Environmental Topology and Water Availability Modulates the Catalytic Activity of β-Galactosidase Entrapped in a Nanosporous Silicate Matrix

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.

Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered β-galactosidase

“…Furthermore, the different structure of water molecules within the polymeric matrix may also modulate enzyme kinetics, as proposed by Burgos et al [47]. Enzyme inactivation upon immobilization has been extensively described with diverse techniques and enzymes [3,5,48].…”

Effect of Polyethylene Glycol-Induced Molecular Crowding on the Enzymatic Activity and Thermal Stability of β-Galactosidase from Kluyveromyces lactis

“…This is confirmed by the fact that under MC conditions, Eadie–Hofstee plots of the hydrolysis of ONPG by E. coli β-Gal was biphasic, , while in the case of K. lactis β-Gal, it exhibited a straight line with a single slope (Figure S3).…”

“…In these situations, macromolecules occupy an important volume fraction that cannot be occupied by other molecules . Not only in the cellular milieu but also in situations of technological interest such as in dairy products and under confined conditions such as lipid vesicles − or silicate matrix , encapsulation, the activity of β-Gal has to be evaluated in crowded media.…”

“…Therefore, the strength of the hydrophobic effect, the main driving force in protein folding, can be altered. , Water activity, solvent ordering and mobility, and polarizability can also affect the enzyme kinetics through its effect on the hydrophobic interaction between the enzyme and the substrate, modifying the thermodynamic activities of substrates, enzymes, and transition state intermediates . Importantly, for hydrolytic reactions, water structuring may affect its availability as a reagent. , …”

“…24 Importantly, for hydrolytic reactions, water structuring may affect its availability as a reagent. 25,26 Another effect of macromolecular crowding is to reduce the rate of diffusion by factors up to 10, depending on the size of the diffusing particle and the degree of occupancy of the medium, compared with the rate in uncrowded solutions. 27 Thus, a biochemical reaction is influenced by macromolecular crowding if its rate is limited by diffusion.…”