Isolation purification and properties of a mammalian glucosidase and a bacterial fructosidase

Abstract

The enzyme acid a-D-glucosidase has been isolated from three batches of pig's liver, and purified by molecular gel filtration. Disc gelelectrophoresis of the purified enzyme established homogeneity. An isoenzyme of acid a-D-glucosidase, purified to homogeneity, was isolated from one batch. Sodium dodecyl sulphate electrophoresis indicated that the enzyme existed in subunits with a molecular weight of 126,000, 101,500, and 87,500 for each. A study of the enzymic properties of the enzyme was carried out. The enzyme liberated D-glucose in a exo-manner from cibrachon blue amylose. The enzymic hydrolysis of phenyl a-D-glucopyranoside yielded the a-anomer of D-glucose, as confirmed by nmr spectroscopy. A series of rate equations were derived and applied to describe the reaction. The enzyme hydrolysed a variety of a-linked glucosyl oligosaccharides, glycosides and polysaccharides. Maltose was the preferred substrate with a K of 4.0 to 4.4 mM. Glycogen was completely hydrolysed to D-glucose. A comparison of the properties of the two isoenzymes showed that they both had similar pH optima, thermal stabilities, and subunit structures. The isoenzymes could be separated by cellulose acetate electrophoresis. One isoenzyme had a diminished affinity for glycogen and dextran T10. PART 2: The extracellular levanase of Streptococcus salivarius strain '51' was isolated from a culture grown on a levan containing medium, gel filtration, ion-exchange and affinity chromatography were used in an attempt to purify the enzyme to homogeneity. A partially purified enzyme preparation was used to study the properties of the enzyme. Heat denaturation studies indicated that two enzymes were present in the preparation, one of which was thermally unstable. Both enzymes had the ability to hydrolyse both levan and inulin. The preparation was also able to hydrolyse raffinose, sucrose, inulin- and levan-oligosaccharides and methyl [beta]-D-fructofuranoside.