Journal of Bioscience and Bioengineering VOL. 119 No. 6, 652e656, 2015 www.elsevier.com/locate/jbiosc

Production and application of a rare disaccharide using sucrose phosphorylase from Leuconostoc mesenteroides Kenji Morimoto,1, * Akihide Yoshihara,1 Toshio Furumoto,2 and Goro Takata1 Rare Sugar Research Center, Kagawa University, Miki-cho, Kagawa 761-0795, Japan1 and Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan2 Received 31 July 2014; accepted 12 November 2014 Available online 8 December 2014

Sucrose phosphorylase (SPase) from Leuconostoc mesenteroides exhibited activity towards eight ketohexoses, which behaved as D-glucosyl acceptors, and a-D-glucose-1-phosphate (G1P), which behaved as a donor. All eight of these ketohexoses were subsequently transformed into the corresponding D-glucosyl-ketohexoses. Of the eight ketohexoses evaluated in the current study, D-allulose behaved as the best substrate for SPase, and the resulting D-glucosyl-D-allu loside product was found to be a non-reducing sugar with a specific optical rotation of ½a20 D D 74.36 . D-Glucosyl-Dalluloside was identified as a-D-glucopyranosyl-(1/2)-b-D-allulofuranoside by NMR analysis. D-Glucosyl-D-alluloside exhibited an inhibitory activity towards an invertase from yeast with a Km value of 50 mM, where it behaved as a competitive inhibitor with a Ki value of 9.2 mM. D-Glucosyl-D-alluloside was also successfully produced from sucrose using SPase and D-tagatose 3-epimerase. This process also allowed for the production of G1P from sucrose and D-allulose from D-fructose, which suggested that this method could be used to prepare D-glucosyl-D-alluloside without the need for expensive reagents such as G1P and D-allulose. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Sucrose phosphorylase; Rare disaccharide; D-Glucosyl-D-alluloside; D-Tagatose 3-epimerase; Invertase inhibition]

Many different types of oligosaccharide have been reported in the literature to possess a broad range of physiological activities such as elicitors of defense reactions in higher plants of chitin-oligosaccharides and chitosan-oligosaccharides (1), and stimulation of calcium absorption in adolescent’s rat of fructooligosaccharide (2,3). Disaccharides are the smallest type of oligosaccharide, and a large number of these compounds, have also been reported to exhibit physiological activity. For instance, palatinose is expecting a material for manufacturing of low glycemic foods and beverages (4) and melibiose is useful for improving symptoms of atopic dermatitis (5). Although oligosaccharides are composed of natural monosaccharide units such as D-glucose, D-fructose, and D-galactose, which are produced abundantly in nature, these units rarely exist in the form of oligosaccharides. For this reason, most of the oligosaccharides used in human health products are produced in an industrial setting using enzymatic processes. Abundant monosaccharides do not exhibit any physiological activity towards humans, and are generally used to produce energy in cells. In contrast, the rare monosaccharide D-allulose (formally known as D-psicose) has been reported to exhibit a variety of interesting physiological activities in humans, including anti-diabetic (6,7), anti-obesity (8), and anti-arteriosclerosis (8) activities. Although D-allulose is rapidly excreted from the human body, it exhibits significant levels of activity. The International Rare Sugar Society (ISRS) defines rare sugars as monosaccharides and their derivatives that exist rarely in nature. Additionally, ISRS discussed

* Corresponding author. Tel./fax: þ81 87 891 3292. E-mail address: [email protected] (K. Morimoto).

the nomenclature of D-psicose (D-ribo-hex-2-ulose) in Rare Sugar Symposium 2014. The usage of D-allulose was recommended as the trivial name for this rare sugar instead of D-psicose (http://isrs. kagawa-u.ac.jp/image/AlluloseNAME-2.pdf). Based on the unique activity of D-allulose, it was envisaged that oligosaccharide units containing this rare sugar would possess interesting physiological activities. To the best of our knowledge, there have been no reports in the literature pertaining to the enzymatic synthesis of oligosaccharides containing rare sugars. To evaluate the validity of this approach, we initially investigated the enzymatic synthesis of oligosaccharides using rare ketohexoses as substrates. Sucrose phosphorylase (SPase, EC 2.4.1.7) was selected as the best enzyme for this particular transformation because some of them have been used previously for the synthesis of unique disaccharides (9e12). Aerts et al. (13) has been reported transglucosylation activity of six sucrose phosphorylases toward different classes of acceptors including rare ketohexoses, D-tagatose, D-allulose. SPase was first reported in 1942 when it was isolated from Leuconostoc mesenteroides ATCC12291 (14), and this enzyme can now be purchased commercially. We recently reported the use of microbial enzymes and whole cell systems for the successful production of six rare ketohexoses, including D-allulose, D-tagatose, D-sorbose, L-allulose, L-tagatose, and L-fructose (15). D-Tagatose-3-epimerase from Pseudomonas cichorii ST-24 was used as a key enzyme in the production of these ketohexoses (16). Given that D-allulose can be readily produced from the inexpensive starting material D-fructose, this process could be used to produce a broad range of rare hexoses. In our current study, we have investigated the synthesis of disaccharides containing one rare ketohexose using SPase and G1P,

