| Along with the development of industry, the demand on petroleum and products of people grow day by day. But at the same time, the massive petroleum and the product ascent environment, have broken the nature original ecological equilibrium, and have caused serious environmental pollution. At present, the strains which were used in strengthening degradation are usually the nature screening mix bacteria colony, the composition proportion in the using process are easy to change, and possibly has the competition, the suppression situation in the practical application. Therefore, using the genetic engineering technology to obtain multi-functional genetic engineering strains which are available in enhancing the entire efficiency of petroleum degeneration.Take petroleum degeneration strains LY-1, LY-2, LY-3, LY-4, LY-5, LY-6, LY-7, LY-8 which were separated and preserved by this laboratory as the object of this study,degradative plasmid were extracted from the strains, and study the degradation ability of this plasmid. c23O gene was cloned and fanctional expression was research carried out.Related literature were consulted to search the degradative gene which possibly existed, on the plasmid,and then target gene (catechol-2,3-dixygenase gene) was determined, homologous sequences of this genes were search on Genbank, and primer was designed, catechol-2,3-dixygenase gene clone by PCR amplification was carried out using plasmid extracted from LY-5 strain as template. Gene sequencing and bioinformatics analysis on the results of sequencing were carried out. c23O gene sequence span 924 bp, codes 307 aa, the protein molecular weight was 34.86 kDa, the theoretical isoelectric point was 5.35. The percentage of G+C content was 54.87%, A+T content was 45.13% in coden area, which was the model basis content characteristic of prokaryotic organism.Amino acid sequence conserved region forecast indicates that there were 3 conserved regions which match to conserved of regions, pfam00903; COG0346; COG2514. The Pfam protein family forecasts indicated that, the protein which this gene codes belonged to the PF00903 family, Glyoxalase/Bleomycin resistance protein/Dioxygenase a supe-rfamily, score: 81.8, E-value: 2.1e-21. The BlastP similarity search result indicated that similarity of the target c23O gene and the Pseudomonas putida gene achieved 99%. c23O gene of the LY-5 strain had a low multi-peptide amino sequence similarity with known c23O gene of Alcaligenes, but had a high multi-peptide amino sequence similarity with c23O gene of Pseudomonas fluorescens and Pseudomonas putida.The cloned c23O was inserted into prokaryotic expression vector, and the recombinant expression vectors was named pGEX-c23O. PCR, extraction of recombinant plasmid and double enzymes digestion were performed for confirming whether the expression vector was constructed successfully. c23O genes were expressed in Escherichia coli induced by IPTG, the protein bands of 64 kDa were observed by SDS-PAGE. It also demonstrated that the protein existed mainly by a intracellular enzyme form in host cell. IPTG induces for 3 hours, the intracellular enzyme activity achieved 476.7 mu/ml to the max. To the total enzyme which are summation of innercellular, outsidecellular, intercellular enzyme activity obtains highest enzyme activity 545 mu/ml. The result conformed to expectation, indicating that c23O gene has been expressed in E. coli, also suggesting that gene from LY-5 could express well in Escherichia coli.Researches of degradative plasmid and c23O gene clone as well as function expression, not only provided solid molecular foundation for the expression of the catechol 2,3-dioxygenase gene and the enzyme activity characteristic research, but also conducted the preliminary study for the degeneration of catechol, which was helpful for the elimination of the catechol pollution in environment. It might help the people to understand the degeneration mechanism and the biodegradation feasibility of other polycyclic aromatic hydrocarbon and provide the corresponding theory basis and the government measure for biological repair of polycyclic aromatic hydrocarbon and the oil pollution in environment.
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