The immobilization of proteins by encapsulation in porous glasses through sol-gel techniques is of special interest for the synthesis of biosensors1 and as an experimental model for the comprehension of the molecular crowding phenomenon2,3. In early studies it has been observed that encapsulated proteins preserved most of their native conformation4,5,6 and retained their native spectroscopic properties and certain functional characteristics5,6,7,8,9,10,11. However, in most cases encapsulated enzymes suffered a partial loss in their catalytic activity, probably due to the high ethanol content resulting from the polymerization process involved in the sol-gel method. Large molecules are trapped within the porous net while small molecules are able to diffuse throughout the gel. Entrapment of protein molecules in the sol-gel matrix apparently occurs because the silicate polymerizes around the biomolecule and physically traps it in the growing oxide network12. It is expected that the macromolecule in this condition would experience a completely different environment from the one found in dilute solutions. Confinement in nanopores would induce diffusional and interfacial effects, local concentration inhomogeneities of the reactants, and changes in the properties of the solvent. Extensive experimental and simulation studies of confined water in nanopores13,14,15,16,17,18,19 revealed that water molecules exhibit properties rather different from bulk water. At least two behaviors of water molecules can be discerned inside the pore. One population corresponds to bound water molecules, i.e. molecules strongly adsorbed at a layer close to the surface having a reduced mobility and specific orientations towards the interface. The other population corresponds to free water molecules, i.e. molecules which are less influenced by the pore walls and which exhibit properties close to the bulk values.

β-Galactosidase from Escherichia coli (β-Gal) is a well-studied tetrameric enzyme whose mechanism of hydrolysis was characterized for its natural and artificial substrates20,21,22,23. In a previous work we encapsulated β-Gal 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.


The silicate matrix is usually formed by hydrolysis of an alkoxide precursor followed by condensation to yield a polymeric oxo-bridged SiO network25. As long as the system remains wet, the structure and properties of the gel so formed suffers a continuous change through a process known as gel ‘aging’. This occurs through polycondensation and cross-linking reactions taking place in the solid amorphous phase. Thus, spontaneous shrinkage and contraction of the gel networks occurs leading to smaller pores26 and this fact may influence on changes in protein activity when confined within it27. It is important to note that acidic medium works as a catalyst, inducing an increase in the rate of tetraethyl-orthosilicate (TEOS) hydrolysis and affecting the condensation reactions.

In the present work, 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. Scanning electron microscopic (SEM) images of dried aerogels revealed the morphology of the wet gel with a good approximation but unfortunately, this methodology did not allow us monitor the effect of aging.

Aerogels morphology

For SEM, it is normally required that samples are completely dry, since the specimen chamber is at high vacuum. In turn, the silicate gel encloses a continuous liquid phase in a solid network. Air-drying causes considerable and irreversible contraction of the gel network and induces a flow of liquid from the interior of the body26,28. At certain point, the capillary forces that arise during the drying process leads to the gel cracking. Therefore, in order to prevent this phenomenon and preserve the structure of the wet samples the silicate gels were dried under supercritical conditions (see the Methods section). The SEM images of aerogel thus obtained, revealed a microscopic structure consistent of agglomerated particles (Fig. 1a). The interstitial spaces between particles (pores) exhibited a wide size distribution within the nanometer scale, between 3 nm and 180 nm diameter, as estimated through the analysis of SEM images (Fig. 1b). Due to resolution limits, pores below 10 nm may have been underestimated however, taking into account that β-Gal diameter is approximately 12–17 nm, smaller pores would not be so relevant for the present work in terms of the environment that pores provide to the enzyme.

Figure 1
figure 1

Aerogel morphology.

(a) SEM image from an aerogel obtained by supercritical drying. Experimental details are described in M&M section. The reference scale bar indicates 200 nm. Inset, equivalent binary image used for the evaluation of porosity. (b) Pore diameter distribution in nm units.