1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.11.011

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which is very expensive (Fig. 1A). Eight disaccharides were synthesized in this way, with D-glucosyl-D-alluloside showing the highest productivity. This disaccharide was subsequently purified and evaluated in terms of its functional behavior. With regard to mass production of this material, we also developed a procedure involving the use sucrose (D-glucopyranosyl-(1 / 2)-b-D-fructofuranoside) as a single substrate with SPase and D-tagatose-3epimerase. Notably, this production method did not require the use of G1P. MATERIALS AND METHODS Chemicals, bacterial strain, plasmid, medium, and culture condition SPase from L. mesenteroides and invertase from Saccharomyces cerevisiae were purchased from Wako Pure Chemical Industries Co., Ltd. (Osaka, Japan) and SigmaeAldrich (St. Louis, MO, USA), respectively. The D-tagatose 3-epimerase (DTE) gene of P. cichorii ST-24 was inserted into pTrc99A, and the resulting product was named as pIK-01. This plasmid was introduced into Escherichia coli JM 105 (17), and the resulting recombinant E. coli was cultured in a medium composed of 3.5% polypeptone, 2.0% yeast extract, 0.5% NaCl, and 100 mg/ml ampicillin at 30 C for 18 h. The recombinant enzyme was subsequently induced by the addition of 0.5 mM of IPTG and 1 mM of MnCl2 to the culture medium. The resulting mixture was incubated at 30 C for 5 h period, and the cells were then collected by continuous centrifugation (8000 g, 4 C). Immobilized DTE (16 U) was prepared by the method reported by Ishida et al. (17). One unit of DTE can produce 1.0 mmol of D-sorbose from D-tagatose per min in phosphate buffer (pH 7.5) at 30 C. D-Allulose, D-tagatose, D-sorbose, L-fructose, L-tagatose, and L-psicose were prepared in our laboratory (18e23). All of the reagents used in the buffers and for the determination of the enzyme activity were purchased from Wako Pure Chemical Industries Co., Ltd. unless stated otherwise. Analytical methods The reducing sugar was determined by the SomogyiNelson method (24,25). Nuclear magnetic resonance (NMR) spectra were recorded on a JNM-ECA600 spectrometer (JEOL, Tokyo, Japan) at 600 and 150 MHz for the 1 H and 13C NMR, respectively. All of the samples (20 mg) for NMR analysis were dissolved in 1.0 mL of D2O and subsequently lyophilized before being dissolved in 0.6 mL of D2O. All of the chemical shifts have been expressed relative to the signal corresponding to the methyl group of sodium 3-(trimethylsilyl)[2,2,3,3-2H4] propionate (TSP-d4), which was set to 0 ppm. Optical rotation data were measured on a JASCO P-1030 optical rotation polarimeter (JASCO, Tokyo, Japan) using ultrapure water. The reaction mixtures were assayed by High-performance liquid-chromatography (HPLC) using a the Hitachi HPLC system (Hitachi, Tokyo,

