The Soluble Prokaryotic Expression Of EstA For Pyrethroid Pesticides Degradation And The Primary Study On The Degradation Of3-phenoxybenzoic Acid

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.