Purification of Peroxidase Enzyme from Water Mimosa by Chromatography Technique.

Abstract

<b>Background and Objective:</b> Peroxidase (POD) is the most widely used enzyme in the manufacture of diagnostic kits, biosensors, immunohistochemistry and different industrial sectors. In this study, the POD was extracted from some local vegetables in Thailand; water mimosa. The POD was biochemically purified and characterized from water mimosa. <b>Materials and Methods:</b> The comparison of the peroxidase enzyme activity from water mimosa using Ion exchange chromatography was analyzed statistically using the Kruskal-Wallis one-way ANOVA non-parametric test. Crude extracted from water mimosa was purified by ion exchange chromatography by two techniques (DEAE-Sepharose chromatographic step and CM-Sepharose chromatographic). <b>Results:</b> The crude enzyme from water mimosa exhibited the highest peroxidase activity at 1,7458.5 U/mL. After purification, the peroxidase enzyme in the DEAE-Sepharose column showed a 1.61-fold increase in purity at a NaCl concentration of 0.0 M in 20 mM Tris-HCl buffer, pH 7.2, with a remaining yield of 46.15%. However, after DEAE-Sepharose and CM-Sepharose columns, the purity increased by 1.64-fold at a NaCl concentration of 0.0 M in 20 mM sodium acetate, pH 5.5, but the remaining yield was only 7.45%. The molecular weight of the POD enzyme was 32.3+2 kDa (n = 5) by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The enzyme activity of POD showed approximately 3,500 U/mL at pH 6.8 and the optimum temperature was 37°C. From these studies, peroxidase activities in water mimosa demonstrated a "high total activity". <b>Conclusion:</b> These results suggested that POD from water mimosa could replace horseradish peroxidase (HRP), the most used peroxidase, which is very valuable to reduce the costs of biosensors or diagnostic kit applications.