Effect Of Ni²⁺ On Promoting The Degradation Of Profenofos By Cupravidus Nantongensis X1^T

Profenofos is a highly effective and broad-spectrum organophosphorus insecticide,which is widely used to control agricultural pests on economic crops such as cotton and corn.Profenofos has neurotoxicity and gengtic toxicity to organisms,potential ecological and health risks caused by profenofos residues exposed to environment have received increasing attention.Microbial degradation is considered as an economical and effective remediation technology for environment pollutants.The degradation of organophosphorus pesticides by microorganisms is mostly accomplished by producing corresponding degrading enzymes.In this thesis,Cupriavidus Nantongensis X1^T,a highly efficient degradation of organophosphorus pesticides isolated and screened in the laboratory as the research object.The mechanism of enhancing the activity of organophosphorus hydrolase in strain X1^T by Ni²⁺was studied.The main results are as follows:1.Different metal ions of 1 mM had different effects on the degradation of profenofos by strain X1^T,K⁺had no significant effect on the degradation of strin X1^T.Cr³⁺and Fe³⁺inhibited the degradation of profenofos by strain X1^T,while Ni²⁺,Mn²⁺,Ca²⁺,and Co²⁺had a promoting effect on the degradation of profenofos by strain X1^T,of which Ni²⁺had the best promoting effect.2.Adding 1 mM Ni²⁺at 6 h,8 h,12 h,and 18 h during the degradation of profenofos by strain X1^T increased the degradation rate constant of profenofos by 7.35,5.38,3.73,and 2.20times,respectively.Adding Ni²⁺at different times in the reaction would increase the degradation of profenofos by strain X1^T.3.Adding different concentrations of Ni²⁺（0,0.00001,0.0001,0.001,0.01,0.1,1,2.5,5,and 10 mM）,the degradation rates of profenofos by strain X1^T were 8.56%,10.83%,30.18%,36.79%,50.52%,53.62%,53.79%,54.23%,54.24%,and 55.26%,respectively.The addition of different concentrations of Ni²⁺promoted the degradation of profenofos by strain X1^T.When the concentration of Ni²⁺was 0-0.1 mM,the degradation rate of profenofos by strain X1^T gradually increased with the increase of Ni²⁺concentration.When the concentration of Ni²⁺was 0.1 mM,continued to increase the concentration of Ni²⁺,and there was no significant change in the degradation rate of profenofos by strain X1^T.4.The results of transcriptome sequencing and RT-q PCR showed that the expression of the organophosphorus hydrolase OpdB in strain X1^T increased after the addition of profenofos,but the addition of Ni²⁺did not promote the expression of the opd B gene,indicating that Ni²⁺did not affect the expression of the gene opd B during the degradation of profenofos by strain X1^T.5.The crude enzyme activity of strain X1^Ttreated with EDTA decreased compared to the original crude enzyme.The metal ions in the crude enzyme of strain X1^T were replaced by 1mM Ni²⁺,Mn²⁺,Ca²⁺,Co²⁺,K⁺,and Fe³⁺,respectively,The crude enzyme activity after refolding with Ca²⁺,K⁺,and Fe³⁺showed no significant change compared to the crude enzyme treated with EDTA.The crude enzyme activity after refolding with Co²⁺,Ni²⁺,and Mn²⁺increased compared to the crude enzyme treated with EDTA.The crude enzyme activity after refolding with Ni²⁺and Mn²⁺was higher than the original crude enzyme.The protein model was calculated by Alphafold2 to simulate the three-dimensional structure of OpdB.Autodock-vina was used to simulate the molecular docking between OpdB and PFF.The binding sites of OpdB and metal ions were verified by point mutations.The results of point mutation and metal replacement show that the activity of OpdB depends on the metal ions in the active center,and the metal ions in the active center can be replaced by different metal ions,and different metal ions have different effects on the activity of the enzyme.