Partial purification and characterization of polyphenol oxidase from sugarcane (Saccharum officinarum L.)

Polyphenol oxidase (PPO) of sugarcane was extracted by using 0.02 mol/L phosphate buffer at pH = 6.8 containing 1.5% polyvinylpolypyrrolidone and 0.5% Triton X-100, and then partially purified by 80% ammonium sulfate fractionation, dialysis, and column chromatography on DEAE-Toyopearl 650M, Sephadex G-100. PPO activity was purified 37.6-fold with a recovery of 18.4%. The PPO showed activity to catechol, chlorogenic acid, 4-methylcatechol, caffeic acid and ferulic acid, but not to l-tyrosine. Optimum conditions (pH value and temperature) for PPO were determined using the five substances. PPO activity is quite thermostable between 20 and 30 °C. After heating for 10 min at 80 °C 90% of the activity is lost. Km and Vmax values of PPO were calculated for each substrate and the best substrate of PPO was chlorogenic acid. PPO was markedly inhibited by metal ions (Cu2+, Al3+, and Mg2+) at 1 and 10 mmol/L, and strongly inhibited by NaHSO3 and ascorbic acid at 1 mmol/L.