| Quizalofop-ethyl(QE)is one of aryloxyphenoxy propanoate(AOPP)herbicides that kills weeds by inhibiting the activity of acetyl-CoA carboxylase.It is mainly applied for broad-leaved crops to control annual or perennial grassy weeds.The commercial raceme consists of(R)-QE and(S)-QE,while its herbicidal activity mainly comes from the(R)-QE.QE is a low-toxic herbicide,but its residue in the environment will cause phytotoxicity to the subsequent crops and affect the ecological safety of non-target organisms.(Rac)-QE,especially its different chiral isomers,have been paied much attention in their residual dynamics in the environment.Studies have shown that microorganisms play an important role in the degradation and metabolism of aryloxyphenoxypropionate(AOPP)herbicides.At present,the microbial degradation of QE is mainly concentrated in the(R)-QE(trade name is quizalofop-P-ethyl),while the degradation of the(S)-QE has not been reported.In addition,the enantioselective degradation of QE by the esterase that responsible for catalyzing the hydrolysis of QE is also rarely reported.This reaserch will isolate and screen microbial strains capable of degrading both chiral isomers of QE,clone the esterase gene responsible for the initial degradation of(Rac)-QE.and study the enantioselective degradation of(Rac)-QE by this esterase and its mechanism.1.Isolation,identification of QE-degrading bacteria and its degradation characteristics and metabolic pathwaysUsing enrichment culture method and cultivation with(Rac)-QE as the selective pressure,a QE-degrading strain,named QE-1,was isolated from the soil of an abandoned pesticide factory in Nanjing.Jiangsu Province.Strain QE-1 is a Gram-negative bacterium.Its optimum growth temperature is 30℃.the optimum growth pH is 7.0.the optimum growth NaCl concentration is 0.5%.On basis of bacterial morphological,physiolog ical and biochemical characteristics and 16S rRNA gene sequence analysis.strain QE-1 was identified as sphingobium sp.(R)-QE was used as the substrate to study the degradation characteristics of strain QE-1.The results showed that the optimal degradation temperature and pH for the degradation of(R)-QE by QE-1 was 30℃ and 7.0.Under the optimal degradation condition,the strain QE-1 could degrade about 98.5%of 20 mg/L(R)-QE within 96 h.Mg2+(0.1 mM)can promote the degradation of(R)-QE by QE-1,while Cu2+,Zn2+,Co2+,Cd2+,Ni2+and Mn2+ can significantly inhibit its degradation.Strain QE-1 is selective for the degradation of the two chiral isomers of QE,which is more inclined to degrade(S’)-QE.The substrate spectrum study found that strain QE-1 also has the abilities to degrade fenoxaprop-P-ethyl,Fluazifop-P-butyl,cyhalofop-butyl,clodinafop-propargyl,fluoroglycofen,lactofen and carbaryl.The LC-MS analysis showed that the pathway of strain QE-1 to degrade QE was to break the ester bond and formed quizalofop-ethyl acid(QA)and ethanol.2.Cloning and functional verification of QE hydrolase geneStrain QE-1 could generate transparent halo on LB plate containing 200 mg/L(Rac)-QE.Using this feature,we successfully cloned the QE hydrolase gene qeH from strain QE-1 by shotgun method.Through constructing qeH into the wide host vector pBBR1MCS-2,heterologous expression in E.coli DH5α.it was confirmed that the qeH gene was responsible for catalyzing hydrolysis of(Rac)-QE.After qeH knocked out through homologous recombination,mutant strain QE-M still has the ability to degrade(Rac)-QE,indicating that besides qeH,there were other QE-degradating enzyme genes in the strain QE-1.Gene qeH was 1.215 bp in length and encoded 404 amino acid.Alignment of amino acid sequence of QeH in UniProtKB/Swiss-Prot(Swissprot)database in NCBI revealed that QeH had the highest homology with β-lactamase MT1414(P9WLZ2)of Mycobacterium tuberculosis CDC1551.which is 37%.QeH did not have the classical esterase/lipase family consensus motif GXSXG,but it possessed a SMTK motif corresponding to the conserved motif(SXXK),which is a feature of the N-terminal region of members of esterase/lipase family Ⅷ.In addition,the LLXHXXG LXXXPGXXW.PLGMXDTXF and GGXG motifs.which have been described in several esterase familyⅧ members,were also found.The phylogenetic tree constructed based on the alignment of amino acids sequences.indicating that QeH belonged to the esterase Ⅷ family.3.Expression,purification and characteristics of QeHThe souble QeH was obtained by constructing the qeH gene into the high-efficiency expression vector pET-28a(+).then heterologous expressed in E.coli BL21(DE3)and purificated by Ni2+ affinity chromatography.Through LC-MS analysis.it was determined that QeH could hydrolyze the eater bond of QE,producing QA and ethanol.In addition,QeH can also hydrolyze fenoxaprop-P-ethyl,fluazifop-P-butyl,clodinafop-propargyl and cyhalofop-butyl.The optimal catalytic temperature for QeH were 40℃ and the optimal catalytic pH was 7.0.After incubating for 30 min at a reaction temperature(<45℃),the enzyme activity of QeH almost stayed constant;while the temperature was higher than 50℃,the enzyme activity suddenly changed to inactivate,indicateing its thermal stability was poor.Compared to acidic and alkaline buff,QeH had the highest enzyme activity and was most stable in neutral environment.SDS,Tween80 and Triton X-100 had obvious inhibition effects on QeH enzyme activity at 1 mM.QeH was more inclined to catalyze the cleavage of short-chain ester bonds,and its catalytic efficiency for p-nitrophenyl esters decreased with carbon chain length increased,which indicated that QeH is a carboxylesterase rather than a lipase.Among the five types of AOPP herbicides tested,QeH had the best affinity for fenoxaprop-P-ethyl and the highest catalytic efficiency for quizalofop-P-ethyl.4.Enantioselective degradation of(Rac)-QE by QeH and analysis of its underlying mechanismQeH showed obvious enantioselectivity for degradation of(R)-QE in the process of degrading(Rac)-QE.Combinating of the results that qeH gene mutant strains still degraded(Rac)-QE and strain QE-1 was more prone to degrade(S)-QE.it was suggested that strain QE-1 also had esterase genes which have higher preference for(S)-QE.Prediction of the three-dimensional structure of QeH and the docking simulation between QeH and(Rac)-QE showed that the binding sites of(R)-QE and(S)-QE were basically consistent with QeH.They mainly relyed on hydrophobic interaction and π-πstacking to bind to the hydrophobic cavity of QeH.and were located near the catalytic triad(Ser78-Lys81-Tyr189).However,due to the change of the ethyl orientation in(S)-QE,both the catalytic distance between the carbon atom of the carbonyl group and the oxygen atom of the catalytic center Ser78 and the catalytic distance between the oxygen atom of the ester group and the oxygen atom of the catalytic center Tyrl89 were significantly larger than those of(R)-QE.which made(S)-QE catalyze more difficultly than(R)-QE.Thus,it was speculated that this is the main reason for the chiral selectivity of QeH to degrade(Rac)-QE.In summary.Sphingohium sp.QE-1.which has the ability to degrade the two chiral isomers of(Rac)-QE.was isolated.A new esterase gene qeH.responsible for catalyzing the initial degradation of(Rac)-QE,was cloned by shotgun method.This research also initially clarified the mechanism of preferential degradation of(R)-QE by QeH,providing theoretical guidance,strains and enzyme gene resources for the microbial remediation of the environment polluted by chiral herbicide QE. |
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