文献类型：期刊文献

英文题名：Stereospecific Reduction of Methyl o-Chlorobenzoylformate at 300 g?L-1 without Additional Cofactor using a Carbonyl Reductase Mined from Candida glabrata

作者：Ma, Hongmin[1];Yang, Linlin[2];Ni, Yan[1];Zhang, Jie[1];Li, Chun-Xiu[1];Zheng, Gao-Wei[1];Yang, Huaiyu[2];Xu, Jian-He[1]

机构：[1]E China Univ Sci & Technol, Lab Biocatalysis & Synthet Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Chinese Acad Sci, Drug Discovery & Design Ctr, State Key Lab Drug Res, Shanghai Inst Mat Med, Shanghai 201203, Peoples R China

年份：2012

卷号：354

期号：9

起止页码：1765

外文期刊名：ADVANCED SYNTHESIS & CATALYSIS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000305209300021)，CCR-EXPANDED(收录号:WOS:000305209300021)】；

基金：This work was financially supported by the National Natural Science Foundation of China (Nos. 20902023 & 31071604), Ministry of Science and Technology, P. R. China (Nos. 2009CB724706 & 2011CB710800), China National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204) and Shanghai Leading Academic Discipline Project (No. B505), Shanghai Committee of Science and Technology (No. 11431921600) and Innovation Program of Shanghai Municipal Education Commission (No. 11CXY24).

语种：英文

外文关键词：bioinformatics; biotransformations; cofactors; oxidoreductases

摘要：In order to search for oxidoreductases suitable for the preparation of methyl (R)-o-chloromandelate [(R)-CMM], the key intermediate for clopidogrel, the homologous proteins of Gre2p were expressed in Escherichia coli, among which CgKR1 showed the most satisfactory activity and stereoselectivity towards methyl o-chlorobenzoylformate (CBFM). Using the crude enzyme of CgKR1 and glucose dehydrogenase (GDH), as much as 300 g center dot L-1 of CBFM was almost stoichiometrically converted to (R)-CMM with excellent enantiomeric excess (98.7% ee). More importantly, the reaction could be performed without external addition of an expensive cofactor. The substrate profile indicates that keto esters serve as the most suitable substrate, which was confirmed by gram-scale preparations. Homology modeling and docking analysis revealed the molecular basis for the high stereoselectivity of CgKR1. These demonstrate not only the feasibility of in silico mining of novel enzymes based on sequence homology but also the applicability of this new reductase for the practical production of optically active (R)-CMM.