Enzymatic synthesis of α-anomer-selective D-glucosides using maltose phosphorylase

Article abstract of DOI:10.1271/bbb.70117

A maltose phosphorylase (EC 2.4.1.8; MPase) showed novel acceptor specificity and transferred the glucosyl moiety of maltose not only to sugars but also to various acceptors having alcoholic OH groups. Salicyl alcohol acted as acceptor for MPase from Enterococcus hirae, and the product, salicyl-O-α-D-glucopyranoside (α-SalGlc) was identified. The yield based on supplied salicyl alcohol was 86% (mol/mol).

Full text of DOI:10.1271/bbb.70117

Biosci. Biotechnol. Biochem., 71 (6), 1598–1600, 2007
Note
Enzymatic Synthesis of ꢀ-Anomer-Selective D-Glucosides
Using Maltose Phosphorylase
Kuniki KINO,y Yu SHIMIZU, Shoko KURATSU, and Kohtaro KIRIMURA
Department of Applied Chemistry, School of Science and Engineering, Waseda University,
Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, Japan
Received February 27, 2007; Accepted March 6, 2007; Online Publication, June 7, 2007
[doi:10.1271/bbb.70117]
A maltose phosphorylase (EC 2.4.1.8; MPase) showed
novel acceptor specificity and transferred the glucosyl
moiety of maltose not only to sugars but also to various
acceptors having alcoholic OH groups. Salicyl alcohol
acted as acceptor for MPase from Enterococcus hirae,
and the product, salicyl-O-ꢀ-D-glucopyranoside (ꢀ-
SalGlc) was identified. The yield based on supplied
salicyl alcohol was 86% (mol/mol).
(Kikkoman, Tokyo), recombinant MPase from Enter-
ococcus sp. (Sigma, St. Louis, MO), and purified MPase
from bacteria (Oriental Yeast, Tokyo) were investi-
gated. Acceptor compounds and the MPase (25 U/ml)
were added to 100 mM citrate-100 mM Na2HPO4 buffer
(pH 6.0, total volume 2.0 ml) containing 1.0 M maltose,
and the reaction mixture was incubated at 40 ꢀC with
shaking at 160 rpm for 24 h. TLC was used to detect
products using silica gel 60 plates (Merck, Boston, MA)
and a solvent system of ethylacetate–acetic acid–water
(3:1:1, v/v/v) for phenolic compounds, or 1-propanol–
water (17:3, v/v) for aliphatic alcohols. Spots were
made visible by spraying with methanol–H2SO4–water
(40:3:17, v/v/v), followed by heating at 160 ꢀC. As
shown in Table 1, the spots of glucosides were detected
using aliphatic alcohols, butandiol, cyclohexanol, benzyl
alcohol, and salicyl alcohol as sugar acceptor. These
results suggest that MPases can catalyze transglycosy-
lation to compounds having an alcoholic OH group,
though the acceptors were slightly different, depending
on the source of the organisms.
Salicyl alcohol was transglycosylated by recombinant
MPase from Enterococcus hirae under the following
conditions: 100 mM salicyl alcohol and MPase from
Enterococcus hirae (210 U/ml) were added to 2 ml of
100 mM citrate–100 mM Na2HPO4 buffer (pH 6.0) con-
taining 1.0 M maltose, and incubated at 40 ꢀC with
shaking at 160 rpm for 24 h. By TLC analysis, one pro-
duct corresponding to a salicyl glucoside was detected.
However, in the reaction mixtures without MPase or
phosphate, the product was not detected. Furthermore,
the product was detected using ꢁ-G1P (Sigma) instead
of maltose as sugar donor, but not using D-glucose.
Hence it is possible that the product was synthesized
from maltose via ꢁ-G1P.
Key words: maltose phosphorylase; ꢀ-glucoside; trans-
glycosylation
Many glycosides have biological activities, and the
acceptor compounds become more soluble and stable
with transglycosylation.1) Maltose phosphorylase (mal-
tose: orthophosphate 1-ꢁ-D-glucosyltransferase, EC
2.4.1.8; MPase) catalyzes the reversible phosphorolysis
of maltose to ꢁ-D-glucose-1-phosphate (ꢁ-G1P) and
D-glucose, with inorganic phosphate as cosubstrate.2)
MPase has been found in various bacterial cells,3–5)
and is thought to be involved in the metabolism of
extracellular sugars. The phosphorolysis reaction pro-
ceeds via a sequential bi bi mechanism,6,7) and in the
reverse reaction, MPase has rather broad acceptor spe-
cificity and transfers the glucosyl moiety of ꢁ-G1P to
various sugars.8,9) However, as the sugar acceptor, only
sugars have been deeply investigated. Among disac-
charide phosphorylases, only sucrose phosphorylase (EC
2.4.1.7, SPase) has been reported to show transfer ac-
tivity not only to sugars but to phenolic compounds.10,11)
The enzyme catalyzes a reversible phosphorolysis of su-
crose and inorganic phosphate to ꢀ-D-glucose-1-phos-
phate and D-fructose, and is different from MPase in
reaction mechanism.1) In this note, we report for the first
time that the some MPases catalyzed anomer-selective
transglycosylation of compounds having alcoholic OH
groups. Furthermore, the transglycosylation of salicyl
alcohol by an MPase from Enterococcus hirae was in-
vestigated in detail.
The product was purified for further study. It was
separated by solvent extraction of the reaction mixture
and then purified by a silica column packed with
Wakogel C-280 (Wako, Osaka). The purified product
was obtained as a white powder by lyophilization.
Recombinant MPase from Enterococcus hirae
y
To whom correspondence should be addressed. Fax: +81-3-3232-3889; E-mail: kkino@waseda.jp
Abbreviations: ꢀ-SalGlc, salicyl-O-ꢀ-D-glucopyranoside; ꢁ-G1P, ꢁ-D-glucose-1-phosphate; MPase, maltose phosphorylase

