| Sulfonylurea herbicides are a class of important herbicides used worldwide for controlling weeds in all major agronomic crops. The use of sulfonylurea herbicides has developed rapidly because of their high efficacies at low dosages and multi-crop selectivities. The sulfonylurea products are now the second largest kind of herbicides after the glyphosate, and more than 30 products have been commercialized. Most sulfonylurea herbicides are weak acids, vulnerable to acid hydrolysis under acidic conditions. However,in neutral to alkaline soils, some of the herbicides such as metsulfuron-methyl,chlorsulfuron, and ethametsulfuron-methyl, are degraded at a very slow rate and persist from several months to more than one year .The residues of herbicides in the soil seriously damage subsequent rotation of sulfonylurea sensitive crops like legumes and oilseeds,which can result serious agricultural loss. Thus, great concern and interest have been raised regarding the environmental behavior and degradation mechanism of sulfonylurea herbicide residues in soil.Great concerns have been raised about the persistence and degradation of sulfonylurea herbicide in the environment. Compared with other physicochemical methods,bioremediation is a cost-effective, convenient and security solution to eliminate the sulfonylurea herbicide residues in soil, sediment, and agricultural products. Although many sulfonylurea herbicide-degrading microorganisms have been isolated from the environment,there was no sulfonylurea herbicide-hydrolyzing enzymes purified and gene of sulfonylurea herbicide-hydrolyzing enzymes was cloned.S113, a bacterial strain, capable of degrading a variety of sulfonylurea herbicides, was isolated from sulfonylurea herbicide-contaminated soil and identified as Hansschlegelia zhihuaiae.Sulfonylurea herbicides with an ester structure in the aryl ring(e.g.,thifensulfuron-methyl, metsulfuron-methyl, bensulfuron-methyl,ethametsulfuron-methyl, and chlorimuron-ethyl) could also be de-esterified to their corresponding herbicidally-inactive parent acid forms by cell-free of S113.The herbicides inhibit acetohydroxyacid synthase (AHAS), a key enzyme in the biosynthesis pathway of branched-chain amino acids valine, leucine, and isoleucine in bacteria, fungi and plants. E. coli DH10B shows moderate resistance to sulfonylurea herbicide because it contains an active isozyme, AHAS I, which is highly sensitive to valine but resistant to sulfonylurea herbicide. Another isozyme,AHAS II,which is highly sensitive to sulfonylurea herbicide, is in an inactive form caused by a frameshift mutation that leads to a premature stop codon.Using Red-mediated recombination, the frame-shift mutation of the inactivated AHAS ? was eliminated to generate E. coli DH10B (ilvG+),which was highly sensitive to sulfonylurea herbicide. When grew on MSGM plate .containing 200 mg·L-1 valine, and 200 mg·L-1 leucine, 50 ?M thifensulfuron-methyl was enough to completely inhibit the growth of E. coli DH10B (ilvG+). If a thifensulfuron-methyl de-esterification gene was introduced and functionally expressed, the transformant could grow well due to its ability to convert thifensulfuron-methyl to thifensulfuron acid..Method to extract high quality total DNA from strain S113 was founded. Gene library of S113 total DNA was constructed by the method of shotgun cloning. Using E. coli DH10B (ilvG+) as the recipient strain for library containing approximately 15,000 transformants, one transformant that was able to develop visible colony on on basic medium plate containing 200 mg · L-1 valine, 50 ?M thifensulfuron-methyl. Degradation experiment showed that this transformant was able to degrade about 85% of the initially added 50 ?M thifensulfuron-methyl within 24 h of incubation and simultaneously produce an equal amount of thifensulfuron acid. The results have suggested that the insert fragment of the transformant contains the sulfonylurea herbicide de-esterification esterase gene. The sequencing result showed that the inserted fragment in the transformant was 5,143 bp; nine complete ORFs were identified by computer analysis. According to the results of the blastp program, one ORF shared similarities with some putative or hypothetical esterases (highest identity 37%). The ORF was subcloned to the linearized vector pMD18-T and transformed into E. coli DH10B. The resting cells of the subclone showed the ability to degrade thifensulfuron-methyl. Therefore, we concluded that this ORF was the target gene encoding the sulfonylurea herbicide de-esterification esterase and being designated as sulE.Sequence analysis indicated that sulE consists of 1194 bp, with a GC content of 51%, and encoding a protein of 398 amino acids. A signal peptide is at the N-terminal with the cleavage site situated between the amino acids Ala 37 and Glu 38, and resulted in a 361 residue mature protein. The results of a blastp search in the NCBI protein databases revealed that SulE showed low sequence similarities (highest identity 37%) with many hypothetical or putative alpha/beta hydrolase fold proteins, whose secondary structures are composed of alternating a-helices and ?