Text

theses

Glycans are the most abundant biomolecules in nature and play a significant role in biological systems. They are involved in diverse biological processes such as intercellular signaling, intracellular metabolism, and protein folding. Hence, while the importance of glycans in the fields of chemistry, biology, and bioengineering is being increasingly recognized, limited progress has been made due to the lack of well-defined glycan based reagents available for research applications. Development of efficient chemoenzymatic routes for synthesis of the designer carbohydrates (or glycans) has therefore gained momentum in recent years. Glycosynthases are mutant glycosidase enzymes that can catalyze the formation of glycosidic bonds (between monosaccharide groups) instead of breaking/cleaving these bonds. But identifying a suitable glycosidase from a library of mutants that can act as an efficient glycosynthase for targeted donor or acceptor sugars is still very challenging. This study focuses on the development of a high-throughput screening (HTS) method to rapidly screen and evolve glycosynthase variants generated using traditional directed evolution techniques to enhance catalytic activity or product specificity. A model glycosynthase was used here for validation of this HTS method. Random mutagenesis (via error-prone PCR) was performed on this model glycosynthase to generate a library of mutants that were then screened using this validated HTS method.

ETD_10163

M.S.

Includes bibliographical references

doi:10.7282/t3-ax6t-5f73

ETD graduate

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