Catalytic activity of β-Gal encapsulated in nanoporous gel

Sol-gel silicate synthesis has been usually performed with a 4:1 H2O:TEOS molar ratio which is near the stoichiometric relation of the global reaction. In this condition Eβ-Gal shows low activity (data not shown) probably due to the fact that ethanol, a secondary product in the hydrolysis of TEOS, is present at high concentrations (>40% V/V) in the reaction media (see Supplementary Fig. S2). It is noteworthy that the catalytic activity of β-Gal decreases up to one third in the presence of 40% V/V ethanol if compared with an ethanol free media (see Supplementary Fig. S1). Previous methods were proposed to prevent ethanol accumulation in presence of the protein29. However, we assayed a 20:1 H2O:TEOS molar ratio which demonstrated that the amount of ethanol produced by the condensation reaction was around 13% V/V (see Supplementary Fig. S2), a condition that preserves Eβ-Gal activity.

The experimental set-up allowed the spectrophotometer light beam to cross the gel and the buffer over it and sense simultaneously the concentration of product molecules formed both inside and outside the gel (see Material and Methods and Fig. 5). In these conditions the reaction kinetics became independent of the lag time in the diffusion of the reaction product out of the gel and calculations of Eβ-Gal activity were simplified compared with our previous work24.

Figure 2
figure 2

Effect of the substrate type and aging times on the catalytic activity of Sβ-Gal and Eβ-Gal.

Panels (a,b), saturation curves for the hydrolysis of PNPG (a) and ONPG (b) in buffer solution, in freshly prepared gels (0 days) and in aged gels (3, 7 and 15 days for PNPG and 7 and 15 days for ONPG). Continuous lines represent the fitting of Michaelis-Menten equation (PNPG) or Eq. 1 (ONPG) to experimental data. Panels (c,d), Eadie Hofstee plots for Sβ-Gal and Eβ-Gal of PNPG (c) and ONPG (d) (linearization of curves shown in a and b, respectively). A single straight line in (c,d) corresponds to the hyperbolic Michaelian behavior in (a,b). Two straight lines in (d) were drawn to highlight the different asymptotic slopes of the non-Michaelian behavior. Red lines correspond to Sβ-Gal. Each point is the average of triplicate determinations in a single experiment and error bars represent the standard deviations. These experiments were repeated at least twice with similar results.

Figure 3
figure 3

Water dynamics in nanosporous gel.

(a) Relaxation distribution profile in freshly prepared gels. Three groups of differentially structured water are identified (T2a, T2b and T2c). (b) Mean relaxation times of the individual T2 populations as a function of aging time.

Figure 4
figure 4

General reaction mechanism of a substrate (S) hydrolysis catalyzed by β-Gal (E).

In the present work S represents PNPG or ONPG. ES is the intermediate complex of the substrate bound to the enzyme; P1 is the aglycone residue (PNP or ONP); ES’ is an intermediate complex of β-Gal covalently bound to the galactosyl residue; P2 in the present work is Galactose, the transfer product resultant from the nucleophilic attack of water (hydrolysis) on ES’.

Figure 5
figure 5

Set up used for spectroscopic measurements.

The initial rates of product formation (V0) as a function of substrate concentration were obtained for the two substrate analogues, PNPG and ONPG using soluble enzyme (Sβ-Gal) as well as Eβ-Gal with different aging times (Fig. 2). Both Sβ-Gal and Eβ-Gal exhibited enzymatic activity in all the conditions tested.

A qualitative analysis evidenced that for PNPG the rate of hydrolysis vs. substrate concentration curves, obtained with Sβ-Gal and Eβ-Gal looked hyperbolic and quite similar to each other (Fig. 2a). Furthermore, the Eadie-Hofstee data analysis of PNPG hydrolysis (Fig. 2c) in both cases (Sβ-Gal and Eβ-Gal) showed a single straight line supporting a Michaelian behavior for the hydrolysis of this substrate within the whole concentration range studied. Interestingly, the slopes of the linear regression of data for Eβ-Gal in aged gels were always steeper than those for Sβ-Gal and for β-Gal in fresh gels (Eβ-Gal,0) which, in turn, were similar to each other.

In the case of ONPG hydrolysis, the initial rate (V0) vs. substrate concentration curves evidenced a qualitatively different behavior between Sβ-Gal and Eβ-Gal (Fig. 2b). While the former exhibited a clear hyperbolic shape, the latter deviated from this behavior. Moreover, for substrate concentrations higher than 0.5 mM, the initial rate values for Eβ-Gal were always higher than those exhibited by Sβ-Gal. At least two slopes were resolved in Eadie-Hofstee plots for ONPG hydrolysis catalyzed by Eβ-Gal (Fig. 2d). This is in accordance with the deviation from the hyperbolic behavior reflected in the V0 vs. [ONPG] plot (Fig. 2b). Therefore, we hypothesized that each slope would reflect the behavior of different Eβ-Gal populations. Each population is sensing different environments within the gel. One of them, where β-Gal molecules are confined in large pores, would allow a behavior that resembles that of Sβ-Gal. Other populations of Eβ-Gal confined in smaller pores, would be those exhibiting higher catalytic activity and would be sensing some effects associated to their proximity to the gel-water interface.

