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Peroxidase was purified to a homogeneous state from Castanea Semen by ammonium sulfate precipitation, DEAF-cellulose column chromatography, gel filtration on sephadex G-100 and HPLC, and the purification fold was 65.3. The molecular weight of the enzyme was estimated to be about 35,000 by HPLC. In properties of the enzyme which was purified up to sephadex G-100 column chromatography, the optimum pH and temperature were 5.0 and 50℃, respectively. By heating the enzyme at 80℃ for 1.73 min., the enzyme activity was decreased to 10%. The enzyme was active toward aromatic amines such as o-phenylenediamine and p-phenylendiamine. Kinetic studies indicated a Km of 2.6mM for o-phenylenediamine at an optimal hydrogenperoxide concentration and a Km of lOmM for hydrogenperoxide at an optimal o-phenylendiamine concentration. Among the reagents tested, L-ascorbic acid and sodium L-ascorbate inhibited significantly the enzyme, while Ca^(++) and Ba^(++) activated the enzyme at the concentration of 1mM and 5mM.
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