棉铃虫羧酸酯酶CarE001C定向进化及突变酶的结构与功能研究

The cotton bollworm, Helicoverpa armigera (Hübner), is a major pest of many agricultural crops around the world, the mechanism of insecticide resistance is always the leading research and hot issues in recent years. Carboxylesterases(CarEs) is the major class of detoxification enzymes involved in the development of insecticide resisitance in insects. Our previous study on CarEs from Helicoverpa armigera showed that esterases had some low activities toward organophosphates (OPs). A mutation 001CA127D has shown increasing 7-folds of the hydrolysis activity of diethyl-phosphates than that of the wild type enzyme. In this research proposal, we are investigating the mutations at the oxyanion hole of CarE001C and enhanced OP hydrolase activity. Mutant libraries of both G126 and A127 positions will be produced by saturation mutagenesis technology. The hydrolysis activities of all mutations will be screened and the kinetic properties of the mutants will be analyzed. The results will provide information for understanding the OP resistance mechanism at the enzymatic level. The aims and significance of this study are: 1) to elucidate the relationship between the amino acids of oxyanion hole of CarE001C and its catalytic function and will be used for rational designing and developing new kind of insecticides; 2) to provide useful data for understanding the molecular mechanism of the development of metabolic resistance to insecticides and benefit to the Integrated Pest Management (IPM) in agriculture; 3) to develop OP degrading enzymes for bioremediation.

棉铃虫(Helicovera armigera.Hübner)是一种世界性农业害虫，其抗药性机制一直是该领域研究的前沿和热点问题。羧酸酯酶是其代谢抗性相关的主要解毒酶系之一。课题组前期对棉铃虫系列羧酸酯酶研究发现，其对有机磷杀虫剂有解毒活性；当CarE001C的127位由丙氨酸（Ala）突变为天冬氨酸(Asp）后，活性提高7倍。在此基础上，本项目拟采用饱和突变技术，构建CarE001C氧阴离子洞126位和127位的突变体库，通过分子定向进化和酶学分析方法研究突变对酶促动力学特性的影响机理。本研究具有以下目的和意义：1）解析棉铃虫羧酸酯酶氧阴离子洞126位和127位氨基酸组成（结构）与酯酶催化活性的关系，为新型农药设计提供理论指导；2）分析和预测棉铃虫羧酸酯酶在抗性发展中可能出现的分子机制，为棉铃虫综合防治提供实践指导；3）为开发昆虫解毒酶羧酸酯酶应用于环境修复奠定前期基础。

棉铃虫是一种世界性的重要农业害虫，其体内的羧酸酯酶是一类重要的代谢解毒酶系，是促使棉铃虫对化学杀虫剂产生代谢抗性的主要酶系之一。前期对棉铃虫系列羧酸酯酶的研究发现其对有机磷杀虫剂具有代谢解毒活性，在此基础上本研究通过饱和突变技术对棉铃虫羧酸酯酶CarE001C酶活相关的氧阴离子洞G126/A127位氨基酸进行突变，在E.coli BL21菌株中进行异源表达后，采用全波长酶标仪和高效液相色谱法分别测定了突变体对α-乙酸奈酯的酶促动力学参数及对乙基对氧磷和毒死蜱等杀虫剂的代谢解毒活性。结果表明其中一个双位点突变体（G126N/A127D）活性与未突变前（001C）相比提高了10倍，进一步分析了这两个位点氨基酸变化对酶催化功能的影响及可能机理。在此基础上继续选择酶活相关的多个位点对酯酶CarE001C进行多位点突变，获得一个五位点突变体OP-5，对2种有机磷的杀虫剂的代谢活性比双位点突变体（G126N/A127D）又提高了2倍。上述研究结果表明，氧阴离子洞氨基酸突变对酶催化功能的影响相对有限，因此棉铃虫仅通过单个羧酸酯酶在氧阴离子洞G126/A127位点的替代产生对杀虫剂抗性的可能性较小，但有可能通过单位点突变累积（多位点突变）而提高对有机磷杀虫剂的代谢活性从而产生抗性。上述研究同时也为昆虫解毒酶羧酸酯酶应用于农药残留污染环境的修复提供了较重要实践参考。