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Japan) equipped with an L-7490 refractive index detector, D-2500 Chromato Integrator and GL-C611 column (I.D. 10.7  300 mm). HPLC analyses were carried out at 60 C using a 104 M solution of NaOH as the mobile phase at a flow rate of 1.0 ml/min. Synthesis and purification of disaccharides Solutions of the ketohexoses (10% w/v) and G1P (10% w/v) were prepared in 25 mM MES buffer (pH 6.5). SPase (7.8 U per 200 ml) was added to each mixture following a pre-incubation period of 10 min at 30 C, and the resulting mixtures were incubated at 30 C for 48 h, unless otherwise stated. The SPase unit was calculated from supplier’s unit in this study. The reaction was stopped by boiling for 5 min. The reaction mixture was then cooled to room temperature and deionized by ion exchange chromatography over a Dianion SK1B Hþ resin (Mitsubishi Chemical Co., Ltd, Tokyo, Japan) followed by an Amberlite IRA411 CO3 2 resin (Dow Chemical Co., Ltd Midland, MI, USA). The deionized mixture was subsequently purified by ligand exchange chromatography using a column (I.D. 1  100 cm) packed with DOWEX 502Ca2þ resin (Dow Chemical Co., Ltd.). The column was eluted with deionized water at a flow rate of 1 ml/min. The fraction containing the disaccharide were concentrated by lyophilization or simple evaporation to give the desired products. For the production of D-glucosyl-D-allulose from sucrose, SPase (2 U) and immobilized DTE (16 U) were added to a 40% (w/w) solution of sucrose in 50 mM sodium phosphate (pH 7.5), and the resulting mixture was reacted at 30 C for 48 h. Kinetics analysis of yeast invertase inhibition assay The enzyme, substrate, and inhibitor were dissolved in 100 mM acetate buffer (pH 5.0). A 1 M solution of sucrose (20 ml) in pure water was mixed with 100 ml of the same acetate buffer, together with solutions containing various concentrations of the inhibitor (40 ml). The reaction was started by the addition 40 ml of the yeast invertase solution (12.5 mg/ml, Grade VII >300 U/mg) to the reaction mixture. The reaction mixture was then incubated for 10 min at 30 C before being stopped by the addition of 100 ml of a 0.2 M Na2CO3 solution following by the boiling of the resulting mixture for 5 min. The concentration of released D-glucose was measured using a Glucose CII Test WAKO (Wako Pure Chemical Industries Co., Ltd.). Water (40 ml) was added instead of the inhibitor solution as a control experiment.

RESULTS Production of oligosaccharides from sucrose and each ketohexose by SPase We investigated the effect of the SPase reaction on the structures of eight ketohexoses and evaluated the specific activity of SPase towards the ketohexoses using HPLC. The results of these experiments are summarized in Table 1. The GLC611 column used in the current study was specifically selected

FIG. 1. (A) Outline of the production of D-glucosyl-D-allulose using the SPase and D-tagatose 3-epimerase (DTE) coupling reaction. (B) HPLC profile. Each number represents a retention time (min): 8.2, ions; 13.2, sucrose; 14.4, D-glucosyl-D-allulose; 15.4, D-glucose; 19.5, D-fructose; and 29.5, D-allulose.

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TABLE 1. Substrate specificity of rare disaccharide and retention times by HPLC. Substrate

Area of HPLC (%)

Productivitya (%)

Retention time of HPLC (min)

D-Allulose

17.8 17.0 8.60 6.82 3.21 1.35 0.22 0.22

105 100 50.6 40.0 18.9 7.9 1.3 1.3

14.2 13.2 14.3 17.7 13.6 14.7 13.0 13.1

D-Fructose L-Sorbose L-Tagatose L-Fructose D-Sorbose L-Allulose D-Tagatose

a Productivity obtained compared with that of the control (D-fructose), which was taken as 100%.

for its ability to retain disaccharides such as sucrose, maltose, and cellobiose with retention times in the range of 13e15 min (data not shown). With the exception of D-glucosyl-L-tagatose, all of the rare disaccharides evaluated in the current study gave retention times in the range of 13e15 min, which effectively confirmed that these peaks belonged to disaccharides. Furthermore, sugar alcohols of disaccharide such as maltitol and lactitol gave retention times of 17.5 and 18.4 min, respectively (data not shown). Taken together, these results suggested that the peak at 17.7 min belonged to D-glucosyl-L-tagatose, although further work would be required to allow for the identification of this compound. SPase from L. mesenteroides can be used to synthesize disaccharides using G1P as a glucosyl donor and a rare ketohexose as an acceptor. These results suggested that the SPase enzyme possessed extremely wide substrate specificity. SPase exhibited a much higher level of productivity toward D-allulose and D-fructose than any of the other ketohexoses tested in the current study. SPase also exhibited weak productivity towards L-sorbose, L-tagatose, L-fructose, and D-sorbose, and showed very little activity towards D-tagatose and L-allulose. One-pot production of D-glucosyl-D-allulose using sucrose as a substrate To establish an inexpensive procedure for the production of D-glucosyl-D-allulose, we investigated the possibility of developing a reaction system that did not require the use of G1P or D-allulose. SPase can phosphorolyze sucrose to G1P and D-fructose, and DTE can be used to convert D-fructose to D-allulose through an epimerization process, as shown in Fig. 1A (16). When 40% (w/v) sucrose was used as a substrate, SPase and DTE were both found to be active in the same sodium phosphate buffer (pH 7.5), and provided D-glucosyl-D-allulose (14.4 min) as shown in Fig. 1B. This result suggested that SPase had converted the sucrose substrate into G1P and D-fructose (19.5 min), and that DTE had converted

