A novel carbonyl reductase with anti-Prelog stereospecificity for the production of t-butyl 6-cyano-(3R, 5R)-dihydroxyhexanoate.

Abstract

A novel gene (crc1) from Candida boidinii was cloned and then overexpressed in a recombinant strain BL21(DE3)/pET30a-crc1 of Escherichia coli. The resulting carbonyl reductase was prepared through fermentations using the recombinant strain. The purified enzyme showed an NADPH-dependent activity and specific activity was 4.65U/mg using t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (ATS-6) as substrate. The enzyme was optimally active at 35°C and pH 7, respectively. The apparent K m and V max of the enzyme for ATS-6 are 1.5mM and 21.1μmol/minmg, respectively, indicating excellent anti-Prelog stereospecificity. Under the optimum condition, t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate (ATS-7) was prepared with the enzyme with high d.e. value (99.9%) and good conversion (94%) in 4h, indicating high stereoselectivity and conversion efficiency in biotransformation of ATS-6 to ATS-7.