Galactose oxidase (GaOx, EC 1.1.3.9) secreted by the fungus Fusarium spp is a 68 kDa radical copper oxidase that oxidizes the primary alcohol of galactose to an aldehyde. Unlike glucose and pyranose oxidases, the comparatively open substrate binding cleft of GaOx allows oxidation of oligomeric and polymeric substrates, including terminal galactose residues of galactoglucomannan, galactomannan, and xyloglucan.
The current study assesses the potential to enhance GaOx activity on polymeric substrates and high viscosity solutions through the construction of GaOx fusions to selected carbohydrate-binding modules (CBMs). In particular, a family 29 CBM from Piromyce equi was separately linked to the N-terminal and C-terminal end of GaOx, and corresponding proteins were recombinantly produced in Pichia pastoris. Purified fusion proteins were characterized in terms of substrate selectivity, specific activity on polymeric substrates, enzyme kinetics, and substrate binding using affinity gel electrophoresis. Results to date indicate that the fusion proteins can be stably produced in P. pastoris in active form, and that binding affinities correlate well with the selectivity of CBM29 and measured enzyme activities.