Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli.

A gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4mg enzyme per L of growth culture with a specific activity of 159Umg(-1). By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 68.5kDa. The pH optimum for the oxidation of galactose was in the range of pH 6-7.5. Optimum temperature for the enzyme activity was 35°C, with a half-life of 11.2min, 5.3min, and 2.7min for incubation at 40°C, 50°C, and 60°C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-β-galactopyranoside (226Umg(-1)) and the highest catalytic efficiency (kcat/Km) for melibiose (2700mM(-1)s(-1)). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN.