Thermal hyperactivation and stabilization of β-galactosidase from

release at 420 nm and 40 °C, under controlled stirring ... Aminex HPX 87 H 300 x 7.8 mm column, during 12 min. ...... (32) Ayers,M. R.; Hunt, A. J. S...
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Thermal Hyperactivation and Stabilization of β‑Galactosidase from Bacillus circulans through a Silica Sol−Gel Process Mediated by Chitosan−Metal Chelates Viviana Ospina,† Claudia Bernal,‡ and Monica Mesa*,†

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Grupo Ciencia de los Materiales, Instituto de Química, Facultad de Ciencias Exactas y Naturales, Universidad de Antioquia, UdeA, Calle 70 no. 52-21, Medellín 1226, Colombia ‡ Instituto de Investigación Multidisciplinario en Ciencia y Tecnología, Tecnología Enzimática para Bioprocesos, Departamento de Ingeniería de Alimentos, Universidad de La Serena, Raul Bitran, La Serena 1305,Chile

ABSTRACT: The research of simple and fast enzyme immobilization methods, preserving the enzyme activity and improving the thermal stability, is in the spotlight. The objective of this work is to develop a β-galactosidase immobilization one-pot route, combining the silica sol−gel encapsulation (SSGE) process with a metal chelation strategy by using chitosan and Ca2+, Zn2+, or Cu2+ cations. The results show that the presence of cations does not affect the encapsulation efficiency (81%) and has positive effects on the maximum catalytic potential, especially at 60 °C and in the presence of Ca2+ ions (MPC = 2203). They enhance the biocatalyst thermal stability and promote hyperactivation with respect to the soluble enzyme at 60 °C (1.6 times higher MPC). The biocatalyst prepared with Zn2+ ions exhibits also thermal hyperactivation in the first 30 min of heating (1.3 times more residual activity), but the enzyme is not stabilized (0.9 times lower MPC); also, the presence of Cu2+ ions does not promote hyperactivation or stabilization of the enzyme (0.3 times lower MPC) at this high temperature. These facts are reflected in the hydrolytic and transgalactosylation activities of the enzyme (33.6−57.4% total lactose conversion), higher than that reported with analogue biocatalysts. The physicochemical characterization of the obtained solid biocatalysts by SEM, TEM, XRF, and XPS indicates that chitosan−metal chelation has an important role in the encapsulation process and that a low metal degree incorporation (8.85 ppm of Ca2+) on the solid biocatalyst favors the thermal hyperactivation and stabilization of the evaluated β-galactosidase. This work contributes to the understanding of the SSGE process mediated by chitosan−metal chelates, which is a simple and fast one-pot immobilization strategy. KEYWORDS: Bacillus circulans β-galactosidase, enzyme thermal hyperactivation, silica sol−gel encapsulation process, chitosan−metal chelates, one-pot immobilization strategy

1. INTRODUCTION The β-galactosidases (EC 3.2.1.23) are glycoside hydrolases. Their Glu catalytic residues are involved in the nucleophilic attack over specific β1−4 links.1 They are commonly used for lactose hydrolysis to glucose and galactose in milk or whey and also for the synthesis of galacto-oligosaccharides (GOS) by lactose transgalactosylation reactions.2,3 Increasing the temperature above 50 °C improves the kinetics of these reactions, © XXXX American Chemical Society

especially for the last one, which requires a high lactose concentration and therefore high temperature for increasing the solubility of this substrate.4 However, this condition is not favorable for the enzyme performance. Therefore, some Received: April 30, 2019 Accepted: July 2, 2019 Published: July 2, 2019 A

DOI: 10.1021/acsabm.9b00371 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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the enzymes in the SSGE reaction medium.25 Some strategies for overcoming this problem are related with the use of Triton X-100 surfactant during SSGE of Bgal16,24,25 and the presence of chitosan for immobilization of manganese peroxidase, inspired by biosilicification processes under mild conditions.30,31 Moreover, the use of chitosan can offer other advantages during the one-pot SSGE because it catalyzes the hydrolysis and condensation of the silica source and mediates the silica particles aggregation,32 improving the mechanical properties of the solid biocatalysts. Based on the chemistry of this polymer and its property of “NH2− metal−protein” coordination bonding,31,33−36 we hypothesize that it serves as a metal chelation platform, for designing a new one-pot SSGE process of Bgal mediated by chitosan−metal chelates. The objectives of this work are related with the development of a Bgal encapsulation route, combining the SSGE process with a chitosan−metal chelation strategy in one-pot: studying the effects of concentration and type of metal salts on the encapsulation efficiency, the physicochemical and biochemical properties of the obtained solid biocatalysts, and their behavior in lactose hydrolysis and GOS synthesis. The sol−gel conditions are modulated in order to have a beneficial balance between encapsulation efficiency and enzyme activity/stability. A complete physicochemical characterization of the metal− chitosan incorporation and its correlation with the Bgal performance is shown.

