High-level Extracellular Secretion Of Methyl Parathionhydrolase Of Plesiomonas Sp. M6 In Pichia Pastoris And Its Immobilization Method

Methyl parathion Hydrolase is derived from a Pseudomonas sp.M6 which capable of degrading methyl parathion.MPH can effectively and specifically degrade methylparathion.Methyl parathion,paraoxon,parathionand other carbon-phosphorus bonds or organic groups containing phosphoric acid derivatives,are the main ingredients of organic phosphorus insecticide,organic phosphorus herbicide,organic phosphorus nerve gas and et al.The toxicity of these components to the organism is mainly manifested in the inhibition of acetylcholinesterase activity,resulting in a large accumulation of acetylcholine on the posterior synaptic membrane,leading to excessive stimulation of the nervous system.Blamed for the widespread use of organophosphorus pesticides in many countries,this situation has led to serious water,soil,atmospheric environmental pollution and ecological damage,a serious threat to human life safety.Although a variety of organophosphorus hydrolases have been found,but the low expression level,high production costs hinder their industrial production.In this study,transformation of methanol nutrition P.pastoris GS115,we got a high yield of methyl parathion hydrolase strain.The results are as follows:1.Gene synthesis of methyl parathion hydrolase MPH-RThe methyl parathion hydrolase MPH gene sequence from Plesiomonas sp.M6 was searched in NCBI and the original signal peptide sequence in the mph gene was removed,and added 6 his tags at the end of this sequence.After translation into the amino acid sequence,overlapping PCR primers was designed according to the codon preference of Pichia pastoris,Synthesized the new gene mph-Ris 912bp in length,encoding a total of 304 amino acids.2.Construction of the recombinant vector pHBM905BDM-mph-Rand expression in P.pastoris GS115The synthesized gene was ligated into the pHBM905BDM expression vector by restriction enzyme digestion and then obtain pHBM905BDM-mph-R.SalI was used to digest the recombinant vector,and transform it to the P.pastoris GS115 competent cells,then select the positive colonies were inoculated on BMGY medium shake flask.After 48 hours of methanol induction,the expression level of MPH-R in 500 m L shake flask increased to 1.9U/mL,and the specific activity which was 15.8 U/mg.And the expression level increased to18.4U/mLin 5 L basal salt high density fermentation medium,and the expression level was increased to 9.7 times.Wild-type MPH was almost unable to achieve secretory expression in P.pastoris,the expressed livel was about 0.03 U/mL.It has been reported that the expression level of MPH is about 0.48U/mL by other methods,but is not reached theindustrial production requirements.We improved the expression of MPH-R by changing the signal peptide of wild-type MPHand optimizing the codon according to the codon preference of P.pastoris.The experimental data shows that the expression level of MPH-R in P.pastoris is 63 times that of wild-type MPH,which is 4 times ofother literatures.3.Purification and Enzymatic Properties of MPH-RNi-NTA affinity chromatography was used to purify MPH-R.The optimal temperature and pH were 39?and 11,respectively.A lot of metal ions were used as additives to detect the variation of the activity of R-MPH,it was apparent that Ni²⁺,Co²⁺,and Mg²⁺at the concentration of 1m M enhanced the activity up to 196%,201%,and 154%.The excellent characteristic provides a good choice for industrialization.4.Study on immobilization of MPH-RSelect chitosan as immobilized carrier material,glutaraldehyde as crosslinking agent immobilization machine Phosphatase MPH-R.Chitosan is a good biocompatible and environmentally friendly material that can be effectively covalently bound to MPH-R.And the MPH-R catalytic efficiency was improved compared with the free enzyme.Compared with the immobilized MPH-R and the immobilized MPH-R,we found that although the immobilized enzyme was similar to the optimum temperature and optimum p H of the free enzyme,it was more adaptable to different temperatures and pH.High activity,and chitosan immobilized enzyme thermal stability and pH tolerance than the free enzyme has been significantly improved.And the immobilized enzyme can be recovered in use,subjecting to repeated reactions.Chitosan immobilized MPH-R retains activity of about 50%or more after100 replicates.The excellent characteristic provides a good choice for industrialization.