Heterologous Expression,Purification And Characterization Of IPU-degrading Enzymes From Sphingobium Sp.Strain YBL2

The phenylurea herbicide(PUH),first discovered and marketed in the mid-20th century,is one of the most important and extensively used herbicides in different regions of the world,including China,Europe and the USA.Ecotoxicological data have suggested that PUHs and their intermediates including aniline derivatives are harmful to animals,plants,aquatic invertebrates,algae and microbes,in addition to humans.Microbial degradation is considered to be the primary mechanism for the dissipation of IPU from the environment.Therefore,considerable attentions have been paid to the microbial degradation of PUH.The majority of commonly used PUHs are either N,N-dimethyl-or N-methoxy-N-methyl-substituted compounds.Dozens of bacterial strains that are able to degrade and even mineralize PUHs have been reported.Interestingly,some genera seemed to have an extraordinary ability to degrade certain PUHs.For instance,despite different geographical origins,most of the linuron-catabolizing isolates belong to the genus Variovorax,while most of the reported strains that could mineralize N,N-dimethyl-substituted PUHs are sphingomonads(bacteria of the genus Sphingomonas and the closely related genera Novosphingobium,Sphingopyxis,and Sphingobium are commonly referred to as sphingomonads).Just recently,the IPU-mineralizing pathway in strain YBL2 is revealed by our lab.IPU was first converted into MDIPU and trace amounts of DDIPU by PdmAB;then,MDIPU and DDIPU were transformed into 4-IA by DdhA;finally,4-IA was converted into 4-IPC by aniline-degrading gene clusters(ado),followed by the catechol meta-cleavage pathway and the TCA cycle.However,PdmAB and DdhA have not been heterogeneously expressed and purified to homogeneity.So,in this study,we focused on the recombinant expression,purification and enzymatic characteristics of PdmAB and DdhA.In addition,the functions of aniline-degrading gene clusters and catechol meta-cleavage pathway were preliminarily investigated.1.Heterologous expression and characterization of PdmAB,PdmC and PdmD1PdmAB have to accept electrons from the reductase or ferredoxin and pass them onto the mononuclear iron for catalysis.However,there was no evidence for an electron carrier gene(a ferredoxin or a reductase)located in the immediate vicinity of pudmAB in the genome of strain YBL2.Phylogenetic analysis showed that PdmA belonged to the type V ROs,in which the members generally contain a hetero-oligomer-type oxygenase,a[3Fe-4S]-type ferredoxin,and a GR(glutathione reductase)-type reductase.A GR-type of reductase PdmC and three[3Fe-4S]-type ferredoxins(PdmDl,PdmD2 and PdmD3)were predicted from the genome of YBL2.PdmAB,PdmC,PdmD1,PdmD2 and PdmD3 were purified to homogeneity as His-tagged proteins.PdmC and PdmD3 could effectively support the catalysis activity of PdmAB.The PdmAB exhibited optimal activity at 30 ?and pH7.5,but was strongly inhibited by 1 mM of Hg2+,Co2+,Al3+,Cu2+ or Zn2+.2.Heterologous expression and characterization of DdhAThe heterologous expression of DdhA in E.coli was improved via co-expressing chaperon protein,changing the expression vector and host,and optimizing the induction conditions.His-tagged DdhA was purified by Ni-affinity chromatography,and SDS-PAGE analysis showed that DdhA was about 80 kDa,which was consistent with the predicted molecular mass of the protein.Enzyme assay and HPLC analyses showed that DdhA was able to convert MDIPU and DDIPU to 4IA.Moreover,DdhA could also transform carbamate herbicide carbaryl and carbofuran to alpha naphthol and furan phenol,respectively.The purified DdhA exhibited optimal activity at 35 ? and pH7.5.1 mm of Fe2+,Ca2+,Mn2+ or Mg2+ had no obvious effect on the enzyme activity,while 1 mm of Hg2+,Cu2+ or Zn2+ strongly inhibited the activity of DdhA.1 mm of Ni2+ or CO2+ could inhibit the 50%activity of DdhA.3.Preliminary study on the aniline-degrading gene clusters(ado)and the catechol meta-cleavage pathwayThe substrate specificity of the glutamine synthetase AdoQ encoded by the first gene of ado was investigated preliminary.We found that the substrate specificity of AdoQ could affect the substrate specificity of ADO.Additionally,it was demonstrated that the catechol meta-cleavage pathway genes adoXEGKLIJC were organized in one transcription unit and this cluster were responsible for the degradation of 4-IPC in strain YBL2.