文献类型：期刊文献

英文题名：Combinatorial Evolution of Phosphotriesterase Toward a Robust Malathion Degrader by Hierarchical Iteration Mutagenesis

作者：Luo, Xiao-Jing[1];Zhao, Jian[1];Li, Chun-Xiu[1];Bai, Yun-Peng[1];Reetz, Manfred T.[2,3];Yu, Hui-Lei[1];Xu, Jian-He[1]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Max Planck Inst Kohlenforsch, Mulheim, Germany;[3]Philipps Univ Marburg, Fachbereich Chem, Marburg, Germany

年份：2016

卷号：113

期号：11

起止页码：2350

外文期刊名：BIOTECHNOLOGY AND BIOENGINEERING

收录：;EI(收录号：20164002871533);WOS:【SCI-EXPANDED(收录号:WOS:000386755000005)】；

基金：Contract grant sponsor: National Natural Science Foundation of ChinaContract grant numbers: 21276082; 21505044; 21536004Contract grant sponsor: Ministry of Science and Technology, P.R. ChinaContract grant numbers: 2012AA022206C; 2011CB710803

语种：英文

外文关键词：biocatalysis; directed evolution; DNA shuffling; malathion; phosphotriesterase

摘要：Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. Themutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening < 9,000 mutants, the best variant PoOPHM9 showed 25-fold higher activity than wild-type PoOPHM2, with a thermostability (T-50(15)) of 67.6 degrees C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. (C) 2016 Wiley Periodicals, Inc.