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Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape
- Zheng, Yongju, Shi, Junling, Pan, Zhongli
- European food research & technology 2012 v.234 no.4 pp. 607-616
- EDTA (chelating agent), L-dopa, Vitis vinifera, activation energy, ascorbic acid, caffeic acid, catechol oxidase, citric acid, fructose, gallic acid, glucose, grapes, guaiacol, heat inactivation, heat stability, metal ions, pH, sodium chloride, sodium metabisulfite, sucrose
- Polyphenol oxidase (PPO) was isolated from Thompson seedless grape (Vitis vinifera âThompson Seedlessâ), and its biochemical characteristics were studied. The PPO showed activity to catechol and D, L-DOPA, but not towards monophenol L: -Tyrosine, diphenols guaiacol and caffeic acid, and triphenols pyrogallic acid and gallic acid. Apparent MichaelisâMenten constant (K m) and maximum velocity of the reaction (V max) values were 45.0Â Â±Â 0.05Â mM and 500.0Â Â±Â 15.3 OD400Â nm/min for catechol, and 34.6Â Â±Â 0.03Â mM and 384.6Â Â±Â 11.7 OD478Â nm/min for D, L-DOPA, respectively. The obtained similar specificity values of V max/K m ratio of catechol and D, L-DOPA indicated their similar affinity to Thompson seedless PPO. The most effective inhibitor was L: -cysteine, followed in decreasing order by ascorbic acid, sodium metabisulfite, EDTA, NaCl, and citric acid. It was discovered that metal ions of Mg2+ and Cu2+ increased, while Zn2+ and K+ reduced the PPO activity. Sugars showed inhibition on the PPO activity, with higher effect by sucrose and lower effect by fructose and glucose. Optimum pH and temperature for grape PPO activity were 6.0 and 25Â Â°C with 10Â mM catechol as substrate. The enzyme was heat stable between 10 and 25Â Â°C, but showed significant activity loss at temperatures higher than 40Â Â°C and completely inactivation at 70Â Â°C for 10Â min. Thermal inactivation of PPO showed a first-order kinetic with an activation energy (E a) of 146.1Â Â±Â 10.8Â kJ/mol at pH 6.0.