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

The β-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 β-glucosidase-catalyzed hydrolysis at 25 °C and pH 5.0. Stoichiometric production of Gn − 1 by the β-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 β-glucosidase of Candida wickerhamii. The subsite structure is quite similar to that of the α-glucosidase of A. niger, whereas the dependence of k0 on n is highly characteristic for β-glucosidase, and decreases with n, suggesting some interaction between the particular subsites.