Study on the Degradation Effect of Three Organophosphorus Hydrolase Mutant on Sarin

Qiamin Gu,Chuanxin Zhao,Hailing Xi,Min Liu,Gaoyun Chen,T Zhu,A Sharifi,M Anpo

doi:10.1051/e3sconf/202127103054

Abstract

Organophosphorus hydrolase can effectively degrade organic phosphorus compounds such as sarin. In this study, we constructed a recombinant Bacillus subtilis mutant expressing organophosphorus hydrolase, measured the effect of the mutant on the degradation rate of nerve agent sarin, and selected the optimal mutation scheme. Three different hydrolase mutant genes, 257L, 257Y and 303T, were ligated to PMA0911 vector and transferred into Bacillus subtilis WB800 to construct the target recombinant strain successfully. The recombinant bacteria secreted the target protein by fermentation. The effect of enzyme protein on the degradation of sarin was determined by the benzidine method. The optimal mutant was screened, and its enzymatic performance was explored. The effects of three organophosphorus hydrolase mutants on the hydrolysis rate of sarin were detected. The results showed that the 257Y mutant accelerated the hydrolysis of sarin significantly. Point mutation can improve the enzyme activity of wild-type organophosphorus hydrolase to a certain extent, laying the foundation for subsequent in-depth research.

Highlights

With the improvement of human life requirements and the increasing development of industrial production, many artificially synthesized pollutant compounds that are difficult to be rapidly degraded and transformed by natural microorganisms have entered the natural world
The transformants were coated on the selective plate containing kanamycin, and cultured overnight at 37°C and 180r/min to obtain the target recombinant bacteria. 2.2.3 Activity determination of recombinant organophosphorus hydrolase The recombinant strain was inoculated into 30ml TB expression medium and incubated at 37°C and 180r/min for 24h
The strain with h257l mutation was numbered as No.1, the strain with 257y mutation was numbered as No.2, the strain with 303t mutation was numbered as No.3, and the strain with non mutant Organophosphorus hydrolase (OPH) was numbered as No

Summary

Introduction

With the improvement of human life requirements and the increasing development of industrial production, many artificially synthesized pollutant compounds that are difficult to be rapidly degraded and transformed by natural microorganisms have entered the natural world. Organophosphate compounds (Ops), which are mass-produced and used on a large scale at home and abroad, have caused environmental pollution and damage to the ecological balance to a point that cannot be ignored. The excessive application of these pesticides pollutes the soil and water, and at the same time destroys the ecological balance of nature. Organophosphate nerve agents (such as sarin and VX) with similar structures are highly toxic and lethal organophosphate compounds. These residual organophosphate compounds can inhibit the activity of acetylcholinesterase, cause the accumulation of acetylcholine, trigger a series of symptoms of neurotoxicity, and even cause death

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Discussion

Conclusion