| Pyrethroids were widely used for control of agricultural and sanitary pests,resulting in the increasingly explosion of their residues and hazards.Microorganisms are an important factor that determines the fate of pesticides in the environment.How to use them to degrade the pesticide residues has become the focus of environmental science.The degradation characteristics of Bacillus subtilis BSF01 were studied and the optimum degradation conditions were investigated on the basis of response surface methodology?RSM?in this study.Moreover,the degradation products of beta-cypermethrin?beta-cp?were identified by GC-MS.Based on the degradation products,the microbial degradation pathway of beta-cp were proposed.Furthermore,the gene involved in pyrethroids degradation was cloned and expressed in Escherichia coli BL21.The key enzyme responsible for degradation was purified,which properties were also studied.Saturation mutagenesis of the degrading enzyme was carried out by the molecular simulation software,and in vitro assay was conducted to verify the function of the degrading enzyme.What is more,the gene of quorum sensing regulator comA was cloned and expressed.Effect of the regulator on the transcriptional level of degrading gene was evaluated.The main results were summarized as below:?1?Study on the degradation characteristics of strain BSF01.The optimal conditions for beta-cp degradation by strain BSF01 were determined to be emperature at 34.5?,pH 6.7,and inocula of 0.11 g/L by response surface methodology?RSM?using Box-Behnken design.Under these conditions,strain BSF01 effectively degraded 50 mg/L of beta-cp,deltamethrin,cypermethrin,cyfluthrin,and lambda-cyhalothrin within 7 days of incubation with degradation rates of 89.4%,89.2%,86.9%,86.5%,and 76.8%,respectively.Furthermore,the degradation reaction followed the first-order kinetics and the degradation rate constant?k?of these pyrethroids were 0.2995?0.3811?0.5164?0.4476 and 0.4476 d-1,respectively.The half live(T1/2)were determined to be 2.31?1.82?1.34?1.55 and 2.99 d,respectively.Strain BSF01 tolerated and degraded high concentration of beta-cp up to 400mg/L.At the initial concentration of 25-400 mg/L,more than 60%of the added dose was degraded within 7 days.The degradation products of the beta-cp were characterized by GC-MS.Based on analysis of the metabolites,the biodegradation pathway of beta-cp in strain BSF01 was proposed.Beta-cp was firstly metabolized by hydrolysis of its ester linkage to yield3-?2,2-dichloroethenyl?-2,2-dimethylcyclopropanecarboxylateand?-hydroxy-3-phenoxybenzeneacetonitrile,which was unstable in the environment and was oxidizedtoform3-phenoxybenzaldehyde.Subsequentoxidizationof3-Phenoxybenzaldehyde resulted in the formation of 3-phenoxybenzoic acid that could be further transformed to 3,5-dimethoxyphenol,leading to detoxification of beta-cp.?2?Cloning of the pyrethroid-degrading gene.Homologous clone was conducted to catch a carboxylesterase gene ybfK?891 bp?from Bacillus subtilis BSF01.The recombinant plasmid pMD19T-ybfK could degrade 48.8%of beta-cp after 7 days of incubation.YbfK protein was classified to the family VI hydrolytic enzyme as?/?hydrolytic enzyme by analyzing its conserved domain and amino acid sequence alignment,and it has a typical pentapeptide motif of“-Gly-X-Ser-X-Gly-”conserved in esterases.?3?Expression and purification of YbfK,and the properties of the purified enzyme.The expression vector pET32a?+?-ybfK was successfully constructed and the recombinant protein YbfK was expressed after induction with IPTG.The recombinant protein YbfK was purified by nickel agarose gel.The concentration of purified YbfK was 210.8 g/mL that possessed 7.5 U/mL of the activity against beta-cp.The YbfK enzyme exhibited excellent activity to beta-cp from 25?to 45?and tolerated a wide range of p H?5.0-9.0?,showing its preferable thermal and pH stability.?4?Study on molecular simulation and point mutation of YbfK protein.Three-dimensional structure of YbfK was constructed by homologous modeling.The?/?/?“sandwich”structure mainly composed bilateral alpha helix and internal beta turn.The deep YbfK binding pocket was in a irregular shape with a wide“mouth”,which helped the ligand enter the pocket and combine firmly.There were hydrophobic amino acid residues like leucine?Leu?and phenylalanine?Phe?near the binding site of YbfK and beta-cp by molecular docking.In addition,there was no hydrogen bonding observed between YbfK and beta-cp but van Edward force and other electrostatic forcesno hydrogen bonding between YbfK and beta-cp,play a role mainly by Van der Wals force and other electrostatic force.Five key amino acids were predicted to be in the interaction of YbfK and beta-cp by alanine scanning mutagenesis and saturation mutagenesis.Afterward,these five point mutations L64P,K92Y,L130R,F161G and L172G decreased their degraded ability significantly,and the degradation rates against beta-cp after 7 days of incubation were determined to be 15.2%,27.9%,13%19.3%,and 34.8%,respectively.?5?The role of quorum sensing?QS?in beta-cp degradation by strain BSF01.Bacillus subtilis QS transcription factor gene comA?645 bp?was cloned.The expression vector pET-SUMO-comA was successfully constructed and the recombinant protein ComA was expressed after induction with IPTG.The concentration of purified ComA was 2000 g/mL.The results of quantitative real-time PCR showed synergistic changes between ybfK and comA mRNA relative expression.When the initial concentration of beta-cp was 50-200mg/L,ybfK and comA mRNA relative expression increased with the increase of concentration,while it reached 400 mg/L,both relative expression of mRNA was significantly decreased.ybfK and comA mRNA relative expression increased significantly compared with control after 1 day of culture.Howere,both ybfK and com A mRNA relative expression declined after 3-7 days of incubation.DNA pull-down assay proved that ComA fusion protein can combine and interact with the sequence of ybfK promoter in vitro. |
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