Genes cloning, sequencing and function identification of recombinant polyphenol oxidase isozymes for production of monomeric theaflavins from Camellia sinensis

Theaflavins (TFs) are important quality compounds in black tea with a variety of biological activities. However, direct extraction of TFs from black tea is inefficient and costly. Therefore, we cloned two PPO isozymes from Huangjinya tea, termed HjyPPO1 and HjyPPO3. Both isozymes oxidized corresponding catechin substrates for the formation of four TFs (TF1, TF2A, TF2B, TF3), and the optimal catechol-type catechin to pyrogallol-type catechin oxidation rate of both isozymes was 1:2. In particular, the oxidation efficiency of HjyPPO3 was higher than that of HjyPPO1. The optimum pH and temperature of HjyPPO1 were 6.0 and 35 °C, respectively, while those of HjyPPO3 were 5.5 and 30 °C, respectively. Molecular docking simulation indicated that the unique residue of HjyPPO3 at Phe260 was more positive and formed a π-π stacked structure with His108 to stabilize the active region. In addition, the active catalytic cavity of HjyPPO3 was more conducive for substrate binding by extensive hydrogen bonding.