Activity and Stability Enhancement of Carbonic Anhydrase Entrapped Within Biomimetic Silica by Methyl-Substituted Silanes

Abstract

Carbonic anhydrase (CA), an enzyme that accelerates CO₂ hydration, is one of the most widely used enzymes in the aid of CO₂ sequestration. We entrapped CA from Sulfurihydrogenibium azorense (SazCA) within biomimetic silica; to enhance the activity of the entrapped enzyme, the microenvironment of the silica particles was modified by using methyltrimethoxysilane (MTMS) or dimethyldimethoxysilane (DMDMS) as part of the precursors. When 10% (mol/mol) MTMS or 20% DMDMS was added to tetramethoxysilane (TMOS), the activity of entrapped SazCA increased by almost threefold when compared with the control group without these methyl-substituted silanes. In addition, all three types of entrapped SazCA, namely, the silica formed with only TMOS, 10% MTMS in TMOS, and 20% DMDMS in TMOS, exhibited improved thermal stability and pH stability. All three types of entrapped SazCA also showed good storage stability, with at least 79% of their initial activities retained after being stored at room temperature for six weeks, while the activity of the free enzyme dropped to 14% after only two days. When all three types of entrapped SazCA were applied to carbon sequestration, the efficiency remained above 90% even after ten cycles of reuse.

1. Introduction

Since the onset of the first Industrial Revolution, technological advancements have led to increased concentrations of greenhouse gases, thereby contributing to global warming and climate change. These environmental changes have exerted profound impacts on society, the economy, and human livelihoods. Carbon dioxide accounts for most greenhouse gas emissions, and, consequently, the reduction of CO₂ emissions and atmospheric concentrations has become an urgent global priority.

Carbon capture, utilization, and storage (CCUS) constitute an emerging technology designed to capture CO₂ and convert it into value-added products or store it in designated sites for long-term sequestration. Mineralization is an effective CCUS strategy for carbon sequestration, wherein CO₂ reacts with calcium- or magnesium-containing minerals to form stable carbonates, and the resulting carbonates have potential applications in construction materials and soil amendments [1]. Mineralization offers several distinct advantages over conventional physical and chemical methods, such as environmental sustainability, low energy demand, efficient resource utilization, and the production of value-added products [2,3]. Carbonate minerals are stable, enabling the permanent storage of CO₂. However, the practical implementation of CO₂ mineralization is constrained by its slow reaction kinetics and the availability of suitable mineral resources. Notably, the rate-limiting step of CO₂ mineralization is its hydration, which can be significantly accelerated by the enzyme carbonic anhydrase (CA, EC 4.2.1.1).

CA is a class of metalloenzymes ubiquitously distributed across mammalian tissues, plants, algae, and bacteria. These enzymes play critical roles in physiological processes, including CO₂ metabolism, ion transport, respiration, and pH regulation [4]. CA catalyzes the reversible hydration of CO₂ according to the following reaction:

CO₂ + H₂O ⇌ HCO³⁻ + H⁺

(1)

CA has been widely applied to enhance CO₂ mineralization rates [5,6]. Based on sequence divergence, CA is classified into eight distinct families (α, β, γ, δ, ζ, η, θ, and ι) [7]. Because carbon sequestration processes often operate under high-temperature and alkaline conditions, thermostable and alkali-resistant CA is desirable. In this context, CA from Sulfurihydrogenibium azorense (SazCA), a thermophilic bacterium, has attracted attention due to its unique properties. SazCA belongs to the α-CA family, which requires Zn²⁺ as a cofactor [7]. This enzyme adopts a dimeric structure, with each subunit exhibiting the characteristic α-CA fold [8]. SazCA displays remarkable catalytic efficiency, with a k_cat of 4.4 × 10⁶ s⁻¹ and a k_cat/K_M of 3.5 × 10⁸ M⁻¹·s⁻¹ [9]. Additionally, SazCA demonstrates excellent thermal stability, retaining approximately one-sixth of its activity after incubation at 100 °C for 180 min [9]. Owing to these favorable attributes, various immobilization strategies have been explored to enhance the stability and reusability of SazCA, including immobilization on microcrystalline cellulose [10], chitin [11], and silica [12].

