Kinetic study of the irreversible thermal deactivation of palmito (Acanthophoenix rubra) polyphenol oxidase and effect of pH

The optimal temperature of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is 30 degrees C. The Arrhenius activation energy was calculated to be 5.41 kJ mol(-1). Standard enthalpy of the reaction is -60.99 kJ mol(-1). At 25 degrees C, standard free energy and standard entropy were, respectively, 16.75 kJ mol(-1) and -260.87 J mol(-1) K(-1). The enzyme heated at temperatures above 30 degrees C loses its activity. Fifty percent inhibition is reached in 18 min at 70 degrees C, in 8 min at 75 degrees C, and in 2.5 min at 80 degrees C. The kinetics of the thermal irreversible denaturation of this enzyme is characterized by two steps: N leads to X(Td) leads to D, where N represents the native form, X represents an intermediate form, the structure of which depends on the deactivation temperature Td, and D is the completely denatured form of the enzyme. Our experimental results rule out a two-isoenzyme (with varying heat sensitivity) model. The thermodynamic parameters of the irreversible denaturation of the intermediate form were 102.70 and 97.10 kJ mol(-1) for activation enthalpy and activation energy, respectively, and 16.85 J mol(-1) K(-1) for activation entropy at 60 degrees C. Furthermore, this paper describes the effect of pH on the activity of the PPO. Studies were carried out with 4-methylcatechol and pyrogallol as substrates. The pH profile was not a function of the nature of the substrate assayed. The pH optimum was 5.2. The plot of logVmax app vs pH indicates that the oxidation of the substrates depended of the ionization of two groups in the enzyme-substrate complex with apparent pK values of 3.06 and 7.29 and 3.44 and 7.12, respectively, for 4-methylcatechol and pyrogallol. The very slight differences between the values suggest the existence of only one site on the molecule for both substrates.