[Purification, properties and specificities of a heteroglycosidase from saracen grains].

Abstract

Summary A heteroglycosidase from seedlings of saracen corn has been isolated and purified about 1,000 fold. Such a glycosidase (rhamnodiastase-like enzyme) hydrolyses rutin to rutinose (α- l -rhamnosyl 1 → 6 glucose) and quercetin, the enzymatic activity was thus determined with p-nitrophenyl-β- d -rutinoside as substrate. The purification involved extraction, ammonium sulfate fractionation, chromatographies on hydroxylapatite, DEAE-Sephadex and acrylamide-agarose Ac-A 4/4. The enzyme hydrolyzing p-nitrophenyl-β- d -rutinoside was only partially purified, but successive steps of column chromatography and thermal denaturation provided a clear distinction beetween this heteroglycosidase and the other glycosidases, especially β-glucosidases and β-galactosidases of germinating kernels of saracen corn. The molecular weight of this heteroglycosidase, calculated from data obtained by disc gel electrophoresis was about 85,000. Isoelectric focusing established the isoelectric point to be 3.7. Heteroglycosidase exibits a broad specificity; the catalytic activity was observed to be specific for holosidic and heterosidic bonds with the same configuration as β- d -glucosyl residue for carbon atoms 1, 2 and 3. The Km at pH 5 was calculated to be 2.6 × 10−4 M for p-nitrophenyl-β- d -rutinoside; 8.8 × 10−5 M for p-nitrophenyl-β- d -glucoside and 8.6 × 10−5 M for p-nitrophenyl-β- d -galactoside. The range of activity was very broad, extending to the cleavage of disaccharides, various flavonoid and related phenolic glycosides, and amyloids. At the present time the functions of this heteroglycosidase can only be a matter of conjectures.