Environmental Topology and Water Availability Modulates the Catalytic Activity of β-Galactosidase Entrapped in a Nanosporous Silicate Matrix.
In the present work we studied the catalytic activity of E. coli β-Gal confined in a nanoporous silicate matrix (Eβ-Gal) at different times after the beginning of the sol-gel polymerization process. Enzyme kinetic experiments with two substrates (ONPG and PNPG) that differed in the rate-limiting steps of the reaction mechanism for their β-Gal-catalyzed hydrolysis, measurements of transverse relaxation times (T2) of water protons through1H-NMR, and scanning electron microscopy analysis of the gel nanostructure, were performed. In conjunction, results provided evidence that water availability is crucial for the modulation observed in the catalytic activity of β-Gal as long as water participate in the rate limiting step of the reaction (only with ONPG). In this case, a biphasic rate vs. substrate concentration was obtained exhibiting one phase with catalytic rate constant (kcA), similar to that observed in solution, and another phase with a higher and aging-dependent catalytic rate constant (kcB). More structured water populations (lower T2) correlates with higher catalytic rate constants (kcB). The T2-kcBnegative correlation observed along the aging of gels within the 15-days period assayed reinforces the coupling between water structure and the hydrolysis catalysis inside gels.
IIByT-UNC CONICET. Cátedra de Química Biológica, Facultad de Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba. Av. Vélez Sársfield 1611, X5016GCA Córdoba, Argentina.
M Ines
MI
Burgos
IFEG-UNC CONICET. FaMAF-Universidad Nacional de Córdoba, 5000 Córdoba, Argentina.
Manuel I
MI
Velasco
IFEG-UNC CONICET. FaMAF-Universidad Nacional de Córdoba, 5000 Córdoba, Argentina.
Rodolfo H
RH
Acosta
IIByT-UNC CONICET. Cátedra de Química Biológica, Facultad de Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba. Av. Vélez Sársfield 1611, X5016GCA Córdoba, Argentina.
María A
MA
Perillo
D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D005782:Gels / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D066244:Proton Magnetic Resonance Spectroscopy / Q000379:methods; D017640:Silicates / Q000378:metabolism*; D014867:Water / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
D005782:Gels; D017640:Silicates; D014867:Water; D001616:beta-Galactosidase