Purification and Characterization of Polyphenol Oxidase from Hemşin Apple (Malus communis L.)

Abstract

The polyphenol oxidase (PPO) enzyme was purified and characterized from Hemşin Apple (Malus communis L.), which was organically grown in Hemşin, in the Rize province of Turkey. Enzyme (PPO) activation was determined with catechol substrate. Apples were homogenized with homogenate buffer (pH 8.5). This process was followed by precipitation with (20–80%) saturated solid (NH₄)₂SO₄ and dialysis. Finally, purification with DE52-Cellulose ion-exchange and Sephadex G-25 columns was performed. Experiments were performed at an optimum pH (5.5) and optimum temperature (30–40°C). The kinetic and thermal parameters Km (3.40 mM), Vmax (333.3 EU/mL.min), Ea (3.57 kcal), ∆H (2.968 kcal/mol), Q₁₀ (1.33), k_cat (24.57 min⁻¹) and V₀ (7.2x10³ mM⁻¹.min⁻¹) were assessed. Additionally, the effects of Mg²⁺, Pb ²⁺, Fe²⁺, Fe³⁺, Cd²⁺, Cu²⁺, Zn²⁺, Co²⁺, Al³⁺, Mn²⁺ and Na⁺ on enzyme activity was recorded, and the IC₅₀ values, K_İ values and inhibition types were determined.

Keywords:

• Hemşin apple
• Malus communis
• Polyphenol oxidase
• enzyme purification
• enzyme characterization
• enzyme inhibition

Additional information

Funding

IG and SE would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research, RGP-VPP-254.