| Fluoroglycofen is one of the most important pesticides in diphenyl ether herbicides,which was developed by Rohm And Haas Company.The molecular formula was C18H9ClF3NO3,and the molecular weight was 447.8.Fluoroglycofen was protoporphyrinogen oxidase inhibitor,and it was applicable to control weeds in wheat,barley,soybean,rice field.Fluoroglycofen had significant residual in the soil and the water,which would threat to ecological environment.Several studies found that the fluoroglycofen have high toxicity to fish and some other non-target organisms.The physical and chemical remediation of fluoroglycofen residue was probably to cause the secondary pollution.Microbial remediation had the advantages of safety,environmental protection,efficient and was regarded as the most valuable method.This paper isolated fluoroglycofen degradation strain,designed as KS-1,from soil collected from a pesticide plant.Based on its physiological and biochemical characters,and the result of the 16S rRNA homologue sequence,the strain was identified as Lysinibacillus sp..The gram staining,methyl red tests,indole test of strain KS-1 were negative,oxidase and catalases were positive.The surface of colony was smooth and bright,and the edge was neat with pale yellow,round.The strain could grow in a temperature range of 15?42?,the optional temperature was 30 ?.The optimal pH was 7.0.The strain KS-1 could grow well in glucose,fructose,lactose,yeast extract and peptone.The strain KS-1 could degrade 85.25%of 50 mg/L fluoroglycofen in 3d.The optimum condition of strain KS-1 degrading fluoroglycofen was 30?,pH 7.0.The strain KS-1 could degrade fluoroglycofen well when the concentration was below 150mg/L.The degradation efficiency was improved when added different carbon and nitrogen source such as glucose,fructose,and yeast extract.Adding different metal ions would influence the degradation of fluoroglycofen.The cell-free extract of strain KS-1 could produce a visible transparent halo on PBS solid plate with 100mg/L fluoroglycofen.Results of metabolic products showed metabolites of the degradation of fluoroglycofen were {5-[2-chloro-4-(trifluoromethyl)phenoxy]-2nitronhenvlacyl}hydroxyacetic acid,acifluorfen,5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate.The strain KS-1 could also degrade cypermethrin,permethrin,lactofen.The 3-phenoxy benzoic acid was identified in the final products of strain KS-1 degraded the cypermethrin,permethrin.Gene library of KS-1 was constructed by shotgun method.A visible transparent halo appeared when fluoroglycofen was degraded as the selection marker.A fluoroglycofen esterase gene was cloned from Lysinibacillus sp.KS-1,named fluE.The fluE gene consisted of 930 nucleotides,the G+C content was 38.28%,the ribosome binding site of GGGAGAAAGGA was located at the start codon ATG upstream of 9bp,which encoded 310 amino acids,no signal peptide sequence was found,the molecular weight was 32KDa.FluE belonged to esterase or lipase superfamily and showed the highest similarity with some esterase/lipase,e.g,esterase or lipase from Bacillaceae bacterium MTCC 10057(61%identity),a esterase or lipase from Bacillus sp.SgZ-8(58%identity),a esterase from Bacillus sp.UNC41MFS5(57%identity),a lipase from Viridibacillus arenosi FSL R5-213(56%identity),a esterase from Viridibacillus arenosi(56%identity).Design the expression primer of fluE,connected the purified PCR products with expression vector pET29a to construct a recombinant plasmid pET-FluE after double enzyme digestion.The recombinant protein FluE was highly efficient expressed in Escherichia coli BL21 and purified using Ni-beads.FluE is active to different length of carbon chain p-nitrophenol esters,the good catalytic activity for p-nitrophenol esters of short chain,with the increase of carbon chain length of the catalytic rate decreased.The FluE protein had catalytic activity for lactofen. |
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