Date of Award
Master of Science (MS)
Hughes, Thomas A
Greene , Annel K
Henson , John M
Kurtz , Harry D
Polyaromatic hydrocarbons (PAHs) are ubiquitous toxic pollutants that are slow to degrade naturally and costly to remediate artificially. Several species of bacteria have been shown to degrade PAHs, including Sphingomonas paucimobilis EPA505. The aerobic bacterial catabolism of the PAH naphthalene has been studied in several PAH metabolizing bacteria but never in EPA505. This study addresses this deficiency by identifying and characterizing one of the genes for naphthalene metabolism--nahD. NahD codes for the enzyme 2-hydroxychromene-2-carboxylate (HCCA) isomerase, which is the fourth of six enzymes in the upper catabolic pathway of naphthalene.
In this study, the gene nahD is identified and sequenced from EPA505. Then it is sub-cloned into Escherichia coli for expression and purification of HCCA isomerase. Sub-cloning was done by utilizing a pET-30 vector with a histidine tagged leader peptide and an enterokinase cleavage site at the insert cloning site. This allows for induction by isopropylthiogalactoside (IPTG), simple purification by Ni2+ affinity chromatography and restoration of the native enzyme form by peptide cleavage with enterokinase. The results suggest that nahD and naphthalene catabolism in EPA505 is very similar to PAH catabolism in other species.
Brown, Aaron, "Sequencing, sub-cloning, expression and purification of 2-hydroxychromene-2-carboxylate isomerase from Sphingomonas paucimobilis EPA505" (2008). All Theses. 449.