Purification, characterization, and gene identification of an α-glucosyl transfer enzyme, a novel type α-glucosidase from Xanthomonas campestris WU-9701

Article abstract of DOI:10.1016/j.molcatb.2012.04.014

The α-glucosyl transfer enzyme (XgtA), a novel type α-glucosidase produced by Xanthomonas campestris WU-9701, was purified from the cell-free extract and characterized. The molecular weight of XgtA is estimated to be 57 kDa by SDS-PAGE and 60 kDa by gel filtration, indicating that XgtA is a monomeric enzyme. Kinetic properties of XgtA were determined for α-glucosyl transfer and maltose-hydrolyzing activities using maltose as the α-glucosyl donor, and if necessary, hydroquinone as the acceptor. The V_max value for α-glucosyl transfer activity was 1.3 × 10^-2 (mM/s); this value was 3.9-fold as much as that for maltose-hydrolyzing activity. XgtA neither produced maltooligosaccharides nor hydrolyzed sucrose. The gene encoding XgtA that contained a 1614-bp open reading frame was cloned, identified, and highly expressed in Escherichia coli JM109 as the host. Site-directed mutagenesis identified Asp201, Glu270, and Asp331 as the catalytic sites of XgtA, indicating that XgtA belongs to the glycoside hydrolase family 13.

Full text of DOI:10.1016/j.molcatb.2012.04.014

Journal of Molecular Catalysis B: Enzymatic 80 (2012) 20–27
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Purification, characterization, and gene identification of an ␣-glucosyl transfer
enzyme, a novel type ␣-glucosidase from Xanthomonas campestris WU-9701
Toshiyuki Sato, Nobukazu Hasegawa, Jun Saito, Satoru Umezawa, Yuki Honda, Kuniki Kino,
Kohtaro Kirimura∗
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The ␣-glucosyl transfer enzyme (XgtA), a novel type ␣-glucosidase produced by Xanthomonas campestris
WU-9701, was purified from the cell-free extract and characterized. The molecular weight of XgtA is
estimated to be 57 kDa by SDS-PAGE and 60 kDa by gel filtration, indicating that XgtA is a monomeric
enzyme. Kinetic properties of XgtA were determined for ␣-glucosyl transfer and maltose-hydrolyzing
activities using maltose as the ␣-glucosyl donor, and if necessary, hydroquinone as the acceptor. The
Vmax value for ␣-glucosyl transfer activity was 1.3 × 10−2 (mM/s); this value was 3.9-fold as much as that
for maltose-hydrolyzing activity. XgtA neither produced maltooligosaccharides nor hydrolyzed sucrose.
The gene encoding XgtA that contained a 1614-bp open reading frame was cloned, identified, and highly
expressed in Escherichia coli JM109 as the host. Site-directed mutagenesis identified Asp201, Glu270, and
Asp331 as the catalytic sites of XgtA, indicating that XgtA belongs to the glycoside hydrolase family 13.
© 2012 Elsevier B.V. All rights reserved.
Received 25 February 2012
Received in revised form 5 April 2012
Accepted 17 April 2012
Available online 24 April 2012
Keywords:
Gene cloning and expression
␣-Glucosidase
Glycoside hydrolase family 13
Transglucosylation
Xanthomonas campestris
1. Introduction
[7], although such oligosaccharide by-products are generally pro-
duced in conventional ␣-glucosidase reactions [8–10].
Polysaccharides such as starch and cellulose are abundant
renewable bioresources, and glycosidases are mostly used for the
enzymatic hydrolysis of biomass. On the other hand, transglyco-
sylation or reverse hydrolysis reactions catalyzed by glycosidases
have been performed for the in vitro synthesis of oligosaccharides
and alkylglycosides [1–4]. Various compounds have been selec-
tively ␣- or ␤-glycosylated with glycosidases such as maltogenic
amylase from Thermoplasma volcanium GSS1 [5] and ␤-xylosidase
from Aspergillus awamori K4 [6]. To develop a method for the ␣-
anomer-selective one-step synthesis of the novel refreshing agent
l-menthyl ␣-d-glucopyranoside (␣-MenG), we presumed the exis-
tence of a microbial enzyme for the synthesis of ␣-MenG. We then
screened for microorganisms that produce the enzymes applicable
to the reaction and succeeded in isolating Xanthomonas campestris
WU-9701 as the desirable strain [7]. Using the lyophilized cells of X.
campestris WU-9701 and maltose as the ␣-glucosyl donor, ␣-MenG
was selectively synthesized from l-menthol with a 99% molar con-
version yield after a 48-h reaction. ␣-MenG mainly accumulated
as crystals in the reaction mixture [7]. In addition, only ␣-MenG
was synthesized as the ␣-glucosyl transfer product, and no mal-
tooligosaccharide such as maltotriose or maltotetraose was formed
Furthermore, the crude enzyme of X. campestris WU-9701 was
applicable to the ␣-glucosylation of (+)-catechin to selectively pro-
duce (+)-catechin 3ꢀ-O-␣-d-glucopyranoside (␣-C-G) with a 57%
molar conversion yield [11]. Moreover, the enzyme selectively
synthesized the whitening agent hydroquinone ␣-glucoside (␣-
arbutin; Scheme 1) [12] and the prodrug eugenol ␣-glucoside [13]
with 93% and 52% molar conversion yields, respectively. Because
the enzyme produced by X. campestris WU-9701 differs from typ-
ical ␣-glucosidases that predominantly catalyze the hydrolysis of
maltooligosides, we believe that studies on the purified enzyme and
the gene encoding the enzyme would be necessary to elucidate its
enzymatic properties.
