| Summary: | In this study, we conducted the characterization and purification of the thermostable mannitol dehydrogenase (MtDH) from <i>Caldicellulosiruptor hydrothermalis</i> 108. Furthermore, a coupling-enzyme system was designed using (MtDH) from <i>Caldicellulosiruptor hydrothermalis</i> 108 and formate dehydrogenase (FDH) from <i>Ogataea parapolymorpha</i>. The biotransformation system was constructed using <i>Escherichia coli</i> whole cells. The purified enzyme native and subunit molecular masses were 76.7 and 38 kDa, respectively, demonstrating that the enzyme was a dimer. The purified and couple enzyme system results were as follows; the optimum pH for the reduction and the oxidation was 7.0 and 8.0, the optimum temperature was 60 °C, the enzyme activity was inhibited by EDTA and restored by zinc. Additionally, no activity was detected with NADPH and NADP. The purified enzyme showed high catalytic efficiency <i>K</i><sub>cat</sub> 385 s<sup>−1</sup>, <i>K</i><sub>m</sub> 31.8 mM, and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> 12.1 mM<sup>−1</sup> s<sup>−1</sup> for D-fructose reduction. Moreover, the purified enzyme retained 80%, 75%, 60%, and 10% of its initial activity after 4 h at 55, 60, 65, and 75 °C, respectively. D-mannitol yield was achieved via HPLC. <i>Escherichia coli</i> are the efficient biotransformation mediator to produce D-mannitol (byproducts free) at high temperature and staple pH, resulting in a significant D-mannitol conversation (41 mg/mL) from 5% D-fructose.
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