Polyphenol oxidase from apple (Malus domestica Borkh. cv Bramley's seedling): Purification strategies and characterization

Polyphenol oxidase (PPO) was isolated from Bramley's Seedling apples with 75.7-fold purification and 26.5% recovery by ammonium sulfate precipitation, phenyl sepharose chromatography, ion exchange chromatography, and hydroxyapatite chromatography. Molecular weight was estimated to be about 45 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Optimum PPO activity was at pH 6.5 and greater than 50% activity was retained during storage for 72 h at pH 5.5 to 6.5. Optimum temperature for activity was 30°C and tine enzyme had residual activity of greater than 50% during storage for 72 h at 20°C to 30°C and for 24 h at 40°C to 50°C. Of the substrates tested, activity was greatest with 4-methylcatechol followed by catechol, pyrogallol, and (-)-epicatechin. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid.