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Mentor

Jason Hurlbert, Ph.D.

College

College of Arts and Sciences

Department

Chemistry, Physics and Geology

Funding

This work was also supported by SC-INBRE and the Winthrop University Department of Chemistry, Physics, and Geology

Abstract

Sustainable sources of energy are growing in demand as fossil fuels are rapidly expended. One such energy source is fuel ethanol generated from the fermentation of plant biomass by engineered bacterial biocatalysts. The creation of a biocatalyst capable of converting nearly any plant matter to fuel ethanol requires the identification of novel enzymes capable of degrading specific carbohydrate polymers and cloning these enzymes into a bacterial host. This study seeks to structurally characterize a novel xylanase of glycosyl hydrolase family 30 (GH30) from Bacteroides vulgatus, a bacterium found in the human gut microbiome, via x-ray crystallography. The gene for B. vulgatus GH30 endoxylanase (BvGH30) was cloned into a pET 28b prokaryotic expression vector which was used to transform a culture of Escherichia coli, and the resulting bacterial strain was used to express the cloned BvGH30 gene. The recombinant protein produced was then purified to homogeneity via Ni2+-Metal Chelating Affinity Chromatography (MCAC) as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified protein was concentrated to 10 mg/mL and used to screen for solution conditions that promoted crystal growth by sparse matrix screening in hanging drop, vapor-diffusion plates. Single rectangular crystals of defined morphology (less than 0.1mm in length) were obtained in 0.2M (NH4)2HPO4, 20% (w/v) PEG 3350, pH 8.0 and large numbers of smaller rectangular crystals were obtained in 0.1M Citric acid, 0.8M (NH4)2SO4, pH 4.0. Grid screening around these two conditions will be employed to increase crystal size and all crystals larger than 0.1mm in length will be subjected to x-ray diffraction analysis.

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