 had similar properties

relative to the amino acid sequence (74% identical), pH- and temperature-dependency of enzyme
activities, and substrate specificity. These twooligo-1,6-glucosidases did not produce glucose
from amylopectin and glycogen, indicating these
enzymes to have no hydrolytic activities against
α-1,6-glucosidic linkages present inside the polysaccharides. B. longum subsp. longum including
the strain JCM 7052 cannot grow on starch,
amylopectin, and pullulan [21], while maltodextrins were shown to be the most favorable carbon
source for bifidobacteria [22]. When B. longum
subsp. longum BBMN68 was grown on maltodextrins as sole carbon source, oligo-1,6-glucosidase
beside α-1,4-glucosidases was observed to be
induced and 2 components of ABC-type sugartransporters to be more abundant [23]. Utilization of carbohydrates have been understood to becontrolled by transcriptional regulation with
DNA- and/or sugar-binding regulatory proteins inbifidobacteria [24, 25]. Such sugar-binding regulators might respond to hydrolysates of gum
arabic, and subsequently activate the expressionof genes involved in hydrolysis of other oligosaccharides.
Oligosaccharides have attracted attention totheir beneficial functions as food ingredients (low

A

B

EG－

EG－

Fru－

－Glu

Pal－

51

Fru－

－Glu

Pal－

1

2

3

4

5

6

7

8

－IＭ

－IＭ

－IＭＴ

－IＭＴ
1

2

3

4

5

6

7

8

Fig. 8. TLC analysis of the transglucosylation activities of the purified oligo-1,6-glucosidases
BlAglA1 [A] and BlAglA2 [B]. Standard carbohydrates used were a mixture of ethyl-α-D-glucosidse(EG), fructose (Fru) and palatinose (Pal) in lane 1, and that of glucose (Glu), isomaltose (IM), andisomaltotriose (IMT) in lane 8. The reaction mixtures containing each purified enzyme, 500 mM
palatinose as a glucosyl donor and the following 1.0 M alcohol: ethanol (lane 2), 1-propanol (lane3), 2-propanol (lane 4), 1-butanol (lane 5), 1-hexanol (lane 6), and 1-octanol (lane 7).
51

52

α-Glucosidases from Bifidobacterium longum

calorie, mildly sweet, and viscous compounds),
prebiotics, dental caries preventives, drug
coatings, and so on. Both oligo-1,6-glucosidases
BlAglA1 and BlAglA2 showed transglycosylation
activity to produce higher oligosaccharides (tetraand pentasaccharides). Since both enzymes
showed to use several α-glucosides as substrate intransglucosylation, a variety of oligosaccharide
would be synthesized by using different sugars as
acceptor molecules.
Alcohols also served as glucosyl acceptor
molecules in transglucosylation of BlAglA1 andBlAglA2. Alkyl glucosides are also attractive
compounds because of their health beneficial
properties. Especially, ethyl-α-D-glucoside has
been known as one of the components in traditional Japanese sake wine [26]. This compound
has been known to have a moisture-retaining
effect on skin through oral ingestion or skin application [27], and hepatoprotective effects against
galactosamine-induced liver injury in mice [28].
Production of ethyl-α-D-glucoside has been performed through alcohol fermentation with rice[29], and transglucosylation of enzymes. α-Glucosidase of Mortierella alliacea transfers glucose
from starch to ethanol [30], while Leuconostoc
mesenteroides dextransucrase uses sucrose toform ethyl-α-D-glucoside [31]. In this study wetested only palatinose as glucosyl donor in thesynthesis of alkyl glucoside by the oligo-1,6glucosidases BlAglA1 and BlAglA2. To confirm
the method for more convenient and effective
production of alkyl glucosides, modification of
enzymes, selection of glucosyl donors, andimprovement of reaction conditions are requiredin further investigations.
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Communicated by Ueno Hiroshi