Purification and characterization of peroxidase from foxtail millet (Setaria italica)

Foxtail millet peroxidase (POD) may contribute to quality deterioration during processing and storage, but its biochemical and thermal properties remain insufficiently characterized. In this study, POD was purified from foxtail millet by ammonium sulfate fractionation, dialysis, and a single SP-Sepharose cation-exchange step and identified by nanoLC-MS/MS. The purified enzyme had an apparent molecular mass of 37.7 kDa and a specific activity of 315.21 ± 11.02 U/mg, corresponding to a 62.74-fold purification and a yield of 32.52%. Its optimal temperature and pH were 40 °C and 6.0, respectively. Thermal inactivation between 60 and 100 °C followed first-order kinetics, with an activation energy of 86.00 ± 1.36 kJ/mol. Heating induced progressive CD and UV spectral changes, and CD-based estimates suggested a decrease in α-helical content and an increase in β-sheet content. Using H2O2 as the variable substrate and guaiacol as the co-substrate, the enzyme exhibited an apparent Km of 1.001 mM and a Vmax of 4.30 µmol·L−1·min−1. Ca2+, Cu2+, and Mg2+ enhanced POD activity, whereas Fe³+, Zn2+, Mn2+, citric acid, and EDTA reduced it under the assay conditions. Among the tested additives, ascorbate produced the greatest decrease in the guaiacol-assay signal. Fluorescence quenching and exploratory docking further suggested a possible POD–ascorbate association, providing complementary spectroscopic and structural insights for investigating its effects on POD-mediated reactions. Overall, these findings extend the characterization of foxtail millet POD and may inform strategies for controlling POD-associated quality changes in millet products.