Study On The Aniline-based Herbicides Microbial Degradation Pathway And The Gene Of Key Enzymes

Aniline-based herbicides such as phenylurea herbicides and amides herbicides were widely used in agricultural production.Many of these types of herbicides have long residual periods,and they can keep stable in soil up to several months or even a year or more.The extensive use of these herbicides,combined with some farmers incorrectly use of these herbicides resulted in its severe resdues in soil and water,which not only endanger the environment,but also do harm to the next stubble crops,and led to huge losses in agricultural production.Otherwise,the molecular structure of these herbicides contains the structure of anilines,and after introduced into the environment they can be degraded into anilines and their relative products to cause further environmental pollution.Although there are many reports on microbial degradation of these pesticides,the degradation of aniline-based herbicides is not very clear,especially on the level of genes.Therefore,it is of great theoretical and practical significance.to study the aniline-based herbicides degradation mechanisms to provide theoretical guidance for bioremediation of contaminated environments and to lift the residual hytotoxicity and environmental pollution.At present,there are many research on aniline-based herbiciedes reported.However,they are mainly in the degrading strains isolation and the preliminary study on degradation pathways.Studies on the genes and enzymes level of aniline-based herbicides biodegradation mechanism are rarely reported.Several phenylurea herbicides degrading strains had been isolated in our laboratory,and had been preliminary studied on the identification and the basic biological characteristics.In addition,the phenylurea herbicides degradation pathway by these strains had also been preliminary studied.However,there is no further study on the degradation mechanism.Sphingobium sp.Strain YBL2 and Sphingomonas sp.strain Y57 were excellent aniline-based herbicides degrading strains,and they can degrade a range of different substrates respectively.Therefore,we use these strains as materials for further research on the degradation mechanism of aniline-based herbicides.1 Research on the degradation pathway level1.1 The degradation pathway of isoproturon in strain YBL2In this work,we use isoproturon as an example to study the metabolic pathways of phenylurea herbicides in strain YBL2.Firstly,three isoproturon degradation intermediate metabolites MDIPU,DDIPU and 4IA were detected during the HPLC and MS/MS analysis.And we futher studied the degradability of these known metabolites by YBL2.The results showed that strain YBL2 was able to degrade these substrates added into the MSM effectively.To study on the aromatic ring cleavage pathway of isoproturon degraded by YBL2,we detected the aniline dioxygenase activity and the catechol dioxygenases activity of isoproturon induced YBL2 resting cells and the cell-free extract.The results showed that the activity of aniline dioxygenase and catechol 2,3-dioxygenase in strain YBL2 can be inducted by isoproturon exposure.However,strain YBL2 showed a high catechol 1,2-dioxygenase activity,but not induced by isoproturon.We inferred that aniline dioxygenase and catechol 1,2-,and 2,3-dioxygenase were involved in the degradation of isoproturon by YBL2.Based on these results above,we proposed a isoproturon degradation pathway in YBL2,which including the key step of aromatic ring cleavage.Two steps of demethylation was followed by the urea side chain cleavage to generate 4IA,then under the catalysis of aniline dioxygenase and catechol 1,2-and 2,3-dioxygenase,4IA was completely degraded through 4-isopropylcatechol via meta-and ortho-cleavage pathway.1.2 The degradation pathway of propanil in strain Y57In this work,we studied the degradation pathway of propanil and 3,4-DCA by strain Y57.The results showed that propanil was degraded by Y57 through intermediates such as 3,4-DCA and 4,5-dichlorocatechol.To study the aromatic ring cleavage pathway of the aniline-based herbicides downstream metabolites by Y57,the catechol dioxygenase activity in the crude enzymes of Strain Y57 was detected.And it is indicating that strain Y57 degraded the aniline-based herbicides downstream metabolites through ortho-cleavage pathway.In addition,the N-acylation and N-deacylation modification process on aniline compounds were detected during the degradation of 3,4-DCA by Y57,among of which,3,4-DCA propionyl modification product has not been reported in bacteria strains.2 Research on the gene level2.1 The anilines degrading gene cluster The key enzyme encoding gene cluster responsible for degrading the downstream metabolites of aniline-based herbicides was obtained from the genomic sequence of strain YBL2,and was designated as yagQTA1A2BRCDEFGHIJKL.The gene cluster has significant differences on genes and genetic organization compared with reported representative aniline degrading gene clusters.It was a novel anilines degrading gene cluster.The anilines dioxygenase gene cluster was expressed in Pseudomonas.putida KT2440,and it can transform 3,4-DCA into 4,5-didichlorocatechol.Besides,the anilines dioxygenase gene cluster also has been amplified from strain Y57,and it was almost the same with that from strain YBL2 under the sequence analysis..2.2 The propanil hydrolase geneTo study the degradation mechanism of propanil by Y57 on the gene level,we cloned the propanil hydrolase gene from the shotgun sequences of strain Y57,and designated the gene as prpH.It was a bacterial histone deacetylase superfamily homologous protein encoding gene.Recombinant PrpH was functional expressed in E.coli,and purified.And the enzymatic properties of PrpH were preliminary studied.