Transglycosylation by Maltose Phosphorylase
1599
Table 1. Synthesis of Various Glucosides by MPases
multiple bond coherence (HMBC, 600 MHz) spectra
were obtained. The data suggested that the product con-
sisted of salicyl alcohol and D-glucose (data not shown).
A doublet signal at 4.7 ppm showed the existence of
an ꢀ-anomeric proton of the glucosyl moiety, since this
signal had a smaller coupling constant (J ¼ 3:8 Hz) than
that for ꢁ-glucoside (J ¼ 7:9 Hz). Moreover, a sequence
of correlation at the C-l (64 ppm)/H-10 (4.7 ppm) po-
sition was clearly detected on the HMBC spectrum, in-
dicating that D-glucose was bonded to the C-l position of
salicyl alcohol. Based on these results, the product was
identified as salicyl-O-ꢀ-D-glucopyranoside (ꢀ-SalGlc).
Hence, as shown in Fig. 1, ꢁ-G1P was synthesized
through phosphorolysis of maltose, and then ꢀ-SalGlc
was synthesized from ꢁ-G1P and salicyl alcohol with the
second inverting of the anomer.
Figure 2 shows the time course of the synthesis of
ꢀ-SalGlc under the following conditions: the reaction
mixture (2 ml) containing 100 mM salicyl alcohol, 1.0 M
maltose, and MPase (210 U/ml) in 100 mM citrate–100
mM Na2HPO4 buffer (pH 6.0) was incubated at 40 ꢀC
with shaking at 160 rpm for 240 h. The amounts of
ꢀ-SalGlc and salicyl alcohol were measured by HPLC
LC-8020 with a differential refractmeter (Tosoh, Tokyo)
with a TSK-Gel ODS 80-TS column (4:6 ꢁ 250 mm,
Tosoh). Elution was done with methanol–water (3:7,
v/v) at a flow rate of l ml/min at 40 ꢀC. The amounts of
D-glucose and maltose were measured by HPLC with an
Asahipak NH2P-50 4E column (4:6 ꢁ 250 mm, Showa
Denko, Tokyo), and the elution was done with acetoni-
trile–water (7:3, v/v) at a flow rate of l ml/min at 40 ꢀC.
Under these conditions, only ꢀ-SalGlc was synthesized,
and byproducts such as maltotriose, maltotetraose, and
ꢁ-glucoside were not detected. Production of ꢀ-SalGlc
increased with a gradual decrease in maltose concen-
tration, and the total amount of ꢀ-SalGlc reached a
maximum (50 mg) at 240 h. Hence, the enzyme was
excellent in thermal stability, and 210 U/ml of highly
active MPase made possible a high yield based on
supplied salicyl alcohol, 86% (mol/mol).
Source
Acceptora
Enterococcus hirae Enterococcus sp. Bacteria
Methanol
Ethanol
ꢂ
ꢂ
+
+
+
ꢂ
+
+
+
+
+
+
+
+
+
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
+
1-Propanol
2-Propanol
1-Butanol
+
+
+
+
+
+
t-Butanol
n-Pentanol
n-Hexanol
ꢂ
+
+
+
+
+
n-Heptanol
1,2-Butanediol
1,3-Butanediol
1,4-Butanediol
Cyclohexanol
Benzyl alcohol
Salicyl alcohol
Catechol
+
+
N.T.b
N.T.
N.T.
N.T.
N.T.
+
N.T.
N.T.
N.T.
N.T.
N.T.
+
ꢂ
ꢂ
Resorcinol
Hydroquinone
ꢂ
ꢂ
ꢂ
ꢂ
aQuantities of acceptor compounds used: aliphatic alcohols, 9 M; butane-
diols, cyclohexanol, benzyl alcohol, 55 mM; phenol derivatives, 90 mM.
bN.T., not tested.
+, detected; ꢂ, not detected.
To analyze the structure of the product, ꢀ- and ꢁ-
glucosidase treatment and NMR analysis (Avance 600
spectrometer (Bruker, Rheinstetten, Germany) in
DMSO-d6 with tetramethylsilane as an internal stand-
ard) were done. The product (5 mg/ml) was hydrolyzed
by treatment with 5 mg/ml of ꢀ-glucosidase (from yeast,
Biozyme Laboratories, Blaenavon, UK), or 5 mg/ml of
ꢁ-glucosidase (from almond, Oriental Yeast, Tokyo) at
40 ꢀC for 24 h. By TLC analysis, salicyl alcohol and D-
glucose was liberated after treatment with ꢀ-glucosi-
dase, but not with ꢁ-glucosidase. Hence the product was
ꢀ-monoglucoside of salicyl alcohol. 13C-NMR (150
MHz), 1H-NMR (600 MHz), 1H–1H correlation spec-
troscopy (COSY, 600 MHz), 1H-detected multiple quan-
tum coherence (HMQC, 600 MHz), and hetero-nuclear
CH2OH
OH
CH2OH
OH
OH
OH
OH
Glucose
Pi
Salicyl alcohol
Pi
6'
CH2OH
OH
CH2OH
CH2OH
CH2OH
O
Pi
5'
HO
4
6
OH
4'
1'
OH
OH
OH
OH
OH
3
2'
1
C
O
5
OH
O
OH
3'
2
H2
OH
OH
OH
7
Maltose
β-Glucose-1-phosphate
Salicyl-O-α-glucopyranoside
Fig. 1. Synthesis of ꢀ-SalGlc by MPase.

