Isolation and characterization of two cyanogenic β-glucosidases from flax seeds

Two cyanogenic β-glucosidases, linustatinase and linamarase, were isolated and purified from flax seeds (Linum ussitatissimum). They catalyze the sequential hydrolysis of linustatin and neolinustatin to yield acetone and methylethyl ketone cyanohydrins, respectively. The purification procedure for linustatinase involved acetone extraction, precipitation by polyethyleneimine and ammonium sulfate (40-80% saturation), and Red A gel, concanavalin A-Sepharose, and PBE 94 column chromatography; that for linamarase was similar except that polyethyleneimine precipitation was eliminated and DE-52 and Sepharose CL-6B replaced Red A gel column chromatography. The native substrates neolinustatin and linamarin were used for the assay during purification. Both proteins were purified to electrophoretic homogeneity. Linustatinase is an αβ dimer (molecular weights of α and β = 39,000 and 19,000, respectively) while linamarase appears to be an α₅β₅ decamer (molecular weights of α and β = 62,500 and 65,000, respectively). Both enzymes contain mannose or glucose. Linustatinase exists in five different isozymic forms (isoelectric points between 7 and 8) whereas linamarase occurs in one major form (isoelectric point 4 to 5). The kinetic parameters of the two enzymes are similar: acidic pH optima, K_m's in the millimolar range, and competitive inhibition by δ-gluconolactone, a transition state analog. The presence of an aglycone structure in the substrates is important for both enzyme activities. In addition, both enzymes are specific towards the β-glycosidic linkage; linustatinase (a β-bis-glucosidase) readily hydrolyzes β-bisglucosides with 1,6 and 1,3 linkages whereas linamarase (a β-monoglucosidase) exhibits little activity towards these substrates.