Directed Evolution And Application Of Bulky Organophosphorus Flame Retardant Degrading Enzymes

Organophosphorus hydrolases(OPHs)are widely used for the catalytic degradation of organophosphorus compounds.OPHs deriving from microorganisms can degrade a variety of organophosphorus pesticides and nerve agents.They have important applications in the fields of pollution management and national defense medicine.However,the common OPHs are unable to hydrolyze bulky organophosphate flame retardants(OPFRs)with challenging leaving groups,and little is known about its catalytic mechanism,which hinders the effective treatment of this new class of environmental pollutants.Therefore,it is of great significance to carry out molecular modification of OPHs through directed evolution and improve its ability of degrading bulky OPFRs.PoOPH is a class of natural lactonase deriving from gene mining.It has no homology with the natural enzyme Sb-PTE which has been found to degrade OPFRs.There are significant differences in some aspects such as protein skeleton and catalytic center structure.Through directed evolution,PoOPH obtained the degradation activity of organophosphate substrate.In this work,we adopt the strategies of semi-rational design and high-throughput screening based on whole-cell biosensors to engineer PoOPHvs,a previously screened enzyme which can degrade triphenyl phosphate(TPHP),to improve the catalytic efficiency of TPHP.The site-saturation mutagenesis library was constructed around the active pocket of PoOPHV5 within 6(?).It was found that the position 56 was a sensitive site affecting enzyme activity,and then site-directed saturation mutation was performed on position 56.The mutant PoOPHV6 was identified to improve the hydrolysis ability of TPHP significantly with a specific activity of 30.23 U g-1,a 3.5-fold increase compared with PoOPHV5.Through molecular docking,the structural mechanism of PoOPH enzyme was further analyzed.By characterizing the enzymatic properties of the best mutant PoOPHV6,we found it displayed the highest activity at 40℃ and pH 9.0 in a 50 mM Tris-HCl buffer,the T5015 values of PoOPHV5 and PoOPHV6 were 64.0℃ and 60.5℃,and the engineering process had little effect on the thermal stability of PoOPH.For TPHP,the kcat and KM values for PoOPHV6 were 12.31×10-4 s-1 and 0.23 mM,respectively,with 4-fold kcat/KM compared to PoOPHV5.In addition to TPHP,PoOPHV6 also showed an improving catalytic activity of lactone substrate dihydrocoumarin(DHC),with a specific activity of 55.95 U mg-1,a 2-fold increase compared with PoOPHV5.This work aims to engineer the lactonase PoOPH with catalytic promiscuity into an OPH of excellent degradation activity for bulky OPFRs.The study result will bring a great breakthrough for the discovery of new OPFRs degradation enzymes,and further promote the development and application of enzymes.