Poster Presentation 1B-08

Improving Biodesulfurization Rates via the 4S Pathway by Mutagenesis of the Desulfinase Enzyme

Choo Y. Hamilton and Abhijeet P. Borole*

Life Sciences Division

Oak Ridge National Laboratory

Bldg. 4505, MS 6226

Oak Ridge, TN 37831-6226

Phone: (865)576-7421

Fax: (865)574-6442

E-mail: borolea@ornl.gov

The desulfinase enzyme in Rhodococcus IGTS8 limits the overall rate of conversion of organosulfur compounds in the 4S pathway. Increasing the rate of this enzyme can improve the overall rate by up to five-fold. Point mutations were introduced into dszB gene via error-prone PCR. Transformant cells were then screened for desulfinase activity. The mutants were screened via rapid-HPLC analysis of the product 2-hydroxybiphenyl. About 800 colonies were created in the first generation and screened in 96-well microplates. Four mutants with improved activity were isolated. One of these four mutants, 6A10 gave about four-fold increase in the desulfurization activity. The conversion was tested in 100 mL cultures with the native enzyme as a positive control. This rate enhancement can thus increase the potential for comercialization of this alternative technology for sulfur reduction.