In this study, the purification, molecular characterization, and
gene identification of the ␣-glucosyl transfer enzyme (XgtA) pro-
duced by X. campestris WU-9701 was performed. To our knowledge,
this is the first report of an enzyme having a hyper ␣-glucosyl
transfer activity toward alcoholic and phenolic compounds, but not
toward saccharides, using maltose as the ␣-glucosyl donor. It is also
noteworthy that sucrose is not hydrolyzed or used as the ␣-glucosyl
donor in the reaction of XgtA.
2. Materials and methods
2.1. Strains and materials
Abbreviations:
␣-MenG, l-menthyl ␣-d-glucopyranoside; HPLC, high-
performance liquid chromatography; IPTG, isopropyl-␤-d-thiogalactopyranoside.
∗
X. campestris WU-9701, which produces the ␣-glucosyl transfer
enzyme, was used as the parental strain [7,11–13]. Hydroquinone
Corresponding author. Tel.: +81 3 5286 3208; fax: +81 3 3232 3889.
E-mail address: kkohtaro@waseda.jp (K. Kirimura).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.04.014

T. Sato et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 20–27
21
Scheme 1. Anomer-selective synthesis of hydroquinone ␣-d-glucopyranoside (␣-arbutin) from hydroquinone and maltose by XgtA, an ␣-glucosyl transfer enzyme from X.
campestris WU-9701.
was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). All the
other chemicals used were of a chemically pure grade and were
commercially available.
filtrate was measured using high-performance liquid chromatogra-
phy (HPLC), using following conditions: column, TSK-Gel ODS 80-TS
(4.6 mm × 250 mm, Tosoh Co., Tokyo); solvent, methanol–water
(10:90, v/v); flow rate, 1.0 ml/min; and temperature, 40 ◦C. One
unit (U) of ␣-glucosyl transfer activity was defined as the amount
of enzyme that produces 1 ␮mol of ␣-arbutin per minute from
hydroquinone. To measure maltose hydrolysis activity, the same
conditions mentioned above for ␣-glucosyl transfer activity were
used, except that hydroquinone was omitted from the reaction mix-
ture. The amount of maltose and released glucose was measured
using HPLC, using following conditions: column, Asahipak NH2P-
50 4E (4.6 mm × 250 mm, Showa Denko Co. Ltd., Tokyo); solvent,
acetonitrile–10 mM tetra-n-propylammonium hydroxide contain-
ing acetic acid (pH 10.0) (70:30, v/v); flow rate, 1.0 ml/min; and
temperature, 40 ◦C. One unit (U) of maltose hydrolysis activity was
defined as the amount of enzyme that produces 1 ␮mol of glucose
per minute.
2.2. Purification of the ˛-glucosyl transfer enzyme (XgtA)
Cells of X. campestris WU-9701 were grown under aerobic con-
ditions with shaking at 30 ◦C for 48 h in 1 l of medium containing
50 g maltose, 2.0 g yeast extract (Difco, USA), 10 g peptone, and
1.0 g MgSO4. The pH was initially adjusted to 7.0 with 2.0 M NaOH.
After a 48-h incubation, cells were harvested by centrifugation
(15,000 × g, 30 min, 4 ◦C) and washed twice with a basal buffer
containing 10 mM citrate and 10 mM Na2HPO4 (pH 7.0). Purifica-
tion steps were performed at 0–4 ◦C unless otherwise indicated.
Harvested cells were suspended in 50 ml of the basal buffer, dis-
rupted by sonication (20 kHz, 120 min, 4 ◦C), and centrifuged again
(25,000 × g, 60 min, 4 ◦C). The resulting supernatant (50 ml) was
collected as the cell-free extract. Next, ammonium sulfate powder
was added to 30% (w/v) saturation, and the solution was stirred at
4 ◦C overnight. The resulting precipitate was removed by centrifu-
gation (25,000 × g, 60 min, 4 ◦C). Subsequently, the supernatant
was adjusted to 60% (w/v) saturation with ammonium sulfate and
stirred at 4 ◦C overnight. The resulting precipitate was collected
by centrifugation (25,000 × g, 60 min, 4 ◦C) and dissolved in 30 ml
of the basal buffer. This solution was dialyzed against the basal
buffer overnight. The dialysate was applied to a DEAE-Toyopearl
650S column (2.5 cm × 6 cm; Tosoh Co., Tokyo); it was equilibrated
with the basal buffer after being washed with 60 ml of the buffer.