-strands along the backbone. Of the characterized proteins, sulE shared only 29% similarity with esterase 731, which catalyzed the hydrolysis of halogenated cyclic compounds from an Alcaligenes strain, and less than 20% similarity with other characterized proteins.The sequence alignment of SulE with alpha/beta-hydrolase fold proteins indicates that the enzyme contains the typical catalytic triad of alpha/beta-hydrolase fold proteins, consisting of Ser245-His369-Glu268. However,the conserved pentapeptide sequence (Gly-XI-Ser-X2-Gly) around the catalytic serine residue of most alpha/beta-hydrolase fold proteins has not been found in SulE. The results suggest that SulE differs from previously reported esterases by the absence of sequence relatedness and substrate difference.The mature SulE was expressed in Escherichia coli BL21 (DE3), and was purified using Ni-nitrilotriacetic acid affinity chromatography. SulE is homologous dimmer with molecular mass 80 kDa. The optimal pH of SulE was observed to be 7.5-8.0. The enzyme was stable at the pH range 6.0 to 9.0, retaining more than 85% of the original activity after preincubation in the buffer at that pH range for 1 h. The activity of SulE was maximal at 40?. The enzyme was fairly stable up to 45 ?, retained approximately 85% of its activity at 45 ? for 1 h, retained 20-40% of its residual activity at 55 ?, and was completely inactivated at 65 ?. The enzymatic activity was inhibited by more than 90% with the metal ions Ag+,Cd2+ and Zn2+ (1.0 mM),the organic phosphorus pesticide methamidophos (2.0 mM), the Ser protease inhibitor PMSF, His modifier DEPC (0.5 mM), and thiol reagent pCMB and iodoacetamide (0.5 mM) and the surfactant SDS (1.0 mM), while the metal ions Ni2+ (1.0 mM) caused 40 to 50% inhibition of the SulE activity. The chelators EDTA and the surfactant Tween 80 failed to inhibit the enzymeThe substrate specificities of the enzyme were tested with various sulfonylurea herbicides as the substrates. All the sulfonylurea herbicides with a methyl or ethyl ester were substrates of the enzyme; the hydrolysis rates descended as follows:thifensulfuron-methyl > metsulfuron-methyl > ethametsul furon-methyl >bensulfuron-methyl > chlorimuron-ethyl. Sulfonylurea herbicides without an ester structure,such as chlorsulfuron and cinosulfuron, were not substrates of the enzyme. The HPLC-MS/MS results demonstrated that sulfonylurea herbicides with a methyl or ethyl ester were de-esterified to corresponding parent acids by the enzyme. SulE was able to hydrolyze p-nitrophenyl acetate and p-nitrophenyl butyrate, but not p-nitrophenyl caproate,indicating that the hydrolysis activities of the enzyme decreased with the increase of the aliphatic chain length of p-nitrophenyl esters.To study the possible physiological function of sulE in strain S113, a sulE-disrupted mutant ?sulE was constructed by insertion of a suicide vector pJQ200SK into the sulE gene.S113 degraded almost 100% of the 200 ?M thifensulfuron-methyl within 48 h and 84.4%of the 50 ?M metsulfuron-methyl within 72 h, respectively; while ?sulE lost the degradation ability, which indicated that sulE was the only gene responsible for sulfonylurea herbicide de-esterification in strain S113. The ?sulE and wt showed no growth differences in MSMM. However, wt showed significantly better growth than AsulE in the presence of thifensulfuron-methyl or metsulfuron-methyl, indicating that wt showed higher resistance to thifensulfuron-methyl and metsulfuron-methyl than AsulE. To evaluate if the sulE gene could be functionally expressed in eukaryote, the sulE gene was ligated into the S.cerevisiae-E. coli shuttle vector pRS 427; and transformed into yeast strain S. cerevisiae BY4741, to generate recombinant strain BYSulE. BYSulE almost completely degraded the 100 ?M thifensulfuron-methyl and 88.1% of the 20 ?M metsulfuron-methyl within 48 h of incubation; and it displayed significantly better growth than BY4741 which could not degrade the two herbicides, suggesting that sulE could increase the sulfonylurea resistance of S. cerevisiae. The overall results suggest that sulE is an excellent candidate for genetic engineering of sulfonylurea herbicide-resistant crops and bioremediation of sulfonylurea herbicide-contaminated environments.The recombinant plasmids pP43sulE was constructed by inserting sulE into the shuttle expression-secretion vector pP43NMK. pP43sulE was transferred into prototrophic B. subtilis BS36 to construct strain B. subtilis sulE.When inoculum size was 1.0×106 CFU·g-1 dry soil, pH was 7.5, temperature was 30?, the degradation rate reached 1 00% in soil which contained 0.1 mg`kg-1 metsulfuron-methyl. |
PDF Full Text Request