Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20

Glucosylation of Acetic Acid by Sucrose

Phosphorylase

Koji NOMURA, Kazuhisa SUGIMOTO, Hiromi NISHIURA, Kohji OHDAN,

Takahisa NISHIMURA, Hideo HAYASHI & Takashi KURIKI

To cite this article: Koji NOMURA, Kazuhisa SUGIMOTO, Hiromi NISHIURA, Kohji OHDAN,
Takahisa NISHIMURA, Hideo HAYASHI & Takashi KURIKI (2008) Glucosylation of Acetic Acid by
Sucrose Phosphorylase, Bioscience, Biotechnology, and Biochemistry, 72:1, 82-87, DOI: 10.1271/
bbb.70429

To link to this article: https://doi.org/10.1271/bbb.70429

Published online: 22 May 2014.

Submit your article to this journal

Article views: 237

Citing articles: 17 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tbbb20
Biosci. Biotechnol. Biochem., 72 (1), 82–87, 2008
Glucosylation of Acetic Acid by Sucrose Phosphorylase
Koji N OMURA,1; y Kazuhisa S UGIMOTO,1 Hiromi N ISHIURA,1 Kohji O HDAN,1
Takahisa N ISHIMURA,1 Hideo H AYASHI,2 and Takashi K URIKI1
1
Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku,
Osaka 555-8502, Japan
2
Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 590-8531, Japan
Received July 6, 2007; Accepted September 18, 2007; Online Publication, January 7, 2008
[doi:10.1271/bbb.70429]
Transglucosylation from sucrose to acetic acid by
sucrose phosphorylase (EC 2.4.1.7) was studied. 1-O-
Acetyl--D-glucopyranose was isolated as the main pro-
duct of the enzyme reaction. We also compared the pH-
dependence of transglycosylation catalyzed by sucrose
phosphorylase toward carboxyl and hydroxyl groups.
With hydroquinone as an acceptor molecule, the trans-
fer ratio of glucose residue was higher at neutral pH.
This pH-activity profile was similar to that of the phos-
phorolysis of sucrose by sucrose phosphorylase, but with
acetic acid as an acceptor molecule, the transfer ratio of
glucose residue was higher at low pH. These findings
suggest that the undissociated carboxyl group is essen-
tial to the acceptor molecule for the transglycosylation
reaction of sucrose phosphorylase. In a sensory test,
the sour taste of acetic acid was markedly reduced by
glucosylation. The threshold value of the sour taste of
acetic acid glucosides was approximately 100 times
greater than that of acetic acid.
Key words: sucrose phosphorylase; transglycosylation;
acetic acid; carboxyl group; dissociation
Sucrose phosphorylase (EC 2.4.1.7; SPase) catalyzes
the reversible conversion of sucrose and inorganic phos-
phate to
-D-glucose-1-phosphate (G-1-P) and D-fruc-
tose.1) SPase is used practically in enzymatic assay of
inorganic phosphate.
SPase transfers the glucosyl moiety of G-1-P and
sucrose to various acceptor molecules. In the trans-
glucosylation reaction of this enzyme, which shows
rather broad acceptor specificity, several
-glucosides
can be synthesized in a one-step reaction from sucrose
and various organic compounds.2–6)
Many kinds of biologically active compounds are
used in food and cosmetic materials, many of which
include a carboxyl group in their structures, but some of
these compounds have a strong smell, sourness, or low
solubility. Hence it is very important to improve these
characteristics to enhance their usefulness as food or
y
To whom correspondence should be addressed. Tel: +81-6-6477-8425; Fax: +81-6-6477-8362; E-mail: nomura-koji@glico.co.jp
cosmetic ingredients.
Glycosylation is known to be able to improve the
characteristics of food materials, such as rutin (increas-
ing solubility),7) neohesperidin (reducing bitterness),8)
and capsaicin (reducing pungency).9) There are many
reports on enzymatic glucosylation to aglycones with a
