Isolation and characterization of a novel α-glucosidase with transglycosylation activity from Arthrobacter sp. DL001

Article abstract of DOI:10.1016/j.molcatb.2012.04.016

A strain of Arthrobacter sp. DL001 with high transglycosylation activity was successfully isolated from the Yellow Sea of China. To purify the extracellular enzyme responsible for transglycosylation, a four-step protocol was adopted and the enzyme with electrophoretical purity was obtained. The purified enzyme has a molecular mass of 210 kDa and displays a narrow hydrolysis specificity towards α-1,4-glucosidic bond. Its hydrolytic activity was identified as decreasing in the order of maltotriose > panose > maltose. Only 3.61% maltose activity occurs when p-nitrophenyl α-d-glycopyranoside serves as a substrate, suggesting that this enzyme belongs to the type II α-glucosidase. In addition, the enzyme was able to transfer glucosyl groups from the donors containing α-1,4-glucosidic bond specific to glucosides, xylosides and alkyl alcohols in α-1,4- or α-1,6-manners. A decreased order of activity was observed when maltose, maltotriose, panose, β-cyclodextrin and soluble starch served as glycosyl donors, respectively. When maltose was utilized as a donor and a series of p-nitrophenyl-glycosides as acceptors, the glucosidase was capable of transferring glucosyl groups to p-nitrophenyl-glucosides and p-nitrophenyl-xylosides in α-1,4- or α-1,6-manners. The yields of p-nitrophenyl-oligosaccharides could reach 42-60% in 2 h. When a series of alkyl alcohols were utilized as acceptors, the enzyme exhibited its transglycosylation activities not only to the primary alcohols but also to the secondary alcohols with carbon chain length 1-4. Therefore, all the results indicated that the purified α-glucosidase present a useful tool for the biosynthesis of oligosaccharides and alkyl glucosides.

Full text of DOI:10.1016/j.molcatb.2012.04.016

Journal of Molecular Catalysis B: Enzymatic 80 (2012) 48–57
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Isolation and characterization of a novel ␣-glucosidase with transglycosylation
activity from Arthrobacter sp. DL001
Kun Zhoua,b, Hong-wei Luana, Ying Hua, Guang-bo Gea, Xing-bao Liua, Xiao-chi Mac,
Jie Houc, Xiu-li Wanga, Ling Yanga,∗
a Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China
b Graduate School of Chinese Academy of Sciences, Beijing 100049, China
c Dalian Medical University, Dalian, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A strain of Arthrobacter sp. DL001 with high transglycosylation activity was successfully isolated from the
Yellow Sea of China. To purify the extracellular enzyme responsible for transglycosylation, a four-step
protocol was adopted and the enzyme with electrophoretical purity was obtained. The purified enzyme
has a molecular mass of 210 kDa and displays a narrow hydrolysis specificity towards ␣-1,4-glucosidic
bond. Its hydrolytic activity was identified as decreasing in the order of maltotriose > panose > maltose.
Only 3.61% maltose activity occurs when p-nitrophenyl ␣-d-glycopyranoside serves as a substrate, sug-
gesting that this enzyme belongs to the type II ␣-glucosidase. In addition, the enzyme was able to transfer
glucosyl groups from the donors containing ␣-1,4-glucosidic bond specific to glucosides, xylosides and
alkyl alcohols in ␣-1,4- or ␣-1,6-manners. A decreased order of activity was observed when maltose,
maltotriose, panose, ␤-cyclodextrin and soluble starch served as glycosyl donors, respectively. When
maltose was utilized as a donor and a series of p-nitrophenyl-glycosides as acceptors, the glucosidase
was capable of transferring glucosyl groups to p-nitrophenyl-glucosides and p-nitrophenyl-xylosides in
␣-1,4- or ␣-1,6-manners. The yields of p-nitrophenyl-oligosaccharides could reach 42–60% in 2 h. When
a series of alkyl alcohols were utilized as acceptors, the enzyme exhibited its transglycosylation activities
not only to the primary alcohols but also to the secondary alcohols with carbon chain length 1–4. There-
fore, all the results indicated that the purified ␣-glucosidase present a useful tool for the biosynthesis of
oligosaccharides and alkyl glucosides.
Received 8 October 2011
Received in revised form 20 April 2012
Accepted 20 April 2012
Available online 2 May 2012
Keywords:
Arthrobacter sp.
Hydrolysis
Transglycosylation
Substrate specificity
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
with glycosyltransferases, the glycosidases are synthetically attrac-
tive due to their accommodation of inexpensive glycosyl donors,
Carbohydrates play a pivotal role in regulating the processes
of the embryogenesis, development and differentiation during
the human lifespan [1]. Unfortunately, due to their complicated
structures and difficulties in chemical synthesis, the role of carbo-
hydrates in detail remain poorly understood [2,3].
Enzymatic-catalyzed transglycosylation is a promising method
for synthesis of carbohydrates due to its mild reaction condition,
high regio- and stereo-selectivity, less by-products and friendly to
environment, especially when generally harsh conditions or toxic
(heavy metals) catalysts are undesirable, such as food or cosmetics
areas [4]. There are two major classes of enzymes used in glycosyla-
tion, the glycosyl transferases and the glycosidases [1,5]. Compared
availability and widespread occurrence in nature [6–8].
The ␣-glucosidases [EC 3.2.1.20, ␣-d-glucoside glucohydrolase]
catalyze not only a cleavage of an ␣-glucosyl residue substrate,
but also transglycosylation reaction to synthesize various ␣-
glucosylated compounds. Based on their substrate specificity,
enzymes are divided into three groups (type I, II, and III). Type I
␣-glucosidase hydrolyzes heterogeneous substrates, such as aryl
␣-glucosides or sucrose, more efficiently than maltose; type II
enzymes prefer maltose and isomaltose to aryl glucosides while
type III enzymes possess similar specificity to type II enzymes, but
they are capable of attacking polysaccharides like amylose and
starch [9]. Although ␣-glucosidases have been recognized as an
ideal candidate for carbohydrate synthesis, challenges still exist for
the scientists to explore more glycosidases with high transglyco-
sylation activity and defined catalysis mechanism. Meanwhile, low
yield of transglycosylation and hydrolysis of products is another
bottleneck needed to be addressed to advance the application of
glycosidases in the biosynthesis [3,8].
∗
Corresponding author at: Laboratory of Pharmaceutical Resource Discovery,
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan
Road, Dalian 116023, China. Tel.: +86 411 84379317; fax: +86 411 84676961.
