Master of Science (MSc)
Faculty of Science
Dr. Michael Suits
Bacteroides thetaiotaomicron is a prolific bacterium found in the distal intestinal tract of humans that possesses the ability to breakdown complex polysaccharides through the release of carbohydrate-active enzymes (CAZymes). Overall, the saccharolytic prowess of B. thetaiotaomicron presents an intriguing model for understanding microbial-host nutrient exchange within the gut and to identify novel mechanisms for accessing carbohydrates. The successful expression, purification, and preliminary kinetic characterization of BT2857 are reported. BT2857 was found to be enzymatically active against the artificial substrate, 4-nitrophenyl β-D-galactopyranoside (pNP-Gal); suggesting its role as a putative β-galactosidase and allowed for the quantification of BT2857 activity by the determination of the Michaelis-Menten parameters kcat, KM, and Vmax. A combinatorial molecular modeling approach of genomic context predicted a two-domain structure formed primarily of beta-sheets and loops. Polysaccharide Utilization Loci (PUL) analysis revealed BT2851-55 collectively belonging to GH36, GH42, GH3 and GH2 providing insight on potential substrates downstream from BT2857. The natural substrate remains unknown despite experiments demonstrating the upregulation of this region of the B.thetaiotaomicron genome in the presence of varying carbohydrates. Crystallization of the truncations of BT2857 will be a key finding for structure and functional characterization.
Streptomyces coelicolor is the representative species within its genus and predominantly found in soil with access to a diverse environment of carbohydrate sources. The main contribution of S. coelicolor is the metabolism of insoluble remains from plants ii and animals indicating positive improvements on plant growth and the rhizosphere. To date, the kinetic characterization of a putative galactosidase, SCO6594 has been explored including the determination of Michaelis-Menten parameters. Solubility screening of SCO6595-67 also display potential expression and characterization of this gene cluster will enable an understanding of protein regulation and carbohydrate interaction.
Ngo, Maria, "THE KINETIC CHARACTERIZATION OF TWO PUTATIVE GALACTOSIDASES, BT2857 AND SCO6594, AND THE PRELIMINARY ANALYSES OF SCO6595-97" (2016). Theses and Dissertations (Comprehensive). 1855.