glycosyl residue, alcoholic OH groups, and phenolic OH
groups using
-glucosidase,10)
-amylase,11,12) neopul-
lulanase,13) cyclodextrin glucanotransferase,14) and su-
crose phosphorylase, but there has been no report on
glucosylation to carboxylic groups in various aglycones
using the transglycosylating reaction of carbohydrate
active enzymes. Recently, we reported that SPase
catalyzed the transfer of the glucosyl moiety of sucrose
to the carboxyl group of carboxylic compounds.15)
In this study, we successfully synthesized acetic acid
glucoside using the transglycosylation reaction of SPase
from Streptococcus mutans and studied the conditions of
the transglucosylation reaction.
Some properties of the transfer product, acetic acid
glucoside, are also described, with special focus on the
reduction of the sour taste by glucosylation. Further-
more, the difference in reactivity of SPase toward
carboxyl and hydroxyl groups was also examined using
acetic acid and hydroquinone as acceptor molecules.
Materials and Methods
Enzymes. Sucrose phosphorylase from S. mutans was
prepared by the method of Fujii et al.16) Escherichia coli
TG-1 (supE, hsd
5, thi,
(lac-proAB)/F0 [traD36,
proABþ , lac Iq , lacZ
M15]) carrying plasmid with the
SPase gene was grown at 37
C for 8 h in LB liquid
medium containing 50 mg/ml of ampicillin. Isopropyl--
D-thiogalactopyranoside was then added to a final
concentration of 0.4 mM, and the culture was grown at
27
C for further 18 h. The cells were harvested and
washed twice with 20 mM Tris–HCl (pH 7.0). Then they
were suspended in the same buffer, disrupted by
sonication, and centrifuged to remove cell debris.
 Transglycosylation to Acetic Acid by Sucrose Phosphorylase
Recombinant SPase was further purified from the cell
extract by chromatography with Q-sepharose (Amer-
sham Bioscience, Uppsala, Sweden), Phenyl-Toyopearl
650 M (Tosoh, Tokyo, Japan) and Source 15Q (Amer-
sham Bioscience).
Chemicals and reagents. Acetic acid, hydroquinone,
and sucrose were purchased from Wako Pure Chemical
Industries (Osaka, Japan). All other chemicals used were
commercially available and chemically of pure grade.
Assay of SPase activity. The activity of SPase was
measured basically as described by Silverstein et al.17)
SPase was assayed in a 50-ml reaction mixture contain-
ing 5% (wt/vol) sucrose, 100 mM sodium phosphate
buffer (pH 7.0), and enzyme. The reaction mixture was
incubated at 37
C for 20 min, and the reaction was
terminated by heating at 100
C for 5 min. The amount
of G-1-P produced was coupled to the reduction of NAD
in the presence of phosphoglucomutase and glucose
6-phosphate dehydrogenase. One unit of SPase was
defined as the amount of enzyme that caused a reduction
of 1 mmol of NAD per min under the assay conditions.
Effect of pH on the glucosylation of acetic acid. A
solution (1 ml) containing 0.2 M acetic acid and 40%
sucrose in distilled water was used, and its pH was
adjusted to 3.0–6.5 with NaOH. SPase (60 units) was
added to the solution, which was incubated at 37
C for
30 min.
Effect of the concentration of acetic acid on the
production of acetic acid glucoside. A solution (1 ml)
containing various concentrations (0.1–1.0 M) of acetic
acid and 40% sucrose in distilled water was used, and its
pH was adjusted to 3.5 with NaOH. SPase (5 units/1 mg
acetic acid) was added to the solution, which was
incubated at 37
C for 16 h.
Effect of the concentration of sucrose on the produc-
tion of acetic acid glucoside. A solution (10 ml)
containing 0.4 M acetic acid and various concentrations
of sucrose (20–60%) in distilled water was used, and its