Elution was performed with a continuous linear gradient of 0–0.3 M
NaCl in 240 ml of the basal buffer. Active fractions were collected
and concentrated by ultrafiltration. The concentrated eluate was
applied to a Superdex 200 column (1.6 cm diameter × 60 cm; GE
Healthcare, NJ, USA) equilibrated with the basal buffer containing
0.15 M NaCl. After dialysis against the basal buffer, active fractions
were collected and stored at 4 ◦C for subsequent characterization
of XgtA.
2.4. Protein analyses
Protein concentrations were determined by the method of
Bradford [14] using the Coomassie Protein Assay Kit (Pierce
Chem. Co., Rockford, USA) with bovine serum albumin as the
standard. For column chromatography, protein concentration
was measured by absorbance at 280 nm using
a Shimadzu
UV-240 spectrophotometer (Kyoto, Japan). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis
was performed using 10% (w/v) polyacrylamide by the method of
Laemmli [15].
2.5. Cloning of the ˛-glucosyl transfer enzyme gene (xgtA)
Total DNA of X. campestris WU-9701 was digested with SalI.
After electrophoresis, 4.0–7.0-kb DNA fragments were excised from
agarose and purified using GFX PCR DNA and a Gel Band Purification
Kit (GE Healthcare). The DNA fragments were ligated into pUC18
(SalI/bacterial alkaline phosphatase treated) and introduced into
Escherichia coli JM109 cells to construct a partial DNA library of X.
campestris WU-9701. For preparing a probe, PCR was performed
with total DNA of X. campestris WU-9701 and two primers: 5ꢀ-
CARACICCITGGTGGMG-3ꢀ (sense) and 5ꢀ-AGIACYTGRTCKATCAT-3ꢀ
(antisense), where I, R, M, Y, and K denote deoxyinosine, A + G,
A + C, C + T, and G + T, respectively. The amplified fragment was
labeled with DIG-dUTP (Röche Diagnostics, Tokyo, Japan) and used
as a probe (designated as XgtA-DIG probe) to screen the partial
DNA library of X. campestris WU-9701 by colony hybridization.
White colonies on Luria–Bertani (LB) agar containing 100 ␮g/ml
ampicillin and 0.1 mM isopropyl-␤-d-thiogalactopyranoside (IPTG)
were screened by colony hybridization at 68 ◦C using a Hybond-
N+ membrane (GE Healthcare) and the labeled probe described
above.
2.3. Enzyme assays
␣-Glucosyl transfer activity through the ␣-arbutin synthesis
reaction (Scheme 1) was measured using maltose and hydro-
quinone as substrates according to the methods described
previously [12]. The concentration of maltose in the enzyme reac-
tion mixture was set up to 1.2 M, since the optimal concentration
of maltose for ␣-arbutin synthesis was 1.2 M in the reaction not
only with lyophilized cells of X. campestris WU-9701 [12] but
also with purified XgtA. The reaction was terminated by heating
the reaction mixture in boiling water (100 ◦C) for 10 min. Next,
0.4 ml of the reaction mixture was diluted with water to a final
volume of 2.0 ml and filtered through a 0.2-␮m cellulose acetate
membrane (Iwaki Glass Co., Ltd.). The amount of ␣-arbutin in the

22
T. Sato et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 20–27
2.6. Expression of the ˛-glucosyl transfer gene (xgtA) in E. coli
Full-length xgtA was amplified by PCR with the oligonu-
cleotide primers 5ꢀ-AGGGGAATTCATGTCGCAGACACCATG-3ꢀ and
5ꢀ-TGCAAGCTTTCAGCCACGACCGACAG-3ꢀ; EcoRI and HindIII cleav-
age sites are underlined. The PCR product was digested with EcoRI
and HindIII, and the resulting DNA fragment was subcloned into
plasmid pKK223-3. The resulting plasmid pKKGTF was used for the
transformation of E. coli JM109 cells. E. coli JM109/pKKGTF cells
were grown under aerobic conditions with shaking at 37 ◦C for
22 h in LB medium (pH 7.0) containing 100 ␮g/ml of ampicillin
and 0.8 mM IPTG. After a 22-h incubation, cells were harvested
by centrifugation (6000 × g, 15 min, 4 ◦C) and washed twice with
10 mM citrate–10 mM Na2HPO4 buffer (pH 7.0). Cells were sus-
pended in 5 ml of 10 mM citrate–10 mM Na2HPO4 buffer (pH 7.0),
disrupted by sonication (20 kHz, 200 W, 2 min × 10 cycles) and cen-
trifuged (20,000 × g, 30 min, 0 ◦C). The resulting supernatant (5 ml)
was used as the cell-free extract. Site-directed mutagenesis was
conducted according to the method of Ito et al. [16]. Each of the
resulting mutant plasmids was used for the transformation of E. coli
JM109 cells, and an enzyme assay was performed for each E. coli
transformant.
