Environmental topology and water availability modulates the catalytic activity of beta-Galactosidase entrapped in a nanosporous silicate matrix

BURGOS I; PERILLO MA; ACOSTA RH; VELASCO MI

scientific reports

Nature Publishing Group

Año: 2016 vol. 6 p. 36593 - 36603

In the present work we studied the catalytic activity of E. coli β-Gal confined in a nanoporous silicatematrix (Eβ-Gal) at different times after the beginning of the sol-gel polymerization process. Enzymekinetic experiments with two substrates (ONPG and PNPG) that differed in the rate-limiting steps ofthe reaction mechanism for their β-Gal-catalyzed hydrolysis, measurements of transverse relaxationtimes (T2) of water protons through 1H-NMR, and scanning electron microscopy analysis of the gelnanostructure, were performed. In conjunction, results provided evidence that water availabilityis crucial for the modulation observed in the catalytic activity of β-Gal as long as water participatein the rate limiting step of the reaction (only with ONPG). In this case, a biphasic rate vs. substrateconcentration was obtained exhibiting one phase with catalytic rate constant (kcA), similar to thatobserved 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 assayedreinforces the coupling between water structure and the hydrolysis catalysis inside gels.