Extraction, partial purification and characterization of polyphenol oxidase from Solanum lycocarpum fruits

In this work a polyphenol oxidase (PPO) from Solanum lycocarpum ripe and unripe fruits was studied. The unripe fruits presented higher activity than ripe for both fresh fruits and dried pulp flours. The purification procedure was based on freezing precipitation and a sixfold purification factor was obtained. The SDS-PAGE of the partially purified PPO showed two bands around 47 and 68 kDa. Optimal conditions for enzymatic studies were determined to be pH 6.0–6.5 and 28 °C. The partially purified PPO presented high activity toward catechol (Vmax 3.42 U mL−1 and Km 6.47 mM) and 4-methylcatechol (Vmax 3.01 U mL−1 and Km 0.15 mM) and low activity toward phenol being classified as a catecholase type polyphenol oxidase. S. lycocarpum PPO was sensitive to inhibitors such as l-cysteine, sodium metabisulfite, ascorbic acid, thiourea and citric acid. l-Cysteine was the most effective inhibitor, presenting a competitive inhibition. The results from kinetic and thermodynamic parameters for the thermal inactivation evidenced that the partially purified PPO is a biocatalyst, whose inactivation process is related to aggregation of partially unfolded enzyme molecules.