Progress Towards a Glycosidases Toolbox for Carbohydrate Sequencing: Assay Development for Putative Glycosidase Characterization and Artificial Glycosidase Design for Efficient Carbohydrate Hydrolysis

The complex nature of carbohydrates obtained from biological sources has made their study and the development of efficient tools to probe their structure and function particularly challenging. Conventional ways to determine carbohydrate structure often include acid-hydrolysis as the first step to break large oligosaccharides into individual monosaccharides units for further analysis by techniques such as liquid chromatography and mass spectrometry. However, the use of such a nonspecific cleavage method obliterates any stereochemical information of the original glycosidic linkage. In contrast to acid-mediated hydrolysis methods, glycosidases can catalyze the hydrolysis process of glycosidic linkages with specificity towards the stereochemical orientation of the linkage. A panel of such enzymes, especially ones that could operate at higher temperatures to unravel secondary and tertiary structures that occlude access to the glycosidic bond, could significantly help with oligosaccharide sequencing if enzymes are chosen with well-characterized specificities. In this dissertation, details of a glycosidase toolbox design is reported as well as two mass spectrometry-based glycosidase assays developed for a well-rounded characterization of putative glycosidases. A hydrogen peroxide-based, CuGGH metallopeptide facilitated artificial glycosidase design for a more efficient and controllable carbohydrate hydrolysis is also introduced in this thesis. We envision these new sets of glycosidase assays and new hydrolysis method to serve as useful complementary tools to the current norm of fluorescent or spectroscopic assays and acidic hydrolysis to set a better standard for future glycosidase and carbohydrate characterization studies.