基于分子对接技术的农药残留检测用酶敏感性研究

Enzyme inhibition method was the most promising of rapid determination of pesticide residues, but the presence of the sensitivity and the accuracy of defect. The discovery and research of new enzymes are the key to the development of enzyme inhibition method. Many types of B esterase widely existed in plants and animals provides a rich candidate enzyme libraries of the enzymes used in detection of pesticide residues, and the deep excavation of their differentiation and diversification of sensitivity to pesticides and molecular modification are important for the development of enzyme inhibition method. Because of limits of the traditional experimental method, only a few candidate enzyme sources are systematically studied. Based on pre-research foundation, this project creatively employ a new strategy of homology modeling and molecular docking technology followed by traditional experiment verification to perform a large-scale analysis of candidate B esterase sensitivity and selectivity against the inhibition of different pesticide molecules, draw the sensitive pesticide spectrum for each esterase, screen the new enzyme source with a broad or specificity sensitivity against pesticides, analyze critical amino acid residues for the sensitivity of against pesticides, and carry out molecule rational design of novel target enzymes using site-specific mutagenesis guided by molecular docking . The aim of this project is to provide new materials and scientific basis for choosing the enzymes in detection of pesticide residues and developing multi-enzyme combined detection strategy，and also to provide new strategies, models and methods for related research.

酶抑制法是最具前景的农药残留快速检测方法，但存在灵敏度和准确性方面的缺陷。新酶源的发掘和研究是酶抑制法发展的关键。动植物广泛存在的多种类型的B-酯酶为农药残留检测用酶提供了丰富的候选酶库，其差异化和多样化的农药敏感性特征的深入挖掘和分子改造是酶抑制法发展的重要基础。但受传统实验方法的限制，仅有极少的候选酶源被系统地研究利用。本项目在预研基础上，创造性地采用同源建模及分子对接技术与传统实验方法验证相结合的新策略，大规模分析候选B-酯酶对不同农药分子抑制的敏感性和选择性，绘制各酯酶的敏感农药谱，筛查对各种农药具有广谱敏感性或特异敏感性的新酶源，分析影响农药敏感性的关键残基，并对目标新酶源采用分子对接指导的定点突变进行分子理性设计。本项目旨在为农药残留检测用酶的选择及多酶组合型检测策略的制定提供新的材料和科学依据，同时也为相关研究提供新的策略、模型和方法。

酶的农药敏感性是影响酶抑制法检测农药残留准确性的关键。本课题构建了652种候选酯酶的分子结构数据库，通过集成了分子对接AutoDock Vina方法及亲和力预测ID-Score方法，建立了一种新的反向对接程序；运用该程序对219种酯酶和37种农药分子进行大规模的分子对接预测不同酯酶的农药敏感性，绘制了各种酯酶的敏感性农药谱及各种农药的敏感酯酶谱；通过回顾性评价与实验验证相结合方法，证明该对接程序能有效地预测酯酶对有机磷农药的敏感性。通过对来自不同物种的137条胆碱酯酶与15种常用有机磷农药和高毒有机磷农药进行分子对接，揭示昆虫和鱼类是敏感性胆碱酯酶的重要来源。通过序列对比和虚拟突变与对接分析，初步确定了17个氨基酸残是可能影响乙酰胆碱酯酶农药敏感性的关键位点。克隆和表达了果蝇乙酰胆碱酯酶12个突变体，并检测了突变型酶对Dichlorvos、Triazophos、Omethoate三种农药的敏感性，结果表明突变体对接评分值与实验值之间具有相关性，其中E107Y、E107W、Y109D、E107W/Y109D、I199V/F368Y、Y408F等6个突变体具有研究和应用价值。本课题对雪豆酯酶、猪肝酯酶、鸡血清丁酰胆碱酯酶、美洲蟑螂乙酰胆碱酯酶等4个不同类型的新型候选酯酶代表进行了系统的实验研究，进一步揭示不同类型酯酶的酶学性质、农药敏感性和重金属敏感性的特征和差异，发现雪豆酯酶在农药残留检测的应用尤其值得进一步深入研究。通过比较研究不同的样品氧化前处理的效果，成功建立了以次氯酸钙为氧化剂的农药残留检测增敏方法，可大大提高酶抑制法的敏感性。本课题的研究成果为农药残留检测用酶的挖掘和改造提供了新的模型和方法，同时发现了一系列新型酶源供进一步研究与应用。