RESUMO

Myrosinase (ß-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 µmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

Assuntos

Brassica/enzimologia , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Brassica/química , Brassica/genética , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo