Isolation,Identification And Degradation Mechanism Of Lactofen-Degrading Strain Bacillus Sp.YS-1

Lactofen is a typical diphenyl ether herbicides and it is widely used to control broadleaf weeds of various crops such as soybeans,cotton,peanut,and tomato.Lactofen has low water solubility,tight binding to the soil,and easily caused residues in the soil.The environmental problems caused by the residues of lactofen have attracted increasing attention of researchers.Microbial degradation plays an important role in the removal of residues of lactofen.Up to now,some lactofen-degrading strains have been isolated,but the reported functional genes involved in lactofen degrading are only two.In this study,a strain,named YS-1,capable of degrading lactofen was isolated,and its degradation characteristics and metabolic pathways were studied.The esterase genes,rhoE and rapE,involved in the degradation were cloned and expressed,and characteristics were studied.Enantioselectivity of RhoE and RapE degrading lactofen,diclofop-methyl and quizalofop-ethyl were analyzed.1.Isolation,degradation characteristics and metabolic pathways of the degradation strain of lactofenIn this paper,a strain capable of degrading lactofen,was isolated and identified from farmland soil contaminated with herbicides.The strain,named as YS-1,was identified as Bacillus sp.by the analysis of physiological and biochemical characteristics,and 16S rRNA gene sequence.Strain YS-1 could degrade about 97.6%of 50 mg/L lactofen within 15 h.The optimum temperature and pH for the degradation of lactofen by YS-1 were 40? and 8.0,respectively.The efficiency of YS-1 degradation lactofen could be promoted by Co2+,Fe3+,Mg2+,Al3+,Zn2+ and Mn2+(0.1 mM).The effects of Al3+ was the most obvious,the relative degradation rate was 119.5%.The efficiency of YS-1 was strongly inhibited by Cu2+(0.1 mM)and the relative degradation rate was 18.9%.The metabolites formed in the degradation of lactofen by strain YS-1 were analyzed by HPLC-MS/MS and the degradation pathway was proposed.At first,the two ester bonds on the side chain were hydrolyzed by YS-1 and formed acifluorfen.Then,strain YS-1 reduced the nitro group to the amino group on the phenyl ring of acifluorfen.At last,the amino group was acetylated to yield acetylated aminoacifluorfen.The results of hydroponic experiments showed that lactofen could significantly inhibit the growth of cucumber and sorghum.But the last metabolite,acetylated aminoacifluorfen,had no obvious influence on the growth of cucumber and sorghum seedings.2.Cloning of esterase genes rhoE and rapE in lactofen-degrading strain YS-1Two esterase genes,rhoE and rapE,were cloned from strain YS-1 by constructing the gene library.The aligned amino acid sequences were aligned in the UniProtKB/Swiss-Prot(Swissprot)database in NCBI.The highest similarities of sequences were 28%and 88%,respectively.Both RhoE and RapE had no signal peptide and belong to intracellular enzymes.RhoE and RapE had the typical conserved motif GXSXG and the catalytic triad of Ser-His-Asp/Glu of esterase.RhoE belonged to the family ? of bacterium lipolytic enzymes while RapE was a member of the family V.The homology of the amino acid sequence of rhoE was low and was a new esterase gene.3.Expression,purification and characteristics of RhoE and RapEThe soluble protein RhoE and RapE were expressed and purified.It was found that RhoE and RapE were active on both ester bonds on the side chain of the lactofen.Lactofen could be hydrolyzed to acifluorfen by RhoE and RapE according to the results of HPLC-MS/MS.Besides,RhoE and RapE could also hydrolyze the ester bonds on the side chain of herbicides diclofop-methyl and quizalofop-ethyl.The optimum temperature and pH for RhoE degradation lactofen were 35? and 8.0,respectively.Enzyme activity of RhoE could be strongly inhibited by Zn2+and Cd2+(0.1 mM).The optimum temperature and pH for esterase RapE degradation lactofen were 25? and 8.0,respectively.Enzyme activity of RapE would be strongly inhibited by Ni2+,Cu2+,and Cd2+(0.1 mM).RhoE could maintain 29.9%relative enzyme activity after incubation for 30 min at 70?,while RapE was completely inactivated.Both RhoE and RapE could maintain over 70.0%enzyme activity when pH is 10.0.RhoE and RapE had higher affinity and catalytic efficiency for short-chain p-nitrophenyl ester with carbon chain length shorter than 10 and lower affinity and catalytic efficiency for long-chain p-nitrophenyl ester with a carbon chain length longer than 10.These findings revealed that RhoE and RapE were esterases rather than lipases.The catalytic efficiency of RhoE for p-nitrophenyl esters determined in this study was significantly higher than that of RapE.However,RapE had a higher affinity for lactofen than RhoE.4.Enantioselectivity analysis of RhoE and RapE for lactofen,diclofop-methyl and quizalofop-ethylBoth RhoE and RapE showed significant enantioselectivity and EF decreased gradually during the degradation of lactofen and preferred to degrade the S-(+)-lactofen.In the degradation of diclofop-methyl and quizalofop-ethyl by esterase RhoE and RapE,EF increased gradually and the degradation rate of S-(-)-enantiomer was quickly,and there showed enantioselectivity.