Enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea

Article abstract of DOI:10.1016/j.carres.2009.04.019

Amylosucrase (ASase, EC 2.4.1.4) is a member of family 13 of the glycoside hydrolases that catalyze the synthesis of an α-(1→4)-linked glucan polymer from sucrose instead of an expensive activated sugar, such as ADP- or UDP-glucose. Transglycosylation rea

Full text of DOI:10.1016/j.carres.2009.04.019

Carbohydrate Research 344 (2009) 1612–1619
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Enzymatic synthesis of salicin glycosides through transglycosylation
catalyzed by amylosucrases from Deinococcus geothermalis
and Neisseria polysaccharea
Jong-Hyun Jung a, Dong-Ho Seo a, Suk-Jin Ha a, Myoung-Chong Song a, Jaeho Cha b, Sang-Ho Yoo c,
Tae-Jip Kim d, Nam-In Baek a, Moo-Yeol Baik a, Cheon-Seok Park a,
*
a Graduate School of Biotechnology, and Institute of Life Science and Resources, Kyung Hee University, Yongin 446-701, Republic of Korea
b Department of Microbiology, Pusan National University, Busan 609-735, Republic of Korea
c Department of Food Science and Technology, Sejong University, Seoul 143-747, Republic of Korea
d Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Amylosucrase (ASase, EC 2.4.1.4) is a member of family 13 of the glycoside hydrolases that catalyze the
Received 20 January 2009
Received in revised form 14 April 2009
Accepted 16 April 2009
synthesis of an
a-(1?4)-linked glucan polymer from sucrose instead of an expensive activated sugar,
such as ADP- or UDP-glucose. Transglycosylation reactions mediated by the ASases of Deinococcus geo-
thermalis (DGAS) and Neisseria polysaccharea (NPAS) were applied to the synthesis of salicin glycosides
with sucrose serving as the glucopyranosyl donor and salicin as the acceptor molecule. Two salicin gly-
coside transfer products were detected by TLC and HPLC analyses. The synthesis of salicin glycosides was
very efficient with NPAS with a yield of over 90%. In contrast, DGAS specifically synthesized only one sal-
Available online 22 April 2009
Keywords:
Amylosucrase
Deinococcus geothermalis
Glucosyl-salicin
Maltosyl-salicin
Salicin
icin transglycosylation product. The transglycosylation products were identified as
a-D-glucopyranosyl-
(1?4)-salicin (glucosyl salicin) and -glucopyranosyl-(1?4)- -glucopyranosyl-(1?4)-salicin
a
-D
a-D
(maltosyl salicin) by NMR analysis. The ratio between donor and acceptor had a significant effect on
the type of product that resulted from the transglycosylation reaction. With more acceptors present in
the reaction, more glucosyl salicin and less maltosyl salicin were synthesized.
Ó 2009 Elsevier Ltd. All rights reserved.
Transglycosylation
1. Introduction
Many enzymes that catalyze transglycosylation reactions have
been employed to synthesize oligosaccharides or glycoconjugate
Transglycosylation is a mechanism for glycosidic bond formation
in which a glycosyl molecule in a donor compound is transferred to a
hydroxyl group of an acceptor molecule.1 Recently, the essential
roles of many glycosyl-linked biomolecules (glycoconjugates) and
oligosaccharides in biological systems have been revealed. They
play important roles in host defense, cell–cell recognition, ligand–
receptor binding, and cell signaling.2–6 The therapeutic use of oligo-
saccharides or glycoconjugates as analogs of biologically important
molecules to eliminate or diminish the symptoms of various dis-
eases has been considered.6,7 Currently, chemical methods of glyco-
conjugate (or oligosaccharide) synthesis are often slow and
regiospecifically nonspecific. These syntheses also generally require
multi-step processes under harsh conditions. On the contrary, enzy-
matic biotransformation has been employed as an alternative meth-
od for manufacturing pharmaceuticals, fine chemicals, and food
ingredients, due to the highly selective catalytic reactions and the
use of environmentally friendly, mild reaction conditions.8–10
molecules.8,9 The resulting glycoside-transfer products exhibited
improved pharmacological and physicochemical properties such
as sweetness, water solubility, stability, and transportation
through membrane barriers or by body fluids, as well as having
better biological activities.11–14 For example, maltogenic amylase
from Bacillus stearothermophilus (BSMA) and Thermus sp. (ThMA)
has been used to synthesize various glycosyl-transfer products
such as naringin glycosides, glucosyl ascorbic acid, and puerarin
glycosides.15–17 Recently, a novel
with 10-fold enhanced selectivity, as compared to acarbose, to-
ward -glucosidase over -amylase activity was synthesized by
ThMA using acarviosine glucose as a donor and 3- -glucopyr-
anosylpropen (
GP) as an acceptor.11,15,16 This final product,
-acarviosinyl-(1?9)-3- -glucopyranosylpropen, was a potent
a-glucosidase-selective inhibitor
a
a
a
-D
a
a
a-D
hypoglycemic agent. Cyclodextrin glycosyltransferase (CGTase)
favorably catalyzes transglycosylation reactions, whereas many
other related
a-amylase family enzymes are hydrolases. Therefore,
the intermolecular transglycosylation activity of CGTase was suc-
cessfully accepted to produce
products.18,19
a variety of glycosyl-transfer
* Corresponding author. Tel.: +82 31 201 2631; fax: +82 31 204 8116.
