文献类型：期刊文献

英文题名：Improved efficiency of a novel methyl parathion hydrolase using consensus approach

作者：Liu, Xu-Yun[1,2,3];Chen, Fei-Fei[1,2];Li, Chun-Xiu[1,2];Luo, Xiao-Jing[1,2];Chen, Qi[1,2];Bai, Yun-Peng[1,2];Xu, Jian-He[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, Shanghai 200237, Peoples R China;[3]Natl Engn Res Ctr Miniaturized Detect Syst, Xian 710069, Peoples R China

年份：2016

卷号：93-94

起止页码：11

外文期刊名：ENZYME AND MICROBIAL TECHNOLOGY

收录：;EI(收录号：20164102883349);WOS:【SCI-EXPANDED(收录号:WOS:000386317700002)】；

基金：This work was financially supported by the National Natural Science Foundation of China (Nos. 21505044, 21536004 & 31500592) and the Ministry of Science and Technology, P.R. China (No. 2012AA022206C).

语种：英文

外文关键词：Biodegradation; Organophosphorus pesticides; Methyl parathion hydrolase; Consensus approach; Site-directed mutagenesis; Catalytic efficiency

摘要：A methyl parathion hydrolase (MPH) gene, bjmpd, was cloned from a newly isolated MP-degrading bacterial strain, Burkholderia jiangsuensis MP-1(T) and heterologously expressed in Escherichia coli BL21 (DE3). Although the amino acid sequence of the bjmpd-encoded enzyme, named BjMPH, differed from that of MPH from Pseudomonas sp. WBC-3 (PsMPH) in only three residues, Ser132, Va1247 and Ala267, a significantly higher specific activity towards MP was exhibited by BjMPH than PsMPH. Among them, Ala267 was identified as a key site affecting the catalytic efficiency, and it was rather conservative (Ala or Ser) in homologous proteins, suggesting that a simple substitution of the residue in conservative site with another conservative residue based on the consensus sequence approach might possibly enhance the catalytic efficiency of the MP-degrading enzyme. Inspired by such an observation, we identified a new mutant, BJMPH(T64N), exhibiting 3.78-fold higher catalytic efficiency (k(cat)/K-M) towards MP than its wild type, reaching 4.20 x 10(6) M-1 s(-1), The mutant BJMPH(T64N) also displayed enhanced reactivities (k(cat)/K-M) towards other organophosphorus pesticides. Homology-modelling analysis indicates that enhanced polar contacts of the 64th residue in this mutant may contribute to stabilizing the structure of the enzyme and promote the interactions between enzyme and substrate. This study generated an efficient MP-degrading enzyme, and provides useful information for enhancing the catalytic efficiency of MPHs via conservative residue substitution based on the consensus approach. (C) 2016 Published by Elsevier Inc.