Abstract
This study aimed to purify and biochemically characterize lactoperoxidase (LPO) enzymes obtained from Jersey bovine milk and starter culture fermented kefir, and to investigate the inhibitory effects of sulfanilamide and sulfaguanidine. LPO enzymes were purified using ammonium sulfate precipitation followed by hydrophobic interaction chromatography (sepharose 4B-L-tyrosine-1-naphthylamine); milk LPO reached 220.8 ± 5.41 fold purification with 24.08 ± 0.30% yield, whereas kefir LPO achieved 399.2 ± 7.51 fold with 39.91 ± 0.45% yield. SDS-PAGE analysis revealed a single band with an approximate molecular weight of 80 kDa for both enzymes. Kinetic studies showed that kefir LPO exhibited a lower Km (0.088 ± 0.016 mM) and a higher apparent Vmax (294 ± 0.9 EU·mL−1) than milk LPO (Km 0.166 ± 0.071 mM; Vmax 238 ± 0.7 EU·mL−1). Inhibition assays demonstrated competitive behavior for both inhibitors; by Ki, sulfaguanidine was more potent than sulfanilamide (milk: 5.79 ± 0.78 vs 12.2 ± 1.23 μM; kefir: 5.08 ± 0.82 vs 25 ± 1.18 μM). Molecular docking reproduced the crystallographic pose (redocking RMSD 0.99 Å), and molecular dynamics (MD) simulations indicated more persistent binding of sulfanilamide at the active site.