pH was adjusted to 3.5 with NaOH. SPase (1,200 units)
was added to the solution, which was incubated at 37
C
for 16 h.
Effect of pH on the glucosylation of hydroquinone. A
reaction mixture (1 ml) of various pHs (4.0–6.5) con-
taining 40% sucrose, 0.1 M hydroquinone, and SPase (60
units) in 100 mM of glysine-NaCl-HCl buffer (pH 4.0) or
100 mM of MES-NaOH buffer (pH 5.0, 5.5, 6.0, 6.5) was
incubated at 37
C for 30 min.
Isolation of transfer product. A reaction mixture (10
ml, pH 3.5) containing sucrose (3 g), acetic acid (240
mg), and SPase (1,200 units) was incubated at 37
C for
16 h. Nine volumes of acetone were added to the
83
reaction mixture, which was then centrifuged (5;000 
g, 10 min) to remove saccharides. The supernatant was
chromatographed on a silica gel column (Wako) eluted
with 100% acetonitrile. The fractions containing the
transfer product were collected and lyophilized.
Spectrometric analysis. Mass spectra were recorded
on a JEOL JMS-700 (JEOL, Tokyo, Japan), and 1 H-
NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were
obtained using a JEOL JNM-A400 spectrometer (JEOL)
in dimethyl sulfoxide-d6 .
High-performance liquid chromatography (HPLC).
To analyze the glucosylation of acetic acid and hydro-
quinone, high- perfomance liquid chromatography
(HPLC) was performed. In the analysis of acetic acid,
HPLC was conducted under the following conditions:
column, TSK-GEL ODS-100 V (250  4:6 mm., Tosoh)
connected to a TSK-GEL G2500PW (300  7:5 mm.,
Tosoh); solvent, water (pH 2.2 with phosphoric acid);
flow rate, 0.5 ml/min; column temperature, 40
C;
detector, Shimadzu UV-monitor at 210 nm (Shimadzu,
Kyoto, Japan). In the analysis of hydroquinone, HPLC
was conducted under the following conditions: column,
LiChrosphere RP-18 (250  4:0 mm., Merck, Darm-
stadt, Germany); solvent, methanol-water (20:80, v/v;
pH 2.2 with phosphoric acid); flow rate, 0.5 ml/min;
column temperature, 40
C; detector, Shimadzu UV-
monitor at 280 nm.
Thin-layer chromatography. To analyze the products
obtained by the reaction of SPase, thin-layer chroma-
tography (TLC) was carried out by the ascending
method using silica gel (Merck) and a solvent system
of acetonitrile-water (85:15, v/v). In TLC analysis, spots
were visualized by spraying the TLC plate with H2 SO4 -
methanol (1:1, v/v), followed by heating at 130
C.
Sensory test of acetic acid and acetic acid glucosides.
The intensity of the sourness of acetic acid glucosides
was estimated by comparison with that of acetic acid by
a panel of a professional taster, as follows: Samples
were dissolved in distilled water and diluted stepwise in
tenths to a final concentration of 103 M. The tests,
which started from a 103 M solution, were continued
until a taster, could detect the sour taste of the sample.
The lowest detectable concentration was defined as the
threshold value.
Results
Transglucosylation reaction of SPase toward acetic
acid
SPase from S. mutans was incubated with sucrose as
donor molecule and acetic acid as acceptor molecule.
The reaction products in the mixture were analyzed by
HPLC and TLC. Some peaks other than acetic acid were
detected by HPLC (Fig. 1). One of them (Fig. 1, peak 3)
84 K. NOMURA et al.
1
Table 1. H- and 13 C-NMR Spectral Data for Acetic Acid Glucoside
A
HMBC
Absorbance at 210 nm
No. C H (Mult. J Hz)
AC (H to C)
1 20.8 2.05 (3H, s)
2 169.4
10 91.9 5.92 (1H, d, 3.6) 2
20 70.4 3.32 (1H, m )
30 72.8 3.42 (1H, m )
40 69.2 3.15 (1H, m )
0 10 20 30 40 50 75.0 3.44 (1H, m )
60 60.4 3.44 (1H, m )
Time (min) 3.56 (1H, m )
B 20 -OH 5.09 (1H, d, 6.4)
30 -OH 4.95 (1H, d, 4.8)
Absorbance at 210 nm

