Catalytic activity of beta–galactosidase entrapped in a silicate matrix: Effect of the substrate type, porous structure and aging time

Abstract

Protein encapsulation in solid matrixes is of interest for biotechnological purposes and it also serves as a model of molecular crowding. We have successfully entrapped the enzyme β-galactosidase (β-gal) in silicate gels via a sol-gel reaction. The catalytic activity of encapsulated β-gal (Eβ-gal) was compared with the activity of the soluble form (Sβ-gal) with two different substrates (ONPG and PNPG) and at different aging times. Data were analyzed with a Michaellis-Menten model. With the substrate ONPG, we found a Vmax value higher for Eβ-gal than for Sβ-gal and this difference increased at longer aging times. On the contrary, with PNPG, Vmax was not affected neither by the enzyme condition (Sβ-gal and Eβ-gal) nor by the aging time. The porous structure of the silicate matrix was also studied as a function of aging time of the gels. Thus, fluorescence correlation spectroscopy (FCS) was applied through the analysis of diffusional properties of fluorescent probes of different sizes, entrapped in the gel matrix. We observed an anomalous diffusion in some conditions, suggesting a self-similar pore structure, and aging time dependent changes in diffusion coefficients. Taken together our result supports the hypothesis that there is a relationship between the catalytic mechanism proposed for β-gal and water availability in the silicate gel since the ratio between bulk and structured water depends on the pore size.