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. 2016 Nov 28:5:189.
doi: 10.4103/2277-9175.192738. eCollection 2016.

Purification of lactoperoxidase from bovine whey and investigation of kinetic parameters

Fatemeh Borzouee  1 , Mohammad Reza Mofid  1 , Jaleh Varshosaz  2 , Seyed Ziyae Aldin Samsam Shariat  1
Affiliations

Purification of lactoperoxidase from bovine whey and investigation of kinetic parameters

Fatemeh Borzouee et al. Adv Biomed Res. .

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 Km value 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.

Keywords: Bovine milk; enzyme; kinetic parameters; lactoperoxidase; purification; whey.

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Conflict of interest statement

There are no conflicts of interest.

Figures

Figure 1
Figure 1
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bovine lactoperoxidase: (Lane 1) standards proteins; aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), ribonoclease A (13.7 kDa), insulin (6.6 kDa) (Lane 2). The purified Lactoperosxidas from bovine milk
Figure 2
Figure 2
Lineweaver-Burk reciprocal plot of lactoperoxidase for four different concentrations of guaiacol (100, 200, 400 and 800 mM). The concentration of H2O2 was constant (25 mM). Data are shown as a mean of the activity ± standard deviation of three independent experiments
Figure 3
Figure 3
Optimum pH of lactoperoxidase activity. Assay was performed using two wide range of pH. 30 mM citrate-sodium (pH: 3.0–5.5) and 30 mM sodium phosphate buffer (pH 6–8). Data are shown as a mean of the activity ± standard deviation of three independent experiments
Figure 4
Figure 4
Optimum temperature of lactoperoxidase activity. Assay was performed in temperature ranges of (20–80°C). Data are shown as a mean of the activity ± standard deviation of three independent experiments
Figure 5
Figure 5
Thermal stability of purified lactoperoxidase from bovine milk. The enzyme was incubated at the 75°C in water bath for different times and it was chilled immediately and its activity was calculated. Data are shown as a mean of the activity ± standard deviation of three independent experiments
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