40 -OH 5.02 (1H, d, 5.6)

1
60 -OH 4.51 (1H, t, 5.6)
AC Taken in DMSO-d6 at 400 MHz (1 H-NMR) and 100 MHz (13 C-NMR).

Signals overlapped.
3 2

0 10 20 30 40 H OH

Time (min)
Fig. 1. Acetic Acid Glucoside Formation with Sucrose Phospho-
rylase.
Acetic acid and sucrose were incubated with sucrose phospho-
rylase at 37
C for 16 h. Products were analyzed by HPLC. A, no
enzyme; B, reaction mixture. AC, acetic acid 1, 1-O-acetyl-
-D-
glucopyranose 2, reaction product 3, fructose.
was identified to be fructose, and the others were
probably glucosyltransfer products. In TLC analysis,
some spots other than sucrose, fructose, and glucose
were detected as dark brown spots, visualized by
spraying with H2 SO4 -methanol reagent. These peaks
and spots were not detected without enzyme or sucrose.
These results suggest that the peaks and spots were
glucosyltransfer products produced by the enzyme
reaction.
Isolation and structure elucidation of transfer product
The main product produced from the reaction mixture
of SPase with sucrose and acetic acid was isolated by
column chromatography. The fractions containing the
transfer product were collected and lyophilized, and
about 20 mg of the transfer product was obtained as a
white powder. The FAB-mass of the isolated product
showed a molecular-related ion peak at m=z 223.0
(C8 H15 O7 ).
Eight signals were observed by 13 C-NMR analysis, as
shown in Table 1. One of them (C 20.8) was assigned to
the methyl carbon of acetic acid, and six (C 60.4-C
91.9) were assigned to glucose. One signal, at C 169.4,
was assigned to the carbonyl carbon. In the 1 H-NMR
data, a doublet signal at H 5.9, assignable to the
anomeric proton of glucose, was also observed. The
small coupling constant (J ¼ 3:6 Hz) of the anomeric
proton in the glucoside also suggests an
-configuration
for the anomeric center. Acetic acid was determined to
6’
4’ H
5’ O
HO
1’
HO 2’ H
3’ H OH
H O
2 1
CH3
O
Fig. 2. Structure of the Transglucosyl Product toward Acetic Acid by
SPase, 1-O-Acetyl-
-D-glucopyranose.
be bound to the C-10 position of
-D-glucose, since the
proton at H 5.9 that was assigned to H-10 in the H-H
COSY spectrum was correlated with the carbonyl
carbon (C 169.4), as observed in the HMBC spectrum.
Based on the results described above, the structure of
the transfer product was determined to be 1-O-acetyl-

-D-glucopyranose (Fig. 2).
Optimization of the glucosylation of acetic acid
The conditions of the transglycosylation reaction by
SPase toward acetic acid were examined in detail. The
transfer efficiency of the reaction was expressed as the
percentage of the peak areas of the transfer products
against the total peak areas of the transfer products and
acetic acid. We investigated the effect of pH on the
efficiency of the glucosylation of acetic acid by SPase.
We think that the use of a buffer solution was
inappropriate for adjusting the pH of the reaction
mixture, because most acidic buffer solutions are
composed of carboxylic compounds, which can be
glucosylated by the enzyme. The pHs of the reaction
mixture that reacted at initial pHs above 5.0 changed to
slightly higher with the production of the transfer
 Transglycosylation to Acetic Acid by Sucrose Phosphorylase 85
Table 2. Effects of Various pH Levels on Transfer Efficiency toward Table 4. Effects of Various Concentrations of Sucrose on Transfer
Acetic Acid Efficiency
pH
Sucrose concentration (%) Transfer efficiency (%)
Initial 30 min Transfer efficiency (%)
20 20.6
3.0 3.1 4.6 30 53.6
3.5 3.5 7.5 40 84.6
4.0 4.0 12.5 50 90.1
4.5 4.6 20.0 60 91.7
5.0 5.4 22.4
5.5 6.2 13.6
6.0 6.8 4.9
6.5 7.2 1.9 120

Relative activity (%)

100
80
Table 3. Effects of Various Concentrations of Acetic Acid on 60
Transfer Efficiency
40
Acetic acid Amount of the 20
Transfer efficiency (%)
concentration (M) transfer product (mg) 0
0.1 94.8 5.7 3.5 4 4.5 5 5.5 6 6.5 7
0.2 93.4 11.2
0.3 91.2 16.4
pH
0.4 86.6 20.8
Fig. 3. Effect of pH on the Activity of SPase toward Phosphate ,
0.5 17.4 5.2
Acetic Acid, and Hydroquinone.
0.6 4.2 1.5
Reaction mixtures (1.0 ml) at various pH levels (4.0, 5.0, 5.5, 6.0,
0.7 1.6 0.7
and 6.5) containing sucrose (40%, w/v), SPase (60 units), and 0.2 M
0.8 — —
acetic acid ( ), or 0.1 M hydroquinone ( ) were incubated at 37
C
0.9 — —
for 30 min. The data for phosphate ( ) were obtained from a study
1.0 — —
by Fujii et al.16) The highest activity is denoted as 100%.

