Isolation,Identification Of Lactofen Degrading Strains And Degradation Mechanism Studies

Lactofen is a member of the diphenyl ether herbicides that was introduced by PPG Industries.It is commonly used to control annual broad-leaved weeds in soybeans,cereal crops,potatoes and peanuts.The use of this herbicide is increasing,resulting in varying degrees of environment pollution.Several studies indicate that this herbicide have high toxicity to some non-target organisms,such as fish and aquatic invertebrates,with LC50 values less than 0.02 mg L-1 to shrimp and 0.46 mg L-1 to fish.Besides,lactofen is likely to be carcinogenic at high doses.The environment fate of lactofen is closely related to microbial degradation and transformation.It is theoretically and practically significant for the removal of lactofen residues to explore the microbial role in this process.Up to now,several papers involving diphenyl ether herbicides-degrading strains and identification of intermediates have been published.However,the biochemical and molecular basis for the microbial metabolism of lactofen has not been illustrated.The aim of this study was to isolate lactofen-degrading strains,study their degrading characteristics and investigate the degradation pathway of lactofen.We also cloned the key gene involved in lactofen degradation and further researched on the enzyme properties,which would be helpful to illustrate the degradation mechanism of lactofen in biochemical and molecular level.Moreover,a study on the application of the isolated strain for remediation of lactofen-polluted soils was also carried out.In this study,two lactofen-degrading bacterial strains,named Za and M-8,were isolated from a herbicides-polluted farmland soil sample by the conventional enrichment culture technique.Based on the phenotypic,physiological,and biochemical tests and phylogenetic analysis,strain Za was characterized as Bacillus sp.16S rRNA gene sequence analysis indicated that strain M-8 was most closely related to Terrimonas ferruginea KACC 11310(97.1%),Terrimonas aquatica LMG 24825T(96.3%)and Terrimonas lutea KACC 13047T(95.3%).Levels of DNA-DNA relatedness between strain M-8 and T.ferruginea KACC 11310T and T.aquatica LMG 24825T were 54.3%and 23.0%,respectively.The major fatty acids of strain M-8 were iso-C15:0(36.67%),iso-C17:0 3-OH(12.39%)and summed feature 3(comprising C16:? 6c and/or C16:1? 7c)(25.05%).The predominant respiratory quinone of strain M-8 was menaquinone-7(MK-7).The DNA G+C content of strain M-8 was 47.0 mol%.On the basis of the phenotypic,physiological,and biochemical tests,chemotaconomic,phylogenetic analysis,DNA G+C content and DNA-DNA hybridization,strain M-8 represents a novel species in the genus Terrimonas,for which the new species name Terrimonas rubra sp.nov.is proposed.The type strain is M-8T(=CCTCC AB 2010401T= KCTC 23299T).The results of degradation experiment revealed that strain Za could degrade 94.8%of the initially added 50 mg L-1 lactofen after 4 days of inoculation and 80.1%of the initially added lactofen was degraded by strain LY-2 within 4 days.However,only a total degradation of 40.2%was observed after 4 days of inoculation with strain M-8.Brevundimonas sp.LY-2 showed enantioselectivity in degradation of lactofen with(R)-(-)-lactofen being degraded faster than the(S)-(+)-form.However,enantiomers of lactofen were observed to be degraded at an approximately equal rate by Za.Two metabolic products were detected and identified as desethyl lactofen and acifluorfen during the degradation of lactofen by M-8 or LY-2.Moreover,strain Za could completely transform lactofen to form aminoacifluorfen with desethyl lactofen and acifluorfen being detected as transitory intermediates.The results of metabolic products showed that lactofen was finally transformed to acifluorfen by M-8 and LY-2 via hydrolysis of the ester bonds.The degradation pathway of lactofen by Za might involve the hydrolysis of the ester bonds and reduction of the nitro group to yield an amino compound.The gene lacH and lacE,encoding lactofen-hydrolase,were cloned from Brevundimonas sp.LY-2 and Brevundimonas sp.Za,respectively.Sequence analysis indicated that lacH consists of 921 bp,encoding a protein of 306 amino acids,and a putative signal peptide at the N-terminal was identified with the most likely cleavage site between Ala 28 and Ala 29.lacE consists of 1419 bp,encoding a protein of 472 amino acids and no potential signal sequence was found.LacH shared moderate identities with some a/p-hydrolase fold proteins and contained a typical Ser128-Asp233-His286 catalytic triad and the conserved pentapeptide GXSXG(residues 126 to 130).The phylogenetic analysis showed that LacH belongs to family V of esterase and lipase.LacE showed the highest similarity with some esterase/lipase and also contained the typical catalytic triad of Ser189-Glu310-His399 and the conserved pentapeptide GXSXG(residues 187 to 191).Based on the phylogenetic analysis,LacE belongs to family ?.LacH and LacE were successfully expressed in Escherichiacoli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography.The molecular mass of LacH and LacE were approximately 30 kDa and 52 kDa,respectively.Both of the recombinant protein exhibited optimal activity around 40 ? at pH 7.0.The activity of LacH was strongly inhibited by more than 70%with the presence of Hg2+ and Zn2+,while the presence of Ni+ caused approximately 35%inhibition.Moreover,the addition of M2+,Mg2+,Cu2+ and Co2+ showed only little effect on the enzyme activity(10 to 20%inhibition).The activity of LacE was strongly inhibited by Al3+ with approximately 80%inhibition,while Li+,Co2+ and Cd2+ showed 30 to 50%inhibition.LacH and LacE showed the highest activity with p-nitrophenyl acetate,and the activities decreased with the increase of the acyl chain lengths.No significant lipolytic activity was observed with esters containing an acyl chain length longer than ten carbon atoms,indicating that both of LacH and LacE should be classified as an esterase and not a lipase.The degradation of lactofen by LacH was enantioselective with(R)-(-)-lactofen being degraded faster than the(S)-(+)-enantiomer,while(S)-(+)-lactofen was preferentially degraded over the(R)-(-)-form by LacE.The phytotoxic effect of aminoacifluorfen on maize was explored by pot experiment and the results showed that aminoacifluorfen in soil had no significant phytotoxicity on the growth of maize.Inoculation with strain Za could mitigate the inhibitive effect of lactofen at 4,6 and 8 mg kg-1 on maize growth to a varying degree.