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 degrees 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 degrees C, respectively. The enzyme was thermoinactivated at temperatures above 60 degrees 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 micromol of H(2)O(2) . ml(-1) . min(-1).
Alberton
Dayane
D
Universidade Estadual de Maringá, Departamento de Bioquímica, Maringá, PR, Brazil.
Silva de Oliveira
Luciana
L
Peralta
Rosane Marina
RM
Barbosa-Tessmann
Ione Parra
IP
D050260:Carbohydrate Metabolism; D003300:Copper; D003470:Culture Media; D005285:Fermentation; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D005670:Fusarium / Q000254:growth & development / Q000378:metabolism*; D005692:Galactose Oxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium; D008345:Manganese; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
D003470:Culture Media; D005656:Fungal Proteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D008345:Manganese; D003300:Copper; D005692:Galactose Oxidase; D008274:Magnesium