Purification of lactoperoxidase from bovine whey and investigation of kinetic parameters

Borzouee, Fatemeh; Mofid, Mohammad Reza; Varshosaz, Jaleh; Shariat, Seyed Ziyae Aldin Samsam

Purification of lactoperoxidase from bovine whey and investigation of kinetic parameters

Document Type : Original Article

Authors

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

Abstract

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 K_mvalue of 178 mM and V_max 0.63 U/ml.min for guaiacol.
Conclusion: Using only one step ion-exchange chromatography, LPO was isolated from bovine whey in high purity.

Keywords

References

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