Study On The Degradation Mechanism Of Nicosulfuron By Bacillus Velezensis

As a broad-spectrum and highly effective sulfonylurea herbicide,nicosulfuron was widely used to protect maize crops from a variety of weeds.Like other sulfonylurea herbicides,nicosulfuron exhibits low volatility and photodegradation,thereby leading to long persistence in soil,surface waters and crops.With the extensive use of nicosulfuron,a series of environmental contamination problems has been emerged,such as the pollution to nearby aquatic systems,damage to crop rotation and effect on soil microbial population.Therefore,it is extremely urgent to develop effective strategies to remove nicosulfuron residues from the environment.Related studies have shown that microbial remediation has become one of the important means to solve the residues of sulfonylurea herbicides in the environment due to its safety,high efficiency,low cost and environmental friendliness.1.In this study,a bacterial strain CF57 was isolated from gut of earthworm(Eisenia fetida)and identified as Bacillus velezensis based on its colony morphological,physio-biochemical characteristics and 16S rRNA gene sequence.2.By single factor test and response surface analysis,it was found that the optimum degradation condition was 30.8?,pH 6.31 and inoculation amount 3.04%of strain CF57 in basal culture medium ?.Under the optimal conditions,the degradation rate of 100?300 mg/L nicosulfuron could reach more than 90%by CF57 in 5 d.In addition,strain CF57 could also tolerate and degrade nicosulfuron with an initial concentration of up to 500 mg/L,and showed a good degradation effect on the six sulfonylurea herbicides tested,including tribenuron-methyl,pyrazosulfuron-ethyl,rimsulfuron,sulfometuron-methyl,bensulfuron methyl and cinosulfuron.The metabolites of nicosulfuron was analyzed by LC/MS and two metabolites were mainly obtained.It was inferred that the possible metabolic pathway was mainly derived from the cleavage of the C-N bond of the sulfonylurea bridge.3.The localization of degradation enzyme indicated that the nicosulfuron degrading-enzyme was extracellular fraction.The extracellular crude enzymes were isolated by acetone precipitation method.DEAE-FF anion exchange chromatography column method and PAGE method and a total of 10 active components were obtained.The P3-4,P4-2 and P4-4 with higher activity were determined by MALDI-TOF-MS and were identified as Sugar phosphate isomerase,Leucine aminopeptidase and Arginase.4.According to the gene sequences of Sugar phosphate isomerase,Leucine aminopeptidase and Arginase,the soluble enzyme solution of three degrading enzymes was obtained by means of prokaryotic expression.The enzymatic properties of the three enzymes were studied by means of activity detection.The results showed that the three enzymes showed good stability at different temperatures and pH values,and all of them could degrade nicosulfuron effectively in a wide range of temperature and pH values.The optimum condition of Sugar phosphate isomerase was 35?,pH 7.0,and the optimum condition of Leucine aminopeptidase and Arginase were 40? and pH 7.5.The Fe2+?K+?Mn2+and Ca2+ can effectively promote the degradation of nicosulfuron by Sugar phosphate isomerase,and the Cu2+and Mn2+ can improve the enzyme activity of Leucine aminopeptidase and Arginase to nicosulfuron,respectively.By analyzing the metabolites of nicosulfuron by three degrading enzymes,it was found that the metabolites degraded by the three enzymes were consistent with the metabolites degraded by CF57,and it could be further determined that the degradation of nicosulfuron by CF57 was mainly through degrading-enzymes.5.In order to obtain nicosulfuron degrading-enzymes with good properties,the molecular modification of Leucine aminopeptidase and Arginase was carried out in combination with computer simulation and site-directed mutation,and the enzyme activity before and after mutation was compared by activity detection method.The results showed that the activities of all Leucine aminopeptidase mutants were decreased compared with the wild-type and the mutant K86P completely lost the degradation activity of nicosulfuron.Therefore,it was inferred that K86 may be the key active site for the catalytic degradation of nicosulfuron by Leucine aminopeptidase.In the Arginase mutants,the activity of N135K and S174L mutants were increased,and the enzyme specific activity was 1.23 and 1.08 times of that of the wild type,respectively.By comparing the Km values of N135K and S174L with those of the wild type,it was found that the Km values of the two mutants were both lower than those of the wild type.Therefore,it was speculated that the increased enzyme activity of these two mutants was probably caused by the change of amino acids that enhanced the affinity between Arginase and nicosulfuron.In summary,this study had preliminarily revealed the degradation effect and mechanism of Bacillus velezensis CF57,and analyzed the enzymatic characteristics and metabolic pathways of three new nicosulfuron degrading-enzymes.It not only enriched the resources of degrading bacteria and enzymes for sulfonylurea herbicides,but also laid a theoretical foundation for the development and application of bacteria and enzymes and the industrial production of degrading enzymes in the future.Furthermore,the molecular modification of Leucine aminopeptidase and Arginase was carried out,and two mutants with good activity were obtained,which provided theoretical support for the acquisition of more excellent sulfonylurea herbicide degrading-enzymes and the further exploration of the interaction between the enzyme and small molecules.