多菌灵是一种广泛应用于作物病害防治的氨基甲酸甲酯类杀菌剂，可在环境中长期残留，并能通过食物链的富集放大作用影响人类健康。目前已报道了多株能够高效降解多菌灵的假单胞菌，但关于其降解多菌灵分子机理的研究尚未见报道。申请人前期研究已证实假单胞菌酯酶参与多菌灵的降解过程，并实现了该基因在大肠杆菌中的克隆表达研究。基于此，本项目拟以假单胞菌dcp-1酯酶基因estE1为研究对象，采用蛋白表达、基因突变、基因敲除及过表达等技术，研究酯酶的生化特征，探讨其降解功能及相关的活性位点和结构域；利用液质联用技术鉴定多菌灵的降解产物，阐明酯酶催化的多菌灵降解过程；结合实时荧光定量PCR、Western blotting及凝胶阻滞等技术，解析环境因子影响酯酶表达调控的分子机理。本项目研究成果不仅能够阐明假单胞菌酯酶降解多菌灵的分子机理，而且可为多菌灵污染环境的生物修复提供有力的理论依据和技术手段。

Carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC) is a carbamate fungicide widely used to control a broad range of fungal diseases in agricultural crops. MBC is chemically stable and relatively persistent in the environment, and poses threat to human health through the food chain amplification. Although several Pseudomonas sp. strains capable of degrading MBC have been isolated, no relevant study on the molecular mechanism of MBC degradation by Pseudomonas sp. has been reported so far. In our previous studies, an esterase from Pseudomonas sp. dcp-1 had been proved to be involved in the MBC degradation, and the esterase gene had been cloned and recombined in Escherichia coli. In this project, we will focus on the molecular mechanism of carbendazim degradation catalyzed by the esterase EstE1 derived from MBC-degrading Pseudomonas sp. strain dcp-1. Protein expression, gene mutation, gene knockout and gene overexpression technologies will be employed to investigate the biochemical characteristics and identify the amino acid residues and domain critical for the catalytic activity of EstE1. HPLC/MS analysis will be used to investigate the MBC degradation pathway catalyzed by EstE1. Real-time PCR, Western blotting and electrophoretic mobility shift analyses will be carried out to elucidate the effects of environmental factors on the expression and regulation of estE1. The result of the present project can not only elaborate the molecular mechanism of MBC degradation catalyzed by esterase EstE1, but also provide theoretical basis and restoration strategy for bioremediation of MBC polluted environment.