products (Table 2). As shown in Table 2, the optimum Table 5. Sensory Test for Sour Taste of Acetic Acid and Acetic Acid
Glucosides
pH of the glucosylation of acetic acid was found to be
approximately 5.0. Concentration (M) Acetic acid Acetic acid glucosides
The transglycosylation activity to acetic acid was 1.0 +++ — (sweet and bitter)
estimated to be approximately 40 times lower than the 0.1 ++ — (sweet and bitter)
phosphorolytic one, because 40 units of SPase was used 0.01 + —
to produce 1 mmol of acetic acid glucosides per min 0.001 — —
under the optimum condition. Table 3 shows the effect +++ very strong, ++ strong, + weak, — not detected
of acetic acid concentration. The transfer efficiency was
very high (> 80%) in the reaction mixture containing
acetic acid at a concentration below 0.4 M, but the Sensory test of acetic acid and acetic acid glucosides
transfer efficiency was markedly decreased at 0.5 M. The Since acetic acid has a strong sour taste, the tastes of
amount of the transfer product was maximized at 0.4 M acetic acid and acetic acid glucosides were compared.
of acetic acid. Glucosylation with 0.4 M of acetic acid The sour taste of acetic acid was markedly reduced by
was the optimum condition if we consider both the glucosylation. The threshold value of the sour taste of
amount of glucoside produced and the glucosylation acetic acid glucosides was more than 1.0 M, whereas that
efficiency. Table 4 shows the effects of sucrose concen- of acetic acid was 102 M. Thus the threshold value for
tration. The transfer efficiency of acetic acid increased in the sour taste of acetic acid glucosides was approx-
parallel with an increase in the sucrose concentration. imately 100 times greater than that for acetic acid. While
The efficiencies of glucosylation were more than 80% at acetic acid glucosides were not very sour, they were
sucrose concentrations, between 40% and 60% (w/v). slightly sweet and bitter (Table 5).

Effect of pH on the glucosylation of hydroquinone Discussion

To determine the pH-dependence of the transglyco-
sylating activity of S. mutans SPase toward hydroxyl
groups, we examined the efficiency of glucosylation
using hydroquinone as an acceptor molecule. The
optimum pH of the glucosylation of hydroquinone was
found to be 6.5 (Fig. 3).
We have found that SPase can catalyze the transfer of
the glucosyl moiety of sucrose to a carboxyl group.15) In
the present study, we investigated the transglycosylation
reaction of SPase from S. mutans toward acetic acid in
detail. To determine the mechanism of the transglyco-
86 K. NOMURA et al.
sylation reaction of this SPase, we also examined the
difference in the pH-dependence of the reaction as
between acetic acid and hydroquinone, as model
carboxylic and phenolic compounds respectively. SPase
from S. mutans was incubated with sucrose and acetic
acid as donor and acceptor molecules respectively.
HPLC and TLC analyses indicated that SPase catalyzed
the transglycosylation reaction to acetic acid. We think
that SPase catalyzes not only the transglycosylation
reaction to acetic acid but also the hydrolysis of sucrose,
because glucose was detected in the TLC analysis. Two
peaks other than acetic acid and fructose were detected
in HPLC chromatograms of the reaction mixture. The
main peak was confirmed to be 1-O-acetyl-
-D-gluco-
pyranose, and another peak was perhaps internal acyl
migration products of 1-O-acetyl-
-D-glucopyranose,
since Sugimoto et al.15) reported that 1-O-benzoyl
-D-
glucopyranose spontaneously changed to 2-O-benzoyl