E-mail addresses: hjie 2004@163.com (J. Hou), yling@dicp.ac.cn (L. Yang).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.04.016

K. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 48–57
49
In our previous study, an effective methodology of character-
2.3. Strain identification
izing transglycosylation activity has been well established with
the aim to find more glycosidases with high transglycosylation
activity from the nature [10–12]. We have demonstrated that
this analysis method could detect the transglycosylation activ-
ities more precisely and accurately even with the hydrolytic
activity. In the present study, we described an isolation of the
strain with transglycosylation activity using the modified ana-
Cell morphology of the isolated strain was examined under light
microscopy (Olympus CK40, Japan). Gram staining was carried out
by using the standard Gram reaction [13].
Identification of the isolated strain was done by sequencing the
16S rDNA, which was conducted by Takara Biotechnology (Dalian)
Co., Ltd. In brief, the 16S rDNA sequence was amplified by TaKaRa
16S rDNA Bacterial Identification PCR Kit (Code No. D310, TaKaRa,
Dalian Co., Ltd). The PCR amplification was conducted in a total
volume of 50 ␮l, containing 1 ␮l DNA template, 0.2 mM forward
primer, 0.2 mM reverse primer and required H2O. We conducted
the PCR amplification as followings: an initial denaturation at 94 ◦C
for 5 min, then 30 cycles of 94 ◦C for 1 min, 55 ◦C for 1 min, and 72 ◦C
1.5 min, followed by a final extension at 72 ◦C for 5 min. For each
reaction, we take 5 ␮l of purified resultant PCR products to conduct
agarose (1%) gel electrophoresis.
lytical method and the purification of
a new ␣-glucosidase
from the supernatant of the strain culture. Specifically, the
substrate specificities, including hydrolytic and donor/acceptor
specificity, as well as the linkage types of the products were
characterized.
2. Materials and methods
The 16S rDNA gene sequence was subjected to BLAST searches
of the National Center for Biotechnology Information (NCBI) Gen-
Bank database for identification. Related sequences were obtained
from the GenBank database. Alignments of sequences were carried
out with Clustal W [14]. A phylogenetic tree was constructed using
the neighbor-joining method and evaluated by bootstrap sampling
(1000 replicates) using the MEGA 4 program [15].
2.1. Chemicals and reagents
Uridine 5ꢀ-diphosphoglucose disodium salt (UDPG), 4-
methylumbelliferyl
␣-d-glucoside
(MUG),
p-nitrophenyl
␣-d-glucopyranoside (pNP ␣-G), pNP ␤-d-glucopyranoside (pNP ␤-
G), pNP ␣-d-xylopyranoside (pNP ␣-X), pNP ␤-d-xylopyranoside
(pNP ␤-X), pNP ␤-d-maltoside, pNP ␣-l-arabinopyranoside,
pNP
␤-d-cellobioside,
pNP
pNP
␣-d-galactopyranoside,
␤-l-fucopyranoside,
pNP
pNP
2.4. Purification of extracellular ˛-glucosidase
␤-d-galactopyranoside,
␣-l-fucopyranoside, pNP ␤-d-mannopyranoside, pNP ␣-d-
mannopyranoside, pNP ␣-l-rhamnopyranoside and some
saccharides (cellobiose, maltose, panose, trehalose, sucrose
and lactose) were obtained from Sigma (St. Louis, MO) and used
as enzyme substrates. Maltotriose is a product of Wako Pure
Chemical Co., Japan. Glucose oxidase-base Kit was obtained from
Rongsheng Biotech Co., Ltd, China. Sephacryl S-200 HR was from
GE Healthcare. Toyopearl DEAE-650M and Toyopearl Butyl 650C
are from TOSOH Corporation (Japan). Methanol and acetonitrile
(HPLC grade) are from Tedia (Fairfield, USA) and millipore water
(Millipore, Bedford, MA) was employed. Other general chemicals
used were of AR grade.
Chromatography was performed at room temperature, and frac-
tions were stored at 4 ◦C upon collection. The following buffers were
used in the purification process: buffer A, 50 mM citrate–phosphate
buffer (pH 5.5); buffer B, buffer A containing 1.0 M ammonium sul-
fate.
Two liters of culture was prepared as described in Section 2.2.
The bacterial cells were removed by centrifugation at 8000 × g,
4 ◦C for 10 min. The supernatant fraction was subjected to 70–90%
saturation ammonium sulfate precipitation. The resulting precipi-
tate was collected and dissolved in buffer B. Then the sample was
applied to a Toyopearl Butyl 650C column (Ф 1.2 cm × 5 cm), which
was pre-treated with buffer B. Proteins were eluted with a 200-
ml linear gradient of 1.0-0 M (NH4)2SO4. The ␣-glucosidase active
fractions between 0.6 and 0.45 M (NH4)2SO4 were pooled, dialyzed
extensively against buffer A at 4 ◦C overnight, and loaded onto a
Toyopearl DEAE-650M column (Ф 1.2 cm × 6 cm) previously equi-
librated with the buffer A. A stepwise elution with the buffer over a
range of NaCl concentrations (0–0.4 M) was undertaken. The frac-
tions eluted by 0.08 M NaCl were collected. The active fractions
eluted by 0.08 M NaCl were combined and concentrated to 0.6 ml
by ultrafiltration using a 3 kDa membrane (Microcon YM-3, Milli-
pore). The concentrated sample was loaded on a Sephacryl S-200
HR column (Ф 1.0 cm × 80 cm) which was pre-equilibrated with
buffer A. Then we eluted proteins in the same buffer at a flow rate
of 0.5 ml/min and collected the fractions with transglycosylation
activity.
2.2. Isolation of the strain with transglycosylation activity
Microorganisms were isolated from the seawater samples of the
Yellow Sea (Dalian, China). The culture medium was composed
of 20 g/l soluble starch, 1 g/l KNO3, 0.5 g/l NaCl, 0.5 g/l K2HPO4,
0.5 g/l MgSO4·7H2O, 0.01 g/l FeSO4·7H2O (pH 7.0). Seawater sam-
ples were added to 100 ml medium and cultured at 30 ◦C for
48 h to enrich bacteria. Then the culture was spread on 1.5% agar
plates (mixed agar with culture medium previously described)
and incubated at 30 ◦C for two days. The appearing colonies were
picked and subcultured to get pure isolates. Each isolate was inoc-
ulated into 5 ml medium and cultivated aerobically at 30 ◦C for
two days. Broth culture was centrifuged at 8000 × g for 10 min
at 4 ◦C, and the cell-free supernatant was stored at −20 ◦C until
use. The transglycosylation activity was determined by incubat-
ing the cell-free supernatant with 2.5 mM maltose and 0.5 mM
pNP ␤-G in 50 mM citrate–phosphate buffer (pH 5.5) in a 200 ␮l
reaction mixture at 30 ◦C for 30 min. The reaction was initiated
by addition of the cell-free supernatant and terminated by adding
200 ␮l methanol. After centrifugation at 21,000 × g for 5 min at
4 ◦C, aliquots of the reaction supernatant were qualitatively and
quantitatively analyzed by ultra-fast liquid chromatography/mass
spectra (UFLC–MS) and high-performance liquid chromatography
(HPLC). The colonies containing transglycosylation activity were
re-streaked onto agar plates to obtain pure cultures for further
study.
