Molecular dynamics provide insights into the purification of tea membrane-bound polyphenol oxidase through three-phase partitioning utilizing an eco-friendly deep eutectic solvent.

Membrane-bound polyphenol oxidase (mPPO) presents significant challenges for fine purification due to its inherently amphiphilic properties and its propensity to adhere to membranes. This study evaluated deep eutectic solvents (DES) as replacements for t-butanol in three-phase partitioning (TPP) for the purification of tea mPPO. The results indicated that DES-5 (menthol/octanoic acid) was the most effective alternative solvent. Optimization experiments revealed that using (NH4)2SO4 at 47%, DES: crude extract 2:1 (v/v), extraction time of 75 min, and a temperature of 30 °C yielded an enzyme activity of 1967 U and a purification fold of 16.64. SEM analysis suggested that this purification system effectively preserved the morphological integrity of mPPO. Molecular docking and molecular dynamics (MD) simulations demonstrated that DES exhibited high compatibility and good affinity with mPPO, contributing to its stabilization during purification. Ultimately, these results contribute to a greener and more effective approach for the utilization of membrane-bound enzymes.