-D-glucopyranose and 2-O-benzoyl -D-glucopyranose
in aqueous solution. Fujii et al. investigated the pH-
dependence of the phosphorolytic activity of SPase from
S. mutans, and reported that the optimum pH for
phosphorolysis of sucrose by this SPase was 6.0.16)
The optimum pH and the pH-activity profile of the
transglycosylation activity of SPase toward acetic acid
we reported here were clearly different from those of the
phosphorolytic activity (Fig. 3). Perhaps this difference
was due to the difference in the acid dissociation state
between the carboxyl group of acetic acid and phosphate
to which the glucosyl moiety was transferred. Hence we
further investigated the efficiency of transglycosylation
to hydroquinone, a model phenolic compound in which
most of the hydroxyl groups were undissociated at
neutral and acidic pH levels. The pH-activity profile of
the glucosylation of hydroquinone was similar to that of
phosphorolysis (Fig. 3). These results suggest that the
undissociated-OH part of the acceptor molecule is
essential to the transglycosylation reaction of this SPase.
This is quite reasonable from the viewpoint of the
catalytic mechanism of the transglycosylation reaction
toward phosphate by SPase.18) It showed the proposed
reaction mechanism of SPase, which suggested that
SPase can react with HPO4 2 or H2 PO4  , but not with
PO4 3 , because protonation of the phosphate is neces-
sary for its binding to the catalytic domain of the
enzyme. The abundance ratios of the dissociated and
undissociated forms of acetic acid at each pH level was
calculated from the pKa value of 4.76. The concen-
tration of the undissociated form of acetic acid around
neutral pH was very low. Perhaps the glucosyltransfer
reaction of SPase was very slow around neutral pH,
since insufficient acetic acid was supplied in form
available to SPase. Furthermore, perhaps an increase in
the concentration of the available form of acetic acid
with a decrease in pH from 7.0 to 5.0 was effective in
increasing the rate of the transglycosylation reaction to
acetic acid. The rate of the reaction at pH below 5.0
decreased with decreasing pH, although sufficient
acceptor molecules were supplied. This decrease in the
rate of the reaction was perhaps highly influenced by a
decrease in the catalytic ability of the enzyme at lower
pH levels.
Glycosylation is considered a useful method to
improve the characteristics of compounds with bio-
logical activities. Acetic acid has various kinds of
biological activities,19–21) but at high concentrations,
solutions of acetic acid are difficult to drink because of a
strong sour taste. In this study, we tried to improve the
sourness of acetic acid by glucosylation, and we
remarkably reduced its sour taste by glucosylation of
its carboxyl group. The threshold value for the sour taste
of acetic acid glucosides was approximately 100 times
greater than that for acetic acid. This suggests that the
carboxyl group plays an important role in the sourness of
acetic acid. Hence, it might be possible to reduce the
sourness of many other carboxylic compounds by
glucosylation of their carboxyl groups. Further inves-
tigation of the physicochemical and physiological
properties of acetic acid glucosides is now in progress.
References
1) Mieyal, J. J., and Ables, R. H., ‘‘Enzymes’’ 3rd ed. Vol. 7,
ed. Boyer, P. D., Academic Press, New York, pp. 515–
532 (1972).
2) Kitao, S., Ariga, T., Matsudo, T., and Sekine, H., The
synthesis of catechin-glucosides by transglycosylation
with Leuconostoc mesenteroides sucrose phosphorylase.
Biosci. Biotechnol. Biochem., 57, 2010–2015 (1993).
3) 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).
4) Kitao, S., Matsudo, T., Sasaki, T., Koga, T., and
Kawamura, M., Enzymatic synthesis of stable, odorless,
and powdered furanone glucosides by sucrose phospho-
rylase. Biosci. Biotechnol. Biochem., 64, 134–141
(2000).
5) Kitao, S., and Sekine, H., Syntheses of two kojic acid
glucosides with sucrose phosphorylase from Leuconos-
toc mesenteroides. Biosci. Biotechnol. Biochem., 58,
419–420 (1994).
6) Kitao, S., and Sekine, H., Transglucosylation catalyzed
by sucrose phosphorylase from Leuconostoc mesenter-
oides and production of glucosyl-xylitol. Biosci. Bio-
technol. Biochem., 56, 2011–2014 (1992).
7) Suzuki, Y., and Suzuki, K., Enzymatic formation of 4G -

