Study on the Synthesis of Theaflavin-3,3′-Digallate Catalyzed by Escherichia coli Expressing Tea Tree Polyphenol Oxidase Isozymes and Its Enzymatic Solution

Polyphenol oxidase and its isoenzymes are crucial enzymes in the tea tree that catalyze synthesis of theaflavins. In this study, polyphenol was used as research object, various protein sequence treatments, such TrxA fusion tag + N-terminal truncation, were tested for prokaryotic expression through Escherichia coli system. Comparative analyses conducted on activities different recombinant enzyme proteins substrates fractions. Additionally, with highest catalytic efficiency TFDG substrate immobilized using polyethylene glycol to investigate yield TFDG. Our results demonstrated after truncation expression, CsPPO1, CsPPO2, CsPPO3, CsPPO4 mostly expressed form inclusion bodies cell exhibited varying degrees enhancement activity. Specifically, CsPPO1 significantly increased activity EC ECG, CsPPO2 showed enhanced towards ECG EGCG, displayed toward substrates. Homology modeling structural analysis isozymes revealed active centers CsPPO3 consisted double copper ion center structures, while conserved histidine residues surrounding formed structures. Furthermore, immobilization recovery 74.41%. summary, our study elucidated is could be expression. Moreover, treatment greatly improved efficiency. These findings offer an important enzymatic basis theoretical support