E-mail address: cspark@khu.ac.kr (C.-S. Park).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.04.019

J.-H. Jung et al. / Carbohydrate Research 344 (2009) 1612–1619
1613
Amylosucrase (ASase, EC. 2.4.1.4) is a member of family 13 of the
glycoside hydrolases (GH13, -amylase family). Among the GH13
a
family, ASase has the remarkable enzymatic property of not only
having an unusual specificity for sucrose hydrolysis, but also
synthesizing an amylose-like polymer without using an expensive
activated sugar, such as ADP- or UDP-glucose as a donor mole-
cule.20–24 Until now, this interesting enzyme has only been reported
in bacteria of the Neisseria and Deinococcus genera, and has been
studied exclusively in Neisseria polysaccharea.20,25,26 ASase from N.
polysaccharea (NPAS) is secreted to produce the polysaccharide out-
side of cells, while ASase of other bacteria is found inside cells.20
Recently, a hypothetical protein from Deinococcus geothermalis
(DGAS), which is highly homologous to NPAS, was found, and the
corresponding gene was cloned and expressed in Escherichia coli.27
The recombinant DGAS synthesized an insoluble amylose polymer
accompanied by side reactions (sucrose hydrolysis, sucrose isomer,
and soluble maltooligosaccharide formation). Most interestingly,
DGAS has exceptionally thermostable characteristics, as demon-
strated by a half-life of 6.8 h at 55 °C, whereas the half-life of NPAS
is only 21 h at 30 °C. Although it is obvious that AS performs a trans-
glycosylation reaction when sucrose is used as the sole substrate,
the transglycosylation activity toward acceptor molecules other
than sucrose has not been investigated. In this study, the transgly-
cosylation activities of recombinant DGAS and NPAS were em-
ployed to synthesize salicin glycosides using sucrose as a glucose
donor and salicin as an acceptor. The high yield synthesis of two
salicin derivatives by ASases was also confirmed. Finally, the molec-
ular structures of salicin transfer products and optimal transglyco-
sylation reaction conditions of ASases are discussed.
Figure 1. Effect of temperature on the hydrolysis activities of rNPAS and rDGAS.
rNPAS is described by closed circles, while rDGAS is shown as open circles.
(Fig. 1). However, its activity was significantly reduced at temper-
atures over 50 °C. In rDGAS, the most favorable hydrolysis reaction
temperature was 45 °C, and its enzymatic activity was maintained
at more than 80% of its optimal activity in the range of 35–45 °C.
Furthermore, the hydrolysis activity of rDGAS was maintained at
about 60% of its maximum activity, even at 50 °C. The half-life of
rNPAS was less than 1 min at 50 °C, whereas that of rDGAS was
28.1 h at the same temperature, indicating that rDGAS is much
more thermostable than rNPAS.27 The optimal pHs for rNPAS and
rDGAS were pH 6 and pH 8, respectively. When the polymerization
activity of rDGAS was investigated by high-performance anion-ex-
change chromatography (HPAEC), more oligosaccharides were de-
tected at lower temperatures (25 °C and 30 °C) than at its optimal
hydrolysis temperature (45 °C).27 Since polymerization activity is
associated with transglycosylation activity, the transglycosylation
reaction of rDGAS was performed at 30 °C. Due to the heat-labile
properties of rNPAS, its polymerization activity could not be ana-
lyzed at higher temperatures (>40 °C). Previously, various syn-
thetic reactions of amylose-like polymers by rNPAS were
performed at 30 °C.28 Therefore, the transglycosylation reaction
of rNPAS was also performed at 30 °C.