-D-glucopyranosyl-rutin. Agric. Biol. Chem., 55, 181–
187 (1991).
8) Kometani, T., Nishimura, T., Nakae, T., Takii, H., and
Okada, S., Synthesis of neohesperidin glycosides by
cyclodextrin glucanotransferase from an alkalophilic
Bacillus species. Biosci. Biotechnol. Biochem., 60,
645–649 (1996).
9) Kometani, T., Tanimoto, H., Nishimura, T., Kanbara, I.,
and Okada, S., Glucosylation of capsaicin by cell
suspension cultures of Coffea Arabica. Biosci. Biotech-
nol. Biochem., 57, 2192–2193 (1993).
 Transglycosylation to Acetic Acid by Sucrose Phosphorylase
10) Takenaka, F., and Uchiyama, H., Synthesis of
-D-
glucosylglycerol by
-glucosidase and some of its
characteristics. Biosci. Biotechnol. Biochem., 64, 1821–
1826 (2000).
11) Nishimura, T., Kometani, T., Takii, H., Terada, Y., and
Okada, S., Purification and some properties of
-
Amylase from Bacillus subtilis X-23 that glucosylates
phenolic compounds such as hydroquinone. J. Ferment.
Bioeng., 78, 31–36 (1994).
12) Nishimura, T., Kometani, T., Takii, H., Terada, Y., and
Okada, S., Accepter specificity in the glucosylation
reaction of Bacillus subtilis X-23
-Amylase towards
various phenolic compounds and the structure of
kojic acid glucoside. J. Ferment. Bioeng., 78, 37–41
(1994).
13) Takata, H., Kuriki, T., Okada, S., Takesada, Y., Iizuka,
M., Minamiura, N., and Imanaka, T., Action of neo-
pullulanase. J. Biol. Chem., 267, 18447–18452 (1992).
14) Nakano, H., Kiso, T., Okamoto, K., Tomita, T., Manan,
M. B., and Kitahata, S., Synthesis of glycosyl glycerol
by cyclodextrin glucanotransferases. J. Biosci. Bioeng.,
95, 583–588 (2003).
15) Sugimoto, K., Nomura, K., Nishiura, H., Nishimura, T.,
Hayashi, H., and Kuriki, T., Novel transglucosylating
reaction of sucrose phosphorylase to carboxylic com-
pounds such as benzoic acid. J. Biosci. Bioeng., 104, 22–
87
29 (2007).
16) Fujii, K., Iibosi, M., Yanase, M., Takaha, T., and Kuriki,
T., Enhancing the thermal stability of sucrose phospho-
rylase from Streptococcus mutans by random muta-
genesis. J. Appl. Glycosci., 53, 91–97 (2006).
17) Silverstein, R., Voet, J., Reed, D., and Abeles, R. H.,
Purification and mechanism of action of sucrose
phosphorylase. J. Biol. Chem., 242, 1338–1346 (1967).
18) Mirza, O., Skov, L. K., Sprogøe, D., van den Broek,
L. A., Beldman, G., Kastrup, J. S., and Gajhede, M.,
Structural rearrangements of sucrose phosphorylase from
Bifidobacterium adolescentis during sucrose conversion.
J. Biol. Chem., 281, 35576–35584 (2006).
19) Kishi, M., Fukaya, M., Tsukamoto, Y., Nagasawa, T.,
Takehana, K., and Nishizawa, N., Enhancing effect of
dietary vinegar on the intestinal absorption of calcium in
ovariectomized rats. Biosci. Biotechnol. Biochem., 63,
905–910 (1999).
20) Fushimi, T., Tayama, K., Fukaya, M., Kitakoshi, K.,
Nakai, N., Tsukamoto, Y., and Sato, Y., Acetic acid
feeding enhances glycogen repletion in liver and skeletal
muscle of rats. J. Nutr., 131, 1973–1977 (2001).
21) Kondo, S., Tayama, K., Tsukamoto, Y., Ikeda, K., and
Yamori, Y., Antihypertensive effects of acetic acid and
vinegar on spontaneously hypertensive rats. Biosci.
Biotechnol. Biochem., 65, 2690–2694 (2001).