Purification and characterization of an intracellular peroxidase from genetically transformed roots of red beet ( Beta vulgaris L.)

Abstract

An intracellular peroxidase (POD) produced by genetically transformed root cultures of red beet ( Beta vulgaris L.) was purified using a combination of (NH 4) 2SO 4 fractionation and ion exchange chromatography resulting in 15-fold enhancement of activity. This enzyme exhibited highest activity (10,500 U mg −1 protein) and stability at pH 5.0 and retained over 70% of the activity for 20 min at 70 °C where horseradish peroxidase (HRP) – a vastly used commercial source, had lost its activity after 11 min. The purified enzyme showed highest preference for H 2O 2 as the substrate ( K m value of 0.1). Among the H donors, the enzyme appeared to have affinity in the order of orthodianisidine > 2,2′-azino-bis(3-ethylbenz-thiazoline)-6-sulfonic acid > guaiacol. The purified POD was completely and competitively inhibited by periodate ( HIO 6 - , K i = 0.2 mM) whereas sodium azide (NaN 3) was a non-competitive inhibitor. The purified POD had a molecular mass of 45 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and activity staining. This is the first report describing purification and characterization of POD from red beet hairy roots showing its better efficacy than commercial HRP.