2.5. Enzyme and protein assay
2.5.1. Transglycosylation activity assay
The transglycosylation activity was determined by using mal-
tose as a sugar donor and pNP ␤-G as a sugar acceptor. The reaction
was conducted by incubating the enzyme solution with 2.5 mM
maltose and 0.5 mM pNP ␤-G in 50 mM citrate–phosphate buffer
(pH 5.5) in a 200 ␮l reaction mixture at 30 ◦C for 30 min. All the
reactions were initiated by the addition of the enzyme and termi-
nated by adding 200 ␮l methanol. After centrifugation at 21,000 × g
for 5 min at 4 ◦C, aliquots of the reaction supernatant (30 ␮l) were
quantitatively analyzed by HPLC. One unit of transglycosylation

50
K. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 48–57
activity was defined as the amount of enzyme catalyzes transg-
lycosylation of 1 ␮mol of pNP ␤-G per minute.
The transglycosylation activity towards non-sugar acceptors
was measured by using maltose as a donor and methanol, ethanol,
1-propanol, 2-propanol, 1-butanol, or 2-methyl-2-propanol as an
acceptor respectively. A 100 ␮l reaction mixture containing 5 ␮l
purified enzyme, 2.5 mM maltose and alcohol acceptor 10% (v/v) in
a 50 mM citrate–phosphate buffer (pH 5.5), was incubated at 30 ◦C
for 1 h. The reaction was stopped by the addition of 100 ␮l ace-
tonitrile. After centrifugation at 21,000 × g for 5 min at 4 ◦C, 5 ␮l of
the reaction supernatants were qualitatively analyzed by UFLC-ESI-
MS and 100 ␮l of the reaction supernatants were also deposited on
Silica gel 60 F 254 plate (Merck, Germany). The reaction products
were visualized by soaking rapidly into methanol solution contain-
ing 10% (v/v) sulfuric acid and heating it at 110 ◦C for 10 min. The
TLC solvent system was 1-butanol/ethyl acetate/isopropanol/acetic
acid/water (1:3:2:1:1, v/v/v/v/v).
2.5.2. Hydrolysis activity assay
The ␣-glucosidase activity was measured by incubating the
enzyme solution with 10 mM maltose in 50 mM citrate–phosphate
buffer (pH 5.5) at 30 ◦C for 10 min. The reaction was stopped by heat
inactivation at 100 ◦C for 5 min. The liberated glucose was mea-
sured by the glucose oxidase–peroxidase method (GOD-POD) using
a glucose oxidase-base kit (Rongsheng Biotech Co., Ltd, China) [16].
One unit of ␣-glucosidase activity was defined as the amount of
enzymes that catalyze hydrolysis of 1 mmol of maltose per minute.
2.5.3. Protein assay
Protein was quantified according to the Bradford method [17],
using bovine serum albumin (BSA) as a standard. The fractions
eluted from all chromatographic runs were monitored for protein
by measuring the absorbance at 280 nm.
2.9. Substrate specificity of hydrolysis
Hydrolytic activity against saccharides (0.5 mM), pNP ␣/␤-G
(0.5 mM), ␤-CD (0.5 mM) and soluble starch (0.5 g/l) were deter-
mined by measuring the amount of released glucose with the
glucose oxidase–peroxidase method (GOD-POD) using a glucose
oxidase-base kit (Rongsheng Biotech Co., Ltd, China) [16].
Activities towards pNP-glycosides were routinely assayed by
using a reaction mixture (total volume 300 ␮l) containing 0.5 mM
of each substrate and 5 ␮l of the purified enzyme in 50 mM
citrate–phosphate buffer (pH 5.5). After incubating at 30 ◦C for
30 min, the reaction was stopped by the addition of 0.25 M NaOH
(1.5 ml), and then the absorbance was read at 405 nm with UV–vis
spectrophotometer (JASCO V-530, Japan). The reference cuvette
contained all reactants except the enzyme. One unit of enzyme
activity was defined as the amount of enzyme liberating 1 mmol of
p-nitrophenol per minute under the above-mentioned conditions.
2.6. Electrophoresis and determination of molecular weight
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed on 8% polyacrylamide gel in a Mini-
Protein III dual-slab cell electrophoresis unit (Bio-Rad) [18], and
proteins were visualized with Coomassie Brilliant Blue R250.
Native-PAGE was performed with 8% polyacrylamide gels without
SDS. Molecular mass of enzymes was estimated by both SDS-
PAGE and gel-filtration on a column of Sephacryl S-200 HR (Ф
1.0 cm × 80 cm). The gel filtration column was equilibrated with
50 mM citrate–phosphate buffer (pH 5.5) and calibrated by elution
of standard protein markers, which included catalase (250 kDa),
BSA (66 kDa), ovalbumin (44 kDa) and cytochrome C (12 kDa).
2.7. Identification of transglycosylation activity of the protein
band
2.10. Isolation, purification and NMR analysis of transfer products
The purified enzyme band without staining on the polyacry-
lamide gel was excised and dissolved in 50 mM citrate–phosphate
buffer (pH 5.5). The extracted protein band was then subjected
to the transglycosylation analysis. The reaction was conducted by
incubating the band with 2.5 mM maltose and 0.5 mM pNP ␤-G
in 50 mM citrate–phosphate buffer (pH 5.5) at 30 ◦C for 1 h. The
transglycosylation products were analyzed by HPLC.
We used the cell free supernatant to prepare sufficient quanti-
ties of the pNP ␤-G transglycosylation products for NMR analysis,
and scaled up the incubation system to 100 ml. The pNP ␤-G
(0.5 mM) was incubated with the cell free supernatant and the mal-
tose (2.5 mM) for 60 min at 30 ◦C, respectively. Reaction mixture
was extracted with 50% ethyl acetate, and the organic layer was
separated after the reaction mixture centrifuging at 5000 × g for
10 min. The extraction was repeated three times, and the organic
layers were combined and dried in a vacuum. The residues were
dissolved in methanol (2.5 ml). The substrates and products were
separated by HPLC, and the eluent containing the transglycosyla-
tion product p1-1 or p1-2 was collected and dried in a vacuum,
respectively. The purity of product p1-1 and p1-2 was approxi-
mately 90% and 95% (HPLC), respectively.
