Expression And Characterization Of Organophosphorus Hydrolase From Pseudomonas Pseudoalcaligenes In Pichia Pastoris

The major ingredients of organophosphorus pesticides?OPPs?are organic phosphorus compounds,which are acetyl choline?ACH?analogue,thus OPPs could effectively inhibit the acetylcholinesterase activity and kill insects.Due to high-efficiency,faster and lower cost,OPPs are widely used as pesticides.OPPs make a great contribution to increase food production and agriculture development,and guarantee for human being's grain safety.However,as universal pesticides,OPPs can also affect the nervous system of humans and animals.Nowadays the ecological environment pollution of OPPs has already threatened the human living and development.With the improvement of living standard,people pay more attention to food security.To reduce the pollutions of organophosphorus pesticides,many approaches have been developed to process pesticide residue.However,all of those methods have flaws,such as high energy consuming,noneconomic and the exist problems in application.Organophosphorus hydrolase?EC 3.1.8.1?is a kind of three phosphoric acid esterase that can specifically degrade organophosphorus compounds,reducing the toxicity of organophosphorus compounds and avoids high costs and secondary pollution.In this study,the coding sequence of organophosphorus hydrolase gene from Pseudomonas pseudoalcaligenes was modified according to the codon preference of Pichia Pastoris,and the optimized coding sequence of organophosphorus hydrolase?ophcM?was synthesized.The synthetized fragment was cloned into the P.pastoris expression vector pHBM905A-BDM.To improve the enzyme production,we describe a novel approach to express OPHCM efficiently with a biobrick assembly method in vitro.Four recombinant plasmids containing 1-4 copies of ophcM-expressing cassettes were constructed and named pHBM905BDM-ophcMl,pHBM905BDM-ophcM2,pHBM905BDM-ophcM3 and pHBM905BDM-ophcM4,respectively.The recombinant plasmids were transformed into P.pastoris GS115 by electrotransformation.Recombinants were screened and cultured with 1%?V/V?methanol induction.The copy number of integrated ophcM was determined by performing qPCR of genomic DNA from recombinant P.patoris clones.Recombinants harboring progressively increasing expression cassettes were named ophcM-1C,ophcM-2C,ophcM-3C,ophcM-4C.In present study,we examine the influence of gene dosage on the expression level of OPHCM.Real-Time PCR was successfully applied to quantify the expression of mRNA in the recombinant P.pastoris.qPCR results demonstrated that increase in ophcM expression cassettes number significantly enhanced the yield of mRNA.The expression levels of OPHCM was maximum in Pichia pastoris strain with 2 expression cassettes and minimum in Pichia pastoris strain harboring 4 expression cassettes.This higher yield may be attributed to the enhanced the yield of recombinant protein.However,the expression level did not always correlate with increasing copy number of expression cassettes,high copy number do not contribute to increase product of recombinant protein.The maximum yield and specific activity in P.pastoris harboring two-copy tandem ophcM-expressing was inoculated in 5-1 fermenter with 2-1 medium,the recombinant grown at 28?,pH 5.0 and induced at 26?,pH 5.0,after a 144 h induction in high-density fermentation with specific activity of 12.85 U/mg.In this study,the we establish a novel approach to purified and the crude enzyme was purified.The recovery of enzyme activity was 70%and purification fold was 2.8.The organophosphorus hydrolase was characterized.An optimum activity for the recombinant trypsin was observed at 50? and pH 11,respectively.Various additives were presented in our study,metal ions,organic solvents showed little affected on the activity of enzyme,and Cd²⁺,Mg²⁺,Glycerine show an activation on the enzyme activity.In present study,the coding sequence of organophosphorus hydrolase from Pseudomonas pseudoalcaligenes was transformed according to the codon bias of Pichia Pastoris,and a series of recombinant plasmids harboring progressively increasing expression cassettes of the ophcM gene and obtained recombinant multicopy P.pastoris strains.Results suggest that gene dosage and codon optimization play a significant role in optimizing OPHCM expression and gene dosage was not always lead to higher expression.The enzyme showed a wide pH and temperature range.Furthermore,the recombinant enzyme performed a tolerance to most organic solvents and metal ions,those properties make it an appropriate candidate for industrial applications and working in a complex environment.