2. Results and discussion
2.1. Expression and purification of rDGAS and rNPAS
The genes corresponding to DGAS and NPAS from D. geothermal-
is DSM 11300 and N. polysaccharea ATCC 43768 were amplified by
PCR. The absence of possible error in the nucleotide sequence of
the PCR-generated product was confirmed by DNA sequencing
analysis. The resulting PCR fragment corresponding to npas was in-
serted into the E. coli expression vector, pRSET-B vector, to gener-
ate pRSET-NPAS, whereas the fragment coding for dgas was added
into the pGEX-4T-1 vector, making pGEX-DGAS. Therefore, the fi-
nal recombinant NPAS (rNPAS) was tagged by six histidines, while
rDGAS was fused to the glutathione S-transferase (GST). An at-
tempt to use the pRSET-B vector to express rDGAS was not success-
ful. The recombinant E. coli cells transformed with either pRSET-
NPAS or pGEX-DGAS showed a blue-staining halo around the col-
ony when the cells were grown in sucrose-containing medium
and were stained with iodine vapor, indicating that an amylose-
like polymer was produced by these cells (data not shown). In
the pRSET-NPAS vector, there were an extra 33 amino acids at-
tached to the N-terminus of the matured NPAS since the expres-
sion cassette contained six-histidine tags for simple purification,
T7 gene 10 leader amino acids to provide protein stability, and
the XpressTM epitope for easy detection of the fusion protein. These
extra 33 amino acids did not eliminate or impair the enzymatic
activity of rNPAS. Similarly, the GST-fused DGAS showed apparent
ASase activity with its tagged GST protein on the N-terminus. Both
rDGAS and rNPAS were efficiently purified using a Ni-NTA affinity
column and a Glutathione–SepharoseTM High-Performance affinity
column, respectively, and then used for further experiments.
2.3. Production of salicin transfer product
Salicin is a naturally occurring plant glycoside found in the bark
of poplar and willow trees. This compound was used as an analge-
sic and antipyretic.29 It has a
D-glucopyranose unit attached by a b-
linkage to the phenolic hydroxyl group of salicyl alcohol. When sal-
icin is employed as an acceptor of transglycosylation reactions of
ASase, transglycosylation products can be easily detected using a
UV detector due to the hydroxy group on the phenyl group of
the compound. In this study, salicin transfer products were enzy-
matically synthesized by employing the transglycosylation activity
of rNPAS and rDGAS. TLC analysis of the salicin transglycosylation
reaction showed that at least one salicin transfer product was syn-
thesized by both enzymes (Fig. 2). However, HPLC analysis re-
vealed that there were two distinct salicin transfer products
(designated as compounds 1 and 2) in the rNPAS reaction, but only
one product (1) in the rDGAS reaction (Fig. 3).
The yield of transfer products was incredibly high, reaching
over 80% in the rNPAS reaction. The total yields of salicin transfer
products (1 and 2) from 0.5%, 1%, and 2% acceptor (salicin) with
4% donor (sucrose) were 99%, 99%, and 97%, respectively, implying
that all acceptors were glycosylated by rNPAS (Table 1). Even when
higher concentrations of acceptor (3% and 4%) were present in the
2.2. Comparison of enzymatic properties of rDGAS and rNPAS
rNPAS showed its highest sucrose hydrolysis activity at 35 °C,
and over 80% of its activity was retained between 30 and 40 °C

1614
J.-H. Jung et al. / Carbohydrate Research 344 (2009) 1612–1619
Figure 2. TLC analysis of salicin transglycosylation reactions of rNPAS and rDGAS. Lane M, G1(glucose)–G7(maltoheptaose) standard; lane Su, 0.05 M sucrose; lane Sa, 2%
(0.069 M) salicin; lanes 1–10 are the transglycosylation reaction mixtures with various concentrations of salicin as acceptors by rNPAS (lanes 1–5) and rDGAS (lanes 6–10).
Lanes 1 and 6, 0.5% (0.017 M) salicin; lanes 2 and 7, 1% (0.03 M) salicin; lanes 3 and 8, 2% (0.069 M) salicin; lanes 4 and 9, 3% (0.1 M) salicin; lanes 5 and 10, 4% (0.14 M) salicin.
All reactions contained 4% sucrose as a donor. The spots representing compounds 1 and 2 are designated by * and **, respectively.
reaction, the yields of transfer products were still close to 85%.
Interestingly, the portion of 1 increased as the concentration of
acceptor increased. The yields of the rDGAS reaction were a little
lower than those of the rNPAS reaction. The yield achieved with
rDGAS was over 70% when less than 1% of acceptor existed in the
reaction. However, the yield decreased to 43%, 38%, and 29% when
the amounts of acceptor in the reaction mixture increased to 2%,
3%, and 4%, respectively (Table 1). Interestingly, only a very small
quantity of 2, if any, was synthesized by rDGAS, as compared to
the rNPAS reaction.
Usually, the synthesis of glycosidic linkages in nature is carried
out by glycosyltransferases (EC 2.4) that use activated glycosides as
the glycosyl donors. Typical glycoside donors are expensive nucle-
otide sugars such as ADP-glucose, UDP-glucose, and UDP-galact-
ose.30,31 In contrast, ASase employs a considerably inexpensive
substrate (sucrose) as a glycoside donor molecule, leading to large
Figure 3. HPLC analysis of salicin transglycosylation reactions of rNPAS and rDGAS. (A) reaction with 4% (0.14 M) salicin; (B) reaction with 3% (0.1 M) salicin; (C) reaction
with 2% (0.069 M) salicin; (D) reaction with 1% (0.03 M) salicin; (E) transglycosylation reaction with 0.5% (0.017 M) salicin.

