果蔬拟除虫菊酯农药残留高效降解酶Cpde分子改造研究

项目名称： 果蔬拟除虫菊酯农药残留高效降解酶Cpde分子改造研究

项目编号： No.30871660

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 胡美英

作者单位： 华南农业大学

项目金额： 30万元

中文摘要： 拟除虫菊酯是农产品主要农药残留类型之一，国际上对其残留限量极其严格，是我国果蔬出口主要阻碍之一。项目组在首次发现链霉菌属和枝孢霉菌属对高效氯氰菊酯等拟除虫菊酯农药的高效降解作用、鉴定菌种的基础上，研究了降解菌对拟除虫菊酯农药的降解产物，分离获得了降解酶Cpde，研究了降解酶的主要性质，通过MALDI―OF―S测定酶蛋白序列，克隆获得Cpde基因部分序列；以扩大降解谱、提高降解率为改造目标，深入研究和分析了Cpde蛋白分子结构与拟除虫菊酯农药降解活性的构效关系；进一步确证降解酶蛋白结构，建立3D模型，构建反相筛选体系；运用易错PCR和DNA改组技术进行体外随机突变，提高有效突变率，并且采用共毒培养―C法快速筛选高效突变株，利用改造获得的高效菌株制备降解酶Cpde。本项目建立的反相筛选体系是突破DNA改组技术瓶颈，提高预期性改造成功率的有益尝试，本研究成果对于研制具有自主知识产权的农药残留高效生物降解剂具有重要的科学意义和实践价值。

中文关键词： 拟除虫菊酯；降解酶；生物降解；DNA改组技术；构效关系

英文摘要： Pyrethroid is one of the main pesticide groups and due to its persistency,agricultural products show high residue ratio of the chemicals belonging to this group. It causes main hindrance in the export of fruits and vegetables from China,because of the strict international rules about the residues in food commodities.We have found that Streptomyces sp.and Cladosporium sp.could efficiently degrade pyrethroid insecticide including beta-cypermethrin. The researches have studied on these strains in our laboratory including identification of the degrading strains,purification of the degrading enzyme(Cpde),cloning of the gene,protein sequencing using MALDI―OF―S and the main characteristics. In order to enlarge the degradation spectrum and increase the degradation efficiency, we have explored the structure-activity relationship between the cpde protein structure and degradation activity to pyrethroid insecticides,confirmed the protein structure and established the 3D model. A reversed-phase screening system has been constructed. Mutations were introduced into genes encoding proteins of degrading enzyme by error-prone PCR or re-combinative methods such as DNA shuffling. Efficient mutation strain have been obtained by co-toxicity culture and gas chromatography (GC). Prepared degrading enzyme(Cpde) from the strain obtained by transformation has been used.Reversed-phase screening system that has been constructed in this project will help to break through the bottleneck of DNA shuffling and to improve the success rate of expected characters transformation. The anticipated results will have important scientific significance,practical value to research the high efficiency pesticide residue biodegradation agent,and will have the intellectual important in this field.

英文关键词： Pyrethroid Insecticide;Degrading Enzyme; Biodegradation;DNA Shuffling;Structure-Activity Relationship