| Lactofen,a member of diphenyl ether herbicides,is widely used in the control of broadleaf weeds in soybeans,cereal crops,potatoes and peanut fields.Due to its high activity and broad substrate spectrum,the field usage of lactofen is still increasing,which has caused part of lactofen to penetrate the environment and cause harm to the environment.It has been reported that microorganisms play an important role in removing pollutant residues in the environment.So far,a series of lactofen-degrading strains have been reported.However,there are few studies on the key genes and enzymes involved in lactofen degrading and molecular mechanism of enantioselective degradation.The enantioselectivity and its molecular mechanism are rarely mentioned.In this study,strain Edaphocola flava HME-24 was isolated for its lactofen-degrading function,and its degradation characteristics and metabolic pathways were further studied.Then the key gene lanE involved in the degradation was successfully cloned by a shot-gun method,and its expression,purification and enzymatic characteristics were studied.Finally,the enantioselectivity of three chiral herbicides by LanE was studied and the enantioselective degradation mechanism of lactofen by LanE was further revealed by molecular docking.The main contents and results of this study are as follows.1.Screening and polyphasic identification of the lactofen degrading strain HME-24In this study,a strain capable of degrading lactofen,was isolated from soil contaminated with herbicides by continuous enrichment.Strain HME-24 was Gram-stain-negative,strictly aerobic,non-sporulating.catalase-and oxidase-positive.Strain HME-24 has only one respiratory quinone MK-7.Polar lipids mainly include lipids(L),amino lipids(AL).phosphatidylethanolamine(PE)and glycolipids(GL).The main fatty acid is iso-C15:0,iso-C15:1 G and iso-C17.0 3-OH.Strain HME-24 has the highest 16S rRNA homology(99.5%)with Edaphocola aurantiacus H2.The ANI and dDDH between strain HME-24 and H2 are 94.8%and 59.2%,respectively.Based on the morphological.physiological.biochemical characteristics,comparative analysis of 16S rRNA gene sequence and chemical classification results,the strain was identified as a new species of the genus Edaphocola and named as Edaphocola flava sp.nov.HME-24.2.Degradation characteristics and metabolic pathways of lactofen by strain HME-24Strain HME-24 could degrade about 96.7%of 50 mg/L lactofen within 72 h but could not grow with lactofen,methanol or ethanol as the sole carbon source and energy source.The optimum temperature and pH for the degradation of lactofen by HME-24 were 35?and 7.0,respectively.1 mM of Mg2+,Cu2+,Ca2+,Ni2+,Zn2+,Fe3+,Co2+or Cd2+ could inhibit the degradation of lactofen by strain HME-24.1 mM of Co2+had the most significantly inhibitory effect,with an inhibition rate of about 60%.The metabolites were analyzed by HPLC-MS/MS,and the metabolic pathway was presumed to be that strain HME-24 first hydrolyzed the ester bond on the outer side chain of lactofen to produce desethyl lactofen,and then hydrolyzed the ester bond on the inner side to generate acifluorfen.3.Cloning of lactofen esterase gene lanEAn esterase gene lanE was obtained by constructing a gene library of strain HME-24 using the shotgun method.Gene lanE contains 1416 bp and encodes a protein containing 471 amino acids.LanE belongs to the family V of bacterium lipolytic enzymes.Compared with other esterases in UniProtKB/Swiss-Prot database,LanE has the highest identity(29%)to esterase EstD(Q9WYH1)in Thermotoga maritima MSB8,which suggested lanE was a novel esterase.Esterase LanE has a cleavage site between Cys23 and Gln24,which indicated that LanE has a signal peptide and was an extracellular enzyme.LanE also has a typical triplet catalytic site(Ser-Asp-His/Glu)and typical conserved motif GXSXG in the?/? hydrolase superfamily.4.Expression,purification and enzy matic characteristics of LanERecombinant esterase LanE was obtained by heterologous expression and purification.The enzymatic reaction showed that LanE can catalyze the cleavage of the ester bond of lactofen to form desethyl lactofen and subsequently acifluorfen,which is consistent with the degradation pathway of strain HME-24.Besides,LanE could also catalyze the hydrolysis of diclofop-methyl and quizalofop-ethyl.The enzymatic characteristics test showed that the optimal reaction temperature of LanE was 40?.LanE could retain more than 60%of the activity when incubated at a temperature of no more than 50? for 30 minutes.But the enzyme stability decreased sharply when the temperature exceeded 60?.The optimal reaction pH of LanE was 8.0 and it could retain above 60%of enzyme activity at pH 6.0-9.0.1 mM of Mg2+,Cu2+,Ni2+ or Al3+ has no significant effect on enzyme activity.1 mM of Mn2+,Ca2+or Co2+ can slightly enhance the enzyme activity of LanE,while the presence of 1 mM of Zn2+,Fe3+ or Cd2+ could severely inhibit the enzyme activity of LanE.The enzymatic kinetics experiments showed that LanE tended to degrade p-nitrophenyl ester with a short carbon chain,which indicated that LanE was an esterase rather than lipase.5.The enantioselective mechanism of lactofen by LanEThe esterase LanE showed enantioselectivity in the process of transforming lactofen,diclofop-methyl and quizalofop-ethyl,and preferred to degrade the S-enantiomers of these three herbicides.The I-TASSER automatic protein structure prediction software was used to simulate the three-dimensional structure of LanE,and the molecular docking between LanE and lactofen.was further simulated using Autodock software.The results indicated that both(S)-(+)-lactofen and(R)-(-)-lactofen could bind to the catalytic active center of LanE,Due to the change of the methyl orientation at the key position,the distance between the ester group and the catalytic key amino acid Ser255 in the(R)-(-)-lactofen and(S)-(+)-lactofen were 3.90 A and 3.18 A,respectively.These made LanE had a stronger affinity for(S)-(+)-lactofen,and it was easier for LanE to hydrolyze(S)-(+)-lactofen. |
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