Gentaro Okada

The α-amylases as glycosylases, with wider catalytic capacities than envisioned or explained by their representation as hydrolases

Abstract In a study undertaken to illustrate the inadequacy of the familiar concept of carbohydrases as hydrolases, crystalline α-amylases from six different sources, as well as crude salivary amylase, were examined and found to catalyze the synthesis of maltose and maltosaccharides from α- d -glucopyranosyl fluoride, a stereoanalog of α- d -glucopyranose. These syntheses apparently involve initial formation of maltosyl fluoride and higher maltosaccharide 1-fluorides, traces of which were found in digests with certain α-amylases. That the reactions are due to the α-amylases themselves and not to some accompanying enzyme(s) appears certain from the purity and diversity of the preparations; their failure (with one exception) to attack α- or β-maltose; the correspondence of the synthesized products with the known specificity of α-amylases for α-1,4- d -glucosidic linkages (and capacity of different α-amylases to hydrolyze saccharides of different sizes). The “saccharifying” α-amylase of B. sublilis var amylosacchariticus was unique in producing maltosaccharides from both α- and β-maltose (i.e., by α- d -glucosyl transfer). However, the entire group of α-amylases had the capacity to promote α- d -glucosyl transfer from α- d -glucosyl fluoride to C4-carbinol sites, demonstrating for the first time that the catalytic range of α-amylase extends beyond hydrolysis and its reversal. Indeed, all transferred the glucosyl group of α- d -glycosyl fluoride preferentially to C4-carbinols rather than water—a finding neither anticipated nor explained by the representation of α-amylases as hydrolases. The results demonstrate the need to recognize that α-amylases and other hydrolases acting on glycosyl compounds, together with the glycosyl transferases, form a great class of interrelated enzymes whose action is precisely defined as the catalysis of glycosylation (i.e., of glycosyl-hydrogen interchange) and which, therefore, may be designated as “Glycosylases.”

Subsite structure of the β-glucosidase from Aspergillus niger, evaluated by steady-state kinetics with cello-oligosaccharides as substrates☆

The beta-glucosidase from a commercially available preparation from Aspergillus niger was highly purified. The Michaelis constant Km and the molar activity K0 for cello-oligosaccharide substrates Gn (n = 2-6) were obtained by steady-state kinetic analysis on the beta-glucosidase-catalyzed hydrolysis at 25 degrees C and pH 5.0. Stoichiometric production of Gn-1 by the beta-glucosidase reaction for Gn was confirmed by HPLC techniques. Based on Km and K0 for Gn, subsite affinities (Ai, i = 1-6) were estimated as follows (kcal/mol): A1 = 1.3, A2 = 5.2, A3 = 0.65, A4 = -0.10, A5 = -0.65, and A6 = -0.26, of which A1-A3 are much higher than those of the beta-glucosidase of Candida wickerhamii. The subsite structure is quite similar to that of the alpha-glucosidase of A. niger, whereas the dependence of k0 on n is highly characteristic for beta-glucosidase, and decreases with n, suggesting some interaction between the particular subsites.

A Glucodextranase Accompanied by Glucoamylase Activity from Arthrobacter globiformis I42

A glucodextranase was highly purified from a cell-free culture broth of Arthrobacter globiformis 142. The glucoamylase to glucodextranase activity ratio was almost the same (ca. 0.88 %) at each purification step. The activity profiles of the stabilities of the glucoamylase to pH and temperature coincided well with those of the glucodextranase. Furthermore, the inactivation profiles of the glucoamylase with various metal ions and inhibitors were almost the same as those of the glucodextranase. From these results, it was strongly suggested that a single enzyme molecule was responsible for both the glucodextranase and glucoamylase activities.