微生物降解吡唑醚菌酯的代谢途径及机理研究

As one of the most used strobilurins, pyraclostrobin now is continuing to widespread spraying in our country and its residuals are of great harm. In natural environment, microbial degradation is the main denature pathway of pyraclostrobin, but its metabolic pathway and molecular mechanism are still unclear. The applicant of this project has obtained one Pseudomonas plecoglossicaida strain (USTBP-01) that can utilize pyraclostrobin as the sole carbon source and energy, on this basis, the culture conditions optimizion and degradation characteristics of this strain will be studied in the next step. During the period, we will also need to separate and identify the metabolites of pyraclostrobin, obtain the reaction kinetics data and its discipline relatively, reveal the reaction steps and the key enzymes in the pathway of pyraclostrobin degradation process and clone the key enzymes gene further. Illuminating the degradation mechanism of pyraclostrobin in molecular biology and biochemical level will provide theoretical basis and technical guidance for the fair use of strobilurins and the biological control and bioremediation of environmental problem caused by strobilurins.

吡唑醚菌酯作为甲氧基丙烯酸酯类杀菌剂的主要类型正持续在我国大范围使用，残留危害大，自然环境中的微生物降解是其脱毒的主要途径，但其代谢途径和分子机理并不清楚。本项目申请者在筛选获得能以吡唑醚菌酯作为唯一碳源和能源生长的假单胞菌USTBP-01等菌株的基础上，拟优化其培养条件并研究其降解特性。同时继续分离识别吡唑醚菌酯降解过程中的中间代谢产物，获得其反应动力学数据，并得出相应规律，揭示吡唑醚菌酯降解过程中的反应步骤及其关键酶，进一步克隆关键酶基因。在分子生物学和生化水平上阐明吡唑醚菌酯微生物降解机理，为此类农药的合理使用及其引起环境问题的生物控制和生物修复提供理论基础和技术指导。

吡唑醚菌酯作为甲氧基丙烯酸酯类杀菌剂的主要类型正持续在我国大范围使用，残留危害大，自然环境中的微生物降解是其脱毒的主要途径，但其代谢途径和分子机理并不清楚。本研究从夏威夷大学采集的土壤中筛选得到两株可高效降解吡唑醚菌酯的混合菌，其最高可降解初始浓度为100 mg/L的吡唑醚菌酯，且降解率可达98%。此外，通过16S扩增子测序对原始土壤样本及其在吡唑醚菌酯为唯一碳源中富集5次后的富集液进行微生物菌群分析，结果表明原本土壤样本中种类丰富的微生物经富集操作后，仅变形菌门和拟杆菌门类别的细菌得以存活，且富集液中微生物菌群在属类别上，丰度最高的均为假单胞菌属，此研究说明混合菌中该属类别的细菌起到协同降解作用。在此基础上，利用LC-MS/MS对降解代谢产物结构进行分析，表明其产物分子式为C17H16ClN3O2，据此得出其降解代谢途径为吡唑醚菌酯的叔胺基团水解脱去与N相连的乙酰基转化成伯胺基团，说明混合菌产生的乙酰水解酶催化了该反应。同时，本研究进行了代谢产物的潜在风险评估，发现代谢产物在相同温度下其在水中的迁移和扩散略低于吡唑醚菌酯。