Kinetic parameters of β-Gal catalyzed PNPG and ONPG hydrolysis

Values of kinetic parameters for Sβ-Gal catalyzed hydrolysis of PNPG and ONPG (Table 1) were obtained by a non-linear regression analysis of the experimental data shown in Fig. 1, assuming the Michaelis-Menten model. The kinetic parameters obtained with Sβ-Gal against both substrates were in good agreement with those found in the literature.

Table 1 Apparent kinetic parameters for the hydrolysis of PNPG and ONPG by β-Gal free and encapsulated in fresh and aged gels.

PNPG hydrolysis

A non-linear fit of Michaelis-Menten equation to data from the hydrolysis of PNPG catalyzed with Eβ-Gal revealed values of Vmax similar to those obtained with Sβ-Gal, with no significant effect of the aging time. However, the apparent KM value with Eβ-Gal was aging dependent and higher than with Sβ-Gal (Table 1) coincidently with the steeper slope observed in the Eadie-Hofstee plot (Fig. 2c). It is well known that KM is directly related to the affinity of the enzyme-substrate interaction hence, it is associated to the binding of the substrate to the active site in the enzyme and the availability of substrate. Vmax is proportional to the amount of active enzyme molecules (or active sites) and kcat, the turnover number, represents the moles of substrate hydrolyzed per mole of enzyme in a unit time. The values of the kinetic parameters determined for PNPG hydrolysis indicate that the main effect of the confinement of β-Gal was probably exerted on the conformation of the active site without a significant effect on the reaction rate constants (thus, on the reaction mechanism).

ONPG hydrolysis

For the analysis of the ONPG hydrolysis catalyzed by Eβ-Gal, taking in mind the hypothesis of two populations of Eβ-Gal suggested by the two slopes observed in the Eadie-Hofstee plot (Fig. 2d), we estimated the kinetic parameters assuming that the V0 measured was the resultant of the additive activity of each of the enzyme populations. The initial rate of ONP formation V0A, represented ‘Sβ-Gal like’ Eβ-Gal and V0B corresponded to different species of Eβ-Gal, both of them following a Michaelian kinetics, and contributing to a total V0 according to eq. 1:

where ni, kci and KMi are the moles, the catalytic rate constant and the Michaelis constant for each Eβ-Gal species, A and B, respectively. From the fitting of Eq. 1 to the experimental data, it follows that more than 50% of Eβ-Gal contributed to the B population term, a proportion which did not vary significantly with aging time. The apparent KMA values, corresponding to the A population of Eβ-Gal at 0 days of aging were not statistically different from the KM for Sβ-Gal but increased at longer aging times. In turn, the apparent KMB values obtained were significantly higher than KMA (Table 1, bold characters). This result suggests that aging affected slightly the apparent affinity of the A component of the enzyme-substrate interaction (compare KM for ONPG with Sβ-Gal and KMA with Eβ-Gal) but induced a strong inhibition of the B component (compare KM for ONPG with Sβ-Gal and KMB with Eβ-Gal) (recall the inverse relationship between KM and affinity, mentioned above). Similarly, while the A component of Vmax and kcat measured with Eβ-Gal did not differed significantly from the values measured with Sβ-Gal, the B component exhibited Vmax and kcat significantly higher and aging-dependent values. In conjunction these data allow propose the existence of two different population (environments) of Eβ-Gal. Strictly, the apparent KM values would synthesize the simultaneous effects of several factors including an uneven distribution of ONPG along the porous gel structure due to diffusional restrictions7. Additionally, conformational changes suffered by the protein upon encapsulation and different populations of water molecules which are affecting the enzyme-substrate interaction would also contribute to the differential KMvalues measured. Thus, both Eβ-Gal populations should not be rationalized merely as different molecular entities but as two kinds of catalytic behaviors. The kcB values were higher than the catalytic rate constant calculated for Sβ-Gal and for ‘Sβ-Gal like’ Eβ-Gal (kcA) in all the conditions studied (Table 1, bold characters). Moreover, the kcB increased with the aging time within the time period under consideration (Table 1). These results indicate that there exists a population of Eβ-Gal associated with a turnover number higher than the value obtained for the soluble enzyme (compare kcB with the kcat for Sβ-Gal in Table 1), and this population suffers the effect of being confined in an environment in continuous evolution (gel aging), while the ‘Sβ-Gal like’ Eβ-Gal population might consist of β-Gal molecules entrapped in pores big enough so that the aging effect is sensed with lower intensity.

