Purification and biochemical characterization of polyphenol oxidase extracted from Kirmizi Kismis grape (Vitis...

The purification of the polyphenol oxidase (PPO) enzyme from Kirmizi Kismis grape (Vitis vinifera L.) was performed 61.23 times using affinity chromatography. The molecular weight of the enzyme was found to be about 38.1 kDa by SDS-PAGE as a single band. The optimum pH and temperature values were revealed to be 5.0 and 30°C, respectively, in the presence of 4-methyl catechol substrate. The thermal stability of PPO was examined and it was observed to maintain its activity at 20°C for 1 hr. K_m and V_max values were determined to be 4.8 mM and 2000,0 EU/ml for 4-methyl catechol as a substrate. IC₅₀ and K_i values and inhibition types were found for various browning agents and ascorbic acid had the strongest inhibitory impact on PPO. The inhibitory impact of Na⁺ , K⁺ , Mg²⁺ , Cu²⁺ , and Al³⁺ metal ions on the enzyme activity at final concentrations of 1 mM and 10 mM was examined. PRACTICAL APPLICATIONS: Grapes grown and processed take a significant place in our life. The grape has antioxidant, anticarcinogenic, antidiabetic and protective properties against bacteria and viruses. Furthermore, it takes an important position in the country's economy and social life due to providing raw materials to the food industry and having high export potential. Polyphenol oxidase, which is the leading actor of enzymatic browning reactions causing serious economic losses every year, was purified and characterized from Kirmizi Kismis grape (Vitis vinifera L.). This ancient grape variety has industrial processing and export potential due to its long storage life and resistance to oxidation. Therefore, the purification and biochemical characterization of polyphenol oxidase from Kirmizi Kismis grape are of great importance.