Influence of buffer systems on PPO activity of Riesling grapes [Vitis vinifera subsp. vinifera cv. Riesling]

Polyphenol oxidases (PPOs) are type-3 copper proteins that are also found in grape berries, which are rich in phenolic compounds that serve as natural substrates for PPO. The influence of buffer system and pH on kinetic parameters of PPO from Riesling grapes (VvPPOr) was investigated. The pH-optimum for VvPPOr for the diphenolic substrate dopamine ranged between 5.0 and 5.6 determined at 25 °C in citrate–phosphate buffer. At pH 5.0, the apparent KM and vmax were 5.5 mM and 24.9 µM/min with dopamine as substrate and 34.4 mM and 329.7 µM/min with catechol as substrate. Compared to citrate–phosphate buffer, KM was lower when acetate, phosphate or acetate–phosphate buffer was used and about the same in citrate buffer. Thus, not only pH but also the buffer components themselves influence VvPPOr activity. Accordingly, we could show the relevance of using one buffer system covering the whole pH range for the determination of the pH-optimum instead of combining two different buffer systems which is common practice in current literature. VvPPOr also showed activity toward monophenolic substrates such as tyramine and p-tyrosol.