J.-H. Jung et al. / Carbohydrate Research 344 (2009) 1612–1619
1615
Total
Table 1
Yield of transglycosylation products by rNPAS and rDGASa
Salicin concentration (%)
Yield (%)
NPAS
DGAS
Compound 1
Compound 2
Total
Compound 1
Compound 2
0.5
1
2
3
4
15
20
46
62
65
84
79
50
22
18
99
99
97
86
84
79
68
43
37
29
5
3
ND
ND
ND
83
70
43
37
29
a
All reactions contained 4% sucrose as a donor.
industrial interest in employing this enzyme for biotechnological
synthesis of glycoconjugates or polysaccharides.21,32 Our results
showed that both rNPAS and rDGAS can efficiently utilize the en-
ergy generated from the cleavage of the glycosidic bond between
glucose and fructose in sucrose to synthesize other glycosidic link-
ages in transglycosylation products, such as salicin transfer
products.
Interestingly, the ratio of transfer products, 1 and 2, varied as
the concentration of acceptor molecule was changed in the rNPAS
reaction. At higher salicin concentrations (3% and 4%), 1 was the
predominant transfer product over 2, while at lower acceptor con-
centrations (0.5%, 1%, and 2%), significant quantities of 2 were gen-
erated. This result indicates that the transfer product could be
controlled by adjusting the concentration of the acceptor molecule
in the rNPAS transglycosylation reaction. In the rDGAS reaction, 1
was the predominant transglycosylation product with only insig-
nificant or minimal amounts of 2 detected, although the yield
achieved with rDGAS was lower than that of the rNPAS transglyco-
sylation reaction. Therefore, rDGAS could be employed in a specific
reaction to make a certain single transglycosylation product.
whether the hydrolysis reaction was a rate-limiting step in the
transglycosylation reaction. The result showed that the salicin
transglycosylation product was not increased at 45 °C compared
to 30 °C, implying that the hydrolysis reaction was not a rate-lim-
iting step to perform transglycosylation in rDGAS (Fig. 4). The ef-
fect of the concentration of donor molecule on the production of
salicin transglycosylation product was examined (Table 2). As the
ratio of donor concentration over acceptor concentration (D/A)
was increased, the yield of salicin transglycosylation product in-
creased. At a D/A of 0.5, the yield was only 23%, but was approxi-
mately 87% at a D/A of 12.
2.5. Purification and structural determination of salicin
transfer products
Two salicin transglycosylation products in the rNPAS reaction
were purified by a preparative recycling HPLC system. Both products
Table 2
The effect of donor/acceptor ratio (D/A) on the yield of salicin transglycosylation
products
2.4. Effect of temperature and the concentration of donor
molecules in the transglycosylation reaction by rDGAS
Donor concn (M)
Acceptor concn
3% (0.1 M)
D/A
Yield (%)
0.05
0.1
0.3
0.6
0.8
1.2
0.5
1
3
6
8
23
30
35
73
80
87
rDGAS was known to have a maximum hydrolysis activity at
45 °C, while its highest transglycosylation activity was observed
at lower temperatures (25–30 °C).27 Since quite a few donor mole-
cules of sucrose remained in the reaction mixture (Fig. 4), the sal-
icin transglycosylation reaction was performed at 45 °C to monitor
12
Figure 4. TLC analysis of salicin transglycosylation reaction of rDGAS performed at two different temperatures (30 °C and 45 °C). Lane M, G1(glucose)–G7(maltoheptaose)
standard; lane Su, 0.05 M sucrose; lane Sa, 2% (0.069 M) salicin; lanes 1–10 are the transglycosylation reaction mixtures with various concentrations of salicin as acceptors by
DGAS at 30 °C (lanes 1–5) and DGAS at 45 °C (lanes 6–10). Lanes 1 and 6, 0.5% (0.017 M) salicin; lanes 2 and 7, 1% (0.03 M) salicin; lanes 3 and 8, 2% (0.069 M) salicin; lanes 4
*
**
and 9, 3% (0.1 M) salicin; lanes 5 and 10, 4% (0.14 M) salicin. The spots representing compounds 1 and 2 are designated by and , respectively.

1616
J.-H. Jung et al. / Carbohydrate Research 344 (2009) 1612–1619
glucose molecule connected to salicin, whereas two glucose units
are attached to the salicin molecule in 2.