Fig. 1. Purification and molecular mass estimation of XgtA. (a) SDS-PAGE of XgtA
purified from X. campestris WU-9701. Lane M, marker proteins; lane P, purified
XgtA. (b) Linear regression curve and estimation of molecular mass of XgtA. Molec-
ular weight was calibrated based on thyroglobulin (Thy 669,000), ferritin (Fer
440,000), catalase (Cat 232,000), albumin (Alb 66,000), ovalbumin (Ova 45,000),
chymotrypsinogen (Chy 25,000), and ribonuclease A (Rib 13,700). Molecular mass
of XgtA was calculated to be 60,000 Da.
2.7. Nucleotide sequence accession number
Table 2
Effects of various metal ions on ␣-glucosyl transfer activity of the purified XgtA.
The nucleotide sequence of xgtA described in this paper appears
in the DDBJ/EMBL/GenBank nucleotide sequence databases with
the Accession No. AB081949.
Reagent
Relative activity (%)
None
KCl
LiCl
100
194
149
181
81
0
NaCl
3. Results
CaCl2
CuCl2
HgCl2
MgCl2
MnCl2
ZnCl2
EDTA
pCMBa
3.1. Purification of the ˛-glucose transfer enzyme (XgtA)
0
56
29
0
53
0
For the 24-h and 48-h cells of X. campestris WU-9701, more
than 90% of ␣-glucosyl transfer activity toward hydroquinone (an
␣-glucosyl acceptor) was detected in the cytosol through cellular
fractionation (data not shown). Therefore, the ␣-glucosyl transfer
enzyme (XgtA) was purified from the cell-free extract as described
in Section 2. Subsequent purification steps are shown in Table 1.
Native XgtA was purified 12.9-fold with a yield of 14.0%, and its
final specific activity was 14.7 U/mg. The purified XgtA produced
a single band on an SDS-PAGE gel, and its molecular weight was
estimated to be 57 kDa (Fig. 1). Moreover, the molecular weight
was calculated to be 60 kDa by Superdex 200 gel filtration (Fig. 1),
confirming that the purified XgtA enzyme was monomeric.
Purified XgtA 0.4 ml (1.9 U) was added to 1.6 ml of 10 mM H3BO3–NaOH–KCl buffer
(pH 7.5) containing hydroquinone 10 mg (45 mM) and 1.5 M of maltose. The reaction
mixture was shaken at 40 ◦C for 30 min. The amount of ␣-arbutin was measured
using HPLC as described in Section 2.
a
p-Chloromercuribenzoic acid.
We confirmed that many alcoholic and phenolic compounds
such as ethanol, propanol, butanol, phenol, o-cresol, m-cresol,
p-cresol, catechol, resorcinol, and p-nitrophenol were used as ␣-
glucosyl acceptor of the purified XgtA; the corresponding ␣- and
mono-glucosylated product was selectively synthesized from each
substrate used.
3.2. Effects of the various reagents on enzyme activity and
substrate specificity
To confirm the hydrolyzing activity for saccharides contain-
ing glucose, reactions of XgtA with various saccharides were
examined, and the amounts of released glucose from various
saccharides were measured. As shown in Table 3, the hydrolyz-
ing activity toward maltose was the highest, and XgtA slightly
hydrolyzed nigerose. Nigerose may be chemically decomposed via
␤-elimination because of the instability of its reducing end 1,3-
linkage [19]. Hence, the amount of glucose released from nigerose
Effects of various metal ions and chemical reagents on ␣-
glucosyl transfer activity (␣-arbutin synthesis) are shown in
Table 2. Enzyme activity was strongly inhibited by bivalent metal
ions such as Cu2+, Hg2+, and Zn2+, indicating that sulfhydryl groups
may be involved at the active site of XgtA. In contrast, XgtA was
activated by K+ and Na+, similar to the ␣-glucosidases of Rhizopus
sp. [17] and Agrobacterium tumefaciens [18].
Table 1
Purification steps of XgtA from X. campestris WU-9701.
Step
Total protein (mg)
Specific activitya (U/mg)
Yield (%)
Purification (fold)
Crude enzyme
4.78 × 102
2.58 × 102
2.30 × 10
5.19
1.13
1.33
6.64
1.47 × 10
100
63
28
1.00
1.16
5.83
1.29 × 10
Ammonium sulfate precipitation (30–60%)
DEAE-Toyopearl 650S (anion-exchange)
Superdex 200 (gel filtration)
14
a
The specific activity was estimated as ␣-glucosyl transfer activity and was measured using hydroquinone as an ␣-glucosyl acceptor.

T. Sato et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 20–27
23
Table 3
Hydrolysis reaction toward various saccharides by the purified XgtA.
100
(a)
Saccharide
Concentration
Hydrolysis (mM)
80
60
40
20
0
Trehalose
Kojibiose
Nigerose
Maltose
Isomaltose
Cellobiose
Maltotriose
Amylose
pNPGa
100 mM
100 mM
100 mM
100 mM
100 mM
100 mM
100 mM
3.6% (w/v)
25 mM
0
0
4.71
1.99 × 10
3.10 × 10−1
0
3.81 × 10−1
6.44 × 10−1
1.75 × 10−1
0
Sucrose
100 mM
Purified XgtA 0.4 ml (1.9 U) was added to 1.6 ml of 10 mM H3BO3–NaOH–KCl buffer
(pH 7.5) containing various saccharides. The reaction mixture was shaken at 40 ◦
20
30
40
50
C
for 30 min. The amount of released glucose was measured using HPLC as described
in Section 2.