Silica formation is typical in diatoms, radiolarians, and sponges. In Cylindrotheca fusiformis, a diatom, proteins known as silaffins, which possess a high density of positive charges, serve as templates promoting silicic acid polymerization [13,14]. Besides silaffins, various amine-containing polymers or molecules, such as polyallylamine hydrochloride (PAA), poly(lysine), and spermine, can induce biomimetic silica formation in vitro. The formation of biomimetic silica is rapid and facile, and the reaction is performed under mild conditions; therefore, biomimetic silica has been used as a support for enzyme immobilization [15].

In our previous work, SazCA was entrapped within biomimetic silica; however, despite nearly complete immobilization, the enzyme experienced significant activity loss [16]. In this study, we attempted to improve the activity of SazCA entrapped in biomimetic silica by incorporating methyltrimethoxysilane (MTMS) or dimethyldimethoxysilane (DMDMS) in addition to the commonly used tetramethoxysilane (TMOS). The methyl substitution cannot be hydrolyzed to a hydroxyl group necessary for forming the siloxane linkage. Hence, the silica’s pore size, surface area, and hydrophobicity are modified. The altered microenvironment may be beneficial for enzyme molecules entrapped within. Such an approach has been successfully applied to lipases and D-amino acid oxidase [17,18]. In this work, we first optimized the silica composition for immobilization efficiency and activity recovery. Then, the thermal, pH, and storage stability of the entrapped SazCA were determined and compared with the free enzyme. Finally, the reusability of the entrapped SazCA in terms of CO₂ hydration and carbon sequestration was evaluated.

2. Results and Discussion

2.1. Characterization of the Recombinant SazCA

The amino acid sequence of the recombinant SazCA is given in Supplementary Figure S1. The SazCA comprises 257 amino acids with a molecular weight of 29.5 kDa and a theoretical pI of 7.9. The recombinant SazCA carries His-tags at its N and C terminals; such an arrangement could provide higher affinity towards the Ni²⁺ immobilized on the purification resin than the conventional single His-tag. As indicated in the SDS-PAGE (Supplementary Figure S2), the desalted SazCA (Lane 9) contained almost no impure proteins, and its molecular weight is consistent with the theoretical value. The yield of SazCA was determined to be 27.3 ± 1.6 mg/L, with a specific activity of 5331 ± 361 WAU/mg.

2.2. Effect of SazCA Concentration on Entrapment

Different SazCA concentrations (2.5, 25, and 50 μg/mL) were used for the immobilization process, and then the immobilization efficiency and the activity recovery were determined. The immobilization efficiency was nearly 100% across all tested SazCA concentrations. The very high immobilization efficiency is typical for enzyme molecules entrapped within biomimetic silica [19,20]. The highest activity recovery of 23% was found at the concentration of 2.5 μg/mL; further increases in concentration to 25 and 50 μg/mL resulted in a decrease in the activity recovery to 10% and 5%, respectively. This decline is likely due to mass transfer limitations when a large amount of enzyme is entrapped within the silica particles. Enzymes located closer to the center of the silica particles may have limited access to carbon dioxide, leading to lower measured activity. Consequently, a SazCA concentration of 2.5 μg/mL was selected for subsequent entrapment.

