Engineering the substrate specificity of galactose oxidase

Biocatalysis, the use of enzymes to catalyse the generation of specific chemicals, has a number of advantages including high specificity, lower energy requirements and greater sustainability over an equivalent chemical process. Several methods exist for optimisation of enzymes for use in industrial processes, including introduction of mutations to generate libraries of variants which are then screened for the desired properties, such as stability or substrate specificity. Galactose oxidase catalyses the oxidation of primary alcohols to the corresponding aldehyde, with concomitant reduction of dioxygen to hydrogen peroxide. It has already been developed for use in a range of biotechnological processes and is an ideal candidate for further development due to features including high stability, a surface exposed active site displaying broad substrate specificity, and an autocatalytically-generated cofactor.
Research presented in this thesis investigates the effect on activity towards a range of alternative substrates of mutations at selected active site residues with the aim of expanding the biotechnological potential of galactose oxidase. Libraries of variants were designed and generated using high quality oligonucleotides constructed using trimer phosphoramidites. Screening assays used by other groups were optimised by varying different components. These assays were then used to identify a number of variants displaying enhanced activity towards D-arabinose, D-glucose, D-xylose or glycerol. A selection of these variants were then further characterised in order to understand the biochemical basis of the altered activities and determine some of the conditions required for potential industrial application of the variants. The most exciting results include identification of a variant displaying higher levels of activity towards glycerol than towards the native substrate D-galactose; determination of the position of oxidation of D-arabinose at the C-4 hydroxyl; and the observation that mutation of Phe194 significantly affects binding of D-glucose in the active site.