ß-sheet to α-helix conversion and thermal stability of ß-Galactosidase encapsulated in a nanoporous silica gel.

The effect on protein conformation and thermal stability was studied for ß-Galactosidase (ß-Gal) encapsulated in the nanopores of a silicate matrix (Eß-Gal). Circular dichroism spectra showed that, compared with the enzyme in buffer (Sß-Gal), Eß-Gal exhibited a higher content of α-helix structure. Heating Eß-Gal up to 75 °C caused a decrease in the content of ß-sheet structure and additional augments on Eß-Gal components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with Sß-Gal. Steady state fluorescence spectroscopy analysis evidenced an Eß-Gal structure less compact and more accessible to solvent and also less stable against temperature increase. While for Sß-Gal the denaturation midpoint (Tm) was 59 °C, for Eß-Galit was 48 °C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65 °C and Eß-Gal did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changes on the tertiary/cuaternary structure of Eß-Gal leading to the loss of thermal stability and enzymatic activity.

Environmental Topology and Water Availability Modulates the Catalytic Activity of ß-Galactosidase Entrapped in a Nanosporous Silicate Matrix.

In the present work we studied the catalytic activity of E. coli ß-Gal confined in a nanoporous silicate matrix (Eß-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 ß-Gal-catalyzed hydrolysis, measurements of transverse relaxation times (T2) of water protons through 1H-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 ß-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-kcB 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.