Purification, partial biochemical characterization and inactivation of polyphenol oxidase from Mexican Golden Delicious apple (Malus domestica)

Polyphenol oxidase (PPO) is a critical molecule because of its participation in enzymatic browning in fruits and vegetables during ripening, handling, storage, and processing. In this contribution, PPO was extracted, purified, and characterized from Golden Delicious apples, PPO activity was increased 319-fold with a yield of 1.6%. A monomer of 58 kDa was obtained by gel filtration and SDS-PAGE; the optimum temperature and pH were 35°C and 6.0, respectively; the best catalytic efficiencies (V_max/K_m ratios [U/(mg·mM)]) were 15,402 for 4-methylcatechol and 5,941 for pyrogallol. The most efficient inhibitors were sodium metabisulfite (≥1 mM) and tropolone (≥10 mM). PPO activity decreased as the temperature increased and thermal inactivation was achieved at 80°C in 5 min. An E_a of 69.2 kJ/mol was estimated from the kinetic inactivation at different temperatures. Microwave irradiation inactivates PPO after 120 s in crude extract and after 60 s in fresh apple juice. Practical applications: According to FAO (Food and Agricultural Organization) data, Mexico produced about 858,608 tons of apple in 2013, Mexico is in 30th place in the world overall apple production. It is important to characterize the Polyphenol Oxidase (PPO) from Golden Delicious apple and provide data on its inactivation. PPO has been considered a crucial molecule because of its participation in enzymatic browning in several fruit and vegetables during ripening, handling, storage, and processing, being responsible for economical loses for producers and industry. In this contribution PPO was extracted, purified, and partially characterized from Mexican Golden Delicious apples by fast protein liquid chromatography. Also some inactivation methods like the use of several inhibitors and antibrowning agents, thermal and microwave inactivation where tested. The information obtained in this research is important for better understanding of PPO from Golden Delicious apple and gives information on ways to optimize inactivation process of the purified enzyme or in apple juice.