Biochemical and Chemoenzymatic Strategies for the Characterisation of Carbohydrate Active Enzymes

Abstract

The growing ramifications of climate change, coupled with a finite pool of natural resources, highlight a pertinent need for a rapid transition to a renewables-based bio-economy. One essential aspect of sustainability is the valorisation of plant carbohydrates into renewable biofuels, value-added chemicals and bio-polymers through the use of Carbohydrate Active Enzymes (CAZymes). Involved in the synthesis and deconstruction of carbohydrates, CAZymes offer excellent regio- and stereoselectivity that can be exploited for the production of desired structures. Although the utility of CAZymes has been demonstrated extensively, technologies for their large-scale characterisation are lacking. Consequently, the vast majority of predicted CAZymes are not biochemically characterised leaving the true scope of enzyme activities unknown. The overall aim of this thesis was to advance the knowledge on, and assess biotechnological application of, microbial enzymes for the processing of lignocellulose and its components. In this project we identified a glycosyltransferase capable of polymerising glucose into beta-1,4-linked oligosaccharides. Polymerisation of glucose onto imidazolium-based tags, coupled with chemoenzymatic derivatisation, enabled a sensitive, mass spectrometry-based assay that could characterise the substrate scope and specificity of glycoside hydrolases and LPMOs. Furthermore, following phylogenetic analysis of the GH43 subfamily 34 and molecular docking studies, a beta-galactofuranosidase (XynD) from Aspergillus niger was biochemically characterised. Incubations against a variety of oligo- and polysaccharides revealed activity against pNP-Galf, suggesting the natural substrate is a disaccharide or a glycan with a different linkage type from beta-(1,5), beta-(1,6) or alpha- (1,2)-linked Galf. Finally, a thermotolerant feruoyl esterase from Thermobacillus xylanilyticus (Tx-Est1) was further characterised, with activities against ferulated oligosaccharides and plant polysaccharides investigated. The enzyme revealed an important role in the processing of nutritionally important, pre-biotic xylooligosaccharides. This thesis demonstrates the versatile nature and utility offered by CAZymes.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jolanda van Munster (Supervisor) & Sabine Flitsch (Supervisor)