Preliminary Studies On Expression Of Organic Phosphorus Degradation Enzyme Gene (opdA) In Escherichia Coli And Bacillus Subtilis

Organphosphorus (OPs) insecticides were usually used as insecticides throughoutthe world and had made a significant contribution to agriculture for its advantages ofhigh efficiency, wide range of prevention and control and low cost. However, Ops did alot of harm to mammals and human health and pollution for environment．Ops led toexcessive pesticide residues of vegetables and fruits and other agricultural products. Theresearch of the degradation of organophosphorus pesticide had been paid more attentionby researchers. It is an effective method to take advantage of microbial probiotics anddigestion enzymes to degrade chemical pesticides for soil and waterSome esearchers showed that some microbial probiotics could efficiently degradeorganophosphorus pesticide by organophosphate digestion enzymes reaction. And it isan effective method to directly take advantage of digestion enzymes than using ofmicrobial probiotics.The gene of opdA was isolated from Agrobacterium radiobacter P230. OpdA isthe most application organophosphate degradation enzymes for its high substrate rangeand catalytic efficiency. OpdA is effective on hydrolyzation of various OPs.In this study, we described the cloning and expression of plasmid pET-28b-opdA inEscherichia coli (E. coli), and experimental programs were carried out to obtain anoptimal transformation efficiency of Bacillus subtilis electroporation experimentalprogram and optimization conditions for electroporation of Bacillus subtilis cells.Thenwe constructed secretion expression system in Bacillus subtilis of opdA to overcome thedifficulties of the recombinant proteins expression in Escherichia coli system in theform of inclusion body.1. Studies on expression of organic phosphorus degradation enzyme gene (opdA) inEscherichia coli.Using restriction enzymes Xho I and BamHI for double enzyme digestion of opdAand vector pET-28b, we inserted opdA gene into vector pET-28b to construct arecombination plasmid pET-28b-opdA, which was subsequently transformed intoBL21(DE3), producing engineering strain BL21(DE3)/pET-28b-opdA. The OD600was 0.5, after induction with1.25mmol/L IPTG at37℃for5h, the Kmof OpdA forparathion-methyl was42.6μmol/L, and the maximum reaction velocity (Vmax) was32.669μmol/L min. The specific activity of OpdA was24.48U/mg.2. Optimization of electroporation Processing of Bacillus subtilis.In order to obtain an optimal transformation efficiency of Bacillus subtiliselectroporation and optimization conditions for electroporation of Bacillus subtilis cells,a shuttle vector for E. coli and Bacillus subtilis, pHY300PLK were used. Factorsincluding growth medium, electroporation medium, recovery Medium, plasmidconcentration and pulse strength were investigated. Results showed that the optimalvalues for concentration of the growth medium, electroporation medium, recoverymedium，plasmid concentration and pulse strength are LB+0.5M sorbitol,0.5Msorbitol+0.5M mannitol+10%Glycero, LB+0.5M sorbitol+0.38Mmannitolrespectively,the pressure was2.0KV/cm and the concentration of DNAplasmid was100ng/μl.the highest electroporation processing of Bacillus subtilis was1.45x10~3个/μg DNA.3. Studies on secreted expression of organic phosphorus degradation enzyme gene(opdA) in Bacillus subtilis.Using restriction enzymes BamHI for enzyme digestion of opdA and vector pHT43,we inserted opdA gene into vector pHT43to construct a recombination plasmidpHT43-opdA, which was subsequently transformed into WB800N, producingengineering strain WB800N/pHT43-opdA. The OD600was0.7, after induction with1.25mmol/L IPTG at37℃for27h, the Kmof OpdA for parathion-methyl was44.2μmol/L, and the maximum reaction velocity (Vmax) was268.39μmol/L min. Thespecific activity of OpdA was107.94U/mg and the kcatwas648.29min(-1). The enzymeactivity of OpdA by secreted expression increased nearly twice times than reportedresults.