Abstract
A gene encoding an NADH-dependent short-chain dehydrogenase/reductase (gox2036) from Gluconobacter oxydans 621H was cloned and heterogeneously expressed in Escherichia coli. The protein (Gox2036) was purified to homogeneity and biochemically characterized. Gox2036 was a homotetramer with a subunit size of approximately 28 kDa. Gox2036 had a strict requirement for NAD+/NADH as the cofactor. Gox2036 displayed preference for oxidation of secondary alcohols and 2,3-diols as well as for reduction of α-diketones, hydroxy ketones, α-ketoesters, and β-ketoesters. However, Gox2036 was poorly active on 1,2-diols and acetoin and showed no activity on primary alcohols, polyols, and aldehydes. The optimum pH values for the oxidation and reduction reactions were 9 and 6, respectively. Gox2036 was highly selective in the reduction of various β-ketones and β-ketoesters. Among the substrates tested, ethyl 4-chloro acetoacetate was reduced to ethyl (R)-4-chloro-3-hydroxybutanoate ester with an excellent conversion yield of 96.9 % and optical purity of >99 % e.e. using an efficient in situ NADH-recycling system involving glucose and a glucose dehydrogenase from Bacillus subtilis (BsGDH).
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Acknowledgments
This work was financially supported by the National Key Basic Research. Development Program of China (“973” Program, No. 2012CB721003), the Natural Science Foundation of China (No. 21002029/B020706 and 21276084/B060804) and National Major Science and Technology Projects of China (No. 2012ZX09304009).
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Liu, X., Chen, R., Yang, Z. et al. Characterization of a Putative Stereoselective Oxidoreductase from Gluconobacter oxydans and Its Application in Producing Ethyl (R)-4-Chloro-3-Hydroxybutanoate Ester. Mol Biotechnol 56, 285–295 (2014). https://doi.org/10.1007/s12033-013-9707-z
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DOI: https://doi.org/10.1007/s12033-013-9707-z