2.8. Donor/acceptor specificity of transglycosylation
To characterize the synthetic abilities of the purified enzyme,
a 200 ␮l reaction system containing 5 ␮l purified enzyme, a vari-
ety of acceptors and donors in a 50 mM citrate–phosphate buffer
(pH 5.5), was incubated at 30 ◦C for 1 h. When we used pNP ␤-G
(0.5 mM) as an acceptor for the purified enzyme, the synthe-
sis of pNP-oligosaccharides was achieved. To evaluate its donor
specificity, pNP ␤-G (0.5 mM) was used as an acceptor and the
reactions were carried out using maltose (0.5, 1, 2.5, 12.5 mM), iso-
maltose (0.5 mM), maltrotriose (0.5 mM), panose (0.5 mM), UDPG
(0.5 mM), sucrose (0.5 mM), ␤-cyclodextrin (␤-CD, 0.5 mM), tre-
halose (0.5 mM), lactose (0.5 mM), cellobiose (0.5 mM), pNP ␣-G
(0.5 mM), pNP ␤-G (0.5 mM) or soluble starch (0.5 g/l) as donors. To
evaluate the sugar-acceptor specificity, reactions were carried out
by using maltose (2.5 mM) as a donor and a series of pNP-glycosides
(0.5 mM) as acceptors. All the reactions were stopped by the addi-
tion of 200 ␮l methanol. After centrifugation at 21,000 × g for 5 min
at 4 ◦C, aliquots of the reaction supernatants (5 ␮l) were qualita-
tively analyzed by UFLC–MS. In the assays with pNP-substrates, if
glycosylation occurred, other 30 ␮l of the supernatants were quan-
titatively analyzed by HPLC.
The product p1-2 and the pNP ␤-G were dissolved in DMSO-d6
for NMR analysis. All NMR data (1H, 13C, HMBC, HMQC, COSY and
NOESY) were acquired on a Bruker AV-500 spectrometer (Bruker,
Newark, DE) operating at 500 MHz for 1H NMR and 125 MHz for 13
NMR.
C
2.11. Chromatography conditions
High-performance liquid chromatography (HPLC) was per-
formed following the protocol provided by Okuyama et al. with
minor modification [19]. Briefly, the HPLC system (Shimadzu,
Kyoto, Japan) consisted of an SCL-10AVP system controller, one
LC-10ADVP quaternary pump, a PGU-14A degasser, an SIL-10ADVP
autoinjector, and an SPD-M10AVP diode array detector. An ODS
column (4.6 mm × 200 mm × 5 ␮m, Kromasil) was adopted as
solid phase, while the mobile phase consisted of H2O (A) and

K. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 48–57
51
CH3OH (B). Two gradient profiles were employed to analyze dif-
ferent samples containing various substrates: (i) pNP ␤-G and
pNP ␣-G: 0.01–20.0 min, 15% A; 20.01–25.00 min, 15–90% A;
25.01–35.00 min, 90% A; 35.01–45.00 min, 15% A; (ii) for pNP ␤-X
and pNP ␣-X, 0.01–20.0 min, 20–40% A; 20.01–25.00 min, 40–90%
A; 25.01–35.00 min, 90% A; 35.01–45.00 min, 20% A. The flow rate
was 0.7 ml/min, and the injection volume was 30 ␮l. The detection
wavelength was set at 300 nm.
The Ultra-fast liquid chromatography/mass spectra (UFLC–MS)
were utilized to qualitatively characterize the products. UFLC
system (Shimadzu Kyoto, Japan) equipped with a CBM-20A com-
munications bus module, an SIL-20ACHT auto sampler, two
LC-20AD pumps, a DGU-20A3 vacuum degasser, a CTO-20AC col-
umn oven and an SPD-M 20A diode array detector. A Shim-pack
XR-ODS (150 mm × 2.0 mm, 2.2 ␮m, Shimadzu) analytical column
together with an ODS guard column (5 mm × 2.0 mm, 2.2 ␮m, Shi-
madzu) were used and kept at 50 ◦C. Mass detection was performed
on a Shimadzu LCMS-2010EV instrument with an ESI interface in
negative ion mode (ESI−) for pNP-glycosides from m/z 200 to 850
and positive ion mode (ESI+) for alkyl-glycosides from m/z 200 to
500. The detector voltage was −1.55 kV and 1.5 kV, respectively. The
temperature of either curved desolvation line (CDL) or the block
heater was 250 ◦C. All the other MS detection conditions were set
as followings: interface voltage, 4 kV; CDL voltage, 40 V; nebuliz-
ing gas (N2) flow, 1.5 l/min; drying gas (N2) pressure, 0.06 MPa. For
pNP-glycosides analysis, the mobile phase consisted of 0.2% formic
acid in H2O (A) and CH3CN (B) and the related parameters were
listed as followings: 0.01–2.00 min, 92% A; 2.01–10.00 min, 92–12%
A; 10.01–12.50 min, 5% A; 12.51–16.00 min, 92% A. The flow rate
was 0.4 ml/min, and the injection volume was 5 ␮l. DAD detec-
tion was performed in a range of 190–370 nm. The wavelength of
measurement was 300 nm. Similarly, a mobile phase composed of
0.2% formic acid in H2O (A) and CH3OH (B) was used to deter-
mine alkyl-glycoside under following condition: 0.01–5.00 min,
100% A; 5.01–10.00 min, 100–20% A; 10.01–12.50 min, 10% A;
12.51–16.00 min, 100% A. The flow rate was 0.3 ml/min, and the
injection volume was 5 ␮l.
Fig. 1. A phylogenetic tree based on 16S rDNA gene sequences showing the positions
of the strain DL001 among species of the genus Arthrobacter. Numbers at branch
points refer to bootstrap percentages (from 1000 resamplings).
monitored throughout the process of purification. The purified
enzyme exhibited approximately 108-fold increase in purity with
a recovery of 19.2% relative to the amount of the crude enzyme in
transglycosylation. The specific activity of the enzyme for the trans-
glycosylation of pNP ␤-G was 27.7 U mg−1 protein. The hydrolysis
activity of the purification was also shown in Table 1. The purified
enzyme appeared as a single band on PAGE either in the presence
(Fig. 2a, lane 4) or absence (Fig. 2b, lane 4) of SDS. Its subunit mass
was estimated to be approximately 107 kDa on SDS-PAGE. Gel fil-
tration of the enzyme in Sephacryl S-200 HR produced an apparent
molecular mass of 210 kDa. Therefore, the enzyme was though to
be a dimer.
