Partial purification and characterization of peroxidases from the leaves of Sapindus mukorossi

Abstract

Peroxidases were isolated from Sapindus mukorossi (Reetha) and partially purified using acetone precipitation, ion-exchange chromatography with a 14-fold purification, 22% recovery and a specific activity of 266× 10 3 units/mg protein. Sapindus peroxidases (SPases) showed six bands after acetone precipitation and one distinct band after ion exchange chromatography on Native-PAGE after zymography. Enzymes purified by ion exchange chromatography were loaded on Sepahdex G-50 superfine column and their molecular weight was reported to be 25 kDa. They showed temperature optima at 50°C and pH optima at 5.0. km for SPases was reported to be 1.05 mM and 0.186 mM for guaiacol and H 2 O 2 respectively. The V max /K m value for o-dianisidine was 449 while for H 2 O 2 it was 5×10 5 . Protocatechuic acid acts as a potent inhibitor for SPases (6.0% relative activity at 4.5 μM) but ferulic acid inhibits its activity at a much lower concentration (0.02 μM). Enzymes were stimulated by metal cations like Cu 2+ , Ca 2+ (145, 168; percentage relative activity respectively) and showed mild inhibition (up to 20%) with Mn 2+ and Mg 2+ . Alanine stimulated the enzyme activity (up to 33%; at 0-100 μM) while other amino acids like cysteine, methionine, tryptophan and tyrosine inhibited the SPases (13-57% at 0-100 μM).