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 ∼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 °C. TCWP1 and TCWP2 isoenzymes were stable up to 50 °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 °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 °C. The Km 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.

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► The peroxidase enzyme found in tender coconut water is isoenzymes (TCWP1 & TCWP2) in nature.
► Though it is an isoenzyme nature, the TCWP isoenzyme has not much difference in their physico-chemical characters.
► The necessity of sodium acetate for peroxidation reaction is studied.
► The TCWP isoenzymes are potential candidates in the biotransformation of genotoxic into non-genotoxic compound.