Changes in the kinetic parameters of -galactosidase encapsulated in silicate matrix: the role of water.

BURGOS I; MARIN JI; PERILLO MA

San Javier, Tucuman

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

Encapsulation of enzymes in silicate matrices is of interest both for technological applications and to simulate intracellular nanoenviroments. The synthesis of the silicate can proceeds through a sol-gel reaction from tetraetilortosilane precursor (TEOS) in the presence of the enzyme leading to an entrapped protein. A drawback of this procedure is that the conditions for the hydrolysis reaction (extreme pH and high percentages of ethanol) produce the enzyme inactivation. In this work, by changing the molar ratio H2O/TEOS we obtained encapsulated b-galactosidase under conditions that preserve its enzymatic activity. We monitored the percentage of ethanol in the silicate gels by absorption spectroscopy using merocyanine as an electrochromic probe. It was observed that, at 20/1 H2O/TEOS molar ratio, the percentage of ethanol was less than 7% 15 minutes after the gel was formed and decreased to 0% after 1h. Instead, we detected 60% ethanol at 4/1 H2O/TEOS molar relation. In turn, the activity parameters at different aging times (1 hour, 3 and 6 days) of the enzyme in the gel were compared and it was found that VmaxApp increases with aging time. 1H-NMR spin-lattice relaxation times (T1) measurements in the silicate gels revealed different populations in the H2O proton system. Taken together these results suggest that the increase in Vmax,App might lie on changes in water structure during gel aging. Acknowledgements: Secyt-UNC, CONICET, ANPCyT.