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

doi:10.13560/j.cnki.biotech.bull.1985.2018-0221

生物技术通报 ›› 2018, Vol. 34 ›› Issue (10): 194-200.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0221

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

任天雷, 杨海泉, 许菲

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,无锡 214122

收稿日期:2018-03-15 出版日期:2018-10-26 发布日期:2018-11-07
作者简介:任天雷,男,硕士,研究方向：生物工程;E-mail：944046660@qq.com
基金资助:
青年千人计划经费资助（1012069999160060）

Directed Evolution of Methyl Parathion Hydrolase Based on the Multi-dimensional Features：Molecular Structure and Bioinformatics

REN Tian-lei, YANG Hai-quan, XU Fei

School of Biotechnology,Jiangnan University/Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Wuxi 214122

Received:2018-03-15 Published:2018-10-26 Online:2018-11-07

摘要/Abstract

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k_cat/K_m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。

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

Abstract: 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,L258V and L273M were identified. Their catalytic efficiency constants k_cat/K_m 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.

Key words: methyl parathion hydrolase, directed evolution based on structure and bioinformation, active centre, evolutionary conservation, catalytic efficiency

任天雷, 杨海泉, 许菲. 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[J]. 生物技术通报, 2018, 34(10): 194-200.

REN Tian-lei, YANG Hai-quan, XU Fei. Directed Evolution of Methyl Parathion Hydrolase Based on the Multi-dimensional Features：Molecular Structure and Bioinformatics[J]. Biotechnology Bulletin, 2018, 34(10): 194-200.

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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

Directed Evolution of Methyl Parathion Hydrolase Based on the Multi-dimensional Features：Molecular Structure and Bioinformatics

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