Investigation of the biochemical properties of persimmon polyphenol oxidase: Three-phase partitioning, kinetic analysis and inhibition mechanism.

Polyphenol oxidase (PPO) was first purified 12-fold from persimmon fruit (Diospyros kaki Thunb.) using the three-phase partitioning method (TPP) with a yield of 150%. The pure PPO had a molecular weight of about 63 kDa and showed activity against o-diphenols, p-diphenols and triphenols, but no monophenolase activity. The enzyme showed high stability over a wide pH range and moderate thermal stability. Kinetic analysis revealed that catechol and 4-methylcatechol (4MC) are the most efficient substrates. The enzyme is sensitive to ascorbic acid, benzoic acid and thiourea, while its activity can be increased by Cu2+. It was also found that the PPO enzyme largely retains its activity in various organic solvents, especially chloroform. The data obtained show that the TPP method can be an effective, economical and environmentally friendly alternative for PPO purification. It also provides important information for the prevention of enzymatic browning in foods containing persimmon.