Purification and Characterization of Myrosinase from Local Broccoli (Brassica oleracea var. Italica)

Abstract

The study including purification and characterization of myrosinase (β-thioglucosidase glucohydrolase; E.C. 3.2.1.147) from the local broccoli. Myrosinase is a type of glycoproteins containing various thiol groups, disulfide, and salty bridges. The enzyme was extracted by using sodium phosphate buffer (pH 6.5), precipitation process was carried out by adding ammonium sulfate salt at saturation ratio (20-60)%. Myrosinase purified by gel filtration chromatography using ÄKTA Pure 25 apparatus with superdex 200 column, three peaks were obtained but one of them having enzymatic activity, and the purity of enzyme identified with the absence of SDS. The optimum pH for myrosinase activity and stability were 5, and (4-7), respectively, the optimum temperature for myrosinase activity and stability were 40°C, and (20-50) °C respectively. The molecular weight of pure myrosinase estimated by electrophoresis with the presence of SDS which found equal to 75.85 KDa. Myrosinase kept most of its activity regarding to magnesium chloride, potassium chloride, and sodium chloride, while loosing half of its activity regarding to zinc sulfate and copper chloride, then the enzyme gradually lost most of its activity regarding to nickel chloride and EDTA salt, whereas ferrous chloride and aluminum chloride having an inhibition role for myrosinase activity. Standard glucoraphanin using as substrate to determination kinetics of myrosinase which are represented by Mechalis-Menton constant Km and Vmax where equal to 0.37 mM and 606.25 µmol/ml/min., respectively by using different plots.