Degradation Of Quizalofop-P-ethyl By Corynebacterium Sp.Z-1 And Cloning And Expression Of Its Gene QfeH

Quizalofop-P-ethyl was an aryloxyphenoxypropanoate(AOPP)herbicides.Quizalofop-P-ethyl was mainly used in the fields of soybean,peanut and cotton that kills gramineous weeds by inhibiting acetyl-CoA carboxylase.Quizalofop-p-ethyl and its metabolites were genotoxic and could affect the liver’s function although quizalofop-P-ethyl is a low-toxic herbicide.Due to its extensive applications,residual quizalofop-P-ethyl in the environment had been caused by widespread concern.Biodegradation was the main pathway of quizalofop-P-ethyl to be degraded in the environment.However,the biodegradation of quizalofop-P-ethyl mainly focuses on the isolation and identification of degrading strains and their metabolic pathways,the degradation characteristics of quizalofop-P-ethyl-degrading strain and the related cloning and expression of enzyme genes are rarely reported.In this study,a quizalofop-P-ethyl-degrading bacterial strain Z-1 was isolated.The esterase gene responsible for initial degradation was cloned,and the characteristic of the enzyme and the mechanism of quizalofop-P-ethyl degradation were researched.This study further enriched the microbial resources for the biodegradation of AOPPs and provided a theoretical basis and technical support for the bioremediation of quizalofop-P-ethyl in the environment.The main results are as follows:1.Strain Z-1 was isolated from the activated sludge of a quizalofop-P-ethyl production plant by the enrichment culture method.Strain Z-1 was preliminarily identified to be a species of genus Corynebacterium by physiological and biochemical identification and 16S rRNA homologue sequence analysis.The optimal growth temperature and pH for strain Z-l were 30℃ and 8.0,respectively.Strain Z-1 was sensitive to ampicillin,gentamicin,erythromycin,penicillin,roxithromycin,chloramphenicol,tetracycline,streptomycin and kanamycin,but it was resistant to low concentrations(25 mg/L)of erythromycin and roxithromycin.2.The optimal degradation condition of quizalofop-P-ethyl by strain Z-1 were analyzed and optimized by response surface methodology.Temperature,pH and inoculation amount were selected as factors and the degradation percentage of quizalofop-P-ethyl was selected as the response.The influence degree of three factors followed the order:temperature>inoculation dose>pH.The result showed that the optimal condition for degrading 20 mg/L quizalofop-P-ethyl in the mineral salt medium was 37℃ pH 7 and 3.5%.The strain Z-1 could degrade 98.8%of 20 mg/L quizalofop-P-ethyl within 3 h in the optimum condition.Strain Z-1 could also efficiently degrade 50 mg/L of fenoxaprop-P-ethyl,clodinafop-propargyl,haloxyfop-P and cyhalofop-butyl.The HPLC and LC-HRMC analysis showed that the probable biodegradation pathway of quizalofop-P-ethyl by strain Z-1 are as follows:The ester bond of quizalofop-P-ethyl was hydrolyzed to form quizalofop acid and ethanol.3.The complete genome of the strain Z-1 was sequenced.According to the metabolic pathways and reported literature,the possible esterase gene qfeH was analyzed.The gene qfeH was successfully cloned by heterologous expression which could hydrolyze the quizalofop-P-ethyl.The esterase encoded by gene qfeH belongs to cutinase.4.The degradation activity of enzyme QfeH was characterized.The optimal temperature and pH for QfeH catalysis were 42℃ and 8.0,respectively.The QfeH activity increased by 20%in the presence of Ni2+and diminished by 80%in the presence of Cr3+.Methyl alcohol,alcohol,isopropanol,EDTA,SDS,Tween-20,TritonX-100 and N-ethylmaleimide all had an inhibitory effect on QfeH activity.The QfeH activity remained about 13%of the maximal activity in the presence of EDTA or Tween-20.The reaction kinetics of QfeH was also determined.The maximum rate constant(Vmax)and the Michaelis constant were 0.08 U/mg and 11.74 μM,respectively.The kcat/Km value was 3.83×103 M-1 s-1.