Isolation, purification and properties of the peroxidase from the hull of Glycine max var HH2

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from s oybean (Glycine max var HH2) hulls was purified to electrophoretic homogene ity by a combination of ammonium sulphate fractionation, DEAE-Sephadex A-50 chromatography, concanavalin A-Sepharose 4B affinity chromatography and Bi o-Gel P-60 gel filtration. The specific activity of purified peroxidase was about 57-fold higher than that of crude extract. The yield was about 16.4% . The molecular weight of the enzyme was estimated to be 38 000 by SDS-poly acrylamide gel electrophoresis. The peroxidase was a glycoprotein containin g about 18.7% carbohydrate, approximate:ly one-quarter of which was shown t o be glucosamine residues. It was found to have an isoelectric point of 3.9 . The enzyme was most active at pH 4.6 and 45 degrees C, and was stable in the pH range 2.5-11.5. The enzyme could tolerate heating for 10 min at 75 d egrees C without being inactivated, and at 85 degrees C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel therm ostable enzyme. Fe2+, Fe3+, Sn2+, CN- and N-3(-) inhibited enzyme activity, while Hg2+, Ag+, Pb2+, Cr3+, EDTA and SDS were not significantly inhibitor y. (C) 1999 Society of Chemical Industry.