Abstract
In the present work we studied the catalytic activity of E. coli β-Gal confined in a nanoporous silicate matrix (Eβ-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 β-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 β-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.
Highlights
In a silicate matrix through the sol-gel method and observed improved stability for the encapsulated protein (Eβ-Gal) compared with the same protein in solution (Sβ-Gal)[24]
Differences in the kinetic parameters associated to the hydrolysis of two artificial substrates 2-nitrophenyl-β-D-galactopyranoside (ONPG) and 4-nitrophenyl-β-D-galactopyranoside (PNPG), and transverse relaxation 1H-NMR data supported the hypothesis that the structure of water confined inside the nanopores of the silicate matrix would be responsible for the enzymatic activity modulation observed
Enzymatic experiments of Eβ-Gal aged in contact with aqueous buffer for different periods of time were accompanied with 1H-NMR measurements of transverse relaxation times to analyze the molecular mobility of water in the system
Summary
In a silicate matrix through the sol-gel method and observed improved stability for the encapsulated protein (Eβ-Gal) compared with the same protein in solution (Sβ-Gal)[24]. The present work focuses on the catalytic activity of Eβ-Gal confined in gels with varied aging times. Differences in the kinetic parameters associated to the hydrolysis of two artificial substrates 2-nitrophenyl-β-D-galactopyranoside (ONPG) and 4-nitrophenyl-β-D-galactopyranoside (PNPG), and transverse relaxation 1H-NMR data supported the hypothesis that the structure of water confined inside the nanopores of the silicate matrix would be responsible for the enzymatic activity modulation observed
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