Agric. Biol Chem., 55 (9), 2327-2335, 1991 Substrate Specificity and Subsite Affinities of Crystalline a-Glucosidase from Aspergillus niger Akihiko Kita, Hirokazu Matsui, Akishige Somoto, Atsuo Kimura, Masuhiro Takata and Seiya Chiba* Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan Received March 14, 1991 Asp. niger a-glucosidase wascrystallized in ammonium sulfate solution after chromatographies on DEAE-Sepharose CL-6B and TOYOPEARL HW-55columns. The crystalline a-glucosidase, which was a glycoprotein containing 25.5% carbohydrate as glucose, gave a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be about 12.5 x 104 by SDS-disc gel electrophoresis. However, the crystalline enzyme consists of two components (M.W. about 3.3 x 104 and 9.8 x 104, respectively) separable only by reverse-phase HPLC causing irreversible inactivation. The optimumpH was 4.3. The enzyme also hydrolyzed a-glucans such as soluble starch and amylose. The ratios of l values (Km values, in parentheses, him of nonreducing terminal) for maltose, kojibiose, nigerose, isomaltose, phenyl a-glucoside, phenyl a-maltoside, malto-triose, -tetraose, -pentaose, -hexaose, -heptaose and -octaose, maltodextrin (DP = 17), and soluble starch were estimated tobe 100(0.75):23(4.6):62(12):35 (8.0): 1.3(0.34): 126(0.87): 126 (0.69): 135 (1.1): 102(1.9): 119 (3.2) : 102 (4.9) :89 (5.3) :89 (ll) : 114 (4.3). The subsite affinities in the active site were 0.790, 5.93 and 0.191 kcal/mol for subsites 1, 2, and 3, respectively. The three subsites were considered to be effective for the binding of substrate. a-Glucosidases [EC 3.2.1.20, a-D-glucoside glucohydrolase] are a group of typical exo-type carbohydrases, which catalyze the splitting of a-glucosyl residue from the nonreducing terminal of substrate to liberate a-glucose.1} Various types of a-glucosidases2) are widely distributed in nature. Many a-glucosidases hydrolyze not only synthetic a-glucoside and oligosaccharides but also a-glucans such as soluble starch and glycogen at a single active site.3"5) Fungal a-glucosidases have been long in- vestigated by many workers.6~18) It has been reported that these enzymes have wide substrate specificities. However, the kinetic studies on the substrate specificities are rarely made in detail. There is only one report of McCleary et al. describing the rate parameters for the hydrolysis of several substrates by a fungal a-glucosidase.18) It seems worthwhile To whomcorrespondence should be addressed. 2327 to investigate the kinetics of crystalline a- glucosidase of Aspergillus niger, to get more satisfactory results for its properties and the substrate specificity of this fungal a-glucosi- dase. Weare interested in learning the relationship between the catalytic action and the primary structure near the active site of a-glucosidase. Kinetic data for the hydrolysis is necessary to understand the catalytic reaction mechanism. However, the catalytic groups directly involved in the hydrolytic reaction of amylases and a-glucosidases are still obscure. The analyses of the primary structure and the catalytic groups of the crystalline a-glucosidase are under way in our laboratory. This paper describes the crystallization, the substrate specificity, and the subsite affinities of Asp. niger a-glucosidase.