Cloning and Purification of a glycerol-specific alditol oxidase for biosensor construction

Abstract

Wine production is dependent on ethanol, but also on optimal glycerol concentrations, both of which are produced by S. cerevisiae fermentation. Wine characteristics like sweetness levels are influenced by glycerol concentrations. Additionally, elevated glycerol levels can be an indication of abnormal blood sugar levels. In both situations, close observations of glycerol levels are essential. One proposed method of measuring glycerol concentrations is through enzymatic oxidation with a glycerol biosensor. Alditol oxidase (AldO) is a recently discovered carbohydrate oxidase in S. coelicolor. Despite specificity for longer-chained polyols, studies have proposed that AldO can be used as a glycerol oxidase. Using random point mutations, an AldO mutant was isolated and had increased specificity for glycerol. These results suggest that potential for AldO with glycerol biosensor development. This project aimed to produce a glycerol specific alditol oxidase to be used as a biosensor. A synthetic alditol oxidase (AldOG) gene was used to produce AldOG via cloning methods. The method will also be utilized in conjunction with Dr. Samuel Mugo’s work in biosensor construction. Finally, the resulting cloning techniques optimized for AldOG will be used in a future laboratory for a 300-level Biochemistry course currently under development by Dr. Bernstein.

Department: Biological Sciences

Faculty Mentor: Dr. Nina Bernstein