Temperature (ºC)
a
p-Nitrophenyl ␣-d-glucopyranoside.
100
80
60
40
20
0
(b)
under the conditions without the purified XgtA was measured, and
the amount (3.33 mM) was used as a blank value.
It is noteworthy that XgtA does not hydrolyze sucrose. On the
other hand, XgtA slightly hydrolyzed maltose and maltotriose con-
taining ␣-1,4-linkages of glucose. This result confirms that XgtA
is different from ␣-glucosidases that hydrolyze sucrose and mal-
tooligosaccharides [8,9].
To confirm the ␣-glucosyl transfer activity using various saccha-
rides containing glucose moiety as ␣-glucosyl donors, we tested
whether or not XgtA would react toward various saccharides
using hydroquinone as an ␣-glucosyl acceptor, and measured the
amounts of ␣-arbutin formed. As shown in Table 4, ␣-arbutin
was produced from maltose but not any other saccharide ␣-
glucosyl donors. Hence, XgtA is confirmed to be different from
the ␣-amylase detected in the periplasm of X. campestris K-11151
[20], which catalyzes maltooligosaccharide synthesis from maltose.
Among the saccharides tested, only maltose having ␣-1,4-linkage
of two glucose molecules was utilized as an ␣-glucosyl donor for
XgtA, suggesting that XgtA possesses a high substrate specificity
toward saccharide as an ␣-glucosyl donor.
0
20
40
60
80
Temperature (ºC)
Fig. 2. Effects of temperature on ␣-glucosyl transfer activity. (a) Effects of temper-
ature on ␣-glucosyl activity. (b) Effects of temperature on ␣-glucosyl stability. After
the purified enzyme was preincubated at the indicated temperature for 1 h, the
remaining activity was measured under standard conditions. The remaining activ-
ity is expressed relative to the activity, taken as 100% when the enzyme was not
preincubated.
while in the presence of hydroquinone ␣-arbutin synthesis (␣-
glucosyl transfer), maltose-hydrolysis and glucose release were
rapidly increased (Fig. 3). Moreover, glucose release from maltose
was markedly increased in the presence of hydroquinone (Fig. 4).
These results suggest that XgtA is an ␣-glucosidase and mainly
catalyzes the ␣-glucosyl transfer reaction.
3.3. ˛-Glucosyl transfer and maltose-hydrolyzing activities
The optimal temperature for ␣-glucosyl transfer activity was
40 ◦C. ␣-Glucosyl transfer activity was stable up to 40 ◦C, and its
activity was not detected after heating at 50 ◦C for 1 h (Fig. 2). To
confirm the detailed properties of XgtA, the effects of the addi-
tion of hydroquinone on the reaction mixture containing maltose
as the ␣-glucosyl donor were examined. In the absence of hydro-
quinone, decrease of maltose concentration was only slight (Fig. 3),
3.4. Kinetic properties
The kinetic constants Km, Vmax, kcat, and kcat/Km of the ␣-glucosyl
transfer reaction were determined according to the amount of
glucose released from maltose (Table 5). The Vmax value for
␣-glucosyl transfer activity was 3.9-fold greater than that of
maltose-hydrolysis. The Km values for maltose were increased in
the presence of hydroquinone as an ␣-glucosyl acceptor. The kcat
and kcat/Km values, indicating the catalytic efficiency of XgtA for
Table 4
␣-Glucosyl transfer reaction toward hydroquinone by the purified XgtA using vari-
ous saccharides as ␣-glucosyl donors.
Saccharide
Concentration
␣-Arbutin (mM)
Trehalose
Kojibiose
Nigerose
Maltose
Isomaltose
Cellobiose
Maltotriose
Amylose
pNPGa
1.2 M
1.2 M
1.2 M
1.2 M
0
0
0
Table 5
2.5 × 10
Kinetic parameters of purified XgtA.
1.2 M
0
0
0
0
0
0
300 mM
300 mM
3.6% (w/v)
300 mM
1.2 M
Hydroquinone
Km (mM)
Vmax (mM/s)
kcat (s−1
)
kcat/Km (s−1 mM−1
)
Absence
Presence
2.1 × 10
6.8 × 10
3.3 × 10−3
4.7 × 103
2.2 × 102
1.3 × 10−2
1.9 × 104
2.8 × 102
Sucrose
Purified XgtA 0.4 ml (1.9 U) was added to 1.6 ml of 10 mM H3BO3–NaOH–KCl buffer
(pH 7.5) containing maltose of various concentrations (final concentrations from 0
to 1.8 M). Especially, hydroquinone 10 mg (45 mM) was also added to the reaction
mixture in the case of ␣-glucosyl transfer reaction. The reaction mixture was shaken
at 180 rpm at 40 ◦C. The amount of released glucose was measured using HPLC as
described in Section 2.
