Peroxidase Can Perform the Hydroxylation Step in the "Oxidative Cascade" during Oxidation of Tea Catechins.

The formation of black tea thearubigins involves at least two of the following oxidation steps: (i) oligomerization, (ii) rearrangement, and (iii) hydroxylation. The first two are mainly catalyzed by polyphenol oxidase (PPO), whereas the enzyme responsible for hydroxylation has not yet been identified. Two main oxidative activities, peroxidase (POD) and PPO, occur in tea leaves. POD was hypothesized to be responsible for hydroxylation. Model systems with horseradish POD and mushroom tyrosinase were used investigating hydroxylation of theaflavins (TFs). POD was found capable of hydroxylation. TFs with up to five extra hydroxyl groups were annotated by their MS2 data. Hydroxylation by POD was also shown for theanaphtoquinones, theatridimensins, and dehydrodicatechins. The H2O2 concentration influenced the extent of hydroxylation, decreasing it at concentrations above 0.01 mM. TFs with up to five extra hydroxyl groups and traces of other hydroxylated oligomeric catechins could be annotated in black tea without any sample pretreatment, using a selective screening method with reversed-phase ultrahigh-performance liquid chromatography mass spectrometry.