Partial Purification and Characterization of Polyphenol Oxidase from the Wild Edible Mushroom Lepiota Procera Using Three-Phase Partitioning

Polyphenol oxidase (PPO) from the wild edible mushroom is partially purified and biochemically characterized using three-phase partitioning (TPP), which is an easily applied and effective method. This method includes ammonium sulfate saturation at different concentrations, t-butanol addition (1:1; 1:5), and adjustment of pH. Optimum purification parameters with 20% ammonium sulfate saturation and 1:1 t-butanol conditions led to the highest activity at bottom phase with 8.4 fold purification. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the molecular weight of the purified enzyme to be 35 kDa. The partially purified PPO presented a high level of activity with L-DOPA (Michaelis-Menten constant, Km – 0.12 mM), followed by caffeic acid (0.27 mM) and 4-methylcatechol (0.46 mM) and it is classified as a catecholase type of PPO. The enzyme had peak activity at a temperature of 40 °C and a pH value of 7.0. These results demonstrate a new enzyme source and easy purification method, useful for various industrial applications.