Novel Stereoselective Carbonyl Reductase from Kluyveromyces marxianus for Chiral Alcohols Synthesis

doi:10.1007/s40242-013-3286-1

Chemical Research in Chinese Universities ›› 2013, Vol. 29 ›› Issue (6): 1140-1148.doi: 10.1007/s40242-013-3286-1

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Novel Stereoselective Carbonyl Reductase from Kluyveromyces marxianus for Chiral Alcohols Synthesis

LI Hai-dong, SUN Zhi-hao, NI Ye

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P. R. China

Received:2013-07-08 Revised:2013-08-30 Online:2013-12-01 Published:2013-09-17
Contact: NI Ye E-mail:yni@jiangnan.edu.cn
Supported by:
Supported by the National Basic Research and Development Program of China(No.2011CB710800), the National Natural Science Foundation of China(No.21276112), the Project of New Century Excellent Talents in University of China(No.NCET-11-0658), the Natural Science Foundation of Jiangsu Province, China(No.BK2011150), the Program of Introducing Talents of Discipline to Universities, China(No.111-2-06) and the Priority Academic Program Development of Jiangsu Higher Education Institutions(China).

Abstract

Abstract:

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℃ 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.

Key words: Carbonyl reductase, Asymmetric reduction, Chiral alcohol, co-Expression, Kluyveromyces marxianus

LI Hai-dong, SUN Zhi-hao, NI Ye. Novel Stereoselective Carbonyl Reductase from Kluyveromyces marxianus for Chiral Alcohols Synthesis[J]. Chemical Research in Chinese Universities, 2013, 29(6): 1140-1148.

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Novel Stereoselective Carbonyl Reductase from Kluyveromyces marxianus for Chiral Alcohols Synthesis

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