Purification of lactoperoxidase from bovine whey and investigation of kinetic parameters

Document Type : Original Article


1 Department of Clinical Biochemistry, Bioinformatics Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Pharmaceutics, School of Pharmacy and Novel Drug Delivery Systems Research Centre, Isfahan University of Medical Sciences, Isfahan, Iran


Background: Lactoperoxidase (LPO) is related to mammalian peroxidase family which contains a wide spectrum of biological activities. Despite the wide studies on the LPO, there is little study has been performed to simplify and shorten the procedure of enzyme purification. The aim of this project was to purify the enzyme through a simple method, and investigating enzyme kinetic parameters.
Materials and Methods: LPO was purified from bovine whey through modified method of Yoshida (1990) using two steps of ammonium sulfate precipitation and ion-exchange chromatography. The purity of isolated enzyme was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Results: The enzyme was purified 59.13-fold with a recovery of 10.26 having a specific activity of 5.78 U/mg protein and an Rz value of 0.8. The enzyme activity was measured using guaiacol as a chromogenic substrate in phosphate buffer pH 6. SDS-PAGE showed a single bond with molecular weight of 78 kDa. The purified enzyme displayed optimum activity at pH 6 in 30 mM phosphate buffer and at a temperature of 50°C, with a Kmvalue of 178 mM and Vmax 0.63 U/ml.min for guaiacol.
Conclusion: Using only one step ion-exchange chromatography, LPO was isolated from bovine whey in high purity.


