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Title: Investigations into novel enzymatic glycosylation methods
Author: Taylor, Thomas Alex
ISNI:       0000 0004 6353 0068
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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Glycosylation is one of the most prevalent post-translational modifications found on human proteins. There is a great interest in developing new methodologies for the synthesis of glycoproteins, both to elucidate the functions of glycans on proteins and to exploit the beneficial properties they can confer on biotherapeutics. Here, research was carried out investigating enzymatic techniques to achieve the formation of homogenous glycoproteins. EndoS was the first enzyme investigated. EndoS natural activity is for the hydrolysis of glycans specifically from IgG, yet there has been research into its use for the opposite reaction and for glycan extension reactions. Here, a number of EndoS mutants were formed and investigated for this reaction, with a number of novel constructs giving a moderate yield of the glycosylated product. Structural investigations of EndoS were also conducted. In addition, research was conducted into the use of PglB in vitro for the direct glycosylation of an asparagine reside on a peptide with GlcNAc. Studies here demonstrate that it is possible to use far simpler glycan donor substrates with PglB. Additional studies showed that it was possible to conduct further enzymatic glycosylation reactions with GalT and EndoA after the initial PglB reaction. Further research was undertaken with EndoA, with it being desired to investigate whether it could catalyse the formation of a thioglycosidic linkage between two glycans, with the thiol bearing glycan acceptor to be attached to a peptide using PglB. After the successful synthesis of all the substrates, disappointingly, neither of the two enzymatic reactions were successful. Finally, the use of a glycal donor in an EndoA catalysed glycosylation was investigated. Unfortunately, no consumption of the glycan was observed in this reaction.
Supervisor: Davis, Benjamin Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available