the D-fructose into D-allulose (29.5 min). The SPase would also be involved in the simultaneous synthesis of D-glucosyl-D-allulose. Purification and structural determination of D-glucosyl-Dallulose D-Glucosyl-D-allulose exhibited the highest productivity of all of the ketohexoses tested in the current study, and this material was isolated with a yield of approximately 2% following purification. Pure D-glucosyl-D-allulose was analyzed by 1D and 2D NMR spectroscopy. As shown in Table 2, the 13C NMR spectrum of Dglucosyl-D-allulose contained 12 signals. The chemical shifts of the six carbon signals of the D-glucose moiety in D-glucosyl-D-allulose almost coincided with those of the glucose moiety in sucrose. In contrast, the chemical shifts of the remaining six carbon signals of the D-allulose moiety in D-glucosyl-D-allulose did not coincide with those of the D-fructose moiety in sucrose. In particular, the chemical shifts of the C2, C3, and C4 carbons were very different between D-glucosyl-D-allulose and sucrose. Valentine et al. (26) and Katayama et al. (27) reported that the 13 C NMR spectrum of D-allulose, which suggested that D-allulose existed as a mixture of four tautomers (i.e., a-allulofuranose, ballulofuranose, a-allulopyranose, and b-allulopyranose). The signals belonging to the D-allulose moiety of D-glucosyl-D-allulose, however, gave only six peaks, which suggested that it was not possible for the D-allulose moiety to tautomerize. The chemical shifts of D-allulose moiety of D-glucosyl-D-allulose was very similar with those of b-fructofuranose moiety of sucrose. Furthermore, when we measured the reducibility of this disaccharide using the Somogyi-Nelson method, it did not exhibit a reducing power in the same way as sucrose. These results indicated that D-glucosyl-D-allulose was a-D-glucopyranosyl(1/2)-b-D-allulofuranoside, as shown in Fig. 2, with the only significant difference between the structure of this disaccharide and sucrose being the stereochemistry of the C3eOH group in the D-fructose moiety of sucrose. The optical rotation of D-glucosyl-D alluloside was determined to be ½a20 D þ 74.36 (c 1, H2O), and the value for sucrose was þ66.78 . Inhibition of D-glucosyl-D-alluloside on invertase D-Glucosyl-D-alluloside was not digested by the invertase from S. cerevisiae. In fact, this invertase was inhibited by D-glucosyl-Dalluloside because the structure of this disaccharide is very similar to that of sucrose. D-Glucosyl-D-alluloside therefore functioned as a substrate analog towards the invertase enzyme. The activity of the invertase enzyme was inhibited by no less than 15.3% by D-glucosyl-D-alluloside at a concentration of 2.5 mM. Furthermore, the activity of the invertase enzyme was inhibited by 50% by 10 mM Dglucosyl-D-alluloside, whereas the same concentration of D-allulose

TABLE 2. NMR data for D-glucosyl-D-allulose and sucrose. Position

Sucrose

dC

dH

dH (nH, mult. J in Hz)

D-Glucose

95.0 73.9 75.4 72.1 75.2 63.0

5.42 3.56 3.76 3.47 3.85 3.82

(1H, d, 3.8) (1H, dd, 9.8, 3.8) (1H, dd, 9.8, 9.5) (1H, dd, 9.6, 9.5) (1H, m) a (2H)

1

64.2

3.68 (2H, s)

2 3 4 5 6

106.5 79.3 76.9 84.2 65.2

4.22 (1H, d, 8.6) 4.06 (1H, dd, 8.6, 8.4) 3.89 (1H, ddd, 8.4, 6.5, 3.7) 3.82 a (2H)

D-Fructose

a

dC

(nH, mult. J in Hz)

D-Glucose

1 2 3 4 5 6

D-Glucosyl-D-allulose

Position

1 2 3 4 5 6

94.8 73.9 75.6 72.0 75.4 63.0

5.39 3.59 3.72 3.47 3.79 3.81

(1H, d, 3.8) (1H, dd, 9.8, 3.8) (1H, dd, 9.8, 9.3) (1H, dd, 9.6, 9.3) (1H, m) a (2H)

1

62.6

3.69 (1H, d, 12.7) 3.84 (1H, d, 12.7)

2 3 4 5 6

111.9 77.1 73.5 86.4 65.4

D-Allulose

Overlapping NMR signals.

