Abstract:

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.

Key words: methyl parathion hydrolase, orthogonal test design method, site-directed mutation

摘要：

分别自假产碱假单胞菌(Pseudomonas pseudoalcaligenes C2-1)和假单胞菌（Pseudomonas sp. 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。

关键词: 甲基对硫磷水解酶;正交实验;定点突变