Analysis of 1H and 13C NMR data was used to determine the
type of glycosidic linkage in two compounds (1 and 2) between
the glycosyl (or two glucosyl groups) and D-glucopyranosyl group
of salicin. Chemical shifts of 1 in the 13C NMR spectra were com-
pared with those of salicin (Table 3). The position for the terminal
glucose linked to the salicin was determined through the glycosyl-
ation shift of the C-40 signal of the glucose moiety in salicin. Com-
pound 1 showed 19 carbon signals including those of a terminal
glucose molecule. The chemical shift of C-40 in the glucose unit
of salicin changed greatly from 71.34 to 80.85 ppm (+9.51 ppm)
in 1, confirming that the transferred glucosyl group was connected
to C-40 in the glucose unit of salicin. Similarly, the chemical shift of
C-40 in the glucose unit of salicin was principally altered from
71.34 to 80.86 ppm (+9.52 ppm) in 2. In addition, the chemical
shift of C-400 in the transferred glucosyl unit of 1 was distinctly
changed from 71.48 to 81.32 ppm (+9.84 ppm) in 2. In addition,
1H NMR analysis revealed that the glucosyl (or maltosyl) residue
was transferred to C-40 in the glucose unit of salicin to give the
Figure 5. TLC analysis of the purified salicin transfer products. Lane M, G1(glu-
cose)–G7(maltoheptaose) standard; lane Su, 0.05 M sucrose (0.5
0.069 M salicin (0.5 L); lane 1, transglycosylation reaction mixture prepared with
rNPAS; lane 2, purified compound 2; lane 3, purified compound 1.
lL); lane Sa,
l
were successfully isolated as single compounds, and con-
firmed as individual clear spots on TLC (Fig. 5). The molecular
masses of the purified salicin transfer products, 1 and 2, were
determined to be 448 Da and 610 Da, respectively, by matrix-as-
sisted laser-desorption/ionization-time-of-flight (MALDI-TOF)
mass spectrometric (MS) analysis. These molecular masses corre-
sponded to the calculated molecular masses of glucosyl salicin
and maltosyl salicin. When various signals of 1 and 2 in their 1H
and 13C nuclear magnetic resonance (NMR) spectra were compared
with those of salicin, seven of them (group 1 in Table 3) were as-
signed to the (hydroxymethyl)phenyl group, and six (group 2 in
Table 3) were assigned as glucose units of salicin. Compound 1
showed 19 carbon signals, including those of the salicin molecule,
and 2 had 25 signals containing those of salicin and 1. This result,
combined with MALDI-TOFMS analysis, implies that 1 contains a
a-anomeric configuration based on the coupling constant
(J = 4.0 Hz) of the glucose anomeric proton signal observed at
5.18 ppm. Two-dimensional HMBC spectra of 1 and 2 confirmed
the molecular structures of both compounds (data not shown).
The structure of 1was identified as
icin, in which the transferred
-glucose was attached to C-40 of the
glucose moiety of salicin by an -(1?4)-linkage. Likewise, 2 was
determined to be -glucopyranosyl-(1?4)- -glucopyranosyl-
a-D-glucopyranosyl-(1?4)-sal-
D
a
a-
D
a-D
(1?4)-salicin (Fig. 6). The transglycosylation product observed in
the rDGAS reaction was confirmed to be the same as 1. These re-
sults demonstrate that the type of glycosidic bond formed in the
transglycosylation products was exclusively the
a-(1?4)-glyco-
sidic linkage, which must be a specific characteristic of ASases
(rNPAS and rDGAS).
Table 3
13C and 1H NMR analyses of salicin transglycosylation products
Carbon atom
13C NMR
1
1H NMR
Salicin
2
Salicin
1
2
Group 1 salicyl alcohol
1
2
3
4
5
6
7
156.99
132.03
129.75
123.62
129.85
116.96
60.98
156.99
132.03
129.75
123.62
129.85
116.96
60.98
156.99
132.03
129.75
123.62
129.85
116.96
60.93
7.31 (dd, J = 7.6, 1.2 Hz)
7.00 (ddd, J = 7.6, 7.6, 1.2 Hz)
7.23 (ddd, J = 7.6, 7.6, 1.2 Hz)
7.18 (dd, J = 7.6, 1.2 Hz)
4.75 (d, J = 13.2 Hz)
7.31 (br d, J = 7.6, 1.2 Hz)
7.00 (ddd, J = 7.6, 7.6, 1.2 Hz)
7.21 (ddd, J = 7.6, 7.6, 1.2 Hz)
