β-Glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of β-glycosidases Original Research Article

An affinity adsorbent for β-glycosidases has been prepared by using β-glycosylamidine as a ligand. β-Glucosylamidine and β-galactosylamidine, highly potent and selective inhibitors of β-glucosidases and β-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a β-glycosylamine and 2-iminothiolane·HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for β-glucosidases and β-galactosidases, respectively. This one-pot procedure enables various β-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a β-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a β-glucosidase and a β-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The β-glucosidase and β-galactosidase were inhibited competitively by a soluble form of the corresponding β-glycosylamidine ligand with an inhibition constant (Ki) of 2.1 and 0.80 μM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.