Water dynamics in nanoporous gel

The mechanism of β-Gal catalytic activity involves a hydrolysis step (see Fig. 4 below) which may be conditioned by the restricted dynamics of water inside the nanometric pores of the gel. The surface to volume ratio in the gel is high, so most water molecules may experience restricted degrees of freedom and/or they could be in a higher energy state if compared with bulk water30. This phenomenon might lead to differential kinetics in processes where water participates in the rate-determining step of the reaction.

Water dynamics in the silicate gel was studied through the transverse relaxation time (T2) of water protons through 1H-NMR. It is well known that water exhibits more than one component for T2 in the presence of a hydrophilic silicon interface19,31. This behavior is usually related to the existence of distinct phases with independent T2 values; where the shortest relaxation times (in the order of a few milliseconds) is associated to water molecules in tight contact with the solid polymeric backbone, while bulk water renders longer relaxation times (in the order of seconds)32,33,34. Additionally, it is expected that in gels a pool of water molecules exhibit an intermediate relaxation behavior (in the order of tens or hundreds of miliseconds), that is, water molecules that are trapped within the gel network or pores, with an intermediate mobility35. In the present work T2 values were determined for protons in the silicate gels employing CPMG pulse sequence36 and subsequently analyzed with an inverse Laplace transformation. Three T2components (T2a, T2b and T2c) were obtained indicating that in the sample, water molecules can be categorized in three different mobility pools (Fig. 3a). It is worth to note that due to experimental parameters (see methods section) 1H belonging to the silanol groups and water of the buffer do not contribute to the CPMG signal. It must also be recalled that during the experimental time, these three populations may exchange molecules between them, in a dynamic process.

The three T2components (T2a, T2b and T2c) can be interpreted as water in the first hydration sphere of the silicon polymer (T2a; 4.8 ms), water within the gel structure (T2b; 40 ms) and water in larger pores or cracks of the gel ball (T2c 600 ms). These values are in good agreement with those reported in literature for similar systems37. The intensity of the water populations with short T2 values are small compared to the water contained within the gel, as there is a limit as to the amount of water that can reside in the hydration sphere of the polymer surface.

Along the aging of gel the magnitude of the three T2components decreased (Fig. 3b). We have previously shown that changes in the matrix structure of the gel leads to variations in the T2 values35. In this way, the decrease of the relaxation rates can be interpreted as a restriction in the mobility of the water molecules of the different populations due to an increase in the silicate matrix crosslinking.


Sol-gel encapsulation of macromolecules has been proposed as a suitable model system for the study of the effects of crowding and confinement in a living cell3.

The gel topology is very sensitive to sol-gel synthesis conditions, for this reason, it is relevant to verify the structure of the gel whenever some of these conditions are changed. In the present work, the sol-gel synthesis was performed, not only at physiological pH, but also at a H2O:TEOS molar ratio at which ethanol concentration was low enough to preserve the enzyme catalytic activity. From the SEM images it could be figured that an encapsulated macromolecule could be confined in closed pores or a network of pores interconnected by funnels of different diameters resulting in a heterogeneous environment far from dilute solution conditions. From the pore diameters distribution it was observed that most of them were in the order of β-Gal tetramer dimensions (roughly 17 × 13 × 9 nm)38 however, some pores above 100 nm were also found.