Purified XgtA 0.4 ml (1.9 U) was added to 1.6 ml of 10 mM H3BO3–NaOH–KCl buffer
(pH 7.5) containing hydroquinone 10 mg (45 mM) and various saccharides. The reac-
tion mixture was shaken at 40 ◦C for 30 min. The amount of ␣-arbutin was measured
using HPLC as described in Section 2.
a
p-Nitrophenyl ␣-d-glucopyranoside.

24
T. Sato et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 20–27
(a)
(a)
40
30
20
10
0
1.20
1.18
1.16
1.14
1.12
0
10
20
30
1.10
0
0
0
1
1
1
2
3
4
4
4
5
Time (min)
Time (h)
(b)
40
30
20
10
0
50
40
30
20
10
0
(b)
0
10
20
30
Time (min)
2
3
5
Time (h)
Fig. 4. The amounts of released glucose from maltose and decrease of maltose
through (a) ␣-glucosyl transfer and (b) maltose-hydrolyzing reactions. Purified XgtA
(0.4 ml, 1.9 U) was added to 1.6 ml of 10 mM H3BO3–NaOH–KCl buffer (pH 7.5) con-
taining 1.5 M maltose. For the ␣-glucosyl transfer reaction, hydroquinone (10 mg;
45 mM) was added to the reaction mixture. The reaction mixture was shaken at
180 rpm at 40 ◦C. The amounts of ␣-arbutin, maltose, and glucose were measured
by HPLC. Symbols: closed circle, maltose decreased; closed triangle, released glucose
from maltose; closed square, ␣-arbutin.
(c) 120
100
80
60
-Thr-Ser-Ile, respectively by Shimadzu (Kyoto, Japan). Based on
these amino acid sequences, we synthesized two oligonucleotide
primers as described in Section 2. Only one DNA fragment of
approximately 180-bp was amplified by PCR using total DNA of
X. campestris WU-9701 as a template. Through colony hybridiza-
tion selection using the DIG-labeled 180-bp PCR product, two
positive clones were isolated. Among them, one clone harboring
the plasmid pUGTF-7 that contained a 4.3-kb SalI fragment was
isolated. The open reading frame (ORF) of xgtA was found to
be located in the same direction of the lac promoter of pUC18,
and ␣-glucosyl transfer activity was detected in a crude extract
of E. coli JM109/pUGTF-7. Another clone was found to harbor
plasmids containing the same 4.3-kb SalI fragment located in the
opposite direction of the lac promoter of pUC18. Therefore, we
used the plasmid pUGTF-7 for further study.
40
20
0
2
3
5
Time (h)
Fig. 3. Effects of hydroquinone (an ␣-glucosyl acceptor) on ␣-glucosyl transfer
(␣-arbutin synthesis) and maltose-hydrolyzing activities of XgtA. The amounts of
maltose (a), ␣-arbutin and hydroquinone (b), and glucose (c). Purified XgtA (0.4 ml;
1.9 U) was added to 1.6 ml of 10 mM H3BO3–NaOH–KCl buffer (pH 7.5) containing
1.5 M maltose. The reaction mixture was shaken at 180 rpm at 40 ◦C. Next, 10 mg
of hydroquinone was added to the reaction mixture after 1-h or 2-h incubation.
The amounts of ␣-arbutin, hydroquinone, maltose, and glucose were measured by
HPLC every hour, as described in Section 2. Symbols: closed circle, no addition of
hydroquinone; closed triangle, addition of hydroquinone 1 h after the beginning of
the reaction; closed square, addition of hydroquinone 2 h after the beginning of the
reaction; opened triangle, the amount of hydroquinone after addition at 1 h after
the beginning of the reaction; and opened square, the amount of hydroquinone
after addition at 2 h after the beginning of the reaction.
3.6. Nucleotide sequence and expression of xgtA in E. coli and
enzyme assay
Several deletion plasmids were constructed from the 4.3-kb SalI
fragment of pUGTF-7. The plasmid containing a 2.5-kb SalI-PstI frag-
ment was designated, a restriction map was constructed, and the
xgtA gene was contained in a 2.5-kb SalI-PstI-digested fragment
(data not shown). The nucleotide sequence of the SalI-PstI fragment
was determined and xgtA was identified to be 1614 bp encoding 538
amino acid residues (Fig. 5).
The expression plasmid containing the xgtA gene was con-
structed and designated as pKKGTF. The cell-free extract of E. coli
JM109/pKKGTF after 22-h cultivation was prepared and used for
maltose, were increased under the conditions in the presence of
hydroquinone.
3.5. Gene cloning of the ˛-glucosyl transfer enzyme (XgtA)
The N-terminal 20 amino acid residues and the internal 12
amino acid residues of the purified XgtA were determined to
be Ser-Gln-Thr-Pro-Trp-Trp-Arg-Gly-Ala-Val-Ile-Tyr-Gln-Ile-Tyr-
Pro-Arg-Ser-Phe-Leu and Val-Met-Ile-Asp-Gln-Val-Leu-Ser-His

T. Sato et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 20–27
25
Fig. 5. Alignment of amino acid sequence of XgtA, an ␣-glucosyl transfer enzyme of X. campestris WU-9701, with several enzymes belonging to the ␣-amylase family.
