Biochemical characterization and thermal inactivation of polyphenol oxidase from radish (Raphanus sativus var. sativus)

Abstract

Polyphenoloxidase (PPO) is the target for the development of several food antibrowning agents. Different substrates (pyrocatechol, gallic acid, chlorogenic acid, caffeic acid, 3,4 dihydroxybenzoic acid, p-cumaric acid, l-tyrosine, pyrogallic acid and phloroglucinol) were analyzed to determine their affinities with radish PPO. Pyrocatechol, gallic acid and pyrogallic acid were the substrates that showed high affinity based on Vmax/Km ratio. The optimum pH for the PPO using these three substrates were pH = 7 and the optimum temperatures were 20, 60 and 20–40 °C for pyrogallic acid, gallic acid and pyrocatechol, respectively. The kinetics of thermal inactivation was successfully modeled by a biphasic model (r2 > 0.888), attributed to the presence of two enzyme fractions, a heat-labile easily inactivated even at low blanching temperatures, and a heat-resistant fraction that requires blanching temperatures above 80 °C to reach 70% of inactivation. The kinetics constants of this model for both heat-labile and heat-resistant increased with temperature in the range from 60 to 90 °C. The activation energy ratio of resistant to labile fraction was found to be 6 (EaL = 142 kJ/mol).