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 4.4 mg enzyme per L of growth culture with a specific activity of 159 U mg(−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 68.5 kDa. 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 11.2 min, 5.3 min, and 2.7 min 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 (226 U mg(−1)) and the highest catalytic efficiency (k(cat)/K(m)) for melibiose (2700 mM(−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, NaN(3), and KCN.