2.3. Effect of MTMS and DMDMS on Entrapped SazCA

To improve the low activity recovery when using TMOS as the only precursor, MTMS was added to TMOS in the 5 to 30% (mol/mol) range, and the mixed silanes were used as precursors. The immobilization efficiency consistently remained nearly 100% across all tested MTMS concentrations. As shown in Figure 1A, the activity recovery increased from 23 to 61% as the MTMS concentration increased from 0 to 10%, respectively; a 2.7-fold increase in activity was observed. The increased activity recovery could be explained by the decreased cross-link density when MTMS was incorporated. The methyl substitution cannot be hydrolyzed, forming a siloxane linkage, leading to lower mass transfer resistance for the entrapped enzyme. Similar enhancement in enzyme activity was reported for D-amino acid oxidase entrapped in biomimetic silica formed when MTMS was added to TMOS [21]. However, the activity recovery decreased when the MTMS concentration increased further from 10%. The decrease in activity caused by increasing MTMS has been reported for entrapped lipase [22]. The lowered activity could be related to the more hydrophobic microenvironment, as the greater the amount of MTMS used during entrapment, the more the hydrophobic microenvironment might repel water molecules necessary for the hydration of CO₂ by SazCA. The more hydrophobic microenvironment may also disrupt the structure of the SazCA due to adsorption through hydrophobic interaction. Because the highest activity recovery of 61% was obtained at 10% MTMS in TMOS (for simplicity, the concentration is referred to as 10% MTMS herein), this concentration was chosen for subsequent characterizations.

After the successful enhancement in activity by MTMS, we increased the degree of methyl substitution by adding DMDMS to TMOS in the 5 to 40% range. Like entrapment using the MTMS/TMOS mixtures, regardless of the DMDMS concentration, the immobilization efficiency was almost 100%. As shown in Figure 1B, activity recovery increased from 23 to 63% as DMDMS concentration increased from 0 to 20%, respectively. The improvement in activity was remarkably close to that observed using 10% MTMS. Activity enhancement by DMDMS has been reported for catechol 1,2-dioxygenase and D-amino acid oxidase [21,23]. Activity recovery also decreased as the DMDMS concentration increased further from 20%. Furthermore, 20% of DMDMS in TMOS (for simplicity, the concentration is referred to as 20% DMDMS herein) was selected for subsequent characterizations as it had the highest activity recovery.

To examine the possible role of MTMS and DMDMS in activity enhancement, the specific surface area and the pore size of SazCA entrapped with TMOS alone, 10% MTMS, and 20% DMDMS were determined and listed in

Table 1. The specific surface area and the pore size of the biomimetic silica.

	Without SazCA		With SazCA
Precursor Used 1	Specific Surface Area (m2/g)	Pore Size (Å)	Specific Surface Area (m2/g)	Pore Size (Å)
100% TMOS	25	71	21	57
10% MTMS and 90% TMOS	25	76	20	96
20% DMDMS and 80% TMOS	21	117	24	75

¹ The concentration is in mole percentage.

. The pore size was in the range of 57 to 117 Å, regardless of the presence of the enzyme, clearly showing that these biomimetic silicas are mesoporous. When SazCA was present, the pore size was in the range of 57 to 96 Å, suggesting that physical entrapment is the likely mechanism for immobilization because the dimeric structure of SazCA (PDB ID 4X5S) has a diameter close to 80 Å [8]. The pore size increased significantly when 10% MTMS or 20% DMDMS was used as part of the precursors to entrap SazCA; this observation is in agreement with decreased cross-linking density when these methyl-substituted silanes were incorporated. The larger pore size should lower the mass transfer resistance of the substrates and the products and thus contribute to the increased activity.

The morphology of SazCA entrapped with TMOS alone, 10% MTMS, and 20% DMDMS was examined with SEM, and the images are shown in Supplementary Figure S3. For all three types of entrapped SazCA, most of the silica nanoparticles formed agglomerates or fused, and the addition of MTMS or DMDMS did not alter the morphology significantly (Figure S3D–F). The negative controls (without SazCA, Figure S3A–C) also exhibited similar morphology compared to their counterparts.

2.4. Thermal Stability

The thermal stability of entrapped and free SazCA is shown in Figure 