The amino acid residues that are identical between all the members are indicated by white letters in black boxes. The amino acid residues that are identical between
more than half of the members are indicated by gray boxes. Numbers indicate the positions of the residues in the complete amino acid sequence of the enzyme. The
asterisks indicate the catalytic sites of the enzymes belonging to the ␣-amylase family. XgtA, ␣-glucosyl transfer enzyme of Xanthomonas campestris WU-9701 (Accession
No. AB081949); GSJ, ␣-glucosidase of Geobacillus sp. HTA-462 (Accession No. AB154818); TreC, trehalose-6-phosphate hydrolase of Escherichia coli (Accession No. U06195);
MalL, oligo-1,6-glucosidase of Bacillus cereus ATCC 7064 (Accession No. P21332); Mal12, ␣-glucosidase of Saccharomyces cerevisiae (Accession No. P53341).
the assay of ␣-glucosyl transfer activity as described in Sec-
tion 2. The ␣-glucosyl transfer activity in the cell-free extract
from E. coli JM109/pKKGTF was 4.8 U/mg, and was 1.4 × 102-fold
greater than that from X. campestris WU-9701 (3.5 × 10−1 U/mg)
after 48-h incubation according to the methods described previ-
ously [7]. Denaturing SDS-PAGE of the cell-free extract of E. coli
JM109/pKKGTF indicated a protein of approximately 57 kDa; iden-
tical to the mass of the ␣-glucosyl transfer enzyme purified from X.
campestris WU-9701. Moreover, the kinetic values (Km and kcat) of
the purified recombinant XgtA were identical to those of the native
XgtA produced by X. campestris WU-9701. These results indicate
that recombinant XgtA produced by E. coli JM109/pKKGTF is cat-
alytically active and has identical enzymatic properties to native
XgtA.
33% with ␣-glucosidase of Saccharomyces cerevisiae [23], and 32%
with oligo-1,6-glucosidase (EC 3.2.1.10) of Bacillus cereus [24]. Sev-
eral regions near the N-terminal are highly conserved among the
enzymes shown in Fig. 5. Moreover, XgtA seems likely to have four
conserved regions in addition to the (␤/␣)8 barrel structure that is
typical of enzymes belonging to the ␣-amylase family [25,26]. How-
ever, the C-terminal region of XgtA is unique, with no homology to
the enzymes belonging to the ␣-amylase family in the region from
amino acids 423 to 538. The signal sequence was not confirmed in
the N-terminal region of XgtA suggesting that XgtA is not secreted
toward the periplasm or exported out of the cell. These results
are in accordance with the observation that more than 90% of ␣-
glucosyl transfer activity of X. campestris WU-9701 was detected in
the cytosol (data not shown).
3.7. Predicted amino acid sequence of XgtA
3.8. Catalytic-site determination of XgtA by site-directed
mutagenesis
The predicted amino acid sequence of XgtA has significant
homology to several enzymes belonging to the ␣-amylase family:
38% with ␣-glucosidase of Geobacillus sp. strain HTA-462 [21], 35%
with trehalose-6-phosphate hydrolase (EC 3.2.1.93) of E. coli [22],
The critical catalytic amino acid residues of XgtA were identified
as Asp201, Glu270, and Asp331 using site directed mutagenesis,
as follows. The predicted amino acid sequence of XgtA suggests

26
T. Sato et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 20–27
that XgtA belongs to the ␣-amylase family and has four highly con-
served regions “Regions 1–4”. The catalytic amino acid residues of
the enzymes belonging to ␣-amylase family were reported to be
Asp in Region 2, Glu in Region 3, and Asp in Region 4 [27]. As shown
in Fig. 5, the alignment of amino acid sequences of XgtA and the
enzymes belonging to ␣-amylase family clearly indicate that the
amino acid residues Asp201 and Asp331 of XgtA must be the cat-
alytic amino acid residues in Regions 2 and 4, respectively. On the
other hand, one of the three amino acid residues Glu247, Glu253,
and Glu270 was predicted to be the catalytic amino acid residue
corresponding to Glu in Region 3. Therefore, site-directed muta-
genesis toward Asp201, Glu247, Glu257, Glu270, and Asp331 was
done for determination of catalytic site of XgtA. Mutations caus-
ing amino acid substitutions of the amino acid residues Asp201,
Glu270, and Asp331 resulted in complete impairment of ␣-glucosyl
transfer and maltose-hydrolysis activity. In addition, the mutant
XgtA enzymes Glu247Gln and Glu253Gln showed both activities,
indicating that neither Glu247 nor Glu253 are catalytically impor-
tant. These results clearly indicate that the critical catalytic amino
acid residues of XgtA are identified as Asp201, Glu270, and Asp331,
and that XgtA is an enzyme belonging to the glycoside hydrolase
family 13 [27].