7.18 (br d, J = 7.6 Hz)
7.31 (br d, J = 7.6, 1.2 Hz)
7.00 (ddd, J = 7.6, 7.6, 1.2 Hz)
7.21 (ddd, J = 7.6, 7.6, 1.2 Hz)
7.18 (br d, J = 7.6 Hz)
4.75 (d, J = 13.2 Hz)
4.75 (d, J = 13.2 Hz)
4.55 (d, J = 13.2 Hz)
4.55 (d, J = 13.2 Hz)
4.55 (d, J = 13.2 Hz)
Group 2 Glc (I)
Group 3 Glc (II)
Group 4 Glc (III)
10
20
30
40
50
60
103.29
75.05
77.96
71.34
78.21
62.53
103.29
74.64
77.69
80.85
76.79
61.99
103.07
74.74
77.67
80.86
76.78
62.02
4.86 (d, J = 8.0 Hz)
4.88 (d, J = 8.0 Hz)
4.88 (d, J = 8.0 Hz)
3.48 (dd, J = 8.0, 7.6 Hz)
3.43 (dd, J = 8.0, 7.6 Hz)
3.38 (dd, J = 8.0, 8.0 Hz)
3.40 (ddd, J = 8.0, 5.2, 2.0 Hz)
3.87 (dd, J = 12.0, 2.0 Hz)
3.68 (dd, J = 12.0, 5.2 Hz)
3.54 (dd, J = 9.2, 8.0 Hz)
3.71 (dd, J = 9.2, 9.2 Hz)
3.62 (dd, J = 9.2, 9.2 Hz)
3.53 (ddd, J = 9.2, 4.4, 2.0 Hz)
3.87 (dd, J = 12.0, 2.0 Hz)
3.83 (dd, J = 12.0, 4.4 Hz)
3.54 (dd, J = 9.2, 8.0 Hz)
3.71 (dd, J = 9.2, 9.2 Hz)
3.62 (dd, J = 9.2, 9.2 Hz)
3.53 (ddd, J = 9.2, 4.4, 2.0 Hz)
3.87 (dd, J = 12.0, 2.0 Hz)
3.83 (dd, J = 12.0, 4.4 Hz)
100
200
300
400
500
600
102.85
74.14
75.05
71.48
74.79
62.53
102.85
73.75
74.90
81.32
73.35
62.13
5.18 (d, J = 4.0 Hz)
5.18 (d, J = 4.0 Hz)
3.43 (dd, J = 9.6, 4.0 Hz)
3.83 (dd, J = 9.2, 9.2 Hz)
3.51
3.43 (dd, J = 9.6, 4.0 Hz)
3.60 (dd, J = 9.6, 9.2 Hz)
3.25 (dd, J = 9.2, 9.2 Hz)
3.68 (dd, J = 9.2, 5.2, 2.0 Hz)
3.82 (dd, J = 12.0, 2.0 Hz)
3.63 (dd, J = 12.0, 5.2 Hz)
3.73
3.87 (dd, J = 12.0, 2.0 Hz)
3.83 (dd, J = 12.0, 4.4 Hz)
1000
2000
3000
4000
5000
6000
102.62
74.23
75.06
71.46
74.62
62.72
5.2 (d, J = 4.0 Hz)
3.43 (dd, J = 9.6, 4.0 Hz)
3.6 (dd, J = 9.6, 9.2 Hz)
3.25 (dd, J = 9.2, 9.2 Hz)
3.68 (ddd, J = 9.2, 5.2, 2.0 Hz)
3.82 (dd, J = 12.0, 2.0 Hz)
3.63 (dd, J = 12.0, 5.2 Hz)

J.-H. Jung et al. / Carbohydrate Research 344 (2009) 1612–1619
1617
OH
OH
O
HO
+
HO
O
OH
OH
O
OH
HO
OH
HO
O
OH
OH
HO
O
O
OH
HO
OH
OH
HO
O
O
O
OH
HO
O
OH
OH
+
O
HO
OH
HO
OH
O
O
OH
HO
OH
OH
O
O
HO
O
OH
Figure 6. Structures of salicin and transglycosylated salicin derivatives [a-D-glucopyranosyl-(1?4)-salicin and a-D-glucopyranosyl-(1?4)-a-D-glucopyranosyl-(1?4)-
salicin].
The transglycosylation activity of rNPAS has been employed to
modify glycogen and starch-related
-glucans.28,33 When rNPAS
geothermalis DSM 11300 was obtained from Dr. Michael Daly at
the Uniformed Services University of the Health Sciences. E. coli
DH10B [Fꢀ araD139
(ara leu)7697 lacX74 galU galK rpsL deoR
80lacZ M15 endA1 nupG recA1 mcrA (mrr hsdRMS mcrBC)]
a
was employed to glucosylate glycogen particles using sucrose as
the donor molecule, the initial sucrose/glycogen (donor/acceptor)
ratio strongly affected the morphology and structure of the result-
ing insoluble glucans. As this ratio was increased, the external gly-
cogen chains were extended by the enzyme, resulting in dendritic
nanoparticles with diameters 4–5 times those of the initial parti-
cles.28 Until now, the only report about small molecules as possible
acceptors of ASases was that of our previous research on monosac-
D
D
U
D
D
and BL21 [Fꢀ, ompT, hsdSB(rBꢀ, mBꢀ), dcm, gal, k(DE3)] used as
hosts for cloning and expression studies were grown in Luria–Ber-
tani (LB) medium containing 1% (w/v) bactotryptone, 0.5% (w/v)
yeast extract, and 0.5% (w/v) NaCl, supplemented with ampicillin
(100 lg/mL). pGEM T-easy vector (Promega, Madison, WI, USA),
pRSET-B vector (Invitrogen, Carlsbad, CA, USA), and pGEX-4T-1
vector (Amersham Biosciences, Buckinghamshire, UK) were used
for PCR-cloning and expression.
charides, such as galactose, xylose, methyl
and methyl b- -glucopyranoside as acceptors for the DGAS trans-
glycosylation reaction.27 In this study, we have shown for the first
time that a molecule other than a monosaccharide or -glucans
a-D-glucopyranoside,
D
a
3.2. Enzymes and chemicals
can be efficiently employed as an acceptor in the transglycosyla-
tion reaction of ASases.