The activity measured for Eβ-Gal was similar to that of Sβ-Gal for the hydrolysis of PNPG and showed higher activity for the hydrolysis of ONPG, which was evidenced by thekcat values obtained after fitting the curves. For the later, saturation curves were better fitted employing two hyperbolic components (Eq. 1). One of the kcat values obtained (kcA) was similar to the one obtained with Sβ-Gal and the other one was significantly higher (kcB). Upon these observations many questions arise. Firstly, why the hydrolysis of ONPG using Eβ-Gal results biphasic with one of the components having a kcat higher than that obtained with Sβ-Gal? and why this behavior is not observed when PNPG is employed as substrate? The mechanism of catalysis for β-Gal was proposed by Wallenfels and Malhotra and lately confirmed by Viratelle and co-workers21,22,23,39. They postulated two intermediate complexes according to Fig. 4.

The amount of PNP or ONP formed over time and the catalytic rate constant (kcat) can be expressed as shown by Eq. 222:

By performing nucleophilic competition experiments with methanol Viratelle and co-workers identified the rate determining step of the reaction (Fig. 4) for many substrates21,22,23. For PNPG the slowest step was the one governed by k2 in which the aglycone was cleaved whereas for ONPG k2and k3 were of the same order of magnitude with k3k2/2. This means that the water nucleophilic attack to the galactosyl residue bound to the enzyme is the rate limiting step when OPNG is the substrate but it has no incidence on the kinetics of PNPG hydrolysis. This differential mechanism against both substrates would explain the dissimilar catalytic activity of Eβ-Gal when compared to Sβ-Gal. For the hydrolysis of PNPG water availability would not be relevant as it does not take part of the rate limiting step. On the contrary, because the hydrolysis step is the slowest for ONPG, it would be expected that changes in water structure and dynamics could influence the net rate in this case.

From T2 measurements it was shown that three different water proton populations could be resolved in the sample. The most abundant was associated with the biggest T2 value (T2c = 573 ms). Although this correspond to a mobility significantly higher than the other two components (T2a and T2b) it also reflects certain degree of immobilization considering that water in bulk buffer solution usually renders T2values around 2.5 seconds30. This group of water molecules with a dynamics closer to that bulk water would account for the ‘Sβ-Gal like’ kinetic hydrolysis of ONPG (kcA) by confined enzymes in large pores. The least mobile water molecules are those with the shortest proton relaxation times (T2a) and can be identified as those located in the adsorbed layers closer to the silicate matrix19. Between the least mobile water molecules and the bulk type ones there would be a group of water molecules which exhibit intermediate dynamics and that, on average, show the relaxation time T2b. Thus, more structured water, associated with the fastest relaxation times (T2a and T2b), would be responsible for the higher kinetic constants obtained for the hydrolysis of ONPG (kcB) when data were fitted with Eq. (1).

Moreover, we found an inverse correlation between the enzymatic assays with ONPG and the relaxation experiments as a function of aging time that allow us to propose the following hypothesis. Certain Eβ-Gal molecules can be confined in big pores (~100 nm) resulting in bulk-like water environment (T2c) and its catalytic parameters resemble those of the free enzyme (kcA and KMA). Within the environment of nano-sized pores (~10 nm) Eβ-Gal is in the presence of a higher proportion of water molecules with more restricted degrees of freedom (T2a and T2b) and it is expected that the kinetic parameters of hydrolysis would differ from those obtained in bulk water (kcA and KMA). This observation is in agreement with the effect of aging both on water dynamics and on the activity of Eβ-Gal. Since the magnitude of transverse relaxation times of all the resolved water proton populations (T2a, T2b and T2c) decrease upon gels aging it can be assumed that all the differentially structured water populations on average became less mobile enhancing the catalytic activity of Eβ-Gal for the hydrolysis of ONPG. Simulation studies of water confined in silica nanopores showed that the first layer of water molecules are expected to be in a specific orientation (named H down orientation)14. This fact may account for a more efficient use of water as the substrate in the hydrolysis step to the catalytic reactions.


In the present work we tried to contribute to the understanding of how the environment within the pore affects the β-Gal catalyzed reaction. It is noteworthy that in bulk, both reactants and catalyst can move freely while in the gel the steric restriction imposed by the pore size to the latter might affect the initial reaction step (enzyme-substrate binding). However, this is not the rate determining step so, it is not surprising that PNPG and ONPG (considering only component A for ONPG) behave in a similar way at least in terms of Vmax and kcat values, either in bulk or in the gel. The difference was observed with component B of ONPG, the substrate whose hydrolysis involves a reaction mechanism that uses water in the rate limiting step. This let us arrive to the conclusion that the environment affects the availability of this reactant (water) in terms of dynamics and, possibly, of orientation and is not just a ‘confinement effect’ of the enzyme.

