Effect of Water Miscible Organic Solvents on Kinetics of a Thermostable β-Glycosidase

Abstract

A thermostable β-D-glycosidase from Sulfolobus solfataricus was used to determine the effect of water-miscible solvents on enzyme activity and kinetics. Various o- and p-nitrophenyl glycosides were used as representative substrates for hydrolysis. Initial enzyme activity was enhanced in the presence of methyl acetate, butan-2-one and acetonitrile. The activation by solvent was found to be transitory. 1,4-Dioxane, formamide and tetrahydrofuran were inhibitory at all concentrations with all substrates used. Kinetic studies in the presence of solvents suggested that KM values for all substrates were unaffected by methyl acetate, but increased or decreased in the presence of 1,4-dioxane or tetrahydrofuran, in a substrate dependent manner. Studies using an competitive inhibitor supported these results. Methyl acetate did not change competitive-type inhibition by D-gluconic acid lactone, while in the presence of 1,4-dioxane altered the inhibition kinetics to mixed-type. Tetrahydrofuran affected the mode of inhibition but not in a identifiable manner. These results suggest that both 1,4-dioxane and tetrahydrofuran may have a direct influence on the active site of the catalyst.