微生物共代谢降解烟碱类杀虫剂吡虫啉的关键酶及其调控机制研究

Neonicotinoid insecticide imidacloprid (IMI) is able to be co-metabolized by several bacterial strains, such as Escherichia coli DH10B, Stenotrophomonas maltophilia CGMCC 1.1788 and Pseudoxanthomonas indica CGMCC 6648.The degradation pathway of IMI metabolized by the above three strains is via hydroxylation of IMI to form the metabolite 5-hydroxy IMI, which is then dehydrated to form olefin IMI. Interestingly, this pathway is regulated by the primary energy substrances carbohydrate and organic acid. The applicants have revealed that the mechanism of the primary energy substrances control the IMI metabolic pathway is related to that carbohydrates and organic acids as well as the cofactors NADH and NADPH produced by the metabolism of carbohydrates and organic acids regulate the enzyme activities of IMI hydroxylase and 5-hydroxy IMI dehydratase.In present study, the applicants plan to clone and express the IMI hydroxylase genes and proceed the bioinformatics analysis as well as purify and identify the 5-hydroxy IMI dehydratase from S. maltophilia CGMCC 1.1788. The present study is useful for exploration of the mechanism of microbial cometabolism and can be used for microbial remediation of the pesticide contamination.

烟碱类杀虫剂吡虫啉(IMI)可被大肠杆菌等细菌共代谢降解，降解途径为羟基化IMI生成5-hydoxy IMI，5-hydroxy IMI进一步脱水生成olefin IMI。申请人发现该途径受不同初级能源物质糖和有机酸的调节，其机制与糖和有机酸及其产生的NADPH和NADH影响IMI羟基化酶和5-hydroxy IMI脱水酶活性有关。本课题拟从嗜麦芽寡养单胞菌中克隆和表达IMI羟基化酶基因并进行生物信息学分析；分离纯化5-羟基脱水酶并鉴定酶。本研究对认识微生物共代谢降解现象和消除农药残留有重要意义。

课题组按照项目研究计划开展了S. maltophilia CGMCC 1.1788的脱水酶的分离纯化；同时开展了大肠杆菌Escherichia coli DH10B 的13个羟基化酶（monooxygenase）基因和18个脱水酶（dehydratase）基因的单敲除或过表达，分析其对酶活性的影响。目前采用凝胶过滤法从S. maltophilia CGMCC 1.1788的盐析蛋白沉淀中分离到一个具有5-hydroxy IMI脱水酶活性的组份,SDS-page显示有两个蛋白条件，目前正在回收这两个条带和进行质谱分析鉴定。基因敲除结果表明，单敲除rutA、ssuD、mhpA、visC、ybiX、ygiN、yqeB、yqeC、ycfD、ydhR等9个单加氧酶基因和过表达ubiB、ubiH和ubiF单加氧酶不影响E. coli DH10B的IMI羟基化活性。单敲除脱水酶基因pheA,tdcG,caiD,dsdA,aroD,acnB,sdaB,yhaM,paaG,fumB,fadB,fadJ,yfaW,acnA,fumA,fumC的突变株没有降低5-hydroxy IMI脱水酶活性；过表达fabA、fabZ脱水酶基因的表达菌株也没有提高5-hydroxy IMI脱水酶的活性。课题组筛选到一株经羟基化和硝基还原途径降解IMI的Psedudomonas putida Z4菌株。课题组共发表4篇SCI论文，获3项中国发明专利。