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D-allose is a rare monosaccharide that possesses some interesting features for enhancing the antitumor effects of chemoradiotherapy and treatment of cancer, however, efficient production of D-allose with high percent yield has not yet been reported. In this study, we proposed a utilization of D-glucoside 3-dehydrogenase together with chemical reaction to improve the D-allose production. A D-glucoside 3-dehydrogenase, which regioselectively dehydrogenates glycosides at the C-3 position to the corresponding 3-ketoglycoside, was newly isolated as Rhizobium sp. L35 and characterized as a flavin adenine dinucleotide-dependent dehydrogenase. Its molecular weight was determined to be 67,000 by SDS-PAGE and 131,000 by size-exclusion chromatography, suggesting that it is a dimeric enzyme. Its optimum pH and temperature with respect to activity were 7.5 and 40°C, respectively. It was stable between pH 6.0 and 11.0, and below 40°C (half-life of 3 h at 40°C and 50 min at 45°C). The enzyme showed broad substrate specificity towards various glycosides, especially β-1,4-linked disaccharides such as cellobiose and lactose. Finally, D-allose production was performed by a three-step process of enzymatic-dehydrogenation, chemical reduction and acid-hydrolysis, using cellobiose as the starting material. The yield of D-allose was estimated to be 30% from cellobiose. This result indicates that D-allose can be produced by this strategy three-fold higher than the conventional method. CMU J. Nat. Sci. (2019) Vol. 18(3) 268

Characterization of D-Glucoside 3-Dehydrogenase from Rhizobium sp. L35 and Its Application for D-Allose Production