Use this URL to cite or link to this record in EThOS:
Title: Engineering improved specificity and activity into oral bacterial sialidases for glycan biotechnology applications
Author: Satur, Marianne
ISNI:       0000 0004 7972 0413
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2019
Availability of Full Text:
Access from EThOS:
Access from Institution:
A wide range of pathogenic and commensal bacteria secrete sialidases which function in the hydrolysis of terminal sialic acid residues from glycoconjugates and can have roles in nutrition or virulence. Sialic acid is connected to the underlying sugar in the glycan chain via a2,3-, a2,6- or a2,8-glycosidic linkages. The literature has reported bacterial sialidases that can hydrolyse all of these linkages and sialidases that are specific to a2,3-linked sialic acid. However, there are currently no known a2,6 specific sialidases. Glycosylation of biopharmaceuticals can affect their efficacy and so must be managed. This often requires the use of recombinant sialidases and there is an increasing demand for linkage specific sialidases in the glycan biotechnology industry. In this study, biochemical assays and X-ray crystallography were used to understand the biochemical and structural properties of the NanH sialidase from Tannerella forsythia before site-directed mutagenesis was employed to generate point mutants in attempt to alter the specificity of NanH. Biochemical analysis showed that wild-type (WT) NanH is a dual specific sialidase as it hydrolyses a2,3- and a2,6-linked sialic acid. A crystal structure was solved for the apo form of NanH, along with a structure for NanH in complex with the sialidase inhibitor oseltamivir. Alongside this, the crystal structure for an inactive NanH variant (NanH-D237A) was solved in complex with sialidase substrates 3- and 6-sialyllactose. These models enabled rational selection of residues for point mutation in attempt to alter the specificity. Overall two regions in the active site of NanH have been highlighted as potentially having importance in the specificity of the enzyme, which could be probed further in the future.
Supervisor: Stafford, Graham ; Rafferty, John ; Spencer, Daniel Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available