CONICET | Buscador de Institutos y Recursos Humanos

Protein encapsulation in solid matrixes is of interest for biotechnological purposes and it also serves as a model of molecular crowding. We successfully entrapped the enzyme b-galactosidase (Eb-Gal) in silicate gels via a sol-gel reaction and obtained comparable levels of hydrolytic activity with those obtained for soluble b-gal (Sb-Gal). From the Michaelis-Menten kinetic analysis employing 2-nitrophenyl-b-D-galactopyranoside (ONPG) as substrate it was observed that both kcat and KM were higher for Eb-Gal than for Sb-Gal, and they increased with the gel aging time (At). In order to understand the enzymatic modulation of b-Gal upon encapsulation in the silicate gels we performed several structural studies of the silicate matrix. A qualitative analysis of the topological structure was performed from SEM images. For this study it was necessary to dry the gels with CO2 in supercritical conditions obtaining aerogels, in order to preserve the original porous structure. The SEM images showed that the gels consisted in the agglomeration of ~32 nm diameter particles (ranging between 3 nm and 180 nm). From the BET isotherms obtained by dynamic water vapor sorption it was observed that the aerogels have an remarkably large surface-to-volume ratio and a corresponding high specific surface area (1.5x107 cm2/g). Wet gels at At = 0 days, submitted to SAXS analysis, exhibited a ~20 nm gyration radius (Rg) which was consistent with mean particles sized estimated by SEM for dried gels. The fractal dimension exponent was D»2. Neither drying, aging time nor the presence of b-Gal affected significantly the porous structure of the gel. Acknowledgements: Foncyt, Conicet, SeCyT-UNC (Argentina) and SAXS1 beamline of LNLS (Brazil), for financial support. BMI, ORG and PMA a career members of CONICET.