In order to clarify whether the protein isolated on PAGE was
responsible for the transglycosylation activity, the corresponding
band of the enzyme on PAGE without staining was extracted and
further incubated with the sugar donor and acceptor. Consequently,
two transglycosylation products of pNP ␤-G were observed, indi-
cating that the purified enzyme possess the transglycosylation
activity.
3. Results
3.1. Identification and characterization of a strain with
transglycosylation activity
Several colonies capable of transferring glucosyl groups to pNP
␤-G were isolated from the seawater. Among them, the isolate
with highest transglycosylation yield (named DL001) was selected
for the following study. The strain DL001 was an aerobic, gram-
negative, motile and rod-shaped bacterium. The colonies appear
circular, smooth and milky white with a diameter of 0.5–2.0 mm
after culture for 2 days at 30 ◦C.
To determine the phylogenetic position, the 16S rDNA gene
sequence of strain DL001 (comprising 1422 nt) was analyzed, and
a dendrogram based on 1422 nucleotides was constructed (Fig. 1).
The 16S rDNA gene sequence of strain DL001 has been submit-
ted in the GenBank database with accession No. HM172535. The
phylogenetic analysis indicated that the strain DL001 was closely
related to the members of the genus Arthrobacter sp. (97–98% 16S
rDNA gene sequence similarity). This isolated strain has been pre-
served in China center for type culture collection (CCTCC) with the
preservation serial number CCTCC No. 2010107.
Fig. 2. SDS-PAGE and PAGE of the purified enzyme at various stages of purifica-
tion. (A) The SDS-PAGE of the enzymes. Lane 1, active fraction after (NH4)2SO4
precipitation; lane 2, active fraction after butyl 650C chromatography; lane 3, active
fraction after Toyopearl DEAE 650M chromatography; lane 4, the sample after gel-
filtration, purified enzyme AG-I; lane M, molecular weight marker. (B) The PAGE of
the enzymes. Lane 4, the sample after gel-filtration, purified enzyme AG-I; lane BSA,
the standard of BSA.
3.2. Enzyme purification, molecular mass determination
A novel ␣-glucosidase was purified through a four-step proce-
dure as described in Table 1. Its transglycosylation activity was

52
K. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 48–57
Table 1
Purification of the ␣-glucosidase from Arthrobacter sp.
Purification step
Total activity (U)a
Total protein (mg)b
Specificity activity (U/mg)c
Yield (%)
Purification (fold)
Hydrolysis activity
(NH4)2SO4 precipitation
HIC
Toyopeal DEAE 650M
Sephacryl S200 HR
Transglycosylation activity
(NH4)2SO4 precipitation
HIC
24.8
2.88
1.71
0.41
125
0.20
0.47
1.23
1.86
100
1.00
2.36
6.16
9.32
6.13
1.40
0.22
11.6
6.91
1.66
32.1
25.8
13.7
6.15
125
0.26
4.20
9.83
27.7
100
1.00
16.3
38.2
6.13
1.40
0.22
80.3
42.8
19.2
Toyopeal DEAE 650M
Sephacryl S200 HR
108
Two liters of cell free supernatants were as used for purification and assays were performed with substrates at pH 5.5 and 30 ◦C.
a
Details of the protocol are described in Sections 2.5.1 and 2.5.2. One unit of ␣-glucosidase hydrolysis activity was defined as the amount of enzyme that catalyzes
hydrolysis of 1 mmol of maltose per minute. One unit of transglycosylation activity was defined as the amount of enzyme that catalyzes the transglycosylation of 1 ␮mol of
pNP ␤-G per minute.
b
Protein was quantified according to the Bradford method, using bovine serum albumin as a standard.
Specific activity represents the amount of enzyme activity in each milligram of protein.
c
3.3. Specificity of transglycosylation activity
␣-X were converted into the corresponding pNP-oligosaccharide
within 2 h, respectively (Fig. 3b–d).
Transglycosylation activity of the enzyme was evidenced by
the fact that we could detect pNP-disaccharides when the enzyme
was incubated with maltose and pNP ␤-G. To further clarify the
substrate specificity for transglycosylation, a series of donors and
acceptors were adopted. The donor specificity of transglycosylation
reaction was investigated by various saccharides when pNP ␤-G
served as an acceptor. As shown in Table 2, a highest donor activ-
ity was observed in maltose, which was followed by maltotriose,
panose, ␤-CD and soluble starch. The UDPG and other substrates
were considered as non-effective donors. Further analysis of the
structures of donors indicated that the saccharides containing
␣-1,4-linked glucoside might be the effective donors. Therefore,
maltose was selected as a standard donor for transglycosylation
analysis. Its optimal concentration for standard tranglycosylation
assay was further identified as 2.5 mM in the reaction mixture
according to the linear range of the donor concentration and the
transglycosylation products (data not shown).
The transglycosylation activity was also investigated by
using maltose as
a donor and six different alcohols as
Table 2
The substrate specificity of ␣-glucosidase.
Substrate
Hydrolysis
specificitya
Sugar donor
specificityb
Sugar acceptor
specificityc
Maltose
Isomaltose
Maltotriose
Panose
␤-CD
Soluble starch
Sucrose
Trehalose
Lactose
Cellobiose
Gentiobiose
MUG
1001a
3.971a
1721a
1261a
15.21a
14.41a
1.441a
1.081a
0.001a
0.361a
13.71a
0.721a
N.A.
100
N.A.d
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
+
N.D.d
79.0
49.8
23.5
16.8
N.D.d
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
2.10
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.A.
The specificity of acceptor was investigated by a series of pNP-
substrates and alkyl alcohols when maltose (2.5 mM in the reaction
mixture) served as the donor. The constituents in the transglycosy-
lation reactions of pNP-glycosides were separated and qualitatively
analyzed by UFLC-ESI-MS.
UDPG
pNP ␣-d-glucopyranoside
pNP ␤-d-glucopyranoside
pNP ␣-d-xylopyranoside
pNP ␤-d-xylopyranoside
pNP ␣-l-fucopyranoside
pNP ␤-l-fucopyranoside
pNP ␣-d-galactopyranoside
pNP ␤-d-galactopyranoside
pNP ␣-d-mannopyranoside
pNP ␤-d-mannopyranoside
pNP ␣-l-arabinopyranoside
pNP ␣-l-rhamnopyranoside
pNP ␤-d-cellobioside
pNP
3.611a
1.441a
0.002a
0.002a
0.002a
0.002a
0.002a
0.002a
0.002a
0.002a
0.002a
0.002a
0.002a
N.A.