strategies for having active and thermally stable biocatalysts must be implemented, being the enzyme immobilization in solid supports one of the preferred and simple methods. This strategy does not require sophisticated equipment and enhances the selectivity or specificity, resistance to inhibitors or chemicals, purity, convenience, and cost-effectiveness of enzymes in catalysis, widening their use in batch and continuous reactors.5−7 The immobilization strategies can be divided in two main groups: (1) stepwise and (2) one-pot ones. The first one (1) implies the preparation of the support before the immobilization process. This can occur through mechanisms such as physical adsorption, covalent binding, and metal chelation, by one- or multipoint interactions.5,7 These are affected by the spacer arm and steric hindrances of the organic reactive groups in the support surface.5 In some cases, metal cations are dispersed in the support for favoring the metal chelation of enzymes by specific regions.8 (2) The onepot strategies include cross-linking (mediated by a covalent linker) and encapsulation/entrapment inside the matrix at the same time that the solid support is formed.6 The βgalactosidases have been immobilized by many of these methods, on different supports, including silica, chitosan, and composite materials, and the advantages of each strategy have been discussed.2,8−12 For example, the stepwise immobilization of β-galactosidases mediated by metal chelates improves their catalytic performance and specificity.8,13,14 It is because the cationic centers direct the enzyme toward specific adsorption sites, such as His residues, by orbital soft−soft acid−base interactions.15 It led the enzyme in an appropriate orientation for catalysis.13 Metal divalent ions such as Ca2+, Zn2+, and Cu2+ modify the enzyme behavior probably due to structural changes.16,17 The presence of metals and amino groups in the support also contributes to the hyperactivation of βgalactosidases, as the case that from A. oryzae immobilized in nanoparticles by adsorption and cross-linking.18 Nowadays, there is an increased interest in one-pot routes for enzyme immobilization because they are reproducible, less time-consuming, and easy-implemented and avoid multiple steps of synthesis and activation of the solid support.19,20 The silica sol−gel encapsulation (SSGE) process is an example, which involves the polycondensation of siliceous species around the enzyme.19 This route leads to biocatalysts where the enzyme is homogeneously distributed throughout the volume of the obtained solid material. It confers thermal stability to the enzymes combined with the advantages of the silica properties, such as microbiological and mechanical stability and easy recovering from the reaction medium.21,22 These are desired characteristics of a solid support for biocatalysts.23 The commercial β-galactosidase from Bacillus circulans (Bgal) is an example of monomeric, nonmetal dependent enzymes1,29 stabilized by SSGE, without further complicated, laborious, time-consuming, and expensive purification processes. This encapsulated Bgal is used for whey hydrolysis and GOS synthesis,24−26 preserving its specific affinity for β 1 → 4 sugar links and the preference for producing β 1 → 6 trisaccharides.26−28 The enzymes also behave as silica templates in the SSGE route, modifying the silica polycondensation, which in turn affect the characteristics of the silica-based biocatalysts.20 In general, the factors that affect the sol−gel process (pH, ionic strength, etc.) also have effects in the enzyme during the encapsulation process.19,24 To date, there are still many challenges in the development of biocatalysts by SSGE, especially those related to the stability of

2. EXPERIMENTAL SECTION 2.1. Reagents. Bgal from B. circulans commercial extract (BIOLACTASA NTL X2, Biocon española S.A. specific activity = 1380 ± 85 IU/mL and, protein concentration = 38 mg/mL), without additional purification was used. Sodium silicate (Na2Si3O7: 27 wt % SiO2, 4 wt % NaOH) and chitosan (85% deacetylation degree and molecular weight 190−375 kDa) were purchased from Sigma. Sodium acetate was purchased from Panreac, and glacial acetic acid was purchased from Merck. The CaCl2·2H2O and CuCl2·2H2O salts were purchased from Sigma; ZnCl2·2H2O was purchased from Merck. oNitrophenyl-β-D-galactopyranoside (o-NPG) was purchased from Sigma. The bicinchoninic acid kit for protein determination was purchased from Sigma. All reagents were analytical grade and used as received. 2.2. Bgal Hydrolytic Activity Assay. The Bgal hydrolytic activity was measured by UV−vis spectroscopy (PerkinElmer Lambda35 UV/ vis spectrophotometer, UV Winlab software) using o-NPG as a substrate and measuring o-NP release at 420 nm and 40 °C, under controlled stirring (PerkinElmer PTP-1 Peltier Temperature Programmer System and Heating by Thermo scientific). In each test, 100 μL of enzyme solution (E) or biocatalyst suspension (CSX-MY) was added into a cell, containing 2 mL of 45 mM o-NPG substrate in 100 mM acetate buffer, pH 6.0. One International unit of Bgal activity (IU) was defined as the amount of enzyme producing 1 μmol of o-NP per minute under the described conditions.1,29 2.3. Bgal Transgalactosylation Activitiy. The transgalactosylation activity of Bgal was evaluated by using Bgal biocatalysts in the GOS synthesis, as described in previous works.27 The reaction was carried out in a 50 mL erlenmeyer flask by dissolving lactose monohydrate (40 wt %) in 100 mM acetate buffer, pH 6. The mixture was heated until lactose was dissolved, and then, it was incubated at 60 °C. The reaction was started by adding the biocatalyst to adjust Bgal activity to 40 IU per g lactose in the reaction media. The reaction was monitored by collecting 500 μL aliquots from the reaction vessel and incubating the sample at 100 °C per 5 min to stop the reaction. The quantification was performed by HPLC with a refractive index detector, equipped with an autosampler and an isocratic pump. The samples (20 μL) were eluted with 5 mM H2SO4 at a flow of 0.6 mL/ min, through an Aminex HPX 87 H 300 × 7.8 mm column, over 12 min. The column and the detector were at 45 and 40 °C, respectively. B

DOI: 10.1021/acsabm.9b00371 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials A typical HPLC chromatogram exhibits five signals corresponding to GOS-4 (6.2 min), GOS-3 (6.5 min), lactose (7.2 min), glucose (8.4 min), and galactose (8.9 min). The wt % of each saccharide was calculated with respect to the total saccharides determined by the peak intensities. The lactose conversion percentage was calculated with respect to the initial (Li = 10 g, 40 wt %) and the final lactose (measured from HPLC) concentration in the reaction medium. 2.4. Bgal Encapsulation. It was carried out by the one-pot SSGE strategy schematized in the Abstract: A 53 mM sodium silicate solution was prepared in 50 mM sodium acetate solution, and the pH was adjusted to 7.2 with glacial acetic acid. Parallel, a 3 wt % chitosan solution was prepared in 300 mM acetic acid solution. Then, 500 μL of chitosan solution was added to 50 mL of sodium silicate solution under magnetic stirring, at room temperature (1:100 volume ratio), and the mixture was stirred for 10 min. The resulting one-pot dispersion had a final pH of 6.0 (total volume = 50.5 mL). After that, the enzyme solution was added to this dispersion, varying the offered enzyme concentration in each experiment (20−200 IU/mL as the final concentration, Table 1). Subsequently, the dispersion was left

This protein quantification was performed by the bicinchoninic acid method (BCA). The protein immobilization yield (IYP, eq 2) relates the immobilized (Pi in mg/g) and offered protein (Pc in mg/g) for the SSGE process. The soluble protein was corrected by the total mass of the obtained solid biocatalyst for having Pc. The Pi was calculated by the difference between offered protein and that measured in the supernatant at the end of the SSGE process.