Our results allow us to propose that in this condition of structured water within the nanopores, silica would act as an additional catalyst due to its big surface area, contributing with oriented water molecules to an improved hydrolytic step expressed as a turnover number (kcB) for the Eβ-Gal catalyzed hydrolysis of ONPG higher than the turnover number obtained with Sβ-Gal. For the hydrolysis of PNPG catalyzed by Eβ-Gal, the quasi independence of the global reaction kinetics from the hydrolysis step, can explain the absence of an aging-dependent biphasic kinetics. Finally, our findings highlight the synergism between the confined enzyme in a water structured environment and silica surface in the catalytic properties of nanostructured media. Aging affects KM possibly through a mechanical (decreasing pores size) or osmotic (decreasing free water availability) phenomenon, not explored in the present paper, but which may be related to changes in the enzyme conformation associated to the aging process. Present results, considered in a biological context, would help understanding other systems where polymerization-depolymerization dymamics reproduce situations of environments with wide pore size distribution (e.g. cell cytoplasm)40.

Material and Methods


β-galactosidase (β-Gal) from Escherichia coli [EC3.2.1.23]. 2-nitrophenyl-β-D-galactopyranoside (ONPG), 4-nitrophenyl-β-D-galactopyranoside (PNPG) and tetraethyl-orthosilicate (TEOS) were purchased from Sigma (St. Louis, MO, USA) and used without further purification.


Silicate Gel Synthesis

Silicate gels were synthesized by the sol-gel method41,42 modified for protein entrapment by Ellerby et al.5,11 water:TEOS ratio was adjusted to 20:1. Briefly, TEOS (3.0 mL) and deionized water (4.5 mL) were mixed at room temperature, followed by the addition of 0.1 M HCl (0.2 mL) which was used to catalyze the hydrolysis reaction. The reaction mixture was sonicated until it became homogeneous (approximately 4 h) then, equal volumes of the sol and sodium phosphate buffer (0.01 M, pH 6.8) were mixed. Gelation becomes evident within 10 min. For a 100 μL final volume of gel 10 μL of buffer were replaced by the enzyme solution (10 μL, 1 μg. mL−1). Samples were stored in polystyrene multi-well plates submerged in buffer, at 4 °C until use. No differences were observed in catalytic activity with or without washing the samples prior to the enzymatic reaction, indicating that enzyme was fully encapsulated. It is important to note that at 20:1 water/TEOS molar ratio the ethanol obtained as secondary product is diluted enough to preserve enzymatic activity (see experiments with the solvatochromic probe merocyanine, as described in the Supplementary Fig. S2).

Silicate Gel drying and topology analysis

Aeorgels were obtained from gels aged in buffer. For water elimination, samples were firstly soaked in solvents with decreasing polarity according to the following protocol: 5% v/v, pH 7 formaldehyde for 48 h; 30, 50, 70, 80, 90 and 96% v/v ethanol:water solutions for 24 h each one, and 100% ethanol for 12 h. Finally, the samples were soaked in pure acetone prior to CO2 replacement and were processed in a critical point dryer (Leica EM CPD030) at the Laboratorio de Microscopía Electrónica y Microanálisis (LABMEM) UNSL-CCT San Luis, San Luis, Argentina. Images of Gold Palladium metalized samples were obtained by SEM (FE-SEM - Carl Zeiss – Sigma operating at 5.00 kV, In-Lens detector) at Lamarx – IFEG (CONICET-Universidad Nacional de Córdoba, Córdoba, Argentina). Pore size distribution was estimated through the analysis of SEM images employing images software analyzer ImageJ (Wayne Rasband, NIH, USA). Pore diameters distribution was estimated by analyzing the SEM image shown in Fig. 1 which is representative of a several SEM images. The procedure employing the open access software Image-J is widely used for these tasks (ref. 43 and refs therein). The bar scale provided in the original picture is used to set the pixels scale in nm. The image was thresholded at T = 45. This level corresponds to the grey value between 0 and 255 for which we considered that, compared to the non-thresholded image, all the pores are taken into account at a right size. The resulting binary image was composed of only white and black pixels, with pores represented in black. The pore diameters were measured from this image within the resolution of the technique.

