Isolation and Purification of Xylose Isomerase for Improved Biofuel Production

Cellulosic ethanol biofuel production is an important contributor for switching to renewable energy sources; therefore optimization of biomass conversation is integral to industrializing the production process. High-yield purification of enzymes required for chemical conversion of biomass is a key aspect of optimization. One of these enzymes, xylose isomerase (XI), converts xylose, a high energy component of cellulose, to xylulose, which is converted more efficiently by 6-carbon utilizing yeast such as Saccharomyces cerevisae (baker’s yeast) and other high ethanol production strains. Produced by its native marine bacteria host, isolation and purification of this enzyme were performed while attempting to maintain a high yield, using altered culturing techniques, ammonium sulfate precipitations, and column chromatography. Level of purity of isolated enzyme was determined using three assays: XI reaction, XDH, and Bradford. Based on assay data and confirmation by high pressure liquid chromatography (HPLC), the greatest isolation of XI in ammonium sulfate precipitations was in the 70-80% saturation fractions. Culture media for benchtop fermentor growth was altered based on the Redfield ratio and data from colorimetric ion assays. Column chromatography was performed using both ion exchange and size exclusion media, purifying enzyme isolated by ammonium sulfate precipitations after dialysis. Fractions of 0.5 to 2.0 mL were collected from the columns for assay analysis. Though the protein has been successfully isolated by ammonium sulfate precipitations, further work is required to purify the enzyme using column chromatography and culture media alterations.