文献类型：期刊文献

英文题名：Substrate channel evolution of an esterase for the synthesis of cilastatin

作者：Luan, Zheng-Jiao[1,2];Li, Fu-Long[1,2];Dou, Shuai[1,2];Chen, Qi[1,2];Kong, Xu-Dong[1,2];Zhou, Jiahai[3];Yu, Hui-Lei[1,2];Xu, Jian-He[1,2]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]E China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, Shanghai 200237, Peoples R China;[3]Chinese Acad Sci, Shanghai Inst Organ Chem, State Key Lab Bioorgan & Nat Prod Chem, Shanghai 200032, Peoples R China

年份：2015

卷号：5

期号：5

起止页码：2622

外文期刊名：CATALYSIS SCIENCE & TECHNOLOGY

收录：;EI(收录号：20151900829843);WOS:【SCI-EXPANDED(收录号:WOS:000353641800010)】；

基金：We are grateful for the access to beamline BL17U1 at Shanghai Synchrotron Radiation Facility and thank the beamline staff for technical support. This work was financially supported by NSFC (no. 21276082), MOST (no. 2011CB710800 and 2011AA02A210), and SKLBE (no. 2060204).

语种：英文

外文关键词：Carboxylation - Enzyme activity

摘要：The esterase RhEst1 from Rhodococcus sp. ECU1013 has been reported for the enantioselective hydrolysis of ethyl (S)-(+)-2,2-dimethylcyclopropane carboxylate, producing the building block of cilastatin. In this work, error-prone PCR and site-directed saturation mutagenesis were applied to RhEst1 for activity improvement, with the pH-indicator assay as a high-throughput screening method. As a result, RhEst1(A147I/V148F/G254A), with mutations surrounding the substrate access channel, showed a 5-fold increase in its specific activity compared with the native enzyme, as well as a 4-fold increase in protein solubility. Combined with the determination of protein structures and computational analysis, this work shows that the amino acids around the substrate channel play a more important role in the activity evolution of RhEst1 than those in the active site.