Isolation and characterization of two cyanogenic beta-glucosidases from flax seeds.
Two cyanogenic beta-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 alpha beta dimer (molecular weights of alpha and beta = 39,000 and 19,000, respectively) while linamarase appears to be an alpha 5 beta 5 decamer (molecular weights of alpha and beta = 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, Km's in the millimolar range, and competitive inhibition by delta-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 beta-glycosidic linkage; linustatinase (a beta-bis-glucosidase) readily hydrolyzes beta-bis-glucosides with 1,6 and 1,3 linkages whereas linamarase (a beta-monoglucosidase) exhibits little activity towards these substrates.
D000678:Amygdalin / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005959:Glucosidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism
D010446:Peptide Fragments; D000678:Amygdalin; C023022:linustatin; C023031:neolinustatin; D005959:Glucosidases; C021514:cyanogenic beta-glucosidase; C047534:linustatinase; D001617:beta-Glucosidase