Date of Award
Master of Science
Abhijeet P. Borole
Brian H. Davison, Paul D. Frymier
Polyaromatic hydrocarbons (PAHs) are ubiquitously present in the environment and have deleterious affects on humans. Cytochrome P450 enzymes have been found to be useful in the biodegradation of the PAHs through an initial hydroxylation step. This makes them more water-soluble and accessible to the action of other oxidases. Cytochrome P450cam is a model P450 enzyme that has been mutated for enabling PAH and alkane hydroxylation. P450s therefore have potential for use in commercial process for production of primary alcohols.
Rhodococcus organisms have a special mechanism for transporting the hydrophobic hydrocarbons into their cells. Cloning of the P450cam gene into Rhodococcus can potentially provide a hydroxylation system with high conversion rates. To enable this, cytochrome P450cam gene (CYP101) was cloned into a Rhodococcus-Escherichia coli shuttle vector. This would provide tools to modify the gene and target specific PAHs. This study is aimed at developing a Rhodococcus-based biocatalyst using cytochrome P450 enzymes.
The cytochrome P450cam gene was synthesized using PCR (polymerase chain reaction); the product was cloned into pKSD6-1 (a Rhodococcus-Escherichia coli shuttle vector) and transformed into Escherichia coli. The recombinant protein in Escherichia coli was produced by induction and the protein extract was analyzed using spectrophotometric analysis, camphor binding and carbon monoxide binding assays.
The results showed a Soret band at 414nm but no carbon monoxide or substrate binding was observed. The control culture grown under similar conditions with only the pKSD6-1 gene did not show any absorption band at 414nm. These results together indicate that the gene has been cloned into the recombinant plasmid, but the enzyme being produced might be in an inactive form.
Chada, Sirisha, "Cloning and Expression of Cytochrome P450cam. " Master's Thesis, University of Tennessee, 2004.