Abstract

Glycoside hydrolase (GH) enzymes are utilised by a variety of organisms to convert polysaccharides into sugars. The second generation biofuel industry exploits the enzymatic deconstruction of waste lignocellulosic biomass to produce bioethanol. Investigation into the structural diversity of GH family ₇ (GH₇) enzymes allows a platform to understand the hydrolytic potential of novel cellobiohydrolases that could be used in industrial enzyme cocktails, potentially reducing production costs. Recently, GH7 enzymes have been discovered in other kingdoms of life besides fungi and bacteria, including in protists and animals. Here the structure of a novel endogenous GH₇ cellulase from an animal, the freshwater crustacean Daphnia pulex, is described (Dpul/Cel₇A). Our collaboration with the United States National Renewable Energy Laboratory produced recombinant Dpul/Cel₇A, with which we predict that Dpul/Cel₇A is a cellobiohydrolase via two high-resolution crystal structures. An apo structure of 1.9 Å resolution and a cellobiose ligand docked structure of 1.65 Å resolution were produced, allowing direct comparison to homologues as only the second ever animal GH₇ structure. Biochemical methods were performed in order to reveal several points of interest: A biomass assay of Dpul/Cel7A was utilised to reveal its hydrolytic performance compared to an industry standard cellulase from Trichoderma reesei. An in-situ hybridisation method was developed to understand DpulCel7A gene expression regulation, and confocal laser scanning microscopy performed to expose anatomical structures of interest in high resolution.

Course: Master of Philosophy - MPhil

Date Deposited: 2018-04-17

URI/permalink: https://library.port.ac.uk/dissert/dis13172.html