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

Abstract

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from soybean (Glycine max var HH2) hulls was purified to electrophoretic homogeneity by a combination of ammonium sulphate fractionation, DEAE-Sephadex A-50 chromatography, concanavalin A-Sepharose 4B affinity chromatography and Bio-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-polyacrylamide gel electrophoresis. The peroxidase was a glycoprotein containing about 18.7% carbohydrate, approximately one-quarter of which was shown to 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°C, and was stable in the pH range 2.5–11.5. The enzyme could tolerate heating for 10 min at 75°C without being inactivated, and at 85°C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel thermostable enzyme. Fe 2+, Fe3+, Sn2+, CN − and N3− inhibited enzyme activity, while Hg2+, Ag+, Pb 2+, Cr3+, EDTA and SDS were not significantly inhibitory. © 1999 Society of Chemical Industry