Degradation Mechanism Of Chlorpyrifos-degrading Strain Cupriavidus Nantongensis X1~T And Molecular Mechanism Of Stepwise Oxydehalogenation Via Enzyme TcpA

Chlorpyrifos is an organophosphorus insecticide,which is widely used to control agricultural pests on economic crops and public health pests in households.For mammals and aquatic organisms,chlorpyrifos is not only neurotoxic but also genotoxic.The continued large-scale use of chlorpyrifos in agriculture poses a threat to the environment,food safety,and public health security.This potential ecological and health risks caused by chlorpyrifos residues exposed to the environment have received increasing attention.Biodegradation is considered as an economical and effective remediation technology for environmental pollutants.Due to the bacteriostatic activity of TCP,most of the currently degrading strains can only degrade chlorpyrifos,and cannot further degrade its metabolite TCP.There are few reports of further degradation products of TCP,as well as the mechanism of microbial degradation,molecular genetic mechanism,degradation genes,and degradation enzymes.Therefore,in this thesis,a degrading strain that can efficiently degrade chlorpyrifos and its metabolite TCP,Cupriavidus nantongensis X1~T(a novel species of the genus Cupriavidus),was selected.Based on the whole genome sequence of strain X1~T,the degradation characteristics of chlorpyrifos and its metabolite TCP were studied.Prokaryotic expression was used to explore the catalytic mechanism and degradation mechanism of TCP degradation enzymes.In terms of environmental pollutant remediation,it provides a theoretical basis for the degradation of TCP and similar organic pollutants.The main results are as follows:1.Degradation kinetics showed that strain X1~Tcapable degrade 92.2%of 100 mg/L chlorpyrifos and 87%of 20 mg/L TCP within 24 hours.2.Analysis of the complete genome sequencing data found that the plasmid of strain X1~T contained an opd B gene,which encodes an organophosphate hydrolase for chlorpyrifos hydrolysis;a TCP degradation gene cluster was found on chromosome 1,where the tcp A(2,4,6-trichlorophenol 4-monooxygenase)and fre genes(NAD(P)H:flavin reductase)were identified to be the key genes for TCP degradation.An RT-q PCR detection method for strain X1~Twas established,and the optimal reference gene was rpo B gene.It was determined that during the TCP degradation process,all genes in the TCP degradation gene cluster were up-regulated.The degradation enzymes2,4,6-trichlorophenol 4-monooxygenase and NAD(P)H:flavin reductase was regulated by upstream Lys R transcriptional regulators.3.TcpA and Fre were expressed in E.coli BL21(DE3)respectively.The expression vector,IPTG concentration,and induction temperature were optimized to obtain soluble fusion proteins of degradation enzymes.The fusion protein was purified using amylose resin and Ni-NAT column,respectively.In vitro degradation experiments show that the purified degrading enzyme has degradation activity,and can degrade 90%of 10 mg/L TCP within 2 hours.Two dechlorinated metabolites,3,6-dichloro-2,5-dihydroxypyridine and3,6-dihydroxypyridine-2,5-dione were detected and identified.The degradation of TCP via TcpA and Fre was a stepwise oxidechlorination process.Ion chromatography and molar ratio analysis further determined that all three chlorine atoms on TCP could be removed by degrading enzymes.4.Degradation kinetics of TCP structural analogs(2,3-DCP,2,4-DCP,2,5-DCP,2,6-DCP,3,4-DCP,3,5-DCP)were detected.The results showed that the degrading enzyme TcpA was preferred to degrade 2,3-DCP,2,4-DCP,2,5-DCP,and 3,5-DCP than2,6-DCP,3,4-DCP.A three-dimensional model of TcpA was obtained by homology modeling.The molecular docking was used to calculate the binding site of FAD and 6kinds of dichlorophenols.An activity pocket of 206Phe,101Arg,and 448Gly amino acid residues were verified by point mutations.Dichlorophenols can form hydrogen bonds with amino acid residues in the active pocket.The shorter the hydrogen bond,the higher the degradation activity.5.The metabolites of dichlorophenols showed that the degradation of chlorophenol via TcpA and Fre was a stepwise oxydehalogenation process,firstly removing the para-chlorine,and then removing the ortho-chlorine.And the oxidation ability is positively related to the electron-withdrawing ability of the paragroup of the benzene ring.Degradation of structural analogs of chlorophenol found that TcpA has dual dehalogenation and denitrification functions.These functions will allow the initial rate-limiting step of the detoxification pathways of halogenated phenols and nitrophenols to be hydroxylated by a single monooxygenase TcpA,which is valuable for industrial pollutants bioremediation.