+
+
+
In the MS chromatography of pNP-substrates, most components
predominantly produced [M+2H2O−H]− ions depending on their
structural features. [M−H]− and [M+HCOOH–H]− ions were also
observed. These ions provided reliable information for confirming
molecular weight of the constituents. With respect to the accep-
tor pNP ␤-G (peak 1 in Fig. 3a) and pNP ␣-G (peak 3 in Fig. 3c),
the dominant ion m/z 336 [M+2H2O–H]− was observed, while the
498 [M+2H2O–H]− ion was observed for their transglycosylation
products p1-1, p1-2, p3-1 and p3-2 (Fig. 3a and c). Therefore the
molecular weight of the products is 463, indicating that a glucosyl
group was introduced. In the case of pNP ␤-X, the dominant ion
m/z is 306 [M+2H2O–H]−(peak 2, Fig. 3b), and their transglycosyla-
tion products are 432 [M−H]−, 468 [M+2H2O–H]− for p2-1 (Fig. 3g)
and 630 [M+2H2O–H]− for p2-2 and p2-3, respectively. These prod-
ucts were recognized as the monoglycosylated, diglycosylated and
diglycosylated products, respectively. The case of pNP ␣-X was
the same as pNP ␤-X. But for other pNP-glycosides, no product
was detected out. Therefore, the glucosides and xylosides were
regarded as the preferred acceptors. Further quantitative analysis
of the transglycosylation reaction indicated that about 47% of the
pNP ␤-G was converted into pNP-disaccharides derivatives (data
not shown) within 2 h, including 34% pNP ␤-isomaltoside and 13%
pNP ␤-maltoside. About 60% pNP ␤-X, 42% pNP ␣-G and 50% pNP
−
−
−
−
−
−
−
−
+
−
The purified enzyme was assayed in the standard assay condition with various com-
pounds. Each value represents the mean of triplicate measurements and varied from
the mean by not more than 10%.
a
The hydrolysis of the substrates in 50 mM citrate–phosphate buffer (pH 5.5) at
30 ◦C was measured. Activity expressed relative to activity measured on maltose
(100%).
1a Hydrolysis activity was determined by measuring the amount of released glucose
with the glucose oxidase–peroxidase method.
2a
Hydrolytic activity was determined by measuring the absorbance at 405 nm with
UV–vis spectrophotometer (JASCO V-530, Japan) due to the liberation of pNP.
b
The sugar donor specificity was expressed relative to activity measured with
maltose. A 100 ␮l reaction mixture, containing 5 ␮l purified enzyme, 0.5 mM pNP
␤-G as a sugar acceptor and various sugar donor in a 50 mM citrate–phosphate buffer
(pH 5.5), was incubated at 30 ◦C for 30 min.
c
Sugar acceptor specificity was qualitatively determined by UFLC–MS using mal-
tose as sugar donor. Activity was noted as positive (+) or negative (−).
d
N.A.: not assay; N.D.: not detected.

K. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 48–57
53
Fig. 3. The chromatography profile of transglycosylation of pNP-glycosides with the purified enzyme. The activity was assayed by incubating the enzyme solution with
2.5 mM maltose and 0.5 mM each substrate (pNP ␣/␤-G or pNP ␣/␤-X) in 50 mM citrate–phosphate buffer (pH 5.5) in a 200 ␮l reaction mixture at 30 ◦C for 30 min. (a) The
HPLC chromatogram of the transglycosylation of pNP ␤-G. Ctrl: The standard pNP ␤-G and pNP ␤-maltoside. p1, The peak of pNP ␤-G; peak 1-2, the peak of pNP ␤-maltoside;
peak 1-2, the peak of pNP ␤-isomaltoside. (b) The HPLC chromatogram of the transglycosylation of pNP ␤-X; p2, the peak of pNP ␤-X; p2-1, the peak of monoglucosylation
product of pNP ␤-X; p2-2 and p2-3, the peak of diglucosylation products of pNP ␤-X. (c) The HPLC chromatogram of the transglycosylation of pNP ␣-G. p3, The peak of
pNP ␣-G; p3-1 and p3-2, the peak of monoglucosylation product of pNP ␣-G; p3-3, the peak of diglucosylation products of pNP ␣-G. (d) The HPLC chromatogram of the
transglycosylation of pNP ␣-X. p4, The peak of pNP ␣-X; p4-1, the peak of monoglucosylation product of pNP ␣-X; p4-2 and p4-3, the peak of diglucosylation product of pNP
␣-X. (e) The mass spectrum (MS) chromatogram of the transglycosylation product pNP ␤-maltoside (p1-1), m/z = 462 [M−H]− and 498 [M+2H2O–H]− were the main ions
observed for pNP ␤-maltoside. (f) The MS chromatogram of the transglycosylation product pNP ␤-isomaltosides (p1-2), m/z = 462 [M−H]− and 498 [M+2H2O–H]− were the
main ions observed for pNP ␤-isomaltoside. (g) The MS chromatogram of the transglycosylation product pNP ␤-X-G (p2-1), m/z = 432 [M−H]− and 468 [M+2H2O–H]−were
the main ions observed for the transglycosylation product of pNP ␤-X.
acceptors (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol
and 2-methyl-2-propanol). TLC and MS analysis were applied to
determine the transglycosylation products. An extra band con-
sidered to be the corresponding transglycosylation product was
detected in TLC in the reactions containing alcohols except 2-
methyl-2-propanol (Fig. 4, lanes 1–6). The MS analysis revealed that
all the transglycosylation products formed the [M+Na]+ ions. More-
over, [M+K]+, [M+H2O+Na]+, [M+3H2O+H]+ ions were detected out.

54
K. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 48–57
Table 3
13C data (500 MHz, DMSO, 30 ◦C) and 1H data (125 MHz, DMSO, 30 ◦C) for pNP ␤-G
and its transfer product (chemical shifts [ppm], coupling constant [Hz]).
