【作者】 刘娜；

【导师】 茆灿泉； 许雷；

【作者基本信息】 西南交通大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 拟除虫菊酯类农药是目前使用广泛的有机农药之一。3-苯氧基苯甲酸是拟除虫菊酯类农药降解的中间产物,其生物学毒性和迁移率较菊酯类农药大。我国农业生产对拟除虫菊酯农药的依赖程度与日俱增,其直接结果是导致由该农药残留及其降解产物所产生的环境问题日益严重,因此,拟除虫菊酯类农药及其降解产物的生物降解已成为环境生物技术领域的研究热点。本研究以课题组前期克隆得到的拟除虫菊酯类农药降解基因estA(genbank: DQ906143)为出发基因,构建诱导表达系统,获得可溶性蛋白,并通过优化发酵条件,获得具有实际应用价值相关应用参数。对于探索本实验室前期筛选所得的拟除虫菊酯降解菌4-D对该农药降解产物3-苯氧基苯甲酸降解性能的研究,为后期获得3-PBA降解相关基因及构建多降解酶基因表达载体及工程菌提供可行性论证。研究结果表明：成功构建重组表达载体pMAL-c2X-estA质粒并转化至宿主菌BL21(DE3),获得酯酶estA基因诱导表达系统；通过SDS-PAGE凝胶电泳、以α-乙酸萘酯为底物的酶活性检测及Western blot方法,确认外源蛋白EstA在大肠杆菌宿主菌中成功表达并具有酯酶活性；在无Ca2+参与诱导的情况下可溶性表达也能进行；菌体细胞液中含有可溶性EstA蛋白及部分不可溶的包涵体蛋白,后者为诱导表达条件的优化,奠定了基础；含pMAL-c2X-estA质粒的重组基因工程菌可溶性原核表达诱导条件的优化实验表明,其最佳诱导条件是：Ca2+浓度1mM,诱导初始OD600为0.7,诱导剂IPTG浓度0.1mM,诱导时pH为7,最佳诱导温度为26℃,诱导时间17.5h。优化后的酯酶酶活较未优化前可提高近2倍；3-苯氧基苯甲酸的降解实验表明：在使用高效液相色谱进行3-PBA检测的过程中,直接采用乙腈作为提取液,与样品以1：1比例混合均匀,经过滤处理直接上机检测,所得结果符合线性关系,且重复性好；实验室前期分离得到的一株可降解拟除虫菊酯农药的4-D菌株在12d内对250mg/L的3-苯氧基苯甲酸的降解率可达到50%。更多还原

【Abstract】 Pyrethroid pesticide is one of the organic pesticides which are widely used at present. However, as an intermediate product in hydrolysis of the ester linkage of pyrethroid pesticide, the3-phenoxybenzoic Acid (3-PBA) has a higher biological toxicity and removal rate than the pyrethroid pesticides themselves. Along with an increasing dependence on the pyrethroid pesticides in agricultural pest control in China, the pyrethroid pesticides and its metabolite3-phenoxybenzoic acid (PBA) have exerted critical biological impacts on the environment. The biodegradation of pyrethroids and3-PBA, therefore, is becoming a hotspot in the research of environment biotechnology.In this paper, we construct an expression system by ligating the esterase gene estA (genbank:DQ906143) for pyrethroid pesticides degradation to obtain the soluble protein. In addition, the correlation application parameters are achieved by optimizing the expression conditions. Moreover, the research of the capacity of4-D strain being capable of degrading pyrethroids pesticides degrading3-PBA provides a feasibility argumentation for obtaining3-PBA degradation gene and constructing expression vectors as well as genetic engineering bacteria with multiple degradation enzyme gene. The main results of the work in this paper are as followed:The recombinant expression vector pMAL-c2X-estA plasmid is constructed and transformed into the host strain BL21(DE3) successfully, which leads to the acquirement of esterase estA gene inducible expression system. Via the SDS-PAGE gel electrophoresis, the enzyme activity assay using the a-naphthyl acetate as substrate and Western blot method, it is confirmed that the target protein EstA in Escherichia coli host strains expressed successful and has a esterase activity as same as in the absence of Ca2+. Besides, the cell liquid contains soluble EstA protein and some insoluble inclusion body protein, which provides the possibility for the optimization of induction and expression condition.The optimization of the soluble prokaryotic expression conditions for genetic engineering strain indicates the optional expression condition:the incubation temperature is26℃; the initial OD value of cell concentration is0.7; the concentration of IPTG is0.1mM; the induction time is17.5h. As a result, double expressed protein can be obtained under the optimal condition.The3-PBA degradation experiment shows that in the process of testing the content of3-PBA by high performance liquid chromatography(HPLC), the results conform to be linear and have a good repeatability by using acetonitrile as the extraction reagent while the ratio of acetonitrile and sample being1:1. Moreover, strain4-D is able to degrade50%of250mg/L3-PBA within12d.更多还原