Study On Surface Display Of Organophosphorus Hydrolase On Pichia Pastoris

Methyl parathion irreversibly inhibits the activity of acetylcholinesterase,leading to the accumulation of acetylcholine,which leads to subsequent neurological loss and eventual death.Therefore,it is widely used as an insecticide,but it also causes environmental pollution in the process.Organophosphorus hydrolase is a kind of enzyme that can destroy the phosphate ester bond of organophosphorus molecules and finally make the toxicity of organophosphorus compounds disappear.In this study,organophosphorus hydrolases from different sources(oph gene from Pseudomonas pseudoalcaligenes and mph gene from Plesiomonas sp.M6 strain)were established in Pichia Pastoris GS115 surface of two display modes.First,construct P.pastoris direct surface display expression vector p PICZ?A-HA-mph-SED1?p PICZ?A-HA-oph-SED1,E.coli expression vector p ET23a-CL7-mph?p ET23a-CL7-oph;then linearize the P.pastoris recombinant vector and transform it into GS115 strain,use zeocin-resistant YPD plate to screen the strains with high activity for shaking flask expression;simultaneously transform the vector p ET23a-CL7-mph and p ET23a-CL7-oph into E.coli expression strain BL21(DE3),purify the fusion protein CL7-MPH?CL7-OPH and bind it to strain GS115/p PICZ?A-HA-IM7-SED1 to form an indirect surface display strains.Flow cytometry and immunofluorescence microscopy were used to test the efficiency of the two surface display methods.The results showed that both methods successfully anchored MPH and OPH to the cell surface,and the direct and indirect display efficiency respectively were 81.17%?57.44%;81.12%?70.07%.The direct and indirect surface display strains were tested for whole-cell viability,and the results showed that the optimal reaction temperatures of HMS and HOS were 20? and 50?,respectively,and the optimal reaction pH was 10 and11.the optimal reaction temperatures of HIS-CM and HIS-CO were 40? and 50?,respectively,and the optimal reaction pH was 8 and 9.5 mmol/L heavy metal ion had little inhibitory effect on the enzyme activity of HMS,HOS and HIS-CM,but slightly greater inhibitory effect on HIS-CO.When the substrate concentration was 50 mmo L/L,the enzyme activities of HMS and HOS were 10.95 U/mg and 13.66 U/mg,respectively.The enzyme activities of HIS-CM and HIS-CO were 6.57 U/mg and 6.51 U/mg,respectively.The enzymatic activity of HOS was 4.54 U/mg different from the maximum activity of 18.2U/mg exhibited by known organophosphorus hydrolase on Saccharomyces cerevisiae surface.Through the study of enzymatic properties,it was found that the direct surface display organophosphorus hydrolase has a wide range of pH and temperature,and is not inhibited by metal ions or even can be activated by some metal ions,indicating that the enzyme is suitable for working in a more complex environment,expanding its application range.In this study,Saccharomyces cerevisiae cell wall protein SED1 was used to display MPH and OPH on the surface of P.pastoris GS115.The strong CL7 /Im7 interaction pair was used to indirectly display MPH and OPH on the surface of P.pastoris GS115.The P.Pastoris GS115 display methods in this study can be effectively used in the biodegradable enzyme system of methyl parathion,and provide an effective expression method for other proteins that are difficult to express and purified,and also provide a reference for the subsequent researches on the display of organophosphorus hydrolase on the cell surface.Furthermore,the results of the study on organophosphorus hydrolase were enriched,and finally,the method and basis for the display of other enzymes in P.pastoris GS115 cells were provided.