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

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. [Display omitted] •Lily bulb polyphenol oxidase (PPO) was purified and analyzed.•An optimized muti-stage separation method was developed for lily bulb PPO.•PPO was identified as a domeric protein with molecular weight of 135 kDa.•PPO consisted of random coil, α-helix, β-folding, and β-corner.•PPO binding sites to catechol and 4-methylcatechol was studied by molecular docking.