Production, purification, and characterization of a novel galactose oxidase from Fusarium acuminatum

Extra‐cellular production of a novel galactose oxidase from Fusarium acuminatum using submerged fermentation was studied. Glucose (1.0% w/v) was used as the sole carbon source. Maximum galactose oxidase production (approximately 4.0 U/ml) was obtained when fermentation was carried out at 25 °C, with orbital shaking (100 rpm) and an initial medium of pH 7.0, for 96 h, using a 2% (v/v) inoculum made from a homogenized four‐day‐old liquid culture, in the presence of copper, manganese, and magnesium. The enzyme was purified by one‐step affinity chromatography, with a recovery of 42% of the initial activity. The purified enzyme ran as a single band of 66 kDa in SDS‐PAGE. Optimal pH and temperature for the enzyme activity were 8.0 and 30 °C, respectively. The enzyme was thermoinactivated at temperatures above 60 °C. The purified enzyme was active toward various substrates, including galactose, dihydroxyacetone, guar gum, lactose, melibiose, methyl‐galactopyranoside, and raffinose. SDS was an inhibitor but EDTA, Tween 80, NH 4 + , Na + , Mg 2+ , K + , and glycerol were not. The Michaelis‐Menten constant ( K m ) for galactose was estimated to be 16.2 mM, while maximal velocity ( V max ) was 0.27 μmol of H 2 O 2 · ml –1 · min –1 . (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)