Extraction, purification and characterization of lipoxygenase from foxtail millet (Setaria italica)

This study established a method for extracting and purifying foxtail millet lipoxygenase (LOX) by combining ammonium sulfate precipitation and ion exchange chromatography, and investigated its enzymatic properties. The purified LOX showed high specific activity (448.82 ± 13.26 U/mg) and a molecular weight of 96.6 kDa. It achieved maximum catalytic activity at 30 °C and pH 7.0, with good stability within the pH range of 5.0-6.0. Circular dichroism and ultraviolet spectral analyses demonstrated that the heat-induced reduction in LOX enzymatic activity derived from alterations in the protein's spatial structure. The enzymatic reaction kinetics of LOX followed the Michaelis-Menten equation, characterized by a K_m of 3.62 ± 0.06 mM and a V_max of 20.97 ± 0.24 mmol L^-1·min^-1. Cu²⁺, Mn²⁺, Ca²⁺, chelators, sulfhydryl reagents, and three antioxidants (ascorbic acid, quercetin, rutin) inhibited LOX activity (P < 0.05). Fluorescence spectroscopy analysis demonstrated that the fluorescence quenching of LOX by quercetin and rutin conformed to a static quenching process. Combined molecular docking and thermodynamic analyses showed that quercetin interacted with LOX mainly through hydrophobic forces, while rutin bound via hydrogen bonds and van der Waals forces. This work lays a theoretical foundation for the functional study of LOX and its application in food processing, quality improvement, and preservation.