Purification of lactoperoxidase enzyme from kefir by affinity chromatography technique and investigation of its effects on some chemicals

Lactoperoxidase enzyme (LPO: E.C.1.11.1.7) is a peroxidase found in mammalian milk. There is a similarity between the peroxidase enzyme obtained from milk and the peroxidases found in saliva and tears. Lactoperoxidase consists of a single polypeptide with a molecular weight of approximately 78 kDa, a glycoprotein of 8-10% carbohydrate, and 612 amino acids. Its structure contains heme as a prosthetic group. LPO is an oxidoreductase group enzyme with antibacterial properties. In this study, it was aimed to purify lactoperoxidase (LPO) enzyme from Jersey milk and kefir obtained from the same milk by developing hydrophobic interaction chromatography method, which is a type of affinity chromatography. For this purpose, partial purification of LPO enzymes was carried out by protein precipitation with toluene and then ammonium sulphate. The ammonium sulphate concentration range in which milk lactoperoxidase enzyme showed the best activity was 60% and the ammonium sulphate concentration range in which kefir lactoperoxidase enzyme showed the best activity was 50%. Then, using Sepharose 4B-L-tyrosine-1-naphthylamine gel, milk LPO enzyme was purified 220.8-fold with 24.08 yield and kefir LPO enzyme 399.20-fold with 39.91 yield. The purity of the enzymes was checked using SDS-PAGE electrophoresis method. A single band around 80 kDa was obtained for kefir lactoperoxidase and milk lactoperoxidase. Km and Vmax values of milk LPO and kefir LPO enzymes were determined using Lineweaver-Burk method. For ABTS substrate, Km value for milk LPO was 0.166±0.071 mM and Vmax value was 238.095±0.71 EU/mL; Km value for kefir LPO was 0.088±0.016 mM and Vmax value was 294.117±0.89 EU/mL. In the last step of the study, the effects of some chemicals on pure milk and kefir LPO enzymes were investigated. The inhibition effects of sulfanilamide and sulfaguanidine, which are specific inhibitors of the enzyme, on milk LPO and kefir LPO enzymes were studied. The IC50 and Ki values of sulfanilamide and sulfaguanidine on milk LPO enzyme were 2.14±0.54, 1.5±0.32 and 0.019.10-4±0.009.10-4, 0.035.10-5±0.003.10-5 µM, respectively. IC50 and Ki values of sulfanilamide and sulfaguanidine on kefir LPO enzyme were 2.75±0.67, 2.5±0.59 and 0.044.10-4±0.002.10-4, 0.065.10-5 ± 0.004.10-5 µM, respectively.