Characterization of polyphenol oxidase from Napoleon grape

Polyphenol oxidase (PPO) from Napoleon grape was isolated using a two-phase partitioning approach with Triton X-114. The enzyme was purified in a latent form and could be optimally activated by the presence of 0.2% of sodium dodecyl sulphate (SDS) at pH 6.0. In the absence of SDS, the enzyme showed maximum activity at acid pH (3.0). The enzyme was kinetically characterized at pH 3.0 and pH 6.0 in the presence of 0.2% of SDS, using 4-tert-butylcatechol (TBC) as a substrate. The Vm/KM ratio showed that Napoleon grape PPO presents greater affinity for TBC at acid pH (0.1 min−1) that at pH 6.0 in the presence of SDS (0.02 min−1). The enzyme was highly heat stable, 80% of activity remaining at 70 °C. Selected inhibitors were also studied, tropolone being the most active with a Ki value of 27 μM at acid pH and pH 6.0 in the presence of 0.2% SDS.