IYP =

samplea

protein concentration (mg/mL)

offered proteinb (mg/gcat)

initial specific activity (IU/ mgprot)

CS CS20 CS50 CS100 CS150 CS200

0 20 50 100 150 200

0 0.6 1.4 2.7 3.9 5.1

0 14.6 30.4 46.6 68.5 87.4

0 33.3 35.7 37.0 38.5 39.2

Aesp =

A = (1 − α)e−k1t + α = f (t ) A0

(3)

(4)

ij k1 k1 yzz −k1t k1 A zze = jjj1 + α −β − (α − β) e−k2t + β = f (t ) A 0 jk k 2 − k1 k 2 − k1 z{ k 2 − k1

(5) where A/A0 represents the residual activity at time t, α and β are the specific activity ratio of the intermediate species regarding the native enzyme species, and k1 and k2 are the transition rate constants from the native to the intermediate and from the intermediate to the final enzyme species, respectively. The measurements were carried out up to 30% of residual activity, considering that when the activity of a biocatalyst is under this percentage, it is not interesting for being used in industrial processes.27 Usually, the enzyme thermal stability has been described considering only the half-time values (time for losing 50% of its activity). In order to consider the thermal stability of biocatalysts and the overall activity (Ao) changes, the maximum catalytic potential (MCP)27 was determined by integration of the area under the thermal deactivation curve corrected by Ao (eq 6).

CSX stands for chitosan−silica and x offered enzyme activity. Calculated with respect to the total weight of the obtained solid biocatalyst.

b

under quiescent conditions for aging for 5 h at 25 °C. Finally, the materials were separated from the supernatant by centrifugation (the end-point of the reaction) and washed twice with 100 mM acetate buffer. The nomenclature for these biocatalyst was CSX, where X went from 20 to 200, according to the offered enzyme to the total solution volume in the final one-pot reaction system (Table 1). A sample prepared under the same conditions but in the absence of the enzyme, for simulating the matrix, was called CS (Table 1). For the Bgal encapsulation in the presence of metal salts, a similar procedure was followed, but the salt solution (500 mM CaCl2, ZnCl2, or CuCl2) was added to the chitosan/silica mixture before the enzyme addition (100 IU/mL), keeping the final volume constant. The final salt concentration in the total volume of the system was 1, 5, and 10 mM. The CS100-MY nomenclature for this kind of solid biocatalyst also includes the metal (M) and its concentration (Y). The CS-MY sample was prepared in the presence of chitosan and 1 mM metal salt, without the enzyme, simulating the matrix formation in the absence of protein. The recovered activity after Bgal immobilization (%RA, eq 1) was the ratio between the expressed activity of the solid biocatalyst (Ai in IU/g) and the offered activity in the one-pot recipient (Ac in IU/g). The Ai was measured for 100 mg of the solid biocatalyst in 1 mL of 100 mM acetate buffer (pH 6.0) by Bgal activity assay. The offered activity (Ac in IU/g) for the SSGE process was the activity of the soluble enzyme (IU/mL) corrected by the total mass of the obtained solid biocatalyst.

Ai × 100 Ac

IUTOT prot (mg)

2.5. Thermal Stability. The thermal stability of the soluble and solid biocatalysts was determined under nonreactive conditions in 100 mM acetate buffer, pH 6.0, at 60 °C. Aliquots of the sample were withdrawn periodically for being submitted to a Bgal activity assay. The residual activity (A/A0) vs time was adjusted to first-order deactivation models,36 according to a classical and series-type mechanism (eqs 4 and 5, respectively) by the iterative nonlinear regression method, using Gnuplot version 5.2 freeware.

a

%RA =

(2)

On the other hand, the ratio between the IUTOT and protein quantity encapsulated in the final solid biocatalyst gives the specific activity (Aesp) for the obtained biocatalysts (eq 3)

Table 1. Nomenclature for the Solid Biocatalysts (CSX) According to the Offered Enzyme Activity and the Equivalent Protein Concentrations in the One-Pot System and the Initial Bgal Specific Activity offered enzyme activity (IU/ mL)

Pi × 100 Pc

MPC = A 0

∫0

tf

f (t )dt

(6)

2.6. Kinetic Parameters. The kinetic parameters, Km and Vmax, were determined for the soluble and solid biocatalysts using a concentration of 1.25 mg prot/mL. For the solid biocatalysts, ∼50 mg were resuspended in 2 mL of 100 mM acetate buffer pH, 6.0, and the specific activity was measured using different o-NPG concentrations (1−40 mM) as described in the Bgal activity assay. 2.7. Lixiviation Test. In a typical test, 100 mg of biocatalysts were dispersed in 1 mL of 100 mM acetate buffer (pH 6.0) at room temperature. The protein was determined in the supernatant every hour by BCA analyses for 7 h. 2.8. Physicochemical Characterization of Samples. The prepared samples were characterized by TGA, SEM-EDX, TEMEDX, XPS, and XRF. The solid biocatalysts and the siliceous matrices obtained in this work were analyzed by TGA (Q500 TGA, TA Instruments). Before TGA analysis, all samples were dried at room temperature. A heating rate of 10 °C/min up to 800 °C under a nitrogen atmosphere was used to follow the thermal decomposition. The morphology and the cation distribution in the materials were determined by SEM (JEOL JSM-7100 instrument with an electron assisted Field Emission Gun, FEG); the microscope was equipped with an EDX analyzer for element mapping. The morphology and the

(1) C

DOI: 10.1021/acsabm.9b00371 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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ACS Applied Bio Materials composition of the CS100-Ca1 sample were determined from TEMEDX (FEI Tecnai G2-F20) analysis. The percentage of each metal was determined by XRF analysis (S2 PicoFox instrument, Bruker), by using a 100 ppm gallium standard for the quantification. The surface elemental analysis was made by XPS (Specs photoelectronic X-ray spectrometer, NAP-XPS, equipped with a PHOIBOS 150 1D-DLD analyzer with monochromated Al Kα X-ray radiation, 1486.7 eV, 13 kV, 100 W) with a pass energy of 100 eV for survey spectra and 40 eV for high-energy resolution. The Kα charge compensation system was employed with energy of 10 eV. All spectra were referenced to the C 1s peak attributed to C−(C,H) at a binding energy of 284.8 eV. 2.9. Statistics. The samples were prepared n triplicate and submitted to the physicochemical and biochemical characterization. The obtained data were reported as mean ± SD. The statistical comparisons of data were carried out with ANOVA and Tukey’s tests for p < 0.05 in all cases, which means that significant differences between samples in the discussion were based on a 95% confidence interval.

