通过正交实验设计突变体组合提高甲基对硫磷水解酶OPHC2对乙基对硫磷的降解能力  

Enhanced Ethyl Parathion Hydrolysis Ability of Methyl Parathion Hydrolase OPHC2 by Mutant Combination from Orthogonal Test Design

作　　者：吕红[1] 田健[1] 初晓宇[1] 伍宁丰[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081

出　　处：《中国农业科技导报》2010年第4期78-83,共6页Journal of Agricultural Science and Technology

基　　金：国家863计划项目(2007AA100605)资助

摘　　要：分别自假产碱假单胞菌(Pseudomonas pseudoalcaligenesC2-1)和假单胞菌(Pseudomonassp.1-7)中克隆得到甲基对硫磷水解酶基因ophc2和ophc3,其编码甲基对硫磷水解酶OPHC2和OPHC3的氨基酸序列相似性达98.5%,仅5个氨基酸残基不同,但酶学性质差异很大,特别是对不同底物降解特性有很大差异。OPHC2对甲基对硫磷的催化效率是OPHC3的2.75倍,对乙基对硫磷的催化效率仅为OPHC3的12.9%。为了提高OPHC2对乙基对硫磷的降解活性,以OPHC3的序列为依据,针对5个不同的氨基酸设计正交实验,对OPHC2进行组合突变,最终获得了一个突变体P165S/A169V,对乙基对硫磷的催化效率是OPHC2的1.19倍,对甲基对硫磷的催化效率是OPHC2的1.36倍。The genes ophc2 and ophc3 were cloned from Pseudomonas pseudoalcaligenes C2-1 and Pseudomonas sp.1-7,respectively.They encode two methyl parathion hydrolases OPHC2 and OPHC3.The amino acid sequence of OPHC2 shows 98.5% identity to OPHC3,and is only 5 residues different from OPHC3.But their characteristics are very different,especially in the substrate acceptance.The catalysis activity of OPHC2 is higher than that of OPHC3 by 2.75-fold using methyl parathion as the substrate,but lower than that of OPHC3 using ethyl parathion as the substrate,and only 12.9% to OPHC3.In order to improve the hydrolysis of OPHC2 against ethyl parathion,a combinatorial site-directed mutagenesis was designed by orthogonal test design method according to the amino acid sequence of OPHC3.As a result,the mutant P165S/A169V has been obtained,whose hydrolysis activity was improved by 1.19-fold against methyl parathion and 1.36-fold against ethyl parathion compared with OPHC2.

关 键 词：甲基对硫磷水解酶 正交实验 定点突变 

分 类 号：Q55[生物学—生物化学]