Novel stereoselective carbonyl reductase from Kluyveromyces marxianus for chiral alcohols synthesis

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摘要：

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. V max and K m 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.

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关键词：

还原酶；手性醇；克鲁维酵母；立体选择性；羰基；重组大肠杆菌；烟酰胺腺嘌呤二核苷酸；醇合成

DOI：

10.1007/s40242-013-3286-1

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年份：

2013