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 (E(β-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 (T(2)) of water protons through (1)H-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 (k(cA)), similar to that observed in solution, and another phase with a higher and aging-dependent catalytic rate constant (k(cB)). More structured water populations (lower T(2)) correlates with higher catalytic rate constants (k(cB)). The T(2)-k(cB) negative 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.