Purification and some properties of polyphenoloxidase from sunflower seeds
Raymond, J. | Rakariyatham, N. | Azanza, J.L.
The polyphenoloxidase from Helianthus annuus was purified by a combination of SP-Sephadex G-50 chromatography, DEAE-cellulose chromatography, Sephadex G-150 gel filtration and Concanavalin-A-Sepharose chromatography. The final product gave a single band on PAGE. The enzyme purification factor was 149 with a 5% recovery and a carbohydrate content of 6.7%. The partially purified enzyme exhibited the pH optimum at pH 7.9 with gallic acid as substrate and a good stability in the pH range 4.8-7.9 and below 45 degrees. The enzyme showed activity towards sigma-diphenols with no detectable monophenolase activity: the Km value for gallic acid was 1.11 mM. It was characterized by response to various inhibitors. L-cysteine, beta-mercaptoethanol, sodium metabisulphite, dithiothreitol and phenyl hydrazine inhibited strongly. Purified PPO is a monomeric enzyme and has a Mr ca 42000 on SDS-PAGE.
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