Research On Cloning And Expression Of Gene Related 2,4-dichlorobenzoxyacetic Acid Degrading

The 2,4-D is a synthetic hormone herbicide belong to phenoxyacetic acid.The residue of 2,4-D after agricultural application certainly harmed to human health and polluted environment.The residual 2,4-D in the environment was mainly degraded by microbes,and the microbial degrading process of 2,4-D was mainly through degrading pathway of the 2,4-dichlorophenol or 4-chlorobenzoxyacetic acid.2,4-D degrading strain Cupriavidus campinensis BJ71 was used in this study,which detailed research results are as follows:The growth on BJ71 strain in the MSM containing 500mg/L 2,4-D was investigated.It was found that BJ71 growed well when sucrose,glucose and sodium citrate was utilized as the auxiliary carbon source and yeast extract and peptone was utilized as the nitrogen sources.The concentration of NaCl higher than 2%had inhibited the growth of BJ71 to a certain extent.50mg/L concentration of Mn²⁺and Pb²⁺didn't inhibited the growth of BJ71,while it was inhibited under the condition of Cu²⁺or Cd²⁺concentrations higher than 50mg/L and 0.1mg/L Hg²⁺,and didn't tolerated more than 0.3mg/L Hg²⁺.We obtained strain BJ71-F35 from BJ71 by subculture 35 generations without the condition of 2,4-D pressure and strain BJ71-DP1 from BJ71 of eliminated plasmid.The growth on BJ71-F35 was lower and the BJ71-DP1 almost had not obvious growth trends compared with the original strain BJ71.It was indicated that the stability of BJ71 strain was slightly weak,and the first gene of 2,4-D degrading was located on plasmids.The 139 different xenobiotics biodegradation metabolism genes were obtained from sequence information of the plasmid DNA and partial nuclear genome,which contained the degrading genes catABC in 2,4-D degrading pathway through4-chlorobenzoxyacetic acid.In this study,catechol dioxygenase gene catA was cloned and expressed.The catA gene was amplified by PCR,and the amplified product was connected with pGEM-TEasy vectortoconvertE.coliDH5?obtain pGEM-T-catA-E.coli DH5?strain.The full-length sequence of the gene was obtained by colonies and plasmid PCR.The catA gene was 939bp,and it encoded 312 amino acids.The results showed catA gene from BJ71 was identities with 77.6%and 77.5%to the catechol 1,2-dioxygenase?NCBI ACESSION:CP026544?fromCupriavidus metallidurans Ni-2 and the hydroxyquinol 1,2-dioxygenase from C.metallidurans CH34?NCBI ACESSION:CP000352?,respectively.The similarity between the CatA protein sequences coded by catA gene from BJ71 and the other proteins'amino acid sequences were 91.7%and the identity were 85.6%.Bioinformatics analysis of CatA protein showed that the molecular weight of protein was about 34KDa,which it was the less stable soluble hydrophilic protein,without signal peptide and curl spiral domain,and no leucine zipper,not transmembrane transport in cells and subcellular localization was possibily periplasm.The enzyme activity loci of catechol degradation possibily located in the 146-174amino acid sequence.The secondary structure of the CatA protein was predicted,in which the?helix was 22.76%,the?bend was 11.22%,the extension chain was25.96%,and random coil was 40.06%,respectively.The tertiary structure of the protein sequence was predicted by RaptorX,and a better structure was obtained.The plasmids of the cloned strain were used as templates.The full length catA,containing the restriction enzyme cutting site,was amplified by PCR.The PCR product and the expression vector pET28a with double digestion were transformed E.coli BL21?DE3?,and the pET28a-catA-E.coli BL21?DE3?was obtained.The expression of catA gene were studied under the different inducing time,temperature and IPTG concentration.The results of SDS-PAGE electrophoresis showed that temperature and IPTG concentration had no great effect on the expression of CatA protein,while the CatA protein content increased with the extension of inducing time,and has a better expressing level after 12h.The pure CatA enzyme was obtained after the extracted crude enzyme was purified by Ni-column.The research results of enzymatic characteristics showed that the optimum reaction temperature of CatA was 35?,and the optimum pH was 8.0.Under this condition,the Michaelis constant K_m of CatA protein was 40.02?moL/L,the maximum reaction rate was 56.5?mol/?L·min?and k_catat was 36.45s^-1.The study of thermal stability showed that CatA protein maintained better enzyme activity at less than 40?and only maintain 28%enzyme activity at 45?,and enzyme activity was completely inactivated more than 50?.The study of pH stability showed that CatA protein was more sensitive to pH,and maintained better enzyme activity from pH 7.0to 9.0.The enzyme activity respectively maintained 30.39%and 23.32%in the pH 6.0and 10.0,and the enzyme was inactivated completely less than pH 6.0 or more than pH 10.0.CatA protein didn't utilize hydroquinone as the substrate,while 4-methylcatechol was used as the substrate and the relative activity remains 87.9%compared with the catechol as the substrate.