Partial Purification and Some Properties of Peroxidase from Malvasia Grapes

Abstract

Peroxidase extracted from Malvasia grapes was purified 124-fold by ammonium sulfate fractionation followed by three successive column chromatographies on Bio-Gel P-100, DEAE-Sephadex A-50 and Phenyl-Sepharose CL-4B. Four anionic isoenzymes were detected by polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme was estimated to be 40 000-41 000 by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The pH and temperature optima of the purified enzyme were 5.5 and 40°C, respectively. At 65°C, it took two minutes to inactivate the peroxidase isoenzymes 50%, and at 85°C, these enzymes were almost completely inactivated after five minutes. Grape peroxidase showed a K_m of 5.5 mM for guaiacol at optimum H₂O₂ concentration and a K_m of 1.6 mM for H₂O₂ at optimum guaiacol concentration.