Some kinetic properties of polyphenol oxidase from Thymbra spicata L. var. spicata
Polyphenol oxidase (PPO) of Thymbra (Thymbra spicata L. var. spicata) was isolated by (NH4)2SO4 precipitation and dialysis. A diphenolase from Thymbra plant, active against 4-methylcatechol, catechol and pyrogallol was characterized in detail in terms of pH and temperature optima, stability, kinetic parameters and inhibition behaviour towards some general PPO inhibitors. 4-Methylcatechol was the most suitable substrate, due to the lowest Km and the biggest Vmax/Km values, followed by catechol and pyrogallol. The Thymbra PPO had maximum activity at pH 5.0, 7.0 and 8.0 with 4-methylcatechol, catechol and pyrogallol substrates, respectively. The optimum temperature of activity for Thymbra PPO was 30, 40 and 50 °C for 4-methylcatechol, catechol and pyrogallol substrates, respectively. It was found that optimum temperature and pH were substrate-dependent studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time. Inhibition of Thymbra PPO was investigated with inhibitors such as l-cysteine and glutathione using 4-methylcatechol, catechol and pyrogallol as substrates. It was found that l-cysteine was a more effective inhibitor than glutathione owing to lower Ki. The type of inhibition depended on the origin of the PPO studied and also on the substrate used. Furthermore, the IC50 values of inhibitors sudied on PPO were determined by means of activity percentage (I) diagrams.
Journal: Process Biochemistry - Volume 41, Issue 12, December 2006, Pages 2379–2385