4.22 4.27 4.09 3.81 3.85

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

d, 4.6) dd, 8.2, 4.6) ddd, 8.2, 7.0, 3.3) dd, 12.4, 7.0) dd, 12.4, 3.3)

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H OH

was 50 mM and D-glucosyl-D-alluloside was behaving as a competitive inhibitor of invertase with a Ki value of 9.2 mM.

H O

HO HO

H

DISCUSSION

OH

H HOH 2C

H

O O

H

H

H HO

655

OH

CH 2OH

FIG. 2. Structure of a-D-glucopyranosyl-(1 / 2)-b-D-allulofuranoside.

inhibited the activity of the enzyme by only 22.1%. It is noteworthy, that the hydrolysis of D-glucosyl-D-alluloside in humans would lead to the formation of D-glucose and D-alluloside, which are both safe for human consumption. Fig. 3 shows Lineweaver-Burk and Dixon plots for the inhibition of invertase by D-glucosyl-D-alluloside. These plots suggested that Km value of invertase toward sucrose

FIG. 3. Invertase inhibition by D-glucosyl-D-alluloside. (A) Lineweaver-Burk plots. (B) Dixon plots. (A) D-Glucosyl-D-alluloside was added to a final concentration of 0 mM (closed diamonds), 2 mM (open circles), 5 mM (closed triangles), 10 mM (open squares), or 20 mM (closed squares). (B) Sucrose was used to be a final concentration of 2 mM (closed diamonds), 5 mM (closed triangles), 10 mM (open squares), or 20 mM (closed squares).

We have developed a new method for the production of a variety of rare sugars from D-glucose, and subsequently used this technique as a platform for the synthesis of novel disaccharides containing rare sugars. SPase from L. mesenteroides was selected as the optimum phosphorylase for this work based on the work of Doudoroff (10), who reported the use of SPase in their synthesis of D-glucosyl-L-sorbose. Furthermore, several research groups have reported that SPase exhibits wide acceptor specificity. Aerts et al. (13), in particular, studied the properties of this acceptor in detail and demonstrated that this enzyme was not only capable of catalyzing the conversion of ketohexoses such as D-fructose, L-sorbose, and D-allulose, but could also catalyze the reaction of various low molecular weight compounds such as pentoses, sugar alcohols, and disaccharides. As shown in Table 1, we confirmed the productivity of SPase towards all of the ketohexoses investigated in the current study. SPase therefore exhibited a broad range of ketohexose specificity, which suggested that it could be used for the production of rare disaccharides. It is important to mention, however, that no kinetic parameters were collected during this study, because the SPase productivity was only evaluated by HPLC using the disaccharide peak area. More detailed kinetics studies are currently being conducted in our laboratory, and the results of these studies will be reported at a later date. Weimberg and Doudoroff (9) reported that the SPase from L. mesenteroides produced a disaccharide using L-sorbose and D-xylulose as acceptors and G1P as a donor, although the enzyme did not produce the same disaccharide with D-tagatose (9), which confirmed that our results were not contradictory. In the current study, SPase exhibited its highest levels of productivity towards D-fructose and D-allulose, and our results revealed that the two monomer units in the disaccharide were bD-fructofuranose and b-D-allulofuranose. Both conformations possessed a b-furanose ring, with the only difference between the two conformations being the stereochemistry of the C3eOH group, which suggested that SPase could potentially recognize the remaining four OH groups of each b-D-fructofuranose and b-Dallulofuranose. Verhaeghe et al. (28) have reported the acceptor site in SPase from Bifidobacterium adolescentis. Although they suggested that His234 of this enzyme is positioned in vicinity of C3 and C3eOH of D-fructose, they mentioned His234 does not seem to make polar contact or direct interactions with either of Dfructose and O-group of phosphate. Since our results suggested O3 orientation of D-fructose had no connection with productivity of disaccharide, SPase may not need the interaction with O3 of Dfructose for binding. Given the use of expensive substrates such as G1P and D-allulose represents a significant disadvantage to the mass production of Dglucosyl-D-alluloside, we investigated the development of a direct method for the synthesis of this disaccharide from sucrose. In summary, we have successfully developed a one-pot procedure for the production of D-glucosyl-D-alluloside using SPase and DTE, which allowed for the coupling of two inexpensive monosaccharide units. It is noteworthy that this reaction mixture consisted of an equilibrium process between five sugars, including sucrose, G1P, Dfructose, D-allulose, and D-glucosyl-D-alluloside, because the two enzymes also catalyzed the equilibrium reaction of the starting materials and intermediates. Unfortunately, D-glucose degradated from G1P was also generated during the reaction. As shown in Fig. 1B, the retention time of D-glucosyl-D-alluloside was between those of sucrose and D-glucose, which made its purification particularly difficult. We anticipate that a more efficient procedure