Figure 2. Quantity of immobilized protein (loading) and its correspondingly expressed activity and specific activity in CSX encapsulated Bgal biocatalysts, prepared with different offered enzyme concentrations (X).

3. RESULTS AND DISCUSSION 3.1. Bgal SSGE Mediated by Chitosan in the Absence of Salts. The one-pot Bgal SSGE experiments mediated by

Figure 3. (a) Specific activity (Aesp), recovered activity (%RA), and protein immobilization yield (%IYP) for CS100-MY biocatalysts prepared with 100 IU/mL offered enzyme in the presence of chitosan and 1, 5, or 10 mM of Ca2+, Zn2+, and Cu2+ salts (MY). (b) Recovered activity for the soluble Bgal (E) in the presence of 10 mM salt, at pH 6.0 and 25 °C.

Figure 1. Thermal behavior of encapsulated CSX biocatalysts, prepared with different offered enzyme concentrations (X): (a) TGA derivative curves and (b) weight loss for each thermal event (172, 300, and 443 peaks).

these conditions promote the activity and thermal/colloidal stability of the Bgal in aqueous environments37,38 and during SSGE, even in the presence different organic additives,24,25 analogues to the chitosan. The reactive species in the one-pot reaction system are discarded at the end of the process by eliminating the supernatant (reaction end-point), avoiding

chitosan in the absence of salts were carried out varying the quantity of offered enzyme to the one-pot reaction system (Table 1). The temperature, pH, and gelation/encapsulation time were fixed in 25 °C, pH 6.0, and 5 h, respectively, because D

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Table 2. Lixiviation Percentage, Biochemical Parameters, and Thermal Stability at 60 °C for the Soluble (E) and CS100-M1 Encapsulated Biocatalysts thermal behavior at 60 °Cc

biochemical parametersb biocatalysts

lixiviationa (%)

Km (mM)

Vmax (mM/min)

mech

k1 (h−1)

k2 (h−1)

α

β

MCP

R2

soluble (E) CS100 CS100-Ca1 CS100-Zn1 CS100-Cu1

2.9 2.3 2.0 2.9

14.91 29.88 20.87 23.08 23.27

41.36 20.03 14.68 14.54 16.18

C ST ST ST C

0.340 0.822 1.424 1.124 1.018

1.641 2.847 2.249

0.081 2.355 5.999 2.629 0.038

0.286 0.388 0.158

1380 2263 2203 1296 553

0.9981 0.9878 0.9981 0.9878 0.9717

a

Protein lixiviation (7 h in 100 mM acetate buffer). bCalculated parameters from kinetic experiments: Michaelis−Menten constant (Km) and maximun velocity (Vmax) cCalculated parameters from the thermal deactivation profiles following classical (C) or series-type (ST) mechanisms (mech): deactivation constants (k1 and k2); ratio of specific activities on intermediate and initial enzyme forms (α and β); maximum catalytic potential (MCP); correlation coefficient (R2)

Figure 5. Typical hydrolytic and transgalactosylation profiles at 60 °C (CS100-Ca1 sample, a). Saccharide concentration (bars) and lactose conversion % (circles) at 30 min into the reaction at 60 °C with different encapsulated biocatalysts (b).

chitosan by decarboxylation, deacetylation, depolymerization, and pyrolytic reactions.40,41 At higher temperatures, the process of condensation and elimination of silanol groups could also take place; however, it is not observed in the evaluated samples. Probably the population of vicinal silanol groups on the silica surface is low, due to the catalytic effect of chitosan on the silica polycondensation42 and/or because the presence of organic matter affects the dehydroxylation reactions, as it has been shown for similar chitosan/silica hybrid materials, where this water loss is neglected.43 The TGA derivative curves for the biocatalysts are better resolved and shifted to higher temperatures compared with the curve for the chitosan−silica matrix (prepared in the absence of protein, CS sample, Figure 1a). Probably, the peak 443

Figure 4. Thermal stability profiles at pH 6.0 and 60 °C for the CS100-M1 encapsulated (a) and E soluble Bgal (b) biocatalysts in the presence of 1 mM Ca2+, Zn2+, and Cu2+ salts (M1).

additional deactivation steps made in covalet stepwise route.39 The obtained solid materials were analyzed by TGA in order to have a first glance to the encapsulation process. The TGA curves under a nitrogen atmosphere exhibit four thermal events between 50−600 °C, being the first one related to physisorbed water in the surface (Figure 1a). The other three are below 200 °C (peak 172), between 200 and 400 °C (peak 300), and above 400 °C (peak 443). These events are associated with the thermal decomposition of the protein and E

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enzyme (Figure 1b). The same tendency is observed in the quantity of immobilized protein (measured by BCA method), which does not reach a plateau (Figure 2). This behavior indicates that the protein loading on the solid can occur until the reactants become depleted because the enzyme encapsulation occurs in the same time scale than the matrix formation, as it has been demonstrated for other biocatalysts obtained by SSGE.25 However, a maximum expressed activity value is reached (553 ± 55 IU/mg) for the CS100 and CS150 biocatalysts, and even the specific activities are lower than for the soluble enzyme (Figure 2), as observed after immobilization process.24,25 Exceeding this offered protein quantity has negative effects on measured activity, probably as a result of the molecular crowding, favoring the agglomeration, which could lead to enzyme conformational restrictions and substrate diffusion limitations. The agglomeration effects on the Bgal structure−activity have been demonstrated for this enzyme24,37,38 due to intramolecular interactions. Knowing that they are dependent on the selected immobilization strategy,45 we know they can be minimized in the selected one-pot route by offering less than 150 IU/mL of the enzyme to the reaction system. Therefore, offering 100 IU/mL under the evaluated Bgal encapsulation conditions leads to a high protein immobilization yield (IYP = 81.0 ± 1.8%), which correspond to the enzyme loaded in the support with a recovered activity around 32.0 ± 1.9 with respect to the soluble form. These values are higher compared to that obtained for the Bgal encapsulated in

Figure 6. Metal concentrations in CS100-M1 biocatalysts and their corresponding matrices (CS-M1), quantified by XRF. M1 stands for each metal cation, 1 mM.

corresponds mainly to the encapsulated enzyme in the inner part of the silica matrix, which confers stability toward thermal degradation. This behavior has been discussed for other βgalactosidases in function of the protein location in the external surface or into the mesopores of the silica used as immobilization support, which offers a different confinement degree to the enzyme and therefore different protection against thermal decomposition.44 The organic weight losses, calculated in dried base from these three TGA thermal events and representing the enzyme loading in the biocatalysts, increase linearly with the offered

