Purification and partial characterization of white radish (Raphanus sativus L. var. Long white) peroxidase from cell suspension culture extract

Abstract

Peroxidase mainly Horseradish peroxidase (HRP) has been widely used as a component of clinical diagnostic reagent for Enzyme Linked Immunosorbent Assay (ELISA) technique. White radish (Raphanus sativus L.) was found as another source of peroxidase . In this study, white radish was used for the production of peroxidase by cell suspension culture technique. Isolation of the enzyme was conducted by ammonium sulfate precipitation followed by purification using DEAE-Cellulose column chromatography eluted with 0.01 M phosphate buffer, pH 7.5 and 0-0.5 M NaCl gradient. A major peak of protein having the highest activity and purity 25 folds compared to the crude enzyme was observed. This protein was partially characterized. SDS-Polyacrilamide gel electrophoresis showed one main band with molecular weight of 47.000 Da. This white radish peroxidase (WRP) is a very efficient enzyme with demonstrated maximum activity at temperature 55°C and pH 7.5 as well as a Km 76.6 μg/mL and Vmax 275 μg/mL/ minute toward hydrogen peroxide as substrate and pyrogallol as hydrogen donor.