Combining Kinetics and In Silico Approaches to Evaluate Lipophilic Piperic Acid Esters as Anti-Rhizopus oryzae Lipase Agents for Olive Oil Preservation

Abstract

Rhizopus oryzae lipase (ROL) is a key enzyme involved in olive oil spoilage and acts as a virulence factor in fungal infections. Natural lipophilic lipase inhibitors are crucial for mitigating economic losses resulting from lipid degradation in stored or decaying olive fruits. This study evaluated a series of enzymatically synthesized piperate esters with varying alkyl chain lengths (butyryl, C4; octyl, C8; dodecyl, C12) for their inhibitory effects on ROL activity. Octyl piperate (C8) demonstrated the highest potency, with IC50 values of 0.05 mg/mL using methods B and C or 0.25 mg/mL using method A. Molecular docking indicated that C8 achieved the most favorable predicted binding energy (Gscore: –11.134 kcal/mol), primarily through hydrophobic interactions (Val329, Ala212, Phe209) and hydrogen bonds with oxyanion hole residues (Ser268, Thr206, Gln241). Molecular dynamics simulations confirmed that C8 maintained stable binding and stabilized the catalytic residues. In comparison, C4 exhibited weaker interactions, and the longer C12 chain induced conformational instability and steric hindrance. These results establish a parabolic structure–activity relationship, identifying the octyl chain (C8) as optimal for ROL inhibition among the tested derivatives. The rational design of lipophilic, biodegradable lipase inhibitors thus positions octyl piperate as a promising candidate for extending olive storage and shelf life, and as a scaffold for developing natural antifungal agents targeting virulent R. oryzae strains.