Biodesulfurization is a promising field that can be applied to the processing of crude oil for removing dibenzothiophene (DBT) and its... Show moreBiodesulfurization is a promising field that can be applied to the processing of crude oil for removing dibenzothiophene (DBT) and its derivatives. A thermophilic bacterial strain Paenibacillus naphthalenovorans (32O-Y) was found to metabolize DBT as sole sulfur source. Another thermophilic strain Paenibacillus apiaries (32O-W) which cannot utilize DBT, however, was found to increase the desulfurization activity of 32O-Y in mixed 32O-Y+W culture in minimal-DBT medium at temperatures between 45 and 50 °C. In order to increase the desulfurization activity of these strains, we genetically engineered strain 32O-Y to express Vitreoscilla hemoglobin (VHb). VHb is the first hemoglobin found in bacteria and has been used to increase the growth and product yields various cells. The VHb gene (vgb) was successfully cloned into shuttle vector pNW33N. 32O-Y was successfully transformed with pNW33N-vgb while 32O-W was not. Compared to untransformed 32O-Y, 32O-Y[pNW33N-vgb] grew slower and reached a lower maximum OD600 when cultured in minimal-DBT medium at 45 °C. However, the Gibbs assay showed that VHb expression in 32O-Y increased its desulfurization activity by 18%. Thus, VHb can help 32O-Y metabolizing DBT while it might not promote growth. A series of high temperatures cultures need to be conducted to select an even more thermophilic strain which can fulfill the 60 °C requirement of industrial process. The VHb mutant (vgbM3) was also studied, which was previously found correlated with increased growth of E. coli DH5α, compared with the effect of wild type VHb. The effect that the mutant amino acids in VHbM3 was determined. Plasmid pUC- vgbM3 was obtained and successfully transformed to DH5α. After sequencing, two copies of vgbM3 were found in the plasmid. In order to study only one copy of vgbM3, x primers were designed for amplification. Fragment vgbM3 was successfully inserted into vector pUC18, followed by successful transformation into DH5α. Cell free extract was obtained from strains DH5α, DH5α[pUC8:16] and DH5α[pUC18-vgbM3] for CO-difference assay. As expected, DH5α expressed no hemoglobin. DH5α[pUC8:16] expressed wild type hemoglobin at 20 nmol/gm wet weight, while DH5α[pUC18-vgbM3] expressed mutant hemoglobin at 4 nmol/gm wet weight. Oxygen dissociation constant determination of VHbM3 will be the next step. M.S. in Biology, May 2014 Show less