oligo-1,6-glucosidase but shows little hydrolysis activity toward
isomaltose and no ␣-glucosyl transfer activity using isomaltose
as an ␣-glucosyl donor (Tables 3 and 4). It is also noteworthy
that XgtA as a monomeric enzyme shows 38% homology to the
Geobacillus sp. ␣-glucosidase (GSJ) which is a dimeric enzyme
[21]. GSJ hydrolyzed ␣-1,4-glycosidic linkages of oligosaccharides
and various glycosylated non-sugar molecules using maltose as a
sugar donor, produced maltooligosaccharides in ␣-glycoside syn-
thesis reactions and synthesized isomaltose from maltose [21]. The
enzyme properties of GSJ are distinctly different to those of XgtA.
In addition, as shown in Table 5, the ␣-glucosyl transfer reac-
tion proceeds faster than the hydrolytic reaction with XgtA. As
shown in Fig. 5, the predicted amino acid sequence of XgtA, espe-
cially in relation to N-terminal and core regions, has significant
homologies to several enzymes belonging to the ␣-amylase family.
On the other hand, the C-terminal region of XgtA is unique in the
sequence of amino acid residues, and shows no homology to other
enzymes belonging to ␣-amylase family. Although the reason why
XgtA mainly catalyzes the ␣-glucosyl transfer reaction is not fully
explained, there is some possibility that the unique conformation
of the C-terminal region of XgtA might be related to the unique
reaction property of XgtA mainly catalyzing ␣-glucosyl transfer
reaction.
Based on the amino acid sequence, the stereostructure of XgtA
was predicted using 3D-JIGSAW software, showing the catalytic
sites positioned at the bottom of the cleft in the (␤/␣)8-barrel (see,
Supplementary data). Since the catalytic sites (Asp201, Glu270, and
Asp331) are located in this cleft, it is suggested that the catalytic
center exists in this cleft. These observations are similar to those of
the known enzymes belonging to the ␣-amylase family [25,30].
4. Discussion
In previous papers, we report successful use of X. campestris
WU-9701 to produce the ␣-glucosyl transfer enzyme XgtA for
selective high yield synthesis of various ␣-glucosides using mal-
tose as an ␣-glucosyl donor [11–13]. Conventional ␣-glucosidases
produce maltooligosaccharides such as maltotriose and maltote-
traose [8–10]. Importantly, in the present ␣-glucoside synthesis
reactions, no such maltooligosaccharides were detected [11–13].
From these results, it was suggested that the enzymatic properties
of XgtA were different from those of typical ␣-glucosidases that
catalyze mainly hydrolysis of maltooligosides [8–10]. Therefore, in
this study, XgtA was purified to homogeneity from the cell-free
extract of X. campestris WU-9701 for enzymatic characterization.
XgtA was concentrated 12.9-fold in this purification process with a
yield of 14.0% from the cell-free extract of X. campestris WU-9701
(Table 1).
In studies of saccharide specificity, XgtA hydrolyzed maltose
with ␣-1,4-glucosidic bonds but hardly hydrolyzed maltotriose
or amylose (Table 3). In contrast with other ␣-glucosidases, XgtA
shows no hydrolysis activity toward sucrose [4,28]. On the other
hand, ␣-arbutin was synthesized using maltose as an ␣-glucosyl
donor (Table 4). These results indicate that XgtA is a kind of ␣-
glucosidase and possesses strict substrate specificity for maltose.
We have cloned the gene xgtA and overexpressed it in E. coli.
Since registration of the gene xgtA in the nucleotide sequence
databases DDBJ in 2003, several putative genes showing high
homologies with xgtA have been sequenced through the genome
sequencing of Xanthomonas strains. For example; X. campestris
pv. campestris str. 8004 (100%, Accession No. CP000050.1) and
X. campestris pv. campestris ATCC 33913 (97.6%, Accession No.
AE012359.1). However, the gene products derived from these puta-
tive genes have not yet been characterized and their functions are
unknown.
5. Conclusion
In this paper, we describe the unique properties of XgtA, as a
novel type ␣-glucosidase from X. campestris WU-9701, by purifi-
cation, molecular characterization, and gene identification. It was
noteworthy that XgtA is a monomeric enzyme, and that the Vmax
value for ␣-glucosyl transfer activity was 1.3 × 10−2 (mM/s) and
3.9-fold as much as that for maltose-hydrolyzing activity. The chro-
mosomal gene xgtA containing 1614-bp open reading frame was
identified and successfully expressed in E. coli. Site-directed muta-
genesis identified Asp201, Glu270, and Asp331 as the catalytic sites,
suggesting that XgtA belongs to the glycoside hydrolase family 13.
Acknowledgment
This work was partially supported by the Global COE program
“Center for Practical Chemical Wisdom” by the Ministry of Educa-
tion, Culture, Sports, Science and Technology (MEXT), Japan.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2012.04.014.
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the ␣-amylase family; specifically the glycoside hydrolase family
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