Lily bulb polyphenol oxidase obtained via an optimized multi-stage separation strategy for structural analysis and browning mechanism elucidation

Haishan Xu; Xinyu Wang; Huan Li; Ying Xie; Ke Ding; Saiqing Xu; Shenghua Ding; Rongrong Wang

doi:10.1016/j.foodchem.2024.141418

Lily bulb polyphenol oxidase obtained via an optimized multi-stage separation strategy for structural analysis and browning mechanism elucidation

Food Chem. 2024 Oct 5;463(Pt 4):141418. doi: 10.1016/j.foodchem.2024.141418. Online ahead of print.

Authors

Haishan Xu¹, Xinyu Wang², Huan Li³, Ying Xie², Ke Ding², Saiqing Xu², Shenghua Ding⁴, Rongrong Wang⁵

Affiliations

¹ College of Food Science and Technology, Hunan Agricultural University, Changsha 410128, China; DongTing Laboratory, Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China.
² DongTing Laboratory, Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China; Longping Branch, College of Biology, Hunan University, Changsha 410125, China.
³ DongTing Laboratory, Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China.
⁴ DongTing Laboratory, Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China; Longping Branch, College of Biology, Hunan University, Changsha 410125, China. Electronic address: [email protected].
⁵ College of Food Science and Technology, Hunan Agricultural University, Changsha 410128, China. Electronic address: [email protected].

PMID: 39427459
DOI: 10.1016/j.foodchem.2024.141418

Abstract

An optimized multi-stage separation strategy was developed to purify lily bulb polyphenol oxidase (PPO) for revealing its molecular structure. The PPO was purified 14.64-fold with high specific activity of 153,900 U/mg via optimized conditions of phosphate buffer pH (6.5), solid-liquid ratio (1:3), PVPP content (2 %), extraction time (4 h), followed by 30 %-50 % ammonium sulfate, diethylaminoethyl ion-exchange chromatography (0.1 M NaCl), and size exclusion chromatography. The PPO was identified as a dimeric protein with molecular weight of 135 kDa, containing 58.79 % random coil, 20.78 % α-helix, 17.41 % β-folding, and 3.02 % β-corner. The three-dimensional structure via homology modeling suggested that active center CuA bound to His151, His172, and His181, CuB bound to His307, His311, and His341. Furthermore, molecular docking indicated that its Phe337 and Tyr312 residues were catalytic cavity gates of catechol and 4-methylcatechol, respectively. Therefore, this study successfully analyzed purified PPO structure and further provided a theoretical foundation for its browning mechanism.

Keywords: Binding sites; Lily bulb; Molecular docking; Polyphenol oxidase; Purification optimization.