Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase).
Myrosinase (β-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.
Department of Chemical Engineering, University of Santiago of Chile , Avenida Libertador Bernardo O'Higgins 3363, Estación Central, Santiago 9170019, Chile.
Andrea
A
Mahn
Alejandro
A
Angulo
Fernanda
F
Cabañas
D000595:Amino Acid Sequence; D001937:Brassica / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D010940:Plant Proteins; D006026:Glycoside Hydrolases; C022739:thioglucosidase
Brassica oleracea var. italica; kinetics; myrosinase; purification process