Mechanistic Insights into the Highly Efficient Degradation of Phenylethylamine by a Multicopper Oxidase from Psychrobacter

Phenylethylamine (PEA) is widely present in fermented foods and seafood, posing potential risks to food safety and human health. In this study, a multicopper oxidase gene ( PsyMCO ) responsible for PEA degradation was identified from an isolated PEA-degrading bacterium, Psychrobacter sp. FJNU-SIO36. Structure-guided mutagenesis targeting residue Asn221, located near the T1 copper site, generated mutant Psy MCO_N221D, which exhibited significantly improved substrate affinity and catalytic turnover. Under optimized conditions, the PEA degradation efficiency increased from 27.9 to 98.5%, achieving nearly complete removal within 72 h. Molecular docking and dynamics simulations revealed that the N221D substitution alters the local electrostatic potential, thereby enhancing substrate binding and favorable positioning near the T1 Cu site. This study establishes a direct link between local electrostatic modulation at the T1 Cu site and enhanced amine oxidation, providing mechanistic insight into MCO catalysis and a rational basis for developing efficient MCO-based biocatalysts for detoxifying biogenic amines in foods.