Figure 7. XPS survey spectra of CS100 (a), CS100-Zn1 (b), CS100-Cu1 (c), and CS100-Ca1 (d) biocatalysts. The HR spectrum in the region for the corresponding metal is in each inset. F

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Figure 8. High-resolution core-level O 1s, N 1s, and C 1s XPS spectra for CS100-M1 Bgal encapsulated biocatalysts. M stands for the metal type.

silica by the sol−gel process assisted by Triton X-100,25 which shows the improvement and practical significance of this onepot SSGE immobilization procedure in the presence of chitosan. Therefore, these will be the conditions for the following SSGE process in the presence of metal salts. 3.2. Bgal SSGE Mediated by Chitosan in the Presence of Metal Salts. The Bgal SSGE process mediated by chitosan in the presence of metal salts was made by offering 100 IU/mL protein to the total volume of the one-pot reaction system and varying each salt concentration (1, 5, and 10 mM). CaCl2, ZnCl2, or CuCl2 were chosen because the chlorides have less chaotropic effect in comparison to other anions such as nitrates.46 The influence of the concentration and type of cation on the activity and stability of the obtained solid biocatalysts were studied. Further characterization related with the biochemical and physicochemical properties of the biocatalysts allowed studying the relationships between

activity/stability and structure, gathering information about the effects of cation incorporation on the catalytic performance. 3.2.1. Effect of Cation-Type and Concentration on the Bgal SGEE Process Efficiency. The SGEE process efficiency is reported in terms of protein immobilization yield (IYP) and recovered activity (%RA), in correlation with the specific activity (Aesp) of the immobilized enzyme in the function of the cation-type and concentration (Figure 3a). The samples prepared in the presence of Zn2+ or Ca2+ do not show significant difference on the IYP values between them or with respect to the CS100 sample, at the evaluated concentration levels. The %RA values do not show statistically significant differences between these samples, and the same occurs for Aesp values, demonstrating there is no significant effect of varying calcium and zinc concentrations in the encapsulated Bgal. Although increasing the Cu2+ concentration lead to G

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Figure 9. SEM-EDX elemental composition maps of CS100-Zn1 (a, b, c), CS100-Cu1 (d, e, f), and TEM-EDS for CS100-Ca1 (g, h, i).

scale as the enzyme encapsulation, which in turn affects the matrix porosity.20 Taking into account that Vmax defines the highest possible velocity when the enzyme is saturated with a substrate, its decrease reflects a different effect of the SSGE process, such as conformational changes of the enzyme, restrictions on the substrate transport, and steric constraints for biomolecular reactions, which slow down the reaction rate. These effects are always present in immobilized biocatalysts,24 and their extent will depend on the immobilization strategy.45 Here, the diffusion limitations were minimized by optimizing the offered enzyme to 100 IU/mL, which is the lowest concentration that still exhibits highly expressed activity (553 ± 55 IU/mg, Figure 2), as explained before. On the other hand, the increase of the apparent Michaelis−Menten constant, Km, for the solid biocatalysts with respect to the soluble (Table 2) is a consequence of the lower substrate− enzyme affinity influenced also by the tortuosity effects on the substrate, as it has been discussed in other reports.25 The comparison of the kinetic parameters for biocatalysts encapsulated in the absence and presence of salts (CS100 and CS100-M1 samples, respectively, Table 2) indicates that the presence of cations increased the substrate−enzyme affinity though the Vmax values decrease (less available enzyme

significantly high IYP values with respect to the other samples, it does not represent an increase in the %RA, and it has a negative effect on the Aesp, decreasing significantly for the CS100-Cu10 sample with respect to CS100 and CS100-Zn10 ones. This indicates that the additional immobilized protein in the presence of a high concentration of copper becomes inactive. It can be correlated with the deactivating effect of the copper on the soluble enzyme (E), which is stronger than with calcium or zinc (Figure 3b). Therefore, the samples for further characterization will be prepared with 1 mM salts (Table 2). This also avoids the negative effects of the high ionic strength on the coagulation of the enzyme and particles aggregation during the SSGE processes, which could affect the control of the molecular interactions.47 3.2.2. Effect of the Cation-Type on the Biochemical Parameters and Thermal Stability/Hyperactivation of the Bgal Biocatalysts. The kinetic parameters (Vmax and Km) calculated from the adjustment of the Michaelis−Menten plot for the soluble Bgal, CS100, and CS100-M1 samples are summarized in Table 2. The decrease of the Vmax value for the solid biocatalysts compared with the soluble enzyme shows that this intrinsic characteristic of the Bgal25,48 is affected by the tortuosity of the porous matrix, produced in the same time H

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Ca2+ cations in the presence of chitosan during the SSGE process. These interactions affect the enzyme loading/ crowding, rigidification degree, and conformational changes under specific inactivation conditions, which are relevant for designing the enzyme stabilization strategies.54 In contrast, during incubation of the biocatalysts at high temperatures, the hydrophobic interactions between encapsulated enzyme molecules could be affected, which in turn affect the thermal stability.45 The thermal stability of the chitosan−silica support at 60 °C described from TGA (Figure 1) could also contribute to these results. On the other hand, the adjustment of the thermal inactivation curves to first-order decay kinetic models shows that the behavior of the soluble enzyme obey a classical model (Table 2), where the deactivation proceeds in a single step, passing from the active to the inactive state (Ei → EF). The encapsulated biocatalysts (except CS100-Cu1, Table 2) follow a series-type deactivation mechanism, which implies the

