Robust NADH-regenerator: improved alpha-haloketone-resistant formate dehydrogenase.

来自 EBSCO

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作者：

H Yamamoto，K Mitsuhashi，N Kimoto，Y Kobayashi，N Esaki

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摘要：

Formate dehydrogenases (FDH) are useful for the regeneration of NADH, which is required for asymmetric reduction by several dehydrogenases and reductases. FDHs have relatively low activity and are labile, especially to α-haloketones, thus FDH cannot be applied to the industrial manufacture of optically active α-haloalcohols. To stabilize a FDH from Mycobacterium vaccae (McFDH) against the α-haloketone ethyl 4-chloroacetoacetate (ECAA), a set of cysteine-mutant enzymes was constructed. Sensitivity to ECAA of mutant C6S was similar to that of the wild-type enzyme, and mutants C249S and C355S showed little activity. In contrast, mutant C256S exhibited remarkable tolerance to ECAA. Surprisingly, mutant C146S was activated by several organic compounds such as ethyl acetate. An optimized mutant, C6A/C146S/C256V (McFDH-26), was obtained by combining several effective mutations. Ethyl ( S )-4-chloro-3-hydroxybutanoate [( S )-ECHB] was synthesized from ECAA to 49.9g/l with an optical purity of more than 99% e.e. using recombinant Escherichia coli cells coexpressing McFDH-26 and a carbonyl reductase (KaCR1) from Kluyveromyces aestuarii .

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关键词：

Escherichia coli Mycobacterium Kluyveromyces Acetates Butyric Acids Acetoacetates Ketones Cysteine NAD Alcohol Oxidoreductases