Partial purification, characterization and immobilization of polyphenol oxidase from bitter leaf (Vernonia amygdalina Delile.)

This study focuses on the partial purification of polyphenol oxidase (PPO) from the leaves of Vernonia amygdalina. The aim is to characterize its physicochemical and kinetic properties for potential industrial applications. The enzyme was extracted and purified using aqueous two-phase partitioning systems (ATPS) and three-phase partitioning systems (TPPS). The physicochemical and kinetic parameters, specifically Vmax and Km of the partially purified PPO, were estimated following established protocols. Additionally, the study investigated the effects of inhibitors, crowding agents (such as polyethylene glycol (PEG) and bovine serum albumin (BSA)), and organic solvents on the enzyme’s activity. The immobilization and characterization of the partially purified enzyme were also explored. Using catechol as the substrate, the enzyme’s Vmax and Km values were determined to be 3543 ± 150.1 µmol/min/mL and 29.6 ± 8.8 mM, respectively, with an optimal pH of 7 and a temperature of 30 °C. Various inhibitors, including glutathione, L-cysteine, β-mercaptoethanol, and L-ascorbic acid, exhibited different Ki values of 333.33, 57.1, 147.9, and 125.0 mM, respectively. Polyethylene glycol (PEG) 4000 and 6000 enhanced the activity of V. amygdalina PPO (vaPPO) by 1.25-fold at a concentration of 30 mg/mL. The vaPPO demonstrated low stability in water-miscible solvents such as methanol and ethanol, retaining only 20% of its activity at a 20% concentration. In contrast, it showed high stability and activation (132%) in 20% ether and retained 81% activity in 20% chloroform. Immobilization on calcium alginate improved the enzyme’s activity, allowing it to retain 50% of its activity after three uses. These findings suggest that vaPPO has potential for biotechnological applications.