Activity and Stability of a Thermostableβ-Glucosidase in the Presence of Water Miscible Organic Solvents
Frank Udo HUNEKE*, and Donald COWAN
University College London, Department of Biochemistry and Molecular Biology, Gower Street, London, WC1E6BT, England
The recombinant thermostableβ-D-glucosidase from Sulfolobus solfataricus was used in experiments to determine the effect of water miscible solvents on enzyme activity and stability.
p-Nitrophenyl-β-D-glucopyranoside (p-NP-β-D-Glcp), p-Nitrophenyl-β-D- fucopyranoside (p-Nph-β-D-Fucp) and o-Nitrophenylβ-D-Galactopyranoside (o-Nph- 13-D-Galp) were used as representative substrates for hydrolysis. Enzyme activity was enhanced in the presence of methyl acetate, acetonitrile, butan-2-one or formamide using solvent concentrations between 5-15% (v/v). 1,4-dioxane was inhibitory at all concentrations with all the substrates used.
Tetrahydrofuran showed both activating and inhibitory effects on the enzyme, these effects being substrate dependent. The possibility that THF affected the substrate specificity of the enzyme was investigated with three additional substrates (p-Nitrophenyl-β-D-Lacto-pyranoside, p-Nph-β-D-Lactop; p-Nitrophenyl-β-D- Mannopyranoside, p-Nph-β-D-Mannop; p-Nitrophenyl-β-D-Xylopyranoside, p-Nph-β-D-Xylop). The glucosidase was activated by this solvent using C-6 substrates but inhibited if C-5 substrates or disaccarides were used.
Kinetic studies suggest that kcat was affected by all the solvents tested, while KM was only affected in the presence of methylacetate or 1,4-dioxane. It is proposed that these solvents may have a direct influence on the active site of the catalyst. The effect of activation by solvent was found to be transitory.