1600
K. KINO et al.
9701. J. Biosci. Bioeng., 89, 138–144 (2000).
120
100
80
60
40
20
0
1.2
1.0
0.8
0.6
0.4
0.2
0
2) Kitaoka, M., and Hayashi, K., Carbohydrate-processing
phosphorolytic enzymes. Trends Glycosci. Glycotech-
nol., 14, 35–50 (2002).
3) Wood, B. J. B., and Rainbow, C., The maltose phos-
phorylase of beer lactobacilli. Biochem. J., 78, 204–209
(1961).
4) Kamogawa, A., Yokobayashi, K., and Fukui, T., Puri-
fication and properties of maltose phosphorylase from
Lactobacillus brevis. Agric. Biol. Chem., 37, 2813–2819
(1973).
5) Hiruma, M., Shirokane, T., and Suzuki, M., Purification
and some properties of maltose phosphorylase from
Enterococcus hirae IFO 3181. Nippon No¯geikagaku
Kaishi (in Japanese), 70, 773–780 (1996).
0
120
240
360
Time (h)
Fig. 2. Time Course of ꢀ-SalGlc Synthesis.
A reaction mixuture (2 ml) containing 100 mM salicyl alcohol,
1.0 M maltose, and MPase (210 U/ml) in 100 mM citrate–100 mM
Na2HPO4 buffer (pH 6.0) incubated at 40 ꢀC with shaking at 160
rpm for 240 h. Symbols: closed circle, ꢀ-SalGlc; closed diamond,
salicyl alcohol; open triangle, D-glucse; open square, maltose.
6) Tsumuraya, Y., Brewer, C. F., and Hehre, E. J.,
Substrate-induced activation of maltose phosphorylase:
interaction with the anomeric hydroxyl group of ꢀ-
maltose and ꢀ-D-glucose controls the enzyme’s gluco-
syltransferase activity. Arch. Biochem. Biophys., 281,
58–65 (1990).
7) Kitaoka, M., Studies of carbohydrate-processing phos-
phorolytic enzyme. J. Appl. Glycosci., 50, 83–87 (2003).
8) Selinger, Z., and Schramm, M., Enzymatic synthesis of
the maltose analogues, glucosyl glucosamine, glucosyl
N-acetylglucosamine and glucosyl 2-deoxyglucose by an
extract of Neisseria perflava. J. Biol. Chem., 236, 2183–
2185 (1961).
9) Aisaka, K., Masuda, T., and Chikamune, T., Properties
of maltose phosphorylase from Propionbacterium freu-
denreichii. J. Ferment. Bioeng., 82, 171–173 (1996).
10) Kitao, S., Ariga, T., Matsuda, T., and Sekine, H., The
syntheses of catechin-glucosides by transglycosylation
with Leuconostoc mesenteroides sucrose phosphorylase.
Biosci. Biotechnol. Biochem., 57, 2010–2015 (1993).
11) Kitao, S., and Sekine, H., ꢀ-D-Glucosyl transfer to
phenolic compounds by sucrose phosphorylase from
Leuconostoc mesenteroides and production of ꢀ-arbutin.
Biosci. Biotechnol. Biochem., 58, 38–42 (1994).
In this study, we found that MPase showed transfer
activity for compounds having an alcoholic OH group.
MPase catalyzed phosphorolysis of maltose to form ꢁ-
G1P, and then transferred the glucosyl moiety of ꢁ-G1P
to salicyl alcohol with a second inverting of the anomer.
The novel acceptor specificities of MPase should be
useful for in synthesis of important anomer-selective
glucosides.
References
1) Nakagawa, H., Dobashi, Y., Sato, T., Yoshida, K.,
Tsugane, T., Shimura, S., Kirimura, K., Kino, K., and
Usami, S., ꢀ-Anomer-selective glucosylation of menthol
with high yield through a crystal accumulation reaction
using lyophilized cells of Xanthomonas campestris WU-