Improving Theaflavin-3,3′-digallate Production Efficiency Optimization by Transition State Conformation of Polyphenol Oxidase

Ying, Hai; Gao, Changzheng; Song, Wei; Wei, Wanqing; Chen, Xiulai; Gao, Cong; Liu, Jia; Wu, Jing; Li, Liming

doi:10.3390/molecules28093831

Cited by 3 publications

(1 citation statement)

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“…The presence of multiple isozymes of PPO in the tea tree plays a crucial role in the formation of theaflavin, a key active ingredient in black tea [27]. Therefore, it is of great significance to develop the tea-tree-derived PPOs and use tea polyphenols as substrates for in vitro enzymatic synthesis of theaflavins [28].…”

Section: Discussionmentioning

confidence: 99%

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

Liu,

Zhou,

Huang

et al. 2023

Fermentation

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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.

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Section: Discussionmentioning

confidence: 99%

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

Liu,

Zhou,

Huang

et al. 2023

Fermentation

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Tyrosinases: a family of copper-containing metalloenzymes

Pretzler,

Rompel

2024

ChemTexts

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Tyrosinases (TYRs) are a family of copper-containing metalloenzymes that are present in all domains of life. TYRs catalyze the reactions that start the biosynthesis of melanin, the main pigment of the animal kingdom, and are also involved in the formation of the bright colors seen on the caps of mushrooms and in the petals of flowers. TYRs catalyze the ortho-hydroxylation and oxidation of phenols and the oxidation of catechols to the respective o-quinones. They only need molecular oxygen to do that, and the products of TYRs—o-quinones—are highly reactive and will usually react with the next available nucleophile. This reactivity can be harnessed for pharmaceutical applications as well as in environmental and food biotechnology. The majority of both basic and applied research on TYRs utilizes “mushroom tyrosinase”, a crude enzyme preparation derived from button mushroom (Agaricus bisporus) fruiting bodies. Access to pure TYR preparations comes almost exclusively from the production of recombinant TYRs as the purification of these enzymes from the natural source is usually very laborious and plagued by low yields. In this text an introduction into the biochemistry of the enzyme TYR will be given, followed by an overview of available structural data of TYRs, the current model for the catalytic mechanism, a survey of reports on the recombinant production of this important metalloenzyme family, and a review of the applications of TYRs for the synthesis of catechols, as biosensors, in bioremediation, for the cross-linking of proteins and medical hydrogels as well as for melanoma treatment. Graphical Abstract

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Research progress on the functions and biosynthesis of theaflavins

Liu,

Wang,

et al. 2024

Food Chemistry

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Improving Theaflavin-3,3′-digallate Production Efficiency Optimization by Transition State Conformation of Polyphenol Oxidase

Cited by 3 publications

References 43 publications

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

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

Tyrosinases: a family of copper-containing metalloenzymes

Research progress on the functions and biosynthesis of theaflavins

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