Restriction endonucleases and modifying enzymes, such as T4
DNA ligase and Pfu DNA polymerase, were purchased from New Eng-
land Biolabs (Beverly, MA, USA), Solgent (Seoul, Korea), or Promega
(Madison, WI, USA). ThePCRproducts and DNArestrictionfragments
were purified using the QIAquick Gel Extraction kit (Qiagen, Valen-
3. Experimental
3.1. Bacterial strains and plasmids
cia, CA, USA). All chemicals used in this study, including
(2-(hydroxymethyl)phenyl- -glucopyranoside), were of reagent
grade and obtained from Sigma Chemical Co. (St Louis, MO, USA).
D
-(ꢀ)-salicin
N. polysaccharea (ATCC 43768) was purchased from ATCC
(American Type Culture Collection, Manassas, VA, USA), while D.
D

1618
J.-H. Jung et al. / Carbohydrate Research 344 (2009) 1612–1619
3.3. Construction of NPAS and DGAS expression plasmids
were determined using the Bradford reagents kit (Bio-Rad, Hercu-
les, CA, USA) with bovine serum albumin as the standard.
The gene corresponding to NPAS was amplified by PCR using
two primers designed based on the N. polysaccharea (ATCC
43768) ASase nucleotide sequence (Genbank accession number,
AJ011781). The two primers matched with the 50 and 30 ends of
the npas gene were NPAS1 (50-GGA TCC GAT GTT GAC CCC CAC
GCA GCA A-30) and NPAS2 (50-GGC AAG CTT CAG GCG ATT TCG
AGC CAC AT-30), containing BamHI and HindIII recognition sites
(underlined), respectively. The standard conditions for PCR were
as follows: one cycle of denaturation at 94 °C for 5 min, 35 cycles
of denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, exten-
sion at 72 °C for 2 min, and extra extension at 72 °C for 10 min. The
PCR product of the npas gene made with Pfu DNA polymerase was
cloned into the pGEM T-easy vector after an A-tailing reaction, and
the nucleotide sequence of the PCR-generated insert was then
determined by using the BigDye Terminator Cycle Sequencing Kit
for ABI377 PRISM (Perkin–Elmer Inc., Boston, MA, USA). The insert
was excised from pGEM T-easy vector using BamHI and HindIII and
ligated into pRSET-B vector treated with the same restriction en-
zymes to create pRSET-NPAS. The npas gene in the pRSET-NPAS
was controlled by a T7 promoter. Therefore, E. coli BL21 was trans-
formed with the resulting recombinant plasmids for efficient
expression. The amplification and cloning of the dgas gene were
described previously.27 The final expression vector in which the
DNA fragment corresponding to the dgas gene was inserted into
pGEX-4T-1 vector was designated as pGEX-DGAS.
3.5. Enzyme assay
The hydrolysis activities of the ASases were determined by the
dinitrosalicylic acid (DNS) method. The reaction mixture included
100 lL of 4% sucrose, 100 lL of deionized water, and 250 lL of
100 mM sodium citrate buffer (pH 6.0) for rNPAS and 100 mM
Tris–HCl buffer (pH 8.0) for rDGAS. After the substrate solution
was preincubated at 30 °C for 5 min, the reaction was initiated
by the addition of 50
ued for 10 min before being terminated by the addition of 500
lL of the enzyme solution, and then contin-
lL
of DNS solution, followed by inactivation by boiling. The absor-
bance of the reducing sugar at 545 nm was measured by a spectro-
photometer (Beckman DU 730, Fullerton, CA, USA). Reducing sugar
concentration was calculated using fructose as the standard. One
unit of AS was defined as the amount of enzyme that catalyzes
the production of 1 lmol fructose/min in the assay conditions.
3.6. Transglycosylation reactions of rNPAS and rDGAS
Transglycosylation reactions of rNPAS and rDGAS were per-
formed with 0.5 mL of substrate solution, containing 4% (0.11 M)
sucrose as
a substrate and 0.5% (0.017 M), 1% (0.03 M), 2%
(0.07 M), 3% (0.1 M), and 4% (0.14 M) salicin as the acceptor or con-
taining 0.05 M, 0.1 M, 0.3 M, 0.6 M, 0.8 M, and 1.2 M sucrose as a
substrate with 3% (0.1 M) salicin as an acceptor in 100 mM sodium
citrate buffer (pH 6.0) for rNPAS reactions and 100 mM Tris–HCl
buffer (pH 8.0) for rDGAS. The reactions were carried out at 30 °C
for 14 h with 60 U/mL of each enzyme, followed by heating in boil-
ing water for 30 min. The transglycosylated products in the reac-
tions were analyzed by TLC and HPLC. The transglycosylation
yield was defined as the ratio of synthesized salicin transglycosy-
lated products to the initial salicin added.
