In vitro enzymatic synthesis of a monomeric theaflavin using a polyphenol oxidase isozyme from tea (Camellia sinensis) leaf

Polyphenol oxidase (PPO) plays an important role in tea production, and there are many studies on the enzymatic synthesis of theaflavins using PPO; however, using a purified PPO isozyme (PPOI) instead of a crude PPO enzyme to obtain monomeric theaflavin in vitro is a novel approach that has rarely been previously reported. In the present study, a tea PPOI was purified and used for the enzymatic synthesis of theaflavin monomers. Tea leaf PPOI was purified 12.90-fold, with recovery yield of 1.30% via the sequential use of precipitation with 30%-80% saturated (NH4)(2)SO4, Q-Sepharose Fast Flow anion exchange chromatography and Sephadex G-75 gel filtration chromatography. Subsequently, PPOI was used in the enzymatic synthesis of theaflavins: under various pH, temperatures, reaction times and catechin substrate composition and concentration, only the amount of theaflavin components produced was changed, and no other theaflavin component apart from the theaflavin monomer was produced. These results highlight the importance of monomeric theaflavin synthesis using purified PPOI isozyme and provide insight into PPO in the context of tea production.