Purification of lactoperoxidase from bovine milk and investigation of the kinetic properties

Lactoperoxidase (LPO) was purified from bovine milk using Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. During the purification steps, the activity of enzyme was measured using 2,2 ' -azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diamonium salt (ABTS) as a chromogenic substrate at pH 6. Optimum pH and optimum temperature values for LPO were determined for ABTS, p-phenylendiamine, catechol, epinephrine, and pyrogallol as substrates, and then K-m and V-max values for the same substrate were obtained by means of Lineweaver-Burk graphics. The purification degree of the enzyme was controlled by SDS-PAGE and R-z (A(412)/A(280)) values.