Substrate Specificity and Subsite Affinities of Crystalline .ALPHA.-Glucosidase from Aspergillus niger.

Abstract

Asp. niger α-glucosidase was crystallized in ammonium sulfate solution after chromatographies on DEAE-Sepharose CL-6B and TOYOPEARL HW-55 columns. The crystalline α-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 × 104 by SDS-disc gel electrophoresis. However, the crystalline enzyme consists of two components (M.W. about 3.3 × 104 and 9.8 × 104, respectively) separable only by reverse-phase HPLC causing irreversible inactivation. The optimum pH was 4.3. The enzyme also hydrolyzed α-glucans such as soluble starch and amylose. The ratios of V values (Km values, in parentheses, mM of nonreducing terminal) for maltose, kojibiose, nigerose, isomaltose, phenyl α-glucoside, phenyl α-maltoside, malto-triose, -tetraose, -pentaose, -hexaose, -heptaose and -octaose, maltodextrin (DP= 17), and soluble starch were estimated to be 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 (11): 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.