UBC Theses and Dissertations
Discovery and engineering of carbohydrate-modifying enzymes using targeted high-throughput approaches Mehr, Kevin
Carbohydrate-modifying enzymes can be used for the enzymatic synthesis or cleavage of complex glycans. In this study, various high-throughput assays were assessed as methods for the discovery of glycoside hydrolases (GHs) and glycosyltransferases (GTs) within environmental samples. Fluorescence-activated cell sorting (FACS) was evaluated as an approach for the functional enrichment of sialyltransferase (ST) genes in environmental samples. A model ST from Campylobacter jejuni, CstI, was successfully enriched from a mixture of genomic DNA (proof-of-principle), but active STs could not be isolated from a set of metagenomic libraries. The same FACS screen was applied to the directed evolution of multifunctional sialyltransferase from the Pasteurella multocida, PmST1, in order to isolate mutants with reduced sialidase activity and improved synthetic efficiency over the wild-type enzyme. Sialidase activity (kcat/KM) of the best mutants was reduced approximately 2-fold, with an approximately 2.5-fold increase in the sialyltransferase activity (kcat/KM). Despite these improvements, the maximum product yield of the mutants did not increase appreciably. While engineering PmST1, a study of the sialidase and trans-sialidase mechanisms of PmST1 and other STs from the glycosyltransferase family 80 was also undertaken. The mechanisms of both these activities were found to follow a reversible sialylation path, varying from that previously proposed in literature. A high-throughput plate-based assay was also evaluated as a functional screen for the identification of blood antigen-cleaving enzymes within the human gut microbiome. One GH enzyme from Bacteroides vulgatus (BvGH109) was found to be capable of converting the blood type A antigen into blood type O, offering a new enzyme for the engineering of universal donor blood. Two enzymes with α-N-acetylgalactosaminidase activity were also isolated and determined to represent a new sub-family of the GH family 31.
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