molecules for catalysis process). The affinity increase is one of the positive effects of the presence of metals such as Ca2+ and Zn2+ instead of Cu2+ during the Bgal SSGE process mediated by chitosan. They also cause different effects on the thermal behavior of the encapsulated Bgal. The thermal deactivation profiles for the encapsulated (CS100-M1 solids) and soluble biocatalysts (E) heated at 60 °C and pH 6.0 are shown in Figure 4a and 4b, respectively. These conditions were chosen because the potential application of these biocatalysts in the GOS synthesis requires these temperature and pH values.26,27,49 The calculated maximum catalytic potential (MCP, Table 2) from these curves were used here instead of the half-times because it is a more global index that includes the thermal stability and activity changes of biocatalysts during the heating time.27 The analysis of the profiles for the encapsulated Bgal shows that thermal hyperactivation during the first hour of heating occurs for some biocatalysts, which depends strongly on the presence and type of cation at the evaluated conditions (Figure 4a). It is evident that the presence of Ca2+ ions in the CS100-Ca1 biocatalyst enhances the enzyme thermal stability and promotes the thermal hyperactivation (215% more active with 1.6 times higher MPC than the soluble enzyme). The biocatalyst prepared with Zn2+ ions is also hyperactivated (125% higher residual activity, Figure 4a) but not stabilized (0.9 times MPC than the soluble enzyme, Table 2), and the presence of Cu2+ ions does not promote hyperactivation or stabilization of the enzyme (0.3 times lower MPC than the soluble form) at this high temperature (Figure 4a). The thermal stability profile of the encapsulated Bgal is very different to that observed for the soluble form at 60 °C in the presence of salts (Figure 4b), for which a strong thermal deactivation is seen, which can be related with conformation changes as it is shown by circular dichroism at pH 6.0 and a temperature higher than 45 °C,38 and this deactivation is faster in the presence of Zn2+ and Cu2+ than with Ca2+. These cations have chaotropic effects, disrupting the hydrogen bonding network between water and proteins, generating a salting-out effect,46 inducing the coagulation process,50 and even promoting protein chemical modifications, such as deamination of Asn/Gln residues, hydrolysis of peptide bonds at Asp residues, and oxidation of the S−S and S−H functional groups under some experimental conditions.51 These copper and zinc effects are minimized by the SSGE process mediated by chitosan−metal interactions (Figure 4a). It is possible that the amine groups and metal ions could contribute to the thermal hyperactivation, as it was reported for β-galactosidase from A. oryzae immobilized onto amino-silane and chitosan magnetic maghemite nanoparticles18 and other enzymes immobilized in polyamines in the presence of inorganic salts.52,53 The Ca2+ ions have a lower influence on the soluble Bgal thermal stability (Figure 4b) due to their salting-in effect at low concentrations and pH values near to the isoelectric point of the enzyme, where negative enzyme−enzyme interactions can be minimized,38,46 and their nule effect on the kinetic parameters of some soluble β-galactosidases.29 The nondeactivating effect of the calcium on the soluble Bgal at pH 6.0 and 25 °C (Figure 3b) also contributes to the Bgal stabilization at high temperatures in the CS100-Ca1 sample (Figure 4a). The observed thermal hyperactivation/stabilization of encapsulated Bgal in the CS100-Ca1 sample in comparison with the soluble form (Figure 4b) suggests changes of the interactions between the enzyme and the

k1

k2

formation of intermediate states (Ei → E1α → E2 β ). The α and β parameters describe the proportion of the intermediate forms with respect to the initial active state, and α > 1 indicates that there is an initial activity rise before the decay in the second step (β < 1).36 The α values (Table 2) are well correlated with the thermal hyperactivation tendency (Figure 4a), which are higher for the CS100-Ca1 sample compared with the other ones. 3.2.3. Effect of the Cation-Type on the Hydrolytic and Transgalactosylation Activities of Bgal Biocatalysts. The hydrolytic and transgalactosylation activities of Bgal biocatalysts prepared in this work were assayed at 60 °C and a high lactose concentration (40 wt %). A typical kinetic profile under these conditions (Figure 5a) shows the decrease in the lactose content with the concomitant increase of the tri- and tetrasacharides (GOS-3 and GOS-4) and the presence of glucose and galactose, similar to the results reported by other authors with commercial soluble and immobilized Bgal.25,28,55−57 The concentration for GOS-3 is higher than that for GOS-4 with all evaluated Bgal biocatalysts (Figure 5b), due to the preference of the β-galactosidase from B. circulans for producing trisaccharides with β 1→6 links, under similar conditions.26−28,49 The glucose concentration is also high because this transgalactosylation reaction releases glucose after the nucleophilic attack of the catalytic glutamic residue.1 Even, the reaction conditions promote the reaction of the galactosyl−enzyme complex with mono-, di-, and trisaccharides,56 the concentration of galactose was not zero, evidencing the contribution of the hydrolysis reaction.57 The maximum GOS concentration is obtained 15 min into the reaction with all of the assayed biocatalysts (Figure 5a). The lactose conversion and saccharide concentration percentages indicate that the biocatalysts behave different in function of the incorporated cation (Figure 5b). The CS100-Ca1 sample is approximately more active for hydrolysis and transgalactosylation than CS100 and CS100-Cu1 ones, respectively (Figure 5b). This is correlated with the thermal hyperactivating and stabilizing effects of the SSGE process mediated by chitosan in the presence of Ca2+ ions seen at 60 °C (Figure 4a) reflected in a 1.6 times higher MPC than for the soluble enzyme (Table 2). This correlation highlights the importance of the thermal stabilization of the enzyme as I

