Isolation and characterization of thermo-tolerant polyphenol oxidases in a black tea infusion

Abstract

Polyphenols are important active components of tea. Tea polyphenol oxidase (PPO) catalyzes polyphenols oxidation, hence it is vitally important to the quality and function of black tea. The enzyme was believed to be mostly deactivated during the thermal processing. Here we report the macromolecular fraction of a Chinese black tea (Camellia. sinensis var. Lapsang souchong) infusion oxidized pyrocatechol even at the enzyme's deactivation temperature. Utilizing urea-assisted extraction and ion-exchange chromatography, the PPOs and/or co-enzymes were isolated from the tea infusion and the cold water extracts of fresh tea leaves. The isolates were found to exhibit PPO activities after removing urea by dialysis. A 66.0 kDa PPO with specific activity of 1200 U/mg (Km = 6.973 mM and Vmax = 15.79 U/min) was purified and characterized to be a glycoprotein with isoelectric point of 4.82. The efficiency of four inhibitors against this PPO are cysteine > EDTA > sodium sulfite > ascorbic acid. The PPOs isolated from black tea infusion reserved 40–80% of activities of their aliquots from fresh tea leaves. It reveals the PPOs can survive the thermal processing of the black tea, possibly in form of PPO-polyphenol complex. Exploring the biochemistry and oxidation mechanism of tea infusion, this study may empower the future development of tea and plant-based beverages.