Date of Award

12-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Microbiology

Advisor

Hughes, Thomas A.

Committee Member

Kurtz, Jr. , Harry D.

Committee Member

Cao , Min

Committee Member

Tzeng , Tzuen-Rong J.

Abstract

A bacterial artificial chromosome (BAC) library was previously constructed from genomic DNA of Sphingomonas paucimobilis EPA505 at Clemson University Genomic institute (CUGI). To isolate and identify the bphB gene from the genome of S. paucimobilis EPA505, a set of primers was designed based on conserved regions of the bphB gene encoding dihydrodiol dehydrogenase of Sphingomonas CHY-1 and Sphingomonas yanoikuyae B1. Routine PCR was performed and a PCR fragment of approximately 500 bp that confirmed the presence of pbhB gene in Sphingomonas paucimobilis EPA505 was produced. Hybridization of the PCR product with BAC library was performed and one hundred two positive hits were spotted from the BAC clones.
A shotgun library was constructed and DNA sequencing was performed on one of the BAC clones (03C01). The sequence analysis of shotgun clones and sequences resulted in 27 contigs. At this point it was decided to add another PAH catabolic enzyme to work within this project because of its close proximity to bphB. This enzyme was determined to be aldehyde dehydrogenase (phnN). After further analysis of all the contigs, using Consed software and programming, located the genes of interest, bphB and phnN, both located on contig 26 next to each other. Sequencing of both genes revealed that the bphB gene consisted of 801 nucleotides, and encodes the dihydrodiol dehydrogenase enzyme harboring 266 amino acid and the phnN gene contained 1500 base paires and encodes a polypeptide approximately 499 amino acid.
Protein expression in E. coli was examined by the EK/LIC Cloning system (Novagen, Madison, WI). For cloning and expression of the dihydrodiol dehydrogenase gene (bphB) and the aldehyde dehydrogenase gene (phnN), the pET-30 EK/LIC cloning kit (Novagen, Madison, WI) was used. The recombinant plasmids were then transformed into the BL21 (DE3) pLysS expression host strain for target gene expression. Expression of the recombinant target DNA (pET-30 EK/LIC vector) was induced by addition of 1 mM IPTG to the OD600=0.5-1.0.
Total cell protein (TCP) analysis was performed for target protein verification on a SDS-polyacrylamide gel according to the pET system manual (Novagen, Madison, WI). Substrate Affinity Chromotography was performed to purify cloned proteins. The SDS polyacrylamide gel exhibited a 28 kD protein for DDH and a 55 kD protein for ALDH.
Enzyme assays were performed for purified proteins to confirm enzyme activity. The enzyme assay was only accomplished for ALDH, due to unavailability of the substrate for DDH. Salicylaldehyde was used as the substrate for ALDH which was commercially available (Sigma, USA). All of the detections were performed spectrophotometrically, based on reduction of NAD+ at 340 nm. Different concentrations of enzyme were used for the assay. The lowest amount of enzyme needed to achieve enzyme activity was 0.1 mM. Optimum temperature and optimum pH were obtained for ALDH. The enzyme displayed the best activity at 45°C and pH 7.0. Steady-state kinetics was performed. The Km value was calculated as 1.43 mM while Vmax was measured as 0.07 U mg-1. Kcat and catalytic efficiency were calculated for ALDH as 0.7 (s-1) and 0.489 (M-1 s-1), respectively.
The study proves that ALDH extracted from S. paucimobilis EPA505 is an active enzyme and can be used in many different aspects such as bioremediation processes and bioengineering. It is the first example of an enzyme able to oxidize salicylaldehyde to salicylic acid by reducing NAD+ to NADH in S. paucimobilis EPA505.

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Microbiology Commons

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