Isolation Of Specific Desulfurization Bacterium LY822 And Construction Of Desulfurizational Gene Engineering Strains

Biodesulfurization (BDS) can selectively remove the sulfur from condensed thiophenes that is recalcitrant to hydrodesulfurization (HDS), and it is promising process that can be complement or alternative to HDS for deep desulfurization of petroleum and its products. The isolation of strains is precondition of BDS. The construction of expression vector and engineering strains is an effective method to improve the activity of desulfurization.The soil sample contaminated with petroleum was collected. DBT was taken as the only sulfur source to screen the non-destructive bacterium. The metabolites was extracted by hexane, and analyzed with HPLC. The result shows that the strain LY822 can selectively remove the sulfur for the modular compound. The morphological properties and 16SrDNA sequence suggested that the isolated strain belongs to the evolution branch of Rhodococcus and shows highest homology to Rhodococcus erythropolis. The strain was named as Rhodococcus sp. LY822.The plasmid DNA of LY822 was extracted and acted as template; three structure gene fragments were amplified out by PCR. They were 1.3, 1.0 and 1.2 kb large respectively. These gene fragments were then purified and ligated into pET-21a vector. SDS-PAGE analysis results of whole-cell protein reveal that the three genes have been expressed effectively and respectively.Three plasmids named pBSGt pBSGd and pBSGk were constructed respectively with two plasmids named pEGH and pBS305. The plasmid pEGH contained gfp gene and pBS305 is an E. coli -Rhodococcus shuttle vector. These constructed plasmids can be used to detect the efficiency of promoter. By detecting and comparing the fluorescent intensity of GFP expressed in the strains with pBSGt, pBSGd or pBSGk, The optimal promoter was screened out, because the GFP in the strain with pBSGt expressed effectively, furthermore the strain were not restrained any more by the sulfate.Plasmids named pBSGt, pBSGd and pBSGk were digested with XbaI and Hind III, and then the full length fragment of dsz gene was inserted. Plasmids named pBSDt, pBSDd and pBSDk were so constructed, and then were transformed into both LY822 and LY-0. The result of the HPLC analysis reveals that the desulfurization activity of LYDt was increased remarkable, furthermore it was not restrained by sulfate anymore. This conclusion was in accord with the result by means of report gene gfp.The recombinant strain LYDt was also applied in the desulfurization process of diesel oil (sulfur content 497 ppm). After 4 h, about 82% of sulfur in the oil was removed, whereas the wild strain only removed about 61% of sulfur in the system...