高效氯氰菊酯降解酶PyDO的活性中心及降解机制研究

Pyrethroid insecticides are widespread pollutants and have received the public concern on potential human health and environmental risks that may result from chronic dietary exposure to pyrethroid residues on agricultural products. Recently, degrading enzymes has received increasing attention as a new approach to clean up pyrethroid residues.In the previous National Natural Science Foundation (30871660), Ochrobactrum lupini DG-S-01 was comfirmed to possess efficient degradation to many pyrethroids including beta-cyperthrin. However, the degrading activity centre and mode of action of enzyme PyDO are unknown yet. In the present project, the mutation library of M1 strain using mariner transposon random mutation will be contructed, and then select the mutants without hydrolysis halo indicated as gentamycin resistant gene. The functional genes associated with pyrethroids degradation were systematically studied and confirmed by bioinformatics, Tail-PCR, HPLC, and other methods.Based on these results, the active centre in PyDO will be clarified through homology modeling and active site mutation. Finnally, the degrading mechanism by PyDO to pyrethroid insecticides will be elaborated through the active centre spot mutation, beta-cyperthrin fragmentation process, and confirme of degrading products and activity. These prospective results will expand the contents of pesticide toxicology and provide the key technology support for pyrethroid residues pollution management.

菊酯类农药残留污染已成为影响农产品和环境安全的重要因素。应用环境微生物或其工程菌，对受污染的农产品和环境进行原位生物修复或采后处理，是控制菊酯类农药残留危害的可行途径。申请者在前期项目(30871660)时发现并深入研究了Ochrobactrum lupini DG-S-01对高效氯氰菊酯的降解作用，获得了突变菌株M1及其降解酶PyDO，但其活性中心、降解机制等关键问题尚不清楚。本项目拟应用转座子随机突变技术，构建菌株M1的突变体文库，利用转座子的庆大霉素抗性基因和高效氯氰菊酯降解过程中产生水解圈与否筛选突变株，通过生物信息学、Tail-PCR、HPLC等手段确定降解功能基因；通过同源建模分析、活性位点突变，确定酶活性中心；通过活性中心点突变和高效氯氰菊酯分子碎片化处理，结合降解产物及活性验证，揭示降解机制。研究结果将丰富农药毒理学内容，为拟除虫菊酯类农药残留污染治理提供关键技术支撑。

本项目在前期研究基础上，筛选与鉴定了10余株拟除虫菊酯降解菌株；研究了氯氰菊酯对映异构体的反相液相分离条件和主要菌株对菊酯类农药的降解特性，优化了其降解条件，开展了降解菌对蔬菜和土壤中残留农药的降解动力学研究；鉴定了代表性菌株的降解产物，分析确定了降解途径；应用转座子随机突变等技术，构建了降解菌的基因组文库和突变体文库，利用转座子的庆大霉素抗性基因和高效氯氰菊酯降解过程中产生水解圈与否筛选突变株，克隆获得了系列的降解酶基因的克隆，研究了PyDO、estZ、ybfK、estA等基因的表达、纯化及其酶学性质，通过生物信息学、Tail-PCR、HPLC等手段确证了相关酶的降解功能；建立了降解酶蛋白分子建模及活性中心模型，研究了高效氯氰菊酯降解分子机制和信号传导机制。全面完成了项目计划内容，研究结果丰富了农药毒理学内容，为拟除虫菊酯类农药残留污染微生物降解治理提供了技术支撑。