Chromatographic isolation and characterization of a novel peroxidase from large lima legumes

Abstract

A novel peroxidase with antifungal activity was isolated from the large lima bean (Phaseolus limensis) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Toyopearl, and gel filtration on Sephadex G-75. The enzyme was adsorbed on Affi-gel blue gel and SP-Toyopearl, and possessed a molecular weight of 34 kDa in SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. There was an almost 110-fold increase in specific activity of the purified peroxidase compared with that of the crude extract. The enzyme exhibited a pI of 8.6 by isoelectric focusing electrophoresis, indicating that it is a basic protein. The optimum pH and the optimum temperature were 5.5 and 30 °C, respectively. The enzyme was stable up to 55 °C. It potently suppressed mycelial growth in Fusarium solani, Mycosphaerella arachidicola, and Pythium aphanidermatum with an IC50 of 76, 103, and 119 μM, respectively. © 2009 Institute of Food Technologists®.