Partial Purification and Kinetic Properties of Myrosinase from Cauliflower (Brassica oleracea var. botrytis)

Myrosinase is an important enzyme of cruciferous vegetables having several biological functions in the plant cells. In order to study its nature and behavior from a rather new source, it has been purified to apparent homogeneity from 5 daysold germinated cauliflower seedlings having a specific activity of 12.71 units/mg protein with 54.6% recovery usingammonium sulfate fractionation and gel filtration chromatography on Sephadex G-100. The native molecular mass of thepurified enzyme was estimated, using gel filtration, to be about 128kDa. The purified enzyme migrated as a single band on SDS-PAGE with molecular mass of about 64 kDa, suggesting that myrosinase from cauliflower seedling consists of twosubunits of similar molecular mass. The enzyme exhibited its highest activity at pH 6.0. The optimum temperature for thepurified enzyme was found to be 50°C but maintained nearly 60% of its activity even at 70°C. The purified enzyme remained stable at 4°C for several months. Using sinigrin as a substrate, the K_m and V_max values for the purified enzyme were estimated to be 117 μM and 550μMol min^–1, respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid. Theresults revealed that the cauliflower myrosinase being homodimer has wider pH and temperature optima, hence issuitable for wider applications.