Affiliation:
1. Key Laboratory of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha 410128, China
2. Tea Refining and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China
3. National Research Center of Engineering and Technology for Utilization of Botanical Functional Ingredients, Hunan Agricultural University, Changsha 410128, China
Abstract
Polyphenol oxidase and its isoenzymes are crucial enzymes in the tea tree that catalyze the synthesis of theaflavins. In this study, tea tree polyphenol oxidase was used as the research object, and various protein sequence treatments, such as TrxA fusion tag + N-terminal truncation, were tested for prokaryotic expression through the Escherichia coli expression system. Comparative analyses were conducted on the activities of the different recombinant enzyme proteins on the substrates of tea polyphenol fractions. Additionally, the enzyme with the highest catalytic efficiency on the TFDG substrate was immobilized using polyethylene glycol to investigate the yield of its synthesis of TFDG. Our results demonstrated that after N-terminal truncation and TrxA fusion expression, CsPPO1, CsPPO2, CsPPO3, and CsPPO4 were mostly expressed in the form of inclusion bodies in the cell and exhibited varying degrees of enhancement in substrate activity. Specifically, CsPPO1 exhibited significantly increased activity in EC and ECG, CsPPO2 showed enhanced activity towards ECG and EGCG, and CsPPO2 displayed the highest activity toward TFDG substrates. Homology modeling structural analysis of the polyphenol oxidase isozymes revealed that the active centers of CsPPO1, CsPPO2, and CsPPO3 consisted of double copper ion center structures, while the conserved histidine residues surrounding the active centers formed different catalytic activity centers in different structures. Furthermore, polyethylene glycol immobilization significantly increased the activity recovery of the CsPPO2 enzyme to 74.41%. In summary, our study elucidated that tea tree polyphenol oxidase is expressed as inclusion bodies in prokaryotic expression, and the activity of the recombinant enzyme towards substrates could be enhanced through N-terminal truncation and TrxA fusion expression. Moreover, immobilization treatment of the CsPPO2 enzyme greatly improved enzyme efficiency. These findings offer an important enzymatic basis and theoretical support for the synthesis of theaflavins.
Funder
Intergovernmental International Innovation Cooperation Project of National Key R&D Program
Open Fund of Sichuan Key Laboratory of Refined Sichuan Tea
Ministry of Finance and Ministry of Agriculture and Rural Development: Funding of National Modern Agricultural Industrial Technology System
Research on the Technology of Efficient Utilization of Excellent Resources of Tea Functional Components
Subject
Plant Science,Biochemistry, Genetics and Molecular Biology (miscellaneous),Food Science