Purification of Bovine Milk Lactoperoxidase and Investigation of Antibacterial Properties at Different Thiocyanate Mediated

Abstract

Bovine lactoperoxidase (LPO) was purified with amberlite CG 50 H+ resin, CM sephadex C-50 ion-exchange chromatography, and sephadex G-100 gel filtration chromatography from skim milk. The activity of lactoperoxidase was measured by using 2.2-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH 6.0. Purification degree for the purified enzyme was controlled with SDS-PAGE and Rz value (A412/A280). Rz value for the purified LPO was 0.8. Km value at pH 6.0 at 20 degrees C for the LPO was 0.20 mM. Vmax value was 7.87 micromol/ml min at pH 6.0 at 20 degrees C. Bovine LPO showed high antibacterial activity in 100 mM thiocyanate--100 mM H2O2 medium for some pathogenic bacteria, such as Aeromonas hydrophila ATCC 7966, Micrococcus luteus LA 2971, Mycobacterium smegmatis RUT, Bacillus subtilis IMG 22, Pseudomonas pyocyanea, Bacillus subtilis var. niger ATCC 10, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 15753, Bacillus brevis FMC3, Klebsiella pneumoniae FMC 5, Corynebacterium xerosis UC 9165, Bacillus cereus EU, Bacillus megaterium NRS, Yersinia enterocolytica, Listeria monocytogenes scoot A, Bacillus megaterium EU, Bacillus megaterium DSM32, Klebsiella oxytocica, Staphylococcus aerogenes, Streptococcus faecalis, Mycobacterium smegmatis CCM 2067 and compared with well known antibacterial substances such as penicilline, ampicilline, amoxicillin-clavulanate and ceftriaxon. The LPO--100 mM thiocyanate--100 mM H2O2 system was purposed as an effective agent against many of the diseases causing organisms in human and animals.