Carbon
pNP ␤-isomaltose
pNP ␤-G
C
H
C
H
H,C-aromatic
1
2,6
3,5
4
162.4 s
–
162.3 s
116.5 d
99.8 d
–
125.8 d
116.8 d
141.8 s
8.30
7.27
–
8.20
7.22
–
141.6 s
H,C-glucose
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
100.3 d
73.2 d
76.6 d
69.9 d
74.9 d
66.4 t
4.98 (d, J = 7.5 Hz)
3.43 m
3.30 m
3.16 m
3.66 m
99.8 d
73.0 d
77.2 d
69.4 d
76.4 d
60.5 t
5.08 (d, J = 7.3 Hz)
3.27 m
3.38 m
3.19 m
3.29 m
3.73 (d, J = 9.65 Hz)
3.61 m
3.70 m
3.48 m
1ꢀꢀ
2ꢀꢀ
3ꢀꢀ
4ꢀꢀ
5ꢀꢀ
6ꢀꢀ
98.1 d
71.8 d
73.0 d
70.1 d
72.6 d
60.7 t
4.69 (d, J = 3.6 Hz)
3.22 m
3.31 m
3.08 m
3.37 m
3.51 m
3.38 m
3.5. Characterization of the pNP ˇ-G transglycosylation products
Although both pNP ␤-G and pNP ␤-X were demonstrated to be
effective acceptors, compared to the only one product for pNP ␤-X,
two transglycosylation products were identified when using pNP ␤-
G as acceptor. Therefore, pNP ␤-G was chosen as the acceptor for the
structure analysis of transglycosylation products. In addition, cell-
free supernatant was used for preparation of the product because
the transglycosylation products catalyzed by either the cell-free
supernatant or the purified enzyme were similar (data not shown)
according to the LC–MS analysis. According to the transglycosyla-
tion analysis, two new peaks (designated as p1-1 and p1-2, Fig. 3a)
were detected and further identified as mono-glycosylated prod-
ucts by UFLC–MS (Fig. 3e and f). The peak of p1-1 was identified as
pNP ␤-maltoside evidenced by the m/z [M+2H2O–H]− 498 and the
retention time exhibited in the UV spectra when compared with the
authentic pNP ␤-maltoside. Due to the limited standard, product of
p1-2 was purified by HPLC and further identified by NMR (Table 3).
Compared with the substrate, 13C NMR spectrum of transformed
product exhibited additional carbon signals of ı60.7, ı72.6, ı70.1,
ı73.0, ı71.8 and ı98.1. This suggests the presence of an additional
sugar residue in the chemical molecule. The anomeric proton sig-
nal of this sugar (ı4.69, J = 3.6 Hz) was also observed. All the data
supported the involvement of the ␣-d-glucopyranosyl residue. In
the HMBC spectrum, the proton signal of ı 4.69 (H-1ꢀꢀ) exhibited
long-range correlation with the carbon signals of ı66.4, ı72.6 and
ı73.0, respectively, suggesting the substitution pattern of the gly-
cosyl moiety. Thus, this transformed product was identified as pNP
␤-D-isomaltoside.
Fig. 4. TLC analysis of the transglycosylation products catalyzed by the purified
enzyme. Enzyme reactions were performed as described in Section 2.8 with or with-
out various organic solvents. Reaction products produced with maltose and alcohols
(final concentration of 10%). Lane S, standards (methyl-glucoside, maltose); lane 1,
methanol; lane 2, ethanol; lane 3, 1-propanol; lane 4, 2-propanol; lane 5, 1-butanol;
lane 6, 2-methyl-2-propanol; lane 7, no alcohol. In all reactions containing alco-
hols, besides the bands of hydrolysis product and substrates, an additional band
was detected and was considered as transfer product (alkyl glycoside) because it
was not detected in the reaction without alcohol.
The molecular weight of ion [M+Na]+ is m/z = 217, 231, 245, 245 and
259, which are corresponding to methyl glucoside, ethyl glucoside,
propyl glucoside, isopropyl glucoside and butyl glucoside (Fig. 5).
3.4. Hydrolytic specificity
No transglycosylation activity was detected when UDPG served
as a sugar donor, which indicated that the enzyme involved in the
transglycosylation was not transferase but hydrolase. Therefore, we
further explored the hydrolysis ability of the enzyme. According
to the data in Table 2, the enzyme displayed a narrow hydrolytic
specificity towards ␣-1,4-linked homogeneous substrate. The opti-
mal hydrolytic substrate was maltotriose, followed by panose and
maltose. The enzyme displayed 12–15% of maltose activity when
gentibiose, ␤-CD and soluble starch were used as the substrates.
Other substrates listed in Table 2 showed only less than 4% of the
maltose activity. The hydrolytic substrate specificity of the enzyme
suggests the enzyme belongs to the type II glucosidase.
Since the purified enzyme exhibited hydrolysis activity as
described above, the data of the hydrolysis activity of the enzyme
solutions throughout the purification process was also provided in
Table 1. The hydrolysis activity/transglycosylation activity ratios
throughout purification steps are not constant, which is probably
due to the fact that the culture broth of DL001 was a mixture with
multiple-glucosidase.
4. Discussion
Glycosidases are increasingly becoming more attractable in the
biosynthesis due to their transglycosylation activities. Nearly 3000
glycosidases in 130 clans has been recorded in CAZy database
(http://www.cazy.org/Home.html) so far. However, only a few of
them were reported to possess the transglycosylation activity.
Moreover, among those glycosidases with transglycosylation activ-
ity, over 90% of them were failed to be utilized for the biosynthesis
due to their pretty low transglycosylation activity [20].
Shortage of the effective methods for product analysis has
become a major bottleneck that severely hinders the isolation

K. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 48–57
55
Fig. 5. The representative mass spectrum (MS) chromatogram of transglycosylation products of alkyl alcohols. The transglycosylation reactions were conducted in a 100 ␮l
reaction mixture containing 50 ␮l enzyme, 2.5 mM maltose and alkyl alcohol acceptors (10%, V/V) in a 50 mM citrate–phosphate buffer (pH 5.5). (a) MS chromatogram of
methyl glucoside, m/z = 217 [M+Na]+. (b) MS chromatogram of ethyl glucoside, m/z = 231 [M+Na]+. (c) MS chromatogram of isopropyl glucoside, m/z = 245 [M+Na]+. (d) MS
chromatography of butyl glucoside, m/z = 259 [M+Na]+.
of enzyme with transglycosylation activity and further clarifica-
tion of the related mechanism. The major problem for the current
transglycosylation analysis methodologies, including qualitatively
TLC and quantitatively HPAEC method, is the difficulty in dis-
criminating the products with only difference of linkage manners.
In addition, for some transglycosylation reactions, especially the
transglycosylation of monosaccharide using maltose as a donor,
the presence of excessive amounts of donor also interfered with
the detection and analysis of transglycosylation products (dis-
accharides) [21]. It has been demonstrated that the pyranosidic
structures possessing several hydroxyl groups often performed as
an most interesting acceptors for the transglycosylation reactions
catalyzed by glycosidases [22]. Therefore, in this research, a transg-
lycosylation activity-oriented method was utilized for glucosidase
discovery. Using the pNP-derivative of pyranose as an acceptor and
maltose as a donor, the glucosdiase-catalyzed transglycosylation
reaction was analyzed by qualitative UFLC–MS and quantitative
HPLC method. Specifically, the utilization of pNP-derivative of pyra-
nose as the acceptor could successfully avoid the interference
between the glycosyl donors and the transglycosylation prod-
ucts. Meanwhile, the transglycosylation products with the different
linkage manner could also be separated effectively. Using this
method, a strain with transglycosylation activity was successfully
isolated, and its enzyme was also evaluated in respect to substrate
specificity.
