Biodesulfurization of Petroleum

Mohamed Turkmani, Chemical Engineering, University of Kentucky College of Engineering Paducah Campus, Paducah, KY and Derek Englert, Chemical and Materials Engineering, University of Kentucky, Paducah, KY

Sulfur oxide, a major component of air pollution, is emitted into the atmosphere every day by the combustion of petroleum based liquid fuels. During the combustion process, the sulfur in the fuel is converted to sulfur dioxide, which once in the atmosphere can lead to acid rain.  Current technology, hydrodesulfurization, is very energy intensive and close to its limits. It is great at removing single bonded sulfur, but ring based sulfur is an issue.  It is less effective and removing cyclic, thiophenic sulfur, and breaks the carbon-carbon double bonds, which results in less stored energy in the fuel.  Thus, hydrodesulfurization of the impurity rich crude leads to higher energy and less energy per gallon of fuel.  Biodesulfurization offers a complimentary approach to hydrodesulfurization.  Biodesulfurization uses enzymes to remove the thiophenic sulfur from the carbon source (coal or petroleum) without breaking the carbon-carbon double bonds, which does not reduce the energy per gallon. Biodesulfurization consists of 4 steps, with the last being the rate limiting step.  Therefore, this research looks at evolving the last enzyme in the process, desulfinase B (DszB), to increase the reaction rate of the rate limiting step.  Rhodococcus erythropoliswas isolated from along sulfur veins in coal mines, where it used the energy provided by the cleaving of the carbon-sulfur bond for growth.  DszB has been isolated from the genome of R. erythropolis.  Polymerase chain reaction (PCR) techniques were used to copy the DszB gene and add the restriction enzyme sites.  The PCR product was digested and ligated with the plasmid vector, pTAC-MAT-2.  Escherichia coli (E. coli) was transformed with the new plasmid along with a chaperone plasmid, which create chaperone enzymes to help properly fold DszB.  Since E. coli is a non-native host, it does not have the ability to properly fold DszB on its own and needs help.  If the protein is improperly folded, it will not be active.  Having a created E. coli with the native DszB, work has continued creating mutations of the DszB enzyme.  This work discusses the techniques involved in creating the P187A mutation.  This means that the Proline at the 187 amino acid has been changed to an Alanine.  By removing the large ring structure, the DszB has more flexibility.  If this causes the enzyme activity to drop drastically, it shows the importance of the 187 site allowing for more targeted research.