Isolation And Identification Of An Efficient Quizalofop-p-ethyl Degrading Bacterium And Cloning,expression Of Key Enzyme Gene

Quizalofop-P-ethyl(QE)is a selective aryloxyphenoxy propanoate(AOPP)herbicide,which is mainly applied to the control of the post-emergence of annual and perennial grassy weeds,such as cotton,soybean,rape and other broadleaf crops by inhibiting acetyl-CoA carboxylase(ACCase)to prevent the formation of fatty acids in grass species.The extensive presence and accumulation of QE and its residues have caused global environment pollution and ecological destruction.Moreover,QE can not only pollute the soil,water and atmospheric environment directly,but also is harm to animals,especially to human health by enrichment through the food chain.Consequently,the degradation of QE and its residue is attracting public attention.In natural environment,QE can be degraded through photolysis,chemical action and biodegradation,among which microbial degradation plays the most important role due to the cost-effective and without secondary pollution.This research aimed at isolating the bacteria that can degrade QE efficiently,studying their degradation characteristics,and providing the theory base for the bioremediation of environment pollution with QE.Meanwhile,the key enzyme encoding gene involved in QE degradation was cloned and over expressed,and the properties and catalytic mechanism of this enzyme were investigated.A highly efficient bacterial strain was isolated from a QE-manufacturing wastewater treatment facility in Anhui Province using enrichment culture method with QE as a sole carbon source.On the basis of the morphological,biochemical characters and alignment analysis of the 16 S rRNA sequence,the bacterium was identified as Ochrobactrum sp.and named QE-9.Strain QE-9 could grow well in the temperature range of 28-32 ?.The optimal pH and temperature for its growth were 7.0 and 30 ?,respectively.Glucose and yeast extract were the best carbon and nitrogen source for its growth,respectively.It was resistant to chloramphenicol.The optimum temperature and pH for the degradation were 30 ? and 8.0,respectively.Strain QE-9 could utilize QE as the sole carbon source for its growth,and metabolize more than 98% of QE(0.5 mmol?L-1)within 6 days.The degradation efficiency of QE showed a positive co-relation to the inoculation level.The high quality genomic DNA of strain QE-9 was extracted by high-salt precipitation and used to construct genomic library by the shotgun method.One positive colony was obtained by transparent halos from approximately 12,000 transformants.Sequence analysis revealed that the strain carried a plasmid with a 4058 bp insert,including three complete ORFs(orf1-orf3),corresponding to genes encoding a beta-lactamase classC family protein,a pyridoxine 5'-phosphate oxidase and a membrane protein,respectively.ORF1,designated estqe,was 1149 bp in length and encoded a putative 382 amino acid protein.A putative promoter region upstream of estqe,including-35(TTCCCC)and-10 regions(TGCAAGAAT),was found,as well as a putative ribosomal bindingsite(AAGAGG)8 bp upstream of the GTG start codon.The putative amino acid sequence of the estqe gene showed 99% identity to a putative beta-lactamase protein from Pseudomonas stutzeri(AHY43609).EstQE possessed an SCTK motif corresponding to the conserved motif(S-X-X-K),which is a feature of the N-terminal region of members of esterase/lipase family VIII,as well as proteins in the class C beta-lactamase family.In addition,the L-L-X-H-X-X-G and YSN motifs,which have been described in several esterase family VIII members,were also found.However,the classical esterase/lipase family consensus motif G-X-S-X-G was not found in Est QE,although this is also not conserved in all esterase family VIII members.A similar phenomenon was observed in previous studies on other members from of esterase family VIII.Since strain QE-9 could not grow on the LB agar plates containing beta lactam antibiotics such as Amp,or penicillin(100 mg?L-1),this suggests that EstQE had no beta-lactamase activity.A possible explanation is that the tertiary structure of the EstQE has changed during the evolutionary process,leading to steric hindrance.Phylogenetic analysis based on multiple alignment of EstQE and other family VIII esterase proteins indicated that EstQE clustered as part of esterase family VIII and was most closely related to esterase EstC(AAC60471)from Pseudomonas fluo-rescens(68% identity).Additionally,the amino acid sequence identity of EstQE and other AOPP herbicide hydrolase : the FE-hydrolyzing enzymes FeH(JF970231,KF601763,KP307927)as well as the CyB-hydrolyzing enzyme ChbH(ADW65729)were 34.7%,34.9%,12.5% and 16.4%,respectively.Full-length estqe gene was amplified by PCR and ligated into expression vector pET-29a(+)for functional expression in E.coli BL21(DE3).The resulting recombinant esterase EstQE was purified with Ni-agarose gel affinity column and subjected to the enzymatic properties study.The pI value of EstQE was estimated to be 6.5.The molecular mass of the native EstQE was about 43 kDa,which was consistent with calculated molecular mass of the deduced amino acid sequence,suggesting that the EstQE was a monomer.LC-MS analysis confirmed that QE was transformed by EstQE to form quizalofop acid(QA)through the hydrolysis of the ester bond,further demonstrating that EstQE was an esterase.Using QE as a substrate,the enzymatic properties of EstQE were measured.EstQE exhibited high activity over the temperature range of 25–55 ?,with an optimum temperature of 45 ?.EstQE retained 93% activity after incubation at 4 ?for 2 months.Even after incubation at 50 ? for 1 h,EstQE remained more than 40% of the original activity,showing that EstQE was highly stable.In addition,EstQE displayed high activity between pH 5.0-9.0,and the optimum esterase activity occurred at pH 8.0.EstQE was relatively stable at pH 7.0-9.0.Enzyme activity was severely inhibited by 1.0 mmol?L-1 Cu2+,whereas,1.0 mmol?L-1 Ca2+ and Mg2+ significantly increased enzyme activity to 142% and 122% of the control,respectively.The 10 mmol?L-1 protein inhibitors PMSF,pCMB,and DEPC as well as 1% detergent SDS strongly inhibited esterase activity.Furthermore,EstQE was capable of hydrolyzing a wide range of other AOPP herbicides,in the following order of catalytic efficiency,quizalofop-P-ethyl > fenoxaprop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > quizalofop-P-tefuryl > haloxyfop-P-methyl.For QE,the overall catalytic rate(Kcat)value of EstQE was 48.2 s-1 with a dissociation constant(Km)of 142.6 ?M.The catalytic efficiency value(Kcat/Km)under optimal conditions was 338 s-1?mM-1.These data suggested that EstQE was a potential candidate for remediation of AOPP herbicide-contaminated environments.