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for the synthesis of D-glucosyl-D-alluloside will be constructed because this material behaves as an inhibitor invertase activity. References 1. Xia, W., Liu, P., Zhang, J., and Chen, J.: Biological activities of chitosan and chitooligosaccharides, Food Hydrocoll., 25, 170e179 (2011). 2. van den Heuvel, E. G., Muys, T., van Dokkum, W., and Schaafsma, G.: Oligofructose stimulates calcium absorption in adolescents, Am. J. Clin. Nutr., 69, 544e548 (1999). 3. Zafar, T. A., Weaver, C. M., Zhao, Y., Martin, B. R., and Wastney, M. E.: Nondigestible oligosaccharides increase calcium absorption and suppress bone resorption in ovariectomized rats, J. Nutr., 134, 399e402 (2004). 4. Holub, I., Gostner, A., Theis, S., Nosek, L., Kudlich, T., Melcher, R., and Scheppach, W.: Novel findings on the metabolic effects of the low glycaemic carbohydrate isomaltulose (Palatinose), Br. J. Nutr., 103, 1730e1737 (2010). 5. Kaneko, I., Hayamizu, K., Tomita, K., Kikuchi, H., Nagura, T., Shigematsu, N., and Chiba, T.: Pilot study of melibiose in patients with adolescent or adult-type atopic dermatitis, J. Appl. Glycosci., 51, 123e128 (2004) (in Japanese). 6. Iida, T., Kishimoto, Y., Yoshikawa, Y., Hayashi, N., Okuma, K., Tohi, M., Yagi, K., Matsuo, T., and Izumori, K.: Acute D-psicose administration decreases the glycemic responses to an oral maltodextrin tolerance test in normal adults, J. Nutr. Sci. Vitaminol. (Tokyo), 54, 511e514 (2008). 7. Hayashi, N., Iida, T., Yamada, T., Okuma, K., Takehara, I., Yamamoto, T., Yamada, K., and Tokuda, M.: Study on the postprandial blood glucose suppression effect of D-psicose in borderline diabetes and the safety of long-term ingestion by normal human subjects, Biosci. Biotechnol. Biochem., 74, 510e519 (2010). 8. Murao, K., Yu, X., Cao, W. M., Imachi, H., Chen, K., Muraoka, T., Kitanaka, N., Li, J., Ahmed, A. R., Matsumoto, K., Nishiuchi, T., and Tokuda, M.: D-Psicose inhibits the expression of MCP-1 induced by high-glucose stimulation in HUVECs, Life Sci., 81, 592e599 (2007). 9. Weimberg, R. and Doudoroff, M.: Studies with three bacterial sucrose phosphorylases, J. Bacteriol., 68, 381e388 (1954). 10. Doudoroff, M.: Disaccharide phosphorylases, pp. 229e236, in: Boyer, P. D., Lardy, H., and Myrbaeck, K. (Eds.), The enzymes, vol. 5, 2nd ed. Academic Press, New York (1961). 11. Kitaoka, M. and Hayashi, K.: Carbohydrate-processing phosphorolytic enzymes, Trends Glycosci. Glycotechnol., 14, 35e50 (2002). 12. Kitao, S. and Sekine, H.: Transglucosylation catalyzed by sucrose phosphorylase from Leuconostoc mesenteroides and production of glucosyl-xylitol, Biosci. Biotechnol. Biochem., 56, 2011e2014 (1992).

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