Novel stereoselective carbonyl reductase from Kluyveromyces marxianus for chiral alcohols synthesis

A novel nicotinamide adenine dinucleotide phosphate(NADPH)-dependent carbonyl reductase from Kluyveromyces marxianus(KmCR) was identified, which can convert various prochiral ketone esters and ketone substrates to their corresponding chiral alcohols. KmCR was over-expressed in E. coli BL21(DE3), purified to homogeneity, and characterized. The purified enzyme exhibits the highest activity at 40 °C and pH=6.0. Based on the gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis, the monomeric protein was determined to have a molecular weight of approximate 39000. Vmax and Km of KmCR are 4.28 μmol·min−1·mg−1 and 0.41 mmol/L for ketone ester substrate ethyl 2-oxo-4-phenylbutyrate(OPBE), 3.09 μmol·min−1·mg−1 and 1.21 mmol/L for cofactor NADPH, respectively. Cofactor recycle was achieved by co-expression of KmCR and glucose dehydrogenase(GDH) in E. coli. Recombinant E. coli harboring KmCR and GDH showed moderate asymmetric reduction activity towards various α- and β-ketoesters, diaryl ketone substrates. In an aqueous/butyl acetate biphasic system, the whole-cell biocatalyst was used to prepare ethyl (R)-2-hydroxy-4-phenylbutanoate[(R)-HPBE] in an e.e. of 99.5% with a space-time yield of 433.6 g·L−1·d−1 and a yield of 80.3% at 270 g/L OPBE.