The ocean is an underdeveloped biosystem distinct from the
earth. A number of studies have indicated that the ocean envi-
ronment furnished different sources of glycosyl hydrolases [23].
Thus, further explorating these enzymes and their transglycosyla-
tion activity are essential for the rational exploitation of the ocean
resources. In the present study, the Yellow Sea of China is consid-
ered as a attractable resource for enzyme isolation. A strain with
transglycosylation activity was isolated and a new ␣-glucosidase
contributed to transglycosylation activity was purified through a
four-step process. Our results demonstrated that this enzyme was
type II glucosidase with narrow hydrolytic specificity toward ␣-
1,4-glucosidic bond. The hydrolytic activity decreased in an order
of maltotriose > panose > maltose, and only 3.61% maltose activity
occurred when pNP ␣-G served as the substrate. These results were
different from most of other ocean original ␣-glucosidase. For the
␣-glucosidase from Aplysia fasciata or strain Geobacillus, the Kcat/Km
for pNP ␣-G is 8 times or 57 times higher than that for maltose
[24,25].
The comprehensive study of the sugar donor and acceptor speci-
ficity of the purified ␣-glucosidase was essential for the related
mechanism study and its application. Our results showed that this

56
K. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 80 (2012) 48–57
enzyme can transfer glucosyl groups from the donors containing
␣-1,4-glucosidic bond to their acceptors. The best transglycosyla-
tion activity occurs when maltose served as a donor, followed by
maltotriose, panose, ␤-CD and starch. The transglycosylation activ-
ity decreased with the increasing length of the sugar chain. The
sugar acceptor specificity researches considered the glucosides and
xylosides as efficacious acceptors. From the comparison of struc-
tures of the substrates that could or could not act as acceptor,
we concluded that an effective acceptor should possess pyranose
structure with a similar configuration of the free C2-, C3-, and C4-
hydroxyl groups to d-glucopyranose, while the configuration of
C1-hydroxyl groups is less important (both pNP ␤-G and pNP ␣-G
could be transglycosylated). Due to the exclusively structural dif-
ference between glucoside and xyloside is a hydroxyl methyl group
on C-6 position, we speculate that C-6 position of the acceptor
has little correlation with the transglycosylation. Several investiga-
tors have reported the formation of oligosaccharides from maltose
catalyzed by ␣-glucosidase or other enzymes [24,26–28]. For exam-
ple, Malaˇı and colleagues compared the substrate specificity of
␣-glucosidase from Bacillus stearothermophilus and Brewer’s yeast
[29]. They found that when xylose, mannose, galactose and sorbose
were used as the acceptors, the ␣-glucosidase from B. stearother-
mophilus exhibited specific transglycosylation activity towards
xylose only, and the yield was about 11%. In contrast, ␣-glucosidase
from Brewer’s yeast, which had a relative wider hydrolytic speci-
ficity, exhibited transglycosylation activity toward all the test
acceptors with different yields varying from 7% to 15%. Based on
their substrate specificity, it was hypothesized that the enzyme
with high hydrolytic specificity usually has narrow acceptor speci-
ficity. Our results reported here are somewhat consistent with this
speculation.
product releasing, if a retaining mechanism is assumed for
our enzyme. Finally, the identification of diglycosylation prod-
ucts indicated that the glycosylated products could serve as or
become better sugar acceptors for the following glycosylation
reaction. Thus, the enzyme could be a robust tool for multi-
glycosylation.
Taken together, a new strain named Arthrobacter sp. DL001 with
high transglycosylation activity was isolated from the Yellow Sea
of China by using the transglycosylation activity-oriented method.
Enzymatic studies of the enzyme purified from the strain culture
indicated it had a narrow hydrolytic specificity and high specific for
sugar donor. The facts that the ␣-glucosidase could transfer 42–60%
pNP-monoglycosides to pNP-oligoglycosides and the glycosylated
products could serve as glycosyl acceptors for further glycosylation
indicate that the enzyme is of great biosynthesis potential. This
work provides not only an improved strategy for transglycosylation
study but also a robust tool for the biosynthesis of oligosaccharides
and alkyl glucosides.
Acknowledgments
We thank Mr. Xuran Fan for his revising work for language orga-
nizing and academic writing checking. This work was supported by
the National High Technology Research and Development Program
of China (863 Program) (No. 2009AA02Z205), the National Natu-
ral Science Foundation of China (No. 81102345) and the National
Key Technology Research and Development Program of China
(2009BADB9B02).
References
The specificity of the transglycosylation activity towards non-
sugar acceptors (alkyl alcohols) was also qualitatively analyzed by
TLC and MS. Our results supported that the enzyme was able to
transfer glucosyl groups from maltose to primary or secondary
alcohol with different length of the carbon chain (C1–C4). It has
been well documented that most of the glucosidases exhibit accept-
able transglycosylation activity towards alkyl alcohols only when
using a donor with an easy leaving group, such as pNP-G [20,30].
The fact that the enzyme could catalyze the transglycosylation
of alkyl alcohols using maltose as a donor suggests the exclusive
advantage of this enzyme during the biosynthesis of the alkyl glu-
coside.
In addition, the enzyme displayed ␣-1,4 (minor) or ␣-1,6 (major)
regioselectivity in transglycosylation and could convert 42–60% of
pNP-monoglycosides to pNP-oligoglycosides even at a relatively
low concentration of glycosyl donor (maltose, 2.5 mM) (Fig. 1b–d).
These transglycosylation yields are significantly higher than those
in the previous reports in which only 20–40% yields could be
achieved at a relatively high sugar donor concentration (>200 mM)
[25,29,31–33], and even comparable to some mutant glucosyn-
thases [1].
It can be concluded that the enzyme be able to transfer glucosyl
group from donors containing ␣-1,4-glucosidic bond specifically to
glucosides, xylosides and alkyl alcohols in ␣-1,4- or ␣-1,6-manners.
Combined with the identification of transglycosylation products,
the specificity study of sugar acceptors and donors provided some
hints to the mechanism of the transglycosylation reaction. Firstly,
the fact that the enzyme accepted substrates containing ␣-1,4-
glucosidic bond as sugar donors suggested that hydrolysis of an
␣-1,4-glucosidic linkage of the sugar donor might be the first
step of the transglycosylation reaction. The donor specificity was
in accordance with its hydrolytic specificity. Secondly, based on
the structure analysis of the products, ␣-d-glucopyranosyl moiety
was transferred to the acceptor. This indicated that sugar accep-
tors could compete with water molecules in the process of the
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