Sharma S, Singh AK, Kaushik S, Sinha M, Singh RP, Sharma P, et al. Lactoperoxidase: Structural insights into the function, ligand binding and inhibition. Int J Biochem Mol Biol 2013;4:108-28.  Back to cited text no. 1
Seidel A, Parker H, Turner R, Dickerhof N, Khalilova IS, Wilbanks SM, et al. Uric acid and thiocyanate as competing substrates of lactoperoxidase. J Biol Chem 2014;289:21937-49.  Back to cited text no. 2
Al-Baarri AN, Ogawa M, Hayakawa S. Application of lactoperoxidase system using bovine whey and the effect of storage condition on lactoperoxidase activity. Int J Dairy Sci 2011;6:72-8.  Back to cited text no. 3
Kussendrager KD, van Hooijdonk AC. Lactoperoxidase: Physico-chemical properties, occurrence, mechanism of action and applications. Br J Nutr 2000;84 Suppl 1:S19-25.  Back to cited text no. 4
Boots JW, Floris R. Lactoperoxidase: From catalytic mechanism to practical applications. Int Dairy J 2006;16:1272-6.  Back to cited text no. 5
Davies MJ, Hawkins CL, Pattison DI, Rees MD. Mammalian heme peroxidases: From molecular mechanisms to health implications. Antioxid Redox Signal 2008;10:1199-234.  Back to cited text no. 6
Andersson LA, Bylkas SA, Wilson AE. Spectral analysis of lactoperoxidase. Evidence for a common heme in mammalian peroxidases. J Biol Chem 1996;271:3406-12.  Back to cited text no. 7
Ozdemir H, Aygul I, Küfrevioglu OI. Purification of lactoperoxidase from bovine milk and investigation of the kinetic properties. Prep Biochem Biotechnol 2001;31:125-34.  Back to cited text no. 8
Mecitoğlu Ç, Yemenicioğlu A. Partial purification and preparation of bovine lactoperoxidase and characterization of kinetic properties of its immobilized form incorporated into cross-linked alginate films. Food Chem 2007;104:726-33.  Back to cited text no. 9
Atasever A, Ozdemir H, Gulcin I, Irfan Kufrevioglu O. One-step purification of lactoperoxidase from bovine milk by affinity chromatography. Food Chem 2013;136:864-70.  Back to cited text no. 10
Ye X, Yoshida S, Ng TB. Isolation of lactoperoxidase, lactoferrin, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A from bovine rennet whey using ion exchange chromatography. Int J Biochem Cell Biol 2000;32:1143-50.  Back to cited text no. 11
Defaie M, Sharieat ZS, Divsalar A. Kinetic investigation on lactoperoxidase upon interaction with lead ion. Int J Environ Sci Dev 2010;1:97.  Back to cited text no. 12
Neyestani TR, Djalali M, Pezeshki M. Isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity. Protein Expr Purif 2003;29:202-8.  Back to cited text no. 13
Scopes R. Ion exchangers-principles, properties and uses. In: Protein Purification: Principles and Practice. New York: Springer-Verlag; 1982. p. 75-101.  Back to cited text no. 14
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.  Back to cited text no. 15
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.  Back to cited text no. 16
Maehly A, Chance B. Assay of catalases and peroxidases. Methods Enzymol 1955;2:764-75.  Back to cited text no. 17
Koksal E. Peroxidase from leaves of spinach (Spinacia oleracea): Partial purification and some biochemical properties. Int J Pharmacol 2011;7:135-9.  Back to cited text no. 18
Aghelan Z, Shariat SZ. Partial purification and biochemical characterization of peroxidase from rosemary (Rosmarinus officinalis L.) leaves. Adv Biomed Res 2015;4:159.  Back to cited text no. 19
[PUBMED]  Medknow Journal  
Aune TM, Thomas EL. Accumulation of hypothiocyanite ion during peroxidase-catalyzed oxidation of thiocyanate ion. Eur J Biochem 1977;80:209-14.  Back to cited text no. 20
Dajanta K, Chukeatirote E, Apichartsrangkoon A. Effect of lactoperoxidase system on keeping quality of raw cow's milk in Thailand. Int Dairy Sci 2008;3:112-6.  Back to cited text no. 21
Tayefi-Nasrabadi H, Asadpour R. Effect of heat treatment on buffalo (Bubalus bubalis) lactoperoxidase activity in raw milk. J Biol Sci 2008;8:1310-5.  Back to cited text no. 22
Atamer M, Kocak C, Cimer A, Odabasi S, Tamucay B, Yamaner N. Some quality characteristics of kasar cheese manufactured from milk preserved by activation of lactoperoxidase/thiocyanate/hydrogen peroxide (LP) system. Milchwissenschaft 1999;54:553-6.  Back to cited text no. 23
Pourtois M, Binet C, Van Tieghem N, Courtois PR, Vandenabbeele A, Thirty L. Saliva can contribute in quick inhibition of HIV infectivity. AIDS 1991;5:598-600.  Back to cited text no. 24
Dumontet C, Rousset B. Identification, purification, and characterization of a non-heme lactoperoxidase in bovine milk. J Biol Chem 1983;258:14166-72.  Back to cited text no. 25
Hahn R, Schulz PM, Schaupp C, Jungbauer A. Bovine whey fractionation based on cation-exchange chromatography. J Chromatogr A 1998;795:277-87.  Back to cited text no. 26
Nandini KE, Rastogi NK. Integrated downstream processing of lactoperoxidase from milk whey involving aqueous two-phase extraction and ultrasound-assisted ultrafiltration. Appl Biochem Biotechnol 2011;163:173-85.  Back to cited text no. 27
Jafary F, Kashanian S, Sharieat ZS, Jafary F, Omidfar K, Paknejad M. Stability improvement of immobilized lactoperoxidase using polyaniline polymer. Mol Biol Rep 2012;39:10407-12.  Back to cited text no. 28
Köksal E, Gülçin I. Purification and characterization of peroxidase from cauliflower (Brassica oleracea L. var. botrytis) buds. Protein Pept Lett 2008;15:320-6.  Back to cited text no. 29
Tenovuo J, Kurkijärvi K. Immobilized lactoperoxidase as a biologically active and stable form of an antimicrobial enzyme. Arch Oral Biol 1981;26:309-14.  Back to cited text no. 30
Boscolo B, Leal SS, Ghibaudi EM, Gomes CM. Lactoperoxidase folding and catalysis relies on the stabilization of the α-helix rich core domain: A thermal unfolding study. Biochim Biophys Acta Proteins Proteomics 2007;1774:1164-72.  Back to cited text no. 31