Glucosylation Of Acetic Acid By Sucrose Phosphorylase.pdf

Bioscience, Biotechnology, and Biochemistry

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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

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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-OAcetyl--D-glucopyranose was isolated as the main product of the enzyme reaction. We also compared the pHdependence of transglycosylation catalyzed by sucrose phosphorylase toward carboxyl and hydroxyl groups. With hydroquinone as an acceptor molecule, the transfer ratio of glucose residue was higher at neutral pH. This pH-activity profile was similar to that of the phosphorolysis 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 essential 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 phosphate to
-D-glucose-1-phosphate (G-1-P) and D-fructose.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 transglucosylation 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

cosmetic ingredients. Glycosylation is known to be able to improve the characteristics of food materials, such as rutin (increasing 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) neopullulanase,13) cyclodextrin glucanotransferase,14) and sucrose 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. Furthermore, 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.

To whom correspondence should be addressed. Tel: +81-6-6477-8425; Fax: +81-6-6477-8362; E-mail: [email protected]

Transglycosylation to Acetic Acid by Sucrose Phosphorylase

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Recombinant SPase was further purified from the cell extract by chromatography with Q-sepharose (Amersham Bioscience, Uppsala, Sweden), Phenyl-Toyopearl 650 M (Tosoh, Tokyo, Japan) and Source 15Q (Amersham Bioscience).

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.

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.

Spectrometric analysis. Mass spectra were recorded on a JEOL JMS-700 (JEOL, Tokyo, Japan), and 1 HNMR (400 MHz) and 13 C-NMR (100 MHz) spectra were obtained using a JEOL JNM-A400 spectrometer (JEOL) in dimethyl sulfoxide-d6 .

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 containing 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 production 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) containing 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

High-performance liquid chromatography (HPLC). To analyze the glucosylation of acetic acid and hydroquinone, 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, Darmstadt, 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 UVmonitor at 280 nm. Thin-layer chromatography. To analyze the products obtained by the reaction of SPase, thin-layer chromatography (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)

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K. NOMURA et al. 1

Table 1.

Absorbance at 210 nm

A

No.

AC

0

10

20 30 Time (min)

40

Absorbance at 210 nm

B

AC

20.8 169.4 91.9 70.4 72.8 69.2 75.0 60.4

2.05 (3H, s) 5.92 3.32 3.42 3.15 3.44 3.44 3.56 5.09 4.95 5.02 4.51

20 30 Time (min)

(1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H, (1H,

d, 3.6) m ) m ) m ) m ) m ) m ) d, 6.4) d, 4.8) d, 5.6) t, 5.6)

2

Taken in DMSO-d6 at 400 MHz (1 H-NMR) and 100 MHz (13 C-NMR).  Signals overlapped.

3 2

10

1 2 10 20 30 40 50 60

HMBC (H to C)

H (Mult. J Hz)

C

20 -OH 30 -OH 40 -OH 60 -OH

1

0

H- and 13 C-NMR Spectral Data for Acetic Acid Glucoside

H OH

40

4’

6’ 5’

Fig. 1. Acetic Acid Glucoside Formation with Sucrose Phosphorylase. Acetic acid and sucrose were incubated with sucrose phosphorylase at 37
C for 16 h. Products were analyzed by HPLC. A, no enzyme; B, reaction mixture. AC, acetic acid 1, 1-O-acetyl-
-Dglucopyranose 2, reaction product 3, fructose.

H

O

HO HO 3’ H H

1’

2’

H

OH O 2

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

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

pH Initial

30 min

Transfer efficiency (%)

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5

3.1 3.5 4.0 4.6 5.4 6.2 6.8 7.2

4.6 7.5 12.5 20.0 22.4 13.6 4.9 1.9

Table 3. Effects of Various Concentrations of Acetic Acid on Transfer Efficiency Acetic acid concentration (M)

Transfer efficiency (%)

Amount of the transfer product (mg)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

94.8 93.4 91.2 86.6 17.4 4.2 1.6 — — —

5.7 11.2 16.4 20.8 5.2 1.5 0.7 — — —

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Table 4. Effects of Various Concentrations of Sucrose on Transfer Efficiency Sucrose concentration (%)

Transfer efficiency (%)

20 30 40 50 60

20.6 53.6 84.6 90.1 91.7

Relative activity (%)

Table 2. Effects of Various pH Levels on Transfer Efficiency toward Acetic Acid

120 100 80 60 40 20 0 3.5 4 4.5 5 5.5 6 6.5 7 pH

Fig. 3. Effect of pH on the Activity of SPase toward Phosphate , Acetic Acid, and Hydroquinone. Reaction mixtures (1.0 ml) at various pH levels (4.0, 5.0, 5.5, 6.0, and 6.5) containing sucrose (40%, w/v), SPase (60 units), and 0.2 M acetic acid ( ), or 0.1 M hydroquinone ( ) were incubated at 37
C for 30 min. The data for phosphate ( ) were obtained from a study by Fujii et al.16) The highest activity is denoted as 100%.

products (Table 2). As shown in Table 2, the optimum pH of the glucosylation of acetic acid was found to be approximately 5.0. The transglycosylation activity to acetic acid was estimated to be approximately 40 times lower than the phosphorolytic one, because 40 units of SPase was used to produce 1 mmol of acetic acid glucosides per min under the optimum condition. Table 3 shows the effect 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 transfer efficiency was markedly decreased at 0.5 M. The amount of the transfer product was maximized at 0.4 M of acetic acid. Glucosylation with 0.4 M of acetic acid was the optimum condition if we consider both the amount of glucoside produced and the glucosylation efficiency. Table 4 shows the effects of sucrose concentration. The transfer efficiency of acetic acid increased in parallel with an increase in the sucrose concentration. The efficiencies of glucosylation were more than 80% at sucrose concentrations, between 40% and 60% (w/v).

Table 5. Sensory Test for Sour Taste of Acetic Acid and Acetic Acid Glucosides

Effect of pH on the glucosylation of hydroquinone To determine the pH-dependence of the transglycosylating 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).

Discussion

Concentration (M)

Acetic acid

Acetic acid glucosides

1.0 0.1 0.01 0.001

+++ ++ + —

— (sweet and bitter) — (sweet and bitter) — —

+++ very strong, ++ strong, + weak, — not detected

Sensory test of acetic acid and acetic acid glucosides Since acetic acid has a strong sour taste, the tastes of acetic acid and acetic acid glucosides were compared. The sour taste of acetic acid was markedly reduced by glucosylation. The threshold value of the sour taste of acetic acid glucosides was more than 1.0 M, whereas that of acetic acid was 102 M. Thus the threshold value for the sour taste of acetic acid glucosides was approximately 100 times greater than that for acetic acid. While acetic acid glucosides were not very sour, they were slightly sweet and bitter (Table 5).

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-

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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-glucopyranose, 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
-Dglucopyranose spontaneously changed to 2-O-benzoyl
-D-glucopyranose and 2-O-benzoyl -D-glucopyranose in aqueous solution. Fujii et al. investigated the pHdependence 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 necessary 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 concentration 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 biological 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 investigation of the physicochemical and physiological properties of acetic acid glucosides is now in progress.

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