Polyphenol oxidase from edible insect Antheraea pernyi pupa: Characterization and browning mechanism

The edible insect Antheraea pernyi pupa, a promising insect protein resource, undergoes endogenous polyphenol oxidase (PPO)-mediated enzymatic browning during processing, severely compromising product quality. To elucidate browning mechanisms, this study purified and characterized A. pernyi pupa PPO. LC-MS/MS analysis identified the enzyme as an 80 kDa protein comprising 683 amino acids, exhibiting optimal activity at 45 °C and pH 6.5. Substrate specificity assays revealed a strong preference for o-diphenolic substrates (L-3,4-dihydroxyphenylalanine and Caffeic acid). Molecular docking further demonstrated high-affinity binding of caffeic acid to the active site via bidentate hydrogen bonds (Ser-628), hydrophobic interactions (Pro-638), and salt bridges (Lys-614), highlighting its role as a critical browning substrate. By integrating enzymatic characterization and molecular interaction analyses, this work delineates A. pernyi pupa PPO's catalytic features and provides a theoretical basis for understanding browning pathways and designing targeted inhibitors.