Characterization And Application Of Recombinant Organophosphorus Hydrolase Azohydromonas Australica OPH

Organophosphorus pesticides（OPs）have the advantages of high insecticidal efficiency and low synthesis cost,so they are widely used in agriculture and are currently the most widely used type of pesticides.However,the effective utilization rate of OPs in the environment is low and the residual OPs can inhibit the activity of acetylcholinesterase in mammals and insects,causing serious environmental and human health problems.Organophosphorus hydrolase（OPH,E.C.3.1.8.1）has many advantages such as high degradation efficiency,low toxicity of degradation products and a clear degradation mechanism for various OPs,so the research on degradation of OPs by OPH has received extensive attention.In this project,a new type of OPH（Azau-OPH）from Azohydromonas australica was identified.Firstly,the engineered bacteria E.coli-Azau-OPH was used to degrade various OPs efficiently.Secondly,the recombinant enzyme Azau-OPH was used to degrade various OPs and its enzymatic properties were studied in detail.Finally,the food-grade expression of Azau-OPH was explored.In this project,the gene of Azau-OPH was screened through the NCBI database and was connected to the expression vector p ET-22b（+）to construct a recombinant plasmid.The engineered bacteria E.coli-Azau-OPH carrying the target gene was used for heterologous expression and degrading OPs at a concentration of 50 mg/L.It was found that the engineered bacteria during the growth process could degrade dichlorvos by 100%within 2 h.The engineered bacteria collected by centrifugation could degrade 100%of dichlorvos and methyl parathion within 2 h,and 40%and 22%of profenofos and chlorpyrifos within 6 h,which has a potential ability to degrade OPs in the environment.The recombinant enzyme Azau-OPH was obtained after the engineering bacteria E.coli-Azau-OPH was purified by nickel column.The molecular weight of Azau-OPH was 35k Da.The results of the identification of the enzymatic properties of Azau-OPH showed that the optimum temperature,p H,and metal ion were 50℃,9.5,and Mn²⁺.The results of thermal stability and structural stability showed that the unfolding temperature（T_m value）of the enzyme and enzyme with 1 mmol/L Mn²⁺were 55.10°C and 62.89°C.The incubation of Mn²⁺could improve the thermal stability of Azau-OPH,allowing the activity of Azau-OPH to increase by more than 60%after 2 h incubation at 50°C.By studying the substrate specificity and reaction kinetics of Azau-OPH,it was found that when methyl parathion was used as the substrate,the specific enzyme activity,kinetic parameter Michaelis constant（K_m）,catalytic efficiency(k_cat),and the k_cat/K_m values were 4.94 U/mg,0.717 mmol/L,4.6 s^-1,and 6.41L/mmol.s,respectively.With 50 mg/L OPs as substrates,the enzyme could degrade 91%dichlorvos,80%profenofos,82%chlorpyrifos,33%malathion and 20%parathion within 10min.Finally,the recombinant plasmid p UB-p43-Azau-dal with the Azau-OPH target gene was constructed by the"simple cloning"cloning method using p UB110 without the resistance gene as the expression vector.Then the B.subtilis 1A751（dal^-）was used as the expression host.This research realized the food-grade expression of the recombinase Azau-OPH and improved the application safety of the enzyme.