The extraction of polyphenol oxidase and the enzymatic synthesis of theaflavin are based on a three-phase separation technolog

Abstract Polyphenol oxidase (PPO) plays a pivotal role in the conversion of catechins into theaflavins (TFs). The yield and purity of TFs produced by purified PPO are superior to those produced by crude enzymes in the enzymatic preparation of TFs. The present study utilizes a potato PPO enzyme as a donor to investigate the optimal purification conditions for three-phase separation, the enzymatic properties of the purified PPO, and its performance in the enzymatic synthesis of theaflavins. The optimal three-phase separation conditions were determined to be as follows: 50% ammonium sulfate saturation, a 1:1.5 extraction solution-to-tert-butanol volume ratio, a system pH of 5, and a 60-minute separation time. Consequently, the PPO enzyme activity attained a value of 251.00 U/mL, exhibiting a purification fold of 5.73. Furthermore, the study yielded preliminary findings regarding the enzymatic properties of the purified PPO and its application in the enzymatic synthesis of TFs. The results demonstrated that the purified enzyme exhibited a higher TF synthesis yield in comparison to the crude enzyme. In addition, the investigation revealed that potato PPO exhibited specificity toward the formation of theaflavin monomers, preferentially generating TFDG while demonstrating weaker synthesis capacity for TF-3'-G. The present study employed a three-phase separation method for potato PPO, with a short purification cycle and high extraction efficiency, advantageous for industrial TFs production.