从牛乳清中纯化乳过氧化物酶并研究其动力学参数。

Purification of lactoperoxidase from bovine whey and investigation of kinetic parameters.

作者信息

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

机构信息

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

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

出版信息

Adv Biomed Res. 2016 Nov 28;5:189. doi: 10.4103/2277-9175.192738. eCollection 2016.

DOI:10.4103/2277-9175.192738

PMID:28028529

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5156962/

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 value of 178 mM and V 0.63 U/ml.min for guaiacol.

CONCLUSION

Using only one step ion-exchange chromatography, LPO was isolated from bovine whey in high purity.

摘要

背景

乳过氧化物酶（LPO）属于哺乳动物过氧化物酶家族，具有广泛的生物学活性。尽管对LPO进行了广泛研究，但很少有研究致力于简化和缩短酶纯化过程。本项目的目的是通过一种简单方法纯化该酶，并研究酶动力学参数。

材料与方法

采用吉田（1990年）改良方法，通过两步硫酸铵沉淀和离子交换色谱法从牛乳清中纯化LPO。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）监测分离酶的纯度。

结果

该酶纯化了59.13倍，回收率为10.26，比活性为5.78 U/mg蛋白质，Rz值为0.8。在pH 6的磷酸盐缓冲液中，以愈创木酚作为显色底物测定酶活性。SDS-PAGE显示一条分子量为78 kDa的单条带。纯化后的酶在30 mM磷酸盐缓冲液中pH 6以及温度为50°C时表现出最佳活性，对愈创木酚的K值为178 mM，V为0.63 U/ml·min。