Abstract D-mannitol is a kind of hexitols widely applied in the food and medicinal fields due to its numerous benefits. Mannitol 2-dehydrogenase (MDH, EC 1.1.1.67) is a kind of oxidoreductase playing a pivotal part in the production of d -mannitol from d -fructose. In this work, we identified a highly thermostable d -mannitol-producing MDH from a thermo-tolerant bacterium, Caldicellulosiruptor morganii Rt8.B8. When using d -fructose as the substrate, the recombinant MDH was activated obviously in the presence of Mn2+ with an optimal pH as 8.0 and temperature at 75 °C. The specific activity, Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) for d -fructose were determined as 115 U mg−1, 18 mM and 8.5 s-1 mM−1. Moreover, the half-life (t1/2) of recombinant MDH at 75, 85 and 95 °C was 19 h, 3.5 h and 1.62 h respectively, which was much higher than that of most MDHs. The optimal condition for the production of d -mannitol was determined to be pH at 7.5, the temperature at 70 °C, and 2:1 ratio of C. morganii MDH and Ogataea parapolymorpha formate dehydrogenase (FDH, EC 1.2.1.2). Meanwhile, approximately 80 % d -mannitol was generated by two enzymes after a 50 h reaction from 400 mM d -fructose, indicating a great potentiality in the industrial preparation of d -mannitol.

中文翻译：

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Caldicellulosiruptor morganii Rt8.B8高热稳定性甘露醇2-脱氢酶的鉴定及其在制备D-甘露醇中的应用

摘要 D-甘露醇是一种己糖醇，因其多种功效而广泛应用于食品和医药领域。甘露醇 2-脱氢酶 (MDH, EC 1.1.1.67) 是一种氧化还原酶，在从 d-果糖生产 d-甘露醇中起关键作用。在这项工作中，我们从耐热细菌 Caldicellulosiruptor morganii Rt8.B8 中鉴定了一种高度耐热的 d-甘露醇生产 MDH。当以d-果糖为底物时，重组MDH在Mn2+存在下明显活化，最佳pH为8.0，温度为75℃。d-果糖的比活性、米氏常数 (Km) 和催化效率 (kcat/Km) 确定为 115 U mg-1、18 mM 和 8.5 s-1 mM-1。此外，重组 MDH 在 75、85 和 95 °C 下的半衰期（t1/2）分别为 19 h、3.5 h 和 1.62 h，这远高于大多数 MDH。生产 d-甘露醇的最佳条件被确定为 pH 7.5、温度 70°C 和 2:1 比例的摩根木霉 MDH 和 Ogataea parapolymorpha 甲酸脱氢酶 (FDH, EC 1.2.1.2)。同时，400 mM d-果糖反应50小时后，两种酶生成了约80%的d-甘露醇，表明d-甘露醇在工业制备中具有很大的潜力。