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.
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