Purification and characterization of catalase from sprouted black gram (Vigna mungo) seeds

► Catalase content was estimated in black gram sprouts, seedlings and leafs. ► Day 4 sprouts showed the maximum catalase activity. ► A single step purification of catalase was achieved using Seph 4B-IDA-Zn(II). ► Purity of catalase was confirmed qualitatively by SDS–PAGE and Zymogram analysis. ► Kinetic and stability studies were performed with purified catalase. Black gram (Vigna mungo) is a legume which belongs to Fabaceae family. It is a rich source of protein. It has been known to have interesting small molecule antioxidant activity. However, its enzymatic antioxidant properties have not been explored much. In the present work we studied catalase, a principal antioxidant enzyme from black gram seeds. Day four sprouted black gram seeds were found to have a significant catalase content approximately of 15,240U/g seeds. IMAC (Seph 4B-IDA-Zn(II)) was used for purifying this catalase, a purification fold of 106 and a high specific activity of 25,704U/mg was obtained. The Km and Vmax of the purified catalase were found to be 16.2mM and 2.5μmol/min. The effect of inhibitors like Sodium azide (NaN3) and EDTA and different metal ions on catalase activity were studied. NaN3, Fe3+and Cu2+ were found to have profound inhibitory effects on the enzyme activity. Other metal ions like Ni2+, Ca2+, Mg2+ and Mn2+ had both enhancing and inhibitory effects. The enzyme showed optimal activity at a temperature of 40°C and pH 7.0. It was stable over a broad range of pH 6.0–10.0 and had a half life of 7h 30min at 50°C.