假单胞菌dcp-1酯酶EstE1降解多菌灵的分子机理研究

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.

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。

获得了多菌灵降解菌假单胞菌dcp-1和芽孢杆菌DJ61。dcp-1以多菌灵为唯一碳源生长，且碳源对dcp-1生长和降解多菌灵的效率有调控。dcp-1以多菌灵为唯一碳源时，生长较慢但降解多菌灵效率高；底物多菌灵的浓度亦对dcp-1降解多菌灵有影响，dcp-1在80 mg/L的多菌灵时，降解效率达到最高。重组表达的酯酶EstE1的酶活力为80 U/mg。EstE1的最佳温度为50°C， pH为8.0，且为耐高温的酯酶。Ca2+, Li+, Mg2+ and Mn2+对EstE1活性影响小，但Cd2+, Co2+, Cu2+, Hg2+, Ni2+, Pb2+和Zn2+均能够显著降低该酶的活性。利用鸟枪法构建了dcp-1的基因文库筛选多菌灵降解相关基因，得到尿卟啉脱羧酶和菌毛亚单位C编码基因。在多菌灵存在时，尿卟啉脱羧酶的RNA转录量情况下上升，说明尿卟啉脱羧酶与假单胞菌dcp-1降解多菌灵过程有关。dcp-1降解多菌灵的中间代谢产物为2-amino benzimidazole，提示dcp-1具有其它细菌降解多菌灵相似的途径。MTT法测定dcp-1降解后的多菌灵代谢产物对MCF-7细胞细胞存活率影响变小，说明dcp-1可将多菌灵降解为毒性较小的代谢产物，至于是否是完全矿化为无毒的CO2和H2O，则还需要进一步的实验数据支持。.降解多菌灵的dcp-1在以丁二酸为碳源的培养基中可产生大量的荧光铁载体pyoverdine，该荧光铁载体的激发波长为410 nm，发射波长为460 nm。呋喃唑酮可特异引起pyoverdine的荧光猝灭，作用时间仅需1 min，。呋喃唑酮的浓度与pyoverdine的荧光猝灭量呈线性正相关关系，因此，利用pyoverdine的荧光猝灭发展了可检测呋喃唑酮并对其进行定量的传感检测方法。该方法的线性范围为2-160 μM，LOD为0.5 μM。.假单胞菌dcp-1分泌的pyoverdine具有强的铁离子螯合力，这一特点会导致其它细菌处于缺铁环境。项目组利用了pyoverdine强螯合铁离子的特点，尝试在灿烂弧菌、哈维氏弧菌、副溶血弧菌和溶藻弧菌等常见病原菌的培养过程中，添加pyoverdine作为螯合剂，pyoverdine可抑制弧菌的生物量、生物膜，刺激弧菌的铁载体和荧光的产生。外源添加的pyoverdine可在一定程度上干扰灿烂弧菌引起的仿刺参病变。