Myrosinase in Brassicaceae

Abstract

Myrosinases (thioglucoside glucohydrolases E.C. 3.2.3.1.), which catalyse the hydrolysis of glucosinolates present in Brassicaceae, were isolated from Sinapis alba L. seeds. The crude enzyme extract was purified using gel and ion-exchange chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis. The separation of two myrosinase isoenzymes was obtained after gel chromatography on Sephadex G-100. Further purification of the main myrosinase components was achieved when the combined isoenzymes were separated on the anion-exchanger DEAE-Sephadex A-50 followed by polyacrylamide gel electrophoresis. A similar purification was obtained when the crude extract was group-fractionated on Sephadex G-50 followed by DEAE-cellulose chromatography on Whatman DE-52 and gel chromatography on Sephadex G-200. The enzyme from the last step was further separated by isolectric focusing into two isoenzymes with isoelectric points 4.9 and 6.2. In order to clarify where the myrosinase was localized in the root tip cells, cell fractionation studies were performed using aldehydes as pre-fixatives to stabilize the enzymes and the cell organelles. Biochemical tests of crude and purified samples of the isolated myrosinases showed that when glutaraldehyde or formaldehyde were used as pre-fixatives at a final concentration of 1% (w/v), they did not inhibit the enzyme activity. Relatively homogeneous cell organelle fractions were obtained using ultracentrifugation and stepwise sucrose gradients. The myrosinase activity expressed on the basis of the protein content was found to be highest in the dictyosome and smooth endoplasmic reticulum fractions