Enzymatic activity determination

The hydrolysis reaction catalyzed by β-Gal was studied with each of two substrates, ONPG and PNPG within concentration ranges 0.05 × 10−3–2.0 × 10−3 M and 0.05 × 10−3–1.25 × 10−3 M, respectively, in 0.10 M Phosphate-buffer, pH 6.8, 10 μL of the substrate solution were incubated at 37 °C in the presence of a silicate gel, with or without (blank) the enzyme entrapped in its interior, and located in a well of a 96 wells-microplate The absorbance of the reaction product was measured at 420 nm (o-nitrophenol, ONP) or at 410 nm (p-nitrophenol, PNP) in a Multiskan Spectrum (Thermo Fisher Scientific, Finland) (Fig. 5) at 37 °C. This method allowed the measurement of the absorbance of the product inside the gel thus preventing problems on reaction kinetics calculations derived from delays in the diffusion of the product molecule out of the gel24.

The molar extinction coefficients for ONP and PNP in buffer (Σb,ONP = 3969 cm−1M−1 and Σb,PNP = 14754 cm−1M−1) and in the gels (Σg,ONP = 3902 cm−1M−1 and Σg,PNP = 15537 cm−1M−1) were determined through interpolation in calibration curves (see Supplementary Fig. S3). Calibration curves were also performed in gels aged for 3, 7 and 14 days and no significant differences were observed. In a typical experiment 50 μL of TEOS hydrolyzed (see Enzymatic activity determination above) were mixed with 50 μL of phosphate buffer (100 mM pH 6.8) with or without β-Gal (0.05 μg mL−1) at room temperature. After 10 min the gel was formed and 100 μL of buffer with substrate were added. Temperature was set at 37 °C and after 15 min in the case of ONPG and 60 min for PNPG the reaction was stopped with the addition of 30 μL of 14% P/V Na2CO3. Samples were incubated in dark at room temperature for 30 min in order to achieve a stable value of absorbance before reading. Non- catalytic hydrolysis was discarded with blank measurements. Initial rate (V0) conditions were established by performing product concentration vs. time and V0vs. β-Gal concentration curves (see Supplementary Fig. S4).

Data analysis

The values of KM and Vmax were determined by fitting the experimental data from the Vo vs. substrate concentration plot to the equation of Michaelis-Menten for PNPG data and to Eq. 1 for OPNG by a computer aided nonlinear regression analysis by the least squares method. For the analysis with Eq. 1 we firstly fit data obtained in buffer and the parameters obtained were employed to feed the fit of Eβ-Gal data making them fixed. Total protein number (nT = nA + nB) is another known value before fit. Therefore, the variable parameters were nB, KMB and kcB.

NMR Relaxometry Measurements

Relaxation measurements were performed in a Bruker minispec spectrometer operating at 20 MHz for 1H equipped with a BVT3000 sample temperature controller with 0.01 °C stability. Silicate gel spheres were placed individually in a 10 mm tube. A piece of paper soaked in buffer was placed in the upper part of the tube, in order to keep the sample moisture. Transverse proton relaxation times (T2) were measured using a Carr−Purcell−Meiboom−Gill (CPMG) sequence which was applied after a magnetization inversion water suppression pulse. The buffer suppression waiting time was adjusted to 1.5 s. The CPMG parameters were: echo time 0.2 ms, number of echoes 5000, 64 scans and the length of the 90° radiofrequency pulse was 2.45 Ds.

With this echo time it can be assured that the detected signal arises only from water molecules and not from the silicate network, whose 1H relaxation times were determined to be on the order of microseconds. The resulting CPMG decay presents a multiple exponential decay, and the T2 distribution functions were obtained by using an inverse Laplace transform (ILT) algorithm based on the Tikhonov regularization method provided by Dr. Petrik Galvosas from the Victoria University of Wellington, New Zealand.

All experiments were repeated at least three times for each sample, and a maximum dispersion of 3% in the processed data was obtained.

Additional Information

How to cite this article: Burgos, M. I. et al. Environmental Topology and Water Availability Modulates the Catalytic Activity of β-Galactosidase Entrapped in a Nanosporous Silicate Matrix. Sci. Rep. 6, 36593; doi: 10.1038/srep36593 (2016).

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