Characterization of D-Glucoside 3-Dehydrogenase from Rhizobium sp. L35 and Its Application for D-Allose Production
Characterization of D-Glucoside 3-Dehydrogenase from Rhizobium sp. L35 and Its Application for D-Allose Production
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DOI：
10.12982/cmujns.2019.0019
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发表时间：
2019-07
影响因子：
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通讯作者：
Akkharapimon Yotsombat;K. Uechi;Shun Onishi;K. Mino;T. Hasegawa;K. Morimoto;Goro Takata
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文献类型：
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作者：
Akkharapimon Yotsombat;K. Uechi;Shun Onishi;K. Mino;T. Hasegawa;K. Morimoto;Goro Takata
D-阿洛糖是一种罕见的单糖，具有一些有趣的特性，可增强放化疗和癌症治疗的抗肿瘤作用，然而，高效生产高产率的D-阿洛糖尚未见报道。在本研究中，我们提出利用D-葡萄糖苷3-脱氢酶结合化学反应来提高D-阿洛糖的产量。一种 D-葡萄糖苷 3-脱氢酶，其区域选择性地将 C-3 位上的糖苷脱氢为相应的 3-酮糖苷，被新分离为根瘤菌 (Rhizobium sp.)。 L35 并被表征为黄素腺嘌呤二核苷酸依赖性脱氢酶。 SDS-PAGE测定其分子量为67,000，尺寸排阻色谱测定其分子量为131,000，表明它是二聚酶。其活性的最适pH和温度分别为7.5和40°C。它在 pH 6.0 和 11.0 之间以及低于 40°C 时保持稳定（半衰期在 40°C 下为 3 小时，在 45°C 下为 50 分钟）。该酶对各种糖苷显示出广泛的底物特异性，尤其是 β-1,4-连接的二糖，例如纤维二糖和乳糖。最后，以纤维二糖为起始原料，通过酶促脱氢、化学还原和酸水解三步工艺生产D-阿洛糖。 D-阿洛糖的产率估计为纤维二糖的 30%。该结果表明，通过该策略可以产生比传统方法高三倍的D-阿洛糖。 CMU J. Nat。科学。 (2019)卷。 18(3) 268
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