Purification and Characterization of Plant Peroxidase from Coccinia Grandis (Ivy Gourd)

Plant peroxidase extracted from Coccinia grandis Lin. (Ivy gourd) was purified from crude extract by ammonium sulfate precipitation, ion-exchange chromatography, and size exclusion chromatography. The purified enzyme preparation exhibited a specific activity of 6106.63 μmol.min^(-1).mg protein^(-1), while purification fold and yield were 17.45 and 34.70%, respectively. The purified peroxidase was homogenous as judged by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight as determined by gel filtration and SDS-polyacrylamide gel electrophoresis was 45 kD, which suggested that the purified peroxidase contained only one subunit. The apparent K(subscript m) and V(subscript max) values of the enzyme against phenol were 93 μM and 561 μmol.min^(-1).mg protein^(-1), respectively. The temperature and pH optimum for purified peroxidase were 45℃ and pH 6.0, respective. However, it was stable at 30-60℃ and pH 4.0-8.0. The presence of metal ions such as Cu(superscript 2+) and Ca(superscript 2+) enhanced peroxidase activity. On the other hand, Cr(superscript 3+) and Hg(superscript 2+) strongly inhibited the enzyme activity at 500 μM. Sodium dodecyl sulfate reduced a half of peroxidase activity at approximately 3 mM. Ivy gourd was stability in the presence of each urea concentrations. The affinity of the enzyme with different substrates showed as the highest relative activities on gallic acid followed by catechin, ascorbic acid and caffeic acid, respectively.