3.4. Purification of recombinant NPAS
Ni-NTA affinity column chromatography was used to purify re-
combinant His6-tagged NPAS. Recombinant E. coli BL21 cells har-
boring pRSET-NPAS were grown in 500 mL of LB containing
100
cells reached approximately 0.6, the cells were induced by adding
0.4 mM isopropyl b- -1-thiogalactopyranoside (IPTG) and grown
lg/mL ampicillin at 37 °C. When the optical density of the
D
for 3 h. The cells were harvested by centrifugation at 10,000 g
for 20 min at 4 °C. The cell pellet was thoroughly suspended in ly-
sis buffer [50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole
(pH 8)] and disrupted by sonication (Sonifier 450, Branson, Dan-
bury, CT, USA; output 4, 10 s ꢁ 6, constant duty) in an ice bath.
The cellular debris was spun down by centrifugation at 10,000
g for 30 min at 4 °C. The filtered supernatant was passed through
a Ni-NTA affinity column (Qiagen). Before eluting His6-tagged
NPAS, the Ni-NTA affinity column was washed with washing buf-
fer [50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole (pH
8.0)] and finally rNPAS protein was eluted with elution buffer
[50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole (pH
8.0)]. The eluted fraction was dialyzed to remove the excess
imidazole.
3.7. Analytical methods
The detection and identification of transglycosylation products
in the reaction were carried out with HPLC and TLC analyses. HPLC
analysis was performed as follows. The reaction mixtures were
boiled for 30 min to inactivate the enzyme and were centrifuged
at 10,000 g for 5 min. The supernatants were filtered using a
0.45-
l
m filter and analyzed with a SUPELCOTM LC-NH2 column
m) connected to a Shimadzu LC-10ADvp sys-
(25 cm ꢁ 4.6 mm, 5
l
tem with SPD-10A UV–vis detector at 265 nm. The mobile phase
(75:25 acetonitrile–deionized water) was delivered at a flow rate
of 1.00 mL/min. All solvents were filtered, degassed, and kept un-
der pressure. TLC analysis was performed with Whatman K5F silica
gel plates (Whatman, Kent, UK) after activating at 110 °C for
The expression of GST-tagged DGAS was performed as de-
scribed above. The purification steps of rDGAS were almost the
same as those of rNPAS, except for the lysis buffer used. The lysis
buffer used to resuspend the pellet for the GST-tagged DGAS was
phosphate-buffered saline (PBS) buffer (140 mM NaCl, 2.7 mM
KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH7.3). The superna-
tant was applied to a Glutathione–SepharoseTM High-Performance
affinity column (Amersham Biosciences) pre-equilibrated with
PBS buffer. The column was washed twice with PBS buffer. The
bound recombinant DGAS was eluted with a 50-mM Tris–HCl buf-
fer (pH 8.0) containing a 30 mM concentration of reduced glutathi-
one. The elimination of fused glutathione S-transferase protein
from the purified recombinant DGAS was carried out using throm-
bin treatment when necessary. The purity of rNPAS and rDGAS was
checked by SDS–PAGE using 10% (w/v) polyacrylamide gels and
Coomassie brilliant blue R-250 for staining. Protein concentrations
30 min. An aliquot (2 lL) of the reaction mixture was loaded onto
a plate and developed with a solvent system of 3:1:1 (v/v/v) n-
BuOH–HOAc–H2O in a TLC tank. Ascending development was re-
peated once at room temperature. The plate was allowed to air-
dry in a hood and then developed by rapidly soaking in 0.3% (w/
v) N-(1-naphthyl)ethylenediamine and 5% (v/v) H2SO4 in MeOH.
The plate was dried and placed in an oven for 10 min at 110 °C
to visualize the reaction spots.
3.8. Preparative recycling HPLC analysis
Each transfer product was fractionated and purified by a pre-
parative recycling HPLC system (LC-9104, JAI, Tokyo, Japan)
equipped with refractive index detection. Three milliliters of
the transglycosylation reaction mixture were loaded onto
a
JAIGEL-W252 column (2 ꢁ 50 cm), which was connected with

J.-H. Jung et al. / Carbohydrate Research 344 (2009) 1612–1619
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3.9. NMR analysis
Salicin and salicin transfer products were dissolved in 0.5 mL of
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Acknowledgments
This work was supported by the Korea Research Foundation
Grant funded by the Korean Government (MOEHRD, Basic Research
Promotion Fund) (KRF-2006-311-F00025). C. S. Park acknowledges
the support of both Kyung Hee University and the LG Foundation
during a sabbatical year at The University of Massachusetts at
Amherst.
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