Purification and characterization of peroxidases from liquid endosperm of Cocos nucifera (L.): Biotransformation

Peroxidases are ubiquitous oxidoreductase enzymes and find application in various physiological/biochemical reactions. In the present study, class III peroxidase enzymes from the liquid endosperm (TCWP - Tender Coconut Water Peroxidase) of Cocos nucifera were identified and purified using ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography resulting in 9.77-fold of purified enzymes to its apparent homogeneity. The purification profile of peroxidase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis had shown two protein bands corresponding to two isoenzymes (TCWP1 and TCWP2) with a molecular weight of similar to 47 and 49 kDa respectively. The purified isoenzymes TCWP1 and TCWP2 exhibited its maximal activity (3.5 and 3.2 U/ml; respectively) at pH 4.5 and 5.0 with o-Dianisidine dihydrochloride (o-D) as substrate at 40 degrees C. TCWP1 and TCWP2 isoenzymes were stable up to 50 degrees C for 1 h. The stability of the enzyme at increased temperature may be attributed to the presence of Ca2+ ions to the enzyme. Addition of excess Ca2+ ions to the enzyme mixture enhanced the stability further to 55 degrees C for both the isoenzymes. The excess addition of H2O2 inhibited the peroxidase activity and the TCWP isoenzymes were stable up to 10 mM of H2O2 concentration at 25 degrees C. The K-m for o-D was determined to be 1.63 mM and 4.0 mM for TCWP1 and TCWP2, respectively. The TCWP activity was enhanced with the addition of carboxylic compound (sodium acetate) at 7 mM and Mn2+ at 1 mM. Sodium cyanide and phenyl hydrazine inhibited the enzyme competitively but sodium azide and hydroxylamine were uncompetitive and non-competitive inhibitor respectively. The TCWP enzyme had the potential to biotransform the genotoxic compound into non-genotoxic compound. The meager difference in biochemical characters of TCWP isoenzymes leads to identify its applications in various industries. (C) 2013 Elsevier B.V. All rights reserved.