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
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).
2ꢂ
SPase can react with HPO4 or H2PO4ꢂ, but not with
PO43ꢂ, 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
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).