Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.761558
Title: The new families of β-(1→3)-glucan phosphorylases : identification, mechanisms and applications in carbohydrate synthesis
Author: Kuhaudomlarp, Sakonwan
ISNI:       0000 0004 7652 6455
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2018
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Abstract:
β-(1→3)-glucans have applications in human food and health supplements, owning to their reported immunomodulatory properties. Enzymatic synthesis of β-(1→3)-glucans are attractive due to stereo- and regio-specificity of the biocatalytic enzymes. Glycoside phosphorylases (GPs) catalyse phosphorolysis of glycan into sugar 1-phosphate and shortened glycan chain, the reaction of which can be reversed for glycan synthesis. In order to apply GPs as biocatalyst of β-(1→3)gluco-oligosaccharide production, this work concentrated on identification of sequences, biochemical characterisation and structural elucidation of GPs acting on β-(1→3)-gluco-oligosaccharides, in order to understand the mechanism underlying the enzyme substrate specificity. A GP from Paenibacillus sp. YM-1 (PsLBP), belonging to glycoside hydrolase (GH) family 94, has been reported for its specificity towards a disaccharide, β-D-glucopyranosyl-(1→3)-D-glucopyranose. In this study, the activity of PsLBP on sugar donors, α-D-mannose 1-phosphate and α-D-glucose 1-phosphate, was investigated. X-ray crystallography and saturation transfer difference NMR were used to investigate the interaction between PsLBP and its donor substrates, and a mechanism underlying the substrate chain length specificity of the enzyme. Two new families, GH149 and GHyyy, were discovered. Two GH149 candidates, a Euglena gracilis phosphorylase 1 and Pro_7066, were characterised to confirm their function as β-(1→3)-glucan phosphorylase acting on disaccharide and longer substrates. A GHyyy from Paenibacillus polymyxa was characterised to confirm its β-(1→3)-glucan phosphorylase activity, albeit with a distinct substrate length specificity from the GH149 enzymes. Multiple alignment of GH94, GH149 and GHyyy proteins identified conserved amino acids located in the enzyme active site, implying a conserved catalytic mechanism. Structural studies of Pro_7066 revealed two additional domains and an oligosaccharide surface binding site, which are unique to GH149 and may contribute to the enzyme preference for long oligosaccharide substrates. Bacterial GH149 or GHyyy genes map to gene clusters containing genes encoding other GHs and membrane transporters, highlighting the involvement of the enzymes in polysaccharide degradation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.761558  DOI: Not available
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