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当前位置: 首页 > 文章 > 基于分子结构与生物信息学等多维度特征的定向进化改造甲基对硫磷水解酶 生物技术通报 2018,34 (10) 194-200
Position: Home > Articles > Directed Evolution of Methyl Parathion Hydrolase Based on the Multidimensional Features :Molecular Structure and Bioinformatics Biotechnology Bulletin 2018,34 (10) 194-200

基于分子结构与生物信息学等多维度特征的定向进化改造甲基对硫磷水解酶

作  者：
任天雷;杨海泉;许菲
单  位：
江南大学生物工程学院糖化学与生物技术教育部重点实验室
关键词：
甲基对硫磷水解酶;基于结构与生物信息的定向进化;活性中心;进化保守性;催化效率
摘  要：
甲基对硫磷水解酶作为环境友好绿色催化剂,可降解甲基对硫磷等在土壤中残留的有机磷农药,这使得如何提高甲基对硫磷水解酶的水解效率成为酶工程中备受关注的问题。基于分子结构与生物信息学等多维度特征,确定了催化活性中心周围的9个关键位点。构建了一个含1 500个突变体的筛选文库,进行定向进化。经过两轮筛选,最终确定2株催化效率提高的突变体,L258V和L273M。其催化效率常数kcat/Km值分别为134.1(mmol/L)-1·s-1和166.5(mmol/L)-1·s-1,与野生酶相比较分别提高了27.3%和58.1%。这种通过半理性设计构建定向进化文库的方法,不仅有效的提高了甲基对硫磷水解酶的筛选效率,更为酶的改造提供了新的思路。
译  名：
Directed Evolution of Methyl Parathion Hydrolase Based on the Multidimensional Features :Molecular Structure and Bioinformatics
作  者：
REN Tian-lei;YANG Hai-quan;XU Fei;School of Biotechnology,Jiangnan University/Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education;
关键词：
methyl parathion hydrolase;;directed evolution based on structure and bioinformation;;active centre;;evolutionary conservation;;catalytic efficiency
摘  要：
As an environmental-friendly green catalyst,methyl parathion hydrolase can degrade organophosphorus pesticides such as methyl parathion remained in soil such,thus improving the hydrolysis efficiency of methyl parathion hydrolase becomes a well-concerned issue. Based on the multi-dimensional features such as structural and bioinformatics,9 key sites around the catalytic active center were determined. A screening library containing 1 500 mutants were constructed for directed evolution. After two rounds of screening,2 mutants with the improved catalytic efficiency,L258 V and L273 M were identified. Their catalytic efficiency constants kcat/Km values were 134.1(mmol/L)~(-1)·s~(-1) and 166.5(mmol/L)~(-1)·s~(-1),respectively,which increased by 27.3% and 58.1% compared with the wild type. This method of constructing a directed evolution library by semi-rational design not only improves the screening efficiency of methyl parathion hydrolase,but also provides a new idea to improve properties of enzyme.

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