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keeps a strong correlation with the Ca2+ hyperactivating/ stabilizing, Zn2+ hyperactivating/nonstabilizing, and Cu2+ nonhyperactivating/nonstabilizing effects at 60 °C in the prepared biocatalysts (Figure 4b), explained in the precedent section. The XPS analyses were made in order to have additional information about the metals incorporation and their interactions at the atomic level. The XPS survey spectrum for CS100 sample exhibits signals for O 1s, N 1s, C 1s, Na 1s, Cl 2p, Si 2s, and Si 2p and KLL auger signals for O and C (Figure 7), which come from organic (N, C, O from chitosan and Bgal) and inorganic (O, Si, Na, Cl from silica) components of this biocatalyst. The C 1s high-resolution spectrum (HRS) of the CS100 biocatalyst is characterized by contributions at 284.49, 286.10, and 287.93 eV (Figure 8), arising from C(C,H) of the organic skeleton and adventitious contamination: C(N,O) in hydroxyl, amine, amide and peptidic link, and OCO of the hemiacetal, respectively. These contributions from amide, hydroxyl, and hemiacetal functional groups are also seen in the deconvoluted O 1s HRS (Figure 8) at 529.74 eV (OCN) and 531.38 eV (COH, COC).63 The later signal is also related with OSi from silica.64 The signal, ∼399.33 eV, in the N 1s HRS is correlated with the amine, amide, and peptidic link.65 The survey and HR XPS spectra for the CS100-Ca1 sample do not show calcium signals in the bonding energies between 345 and 365 eV (Figure 7), indicating that the calcium is not exposed in the surface of the biocatalyst (∼10 nm depth, which is the limit of the XPS technique). By the contrary, the presence of zinc and copper in CS100Zn1 and CS100-Cu1 samples is evidenced by Cu 2p3/2, Cu 2p1/2 signals (932.17 eV, 951.96 eV with Δspin‑split = 19.79 eV), and Zn 2p3/2, Zn 2p1/2 ones (1018.26 eV, 1041.11 eV, with Δ spin‑split = 22.85 eV), respectively (Figure 7). It is important to notice that these signals are shifted toward lower binding energies with respect to those reported for the ions in the salts,64 indicating a change in the charge density of them when they are incorporated in the biocatalysts. This is due to their chelation with the electron-donating atoms in the hydroxyl, amine, and amide functionalities of chitosan and enzyme, which in turn shifts the O 1s and N 1s HR signals, especially in the CS100-Cu1 sample (Figure 8). The “bridge” and “suspension or pendant” copper or zinc coordination models reported with chitosan59,60 could be formed in CS100-Cu1 and CS100-Zn1 biocatalysts, involving also inter- and intramolecular complexation with the enzyme, due to the preference of Cu2+ and Zn2+ for nitrogen in His residues.66 It is interesting to notice that the absence of satellite signals in the Cu 2p high-resolution spectra indicates that copper is mainly Cu+ ions instead of Cu2+ ones,65,67 which is explained by the reducing effect of chitosan.68 This reduction does not occur for Ca2+ and Zn2+ ions because their reduction potential (−2.87 and −0.76 V, respectively) is lower than the value for Cu2+ (+0.15 V).15 This is further evidence of the strong interaction and complexation of copper with chitosan, related with its deleterious effects on the activity and thermal behavior of the Bgal, as discussed in precedent sections. These XPS results confirm that metal ions are incorporated in the obtained samples as metal chelates, which has a profound impact on the Bgal immobilization by SSGE mediated by chitosan. The EDX from SEM analysis showed that zinc and copper are homogeneously distributed throughout regions of the CS100-Zn1 and CS100-Cu1

criteria for defining the application, as it has been shown for other enzymes.58 By the contrary, the CS100-Cu1 exhibits the lowest lactose conversion percentage (33.6%, Figure 5b) and a lower activity for transgalactosylation and hydrolysis reactions, leading to the smaller saccharide percentage (Figure 5b). This behavior is expected from its low thermal stability (Figure 4b) and lowest MCP (Table 2). However, the lactose conversion is still higher with respect to the 26% reported value with Bgal encapsulated by SSGE in the presence of sugars,25 highlighting the practical significance of the SSGE process mediated by chitosan−metal chelates for producing solid biocatalysts, which can be used in applications such as whey hydrolysis and GOS production, in batch and continuous reactors. On the other hand, the lixiviation degree from the solid biocatalysts after 7 h of orbital agitation at pH 6.0 and 25 °C is almost negligible (∼3%, Table 2), indicating that the enzyme is well encapsulated inside the formed matrix. This result, in conjunction with the thermal behavior observed specially for the CS100-Ca1 sample, will contribute to the operational and storage stability of the biocatalysts obtained in this work, in analogue form that was reported for Bgal encapsulated by the SSGE process in the presence of Triton X-100.24 3.3. Metal Incorporation on the Bgal Solid Biocatalysts. The presence of the metal ions and their interactions with the Bgal and chitosan−silica matrix play important roles in the biochemical, thermal, and hydrolytic/ transgalactosylation behaviors of the CS100-M1 samples, as described before. Therefore, the metal incorporation degree and the chemistry involved in these interactions will be discussed from XRF, XPS, SEM-EDS, and TEM-EDS results. The quantity of each metal was determined by XRF. The total concentration of calcium is significantly lower (ca. 8.9 ppm) in comparison with zinc and copper in CS100-Ca1 < CS100-Zn1 < CS100-Cu1 samples, respectively (Figure 6). This order of incorporation degree is also seen in the matrices prepared in the absence of the enzyme (CS-M1 samples), but it is interesting to highlight that the metal incorporation degree was 2.7−3.2 times higher in these matrices than in the biocatalysts, independent of the metal type (Figure 6, CS-M1 and CS100-M1 samples, respectively). This indicates that although the cations interact with a variety of species (siliceous, chitosan, and enzyme) during the Bgal SSGE process, the degree of incorporation of them in the final biocatalyst is very dependent on the metal−chitosan interactions, which in turn influence the degree of immobilization and the stability of the protein (Figure 4). A direct correlation between the incorporation degree quantified by XRF in the samples prepared in this work (Figure 6) and the reported order of selectivity for adsorption of the metal ions by chitosan at neutral pH (Cu2+ ≫ Zn2+ > Ca2+)34 supports the idea that chitosan has an important role for the incorporation of these metals. This is due to its high chelating capacity by NH2 and OH donor groups and its flexible structure, allowing the formation of metal−chitosan complexes.59,60 Probably, the presence of d orbitals in Zn2+ and Cu2+ ions favors the interaction with the chitosan donor groups61,62 unlike the alkaline and alkaline-earth cations, explaining the low incorporation degree of Ca2+. Chelated Zn2+ and Cu2+ species with chitosan−silica supports promotes metal−protein interaction forces, which have been correlated with the activity and stability behavior of other immobilized hydrolases.33 This can help to explain why the incorporation degree of each metal J

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IYp, protein immobilization yield; RA, recovered activity; Aesp, specific activity; Vmax, maximum velocity; Km, Michaelis− Menten constant; HRS, high-resolution spectrum.

samples, respectively (Figure 9a−f). The calcium, which is incorporated in a lower concentration, is located in some specific areas of the CS100-Ca1 sample, detectable by TEMEDX (Figure 9g−i), probably, making part of the silica network as in Ca silicate gels.69 Moreover, the SEM and TEM images show that all materials are constituted by aggregated particles and exhibited textural porosity (Figure 9), which contributes to the tortuosity of the porous matrix. This tortuosity affects the biochemical parameters of the encapsulated Bgal biocatalyst with respect to the soluble form as described before (Table 2).



(1) Bultema, J. B.; Kuipers, B. J. H.; Dijkhuizen, L. Biochemical characterization of mutants in the active site residues of the βgalactosidase enzyme of Bacillus circulans ATCC 31382. FEBS Open Bio 2014, 4, 1015−1020. (2) Panesar, P. S.; Kumari, S.; Panesar, R. Potential Applications of Immobilized β-Galactosidase in Food Processing Industries. Enzyme Res. 2010, 2010, 1−16. (3) Warmerdam, A.; Paudel, E.; Jia, W.; Boom, R. M.; Janssen, A. E. M. Characterization of β-Galactosidase Isoforms from Bacillus circulans and Their Contribution to GOS Production. Appl. Biochem. Biotechnol. 2013, 170, 340−358. (4) Mueller, I.; Kiedorf, G.; Runne, E.; Seidel-Morgenstern, A.; Hamel, C. Synthesis, kinetic analysis and modelling of galactooligosaccharides formation. Chem. Eng. Res. Des. 2018, 130, 154−166. (5) Sheldon, R. A.; van Pelt, S. Enzyme immobilization in biocatalysis: why, what and how. Chem. Soc. Rev. 2013, 42, 6223− 6235. (6) Lee, C. H.; Lin, T. S.; Mou, C. Y. Mesoporous materials for encapsulating enzymes. Nano Today 2009, 4, 165−179. (7) dos Santos, J. C.S.; Garcia-Galan, C.; Rodrigues, R. C.; de Sant’Ana, H. B.; Goncalves, L. R.B.; Fernandez-Lafuente, R. Stabilizing hyperactivated Lecitase structures through physical treatment with ionic polymers. Process Biochem. 2014, 49 (9), 1511−1515. (8) Pessela, B. C. C.; Mateo, C.; Filho, M.; Carrascosa, A.; Fernández-Lafuente, R.; Guisan, J. M. Selective adsorption of large proteins on highly activated IMAC supports in the presence of high imidazole concentrations: Purification, reversible immobilization and stabilization of thermophilic a- and β-galactosidases. Enzyme Microb. Technol. 2007, 40, 242−248. (9) Barbosa, O.; Ortiz, C.; Berenguer-Murcia, Á .; Torres, R.; Rodrigues, R. C.; Fernandez-Lafuente, R. Strategies for the one-step immobilization-purification of enzymes as industrial biocatalysts. Biotechnol. Adv. 2015, 33, 435−456. (10) de Albuquerque, T. L.; Peirce, S.; Rueda, N.; Marzocchella, A.; Goncalves, L. R.B.; Rocha, M. V. P.; Fernandez-Lafuente, R. Ion exchange of β-galactosidase: The effect of the immobilization pH on enzyme stability. Process Biochem. 2016, 51, 875−880. (11) Urrutia, P.; Bernal, C.; Wilson, L.; Illanes, A. Use of chitosan heterofunctionality for enzyme immobilization: β-galactosidase immobilization for galacto-oligosaccharide synthesis. Int. J. Biol. Macromol. 2018, 116, 182−193. (12) Ricardi, N. C.; de Menezes, E. W.; Valmir Benvenutti, E.; da Natividade Schoffer, J.; Hackenhaar, C. R.; Hertz, P. F.; Costa, T. M. H. Highly stable novel silica/chitosan support for β-galactosidase immobilization for application in dairy technology. Food Chem. 2018, 246, 343−350. (13) Ma, J.; Hou, C.; Liang, Y.; Wang, T.; Liang, Z.; Zhang, L.; Zhang, Y. Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith. Proteomics 2011, 11, 991−995. (14) Pessela, B. C. C.; Mateo, C.; Carrascosa, A. V.; Vian, A.; Garcia, J. L.; Rivas, G.; Alfonso, C.; Guisan, J. M.; Fernandez-Lafuente, R. One-step purification, covalent immobilization, and additional stabilization of a thermophilic poly-his-tagged β-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate - Epoxy sepabeads. Biomacromolecules 2003, 4 (1), 107−113. (15) Housecroft, C. E.; Sharpe, A. G. Inorganic Chemistry, 2nd ed; Pearson, Prentice Hall, 2005; pp 166−210. (16) Guerrero, C.; Vera, C.; Serna, N.; Illanes, A. Immobilization of Aspergillus oryzae β-galactosidase in an agarose matrix functionalized by four different methods and application to the synthesis of lactulose. Bioresour. Technol. 2017, 232, 53−63.

4. CONCLUSIONS The immobilization of β-galactosidase from B. circulans was carried out by a one-pot silica sol−gel encapsulation (SSGE) process, in the presence of chitosan acting as a chelating agent of Ca2+, Zn2+, and Cu2+ cations. The metal ions did not affect the encapsulation efficiency, and the Bgal hydrolytic activity is highly preserved. A differential thermal behavior at 60 °C was observed for the obtained biocatalysts according to the metal type: Ca2+ hyperactivating/stabilizing, Zn2+ hyperactivating/ nonstabilizing, and Cu2+ nonhyperactivating/nonstabilizing effects. These results were highly correlated with the hydrolytic and transgalatosylation activities of the enzyme at this temperature. The physicochemical characterization by SEMEDX, TEM-EDX, XPS, and XRF demonstrated that chitosan− metal chelation during the SSGE process had an important role in the encapsulation process and Bgal activity. A low metal degree incorporation on the solid biocatalyst favored the enzyme thermal hyperactivation and stabilization of the evaluated β-galactosidase. This work contributed to the understanding of the SSGE process mediated by chitosan− metal chelates, which is a simple and fast one-pot immobilization strategy.



REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. ORCID

Claudia Bernal: 0000-0001-9117-0483 Monica Mesa: 0000-0002-6175-6384 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank the University of Antioquia for the scholarship through the “Estudiante instructor” Program and the financial support by the ES84170135 CODI project “preparación de ́ polvos multifuncionales mediante proceso sol−gel de silice, y su evaluación in vitro como sistemas de dosificación de suplementos dietarios enzimáticos y minerales”. We also thank the UdeA for the NAPS-XPS facilities.



ABBREVIATIONS SSGE, silica sol−gel encapsulation; Bgal, β-galactosidase from Bacillus circulans; MCP, maximum catalytic potential; o-NPG, o-nitrophenyl-β-D-galactopyranoside; BCA, bicinchoninic acid; K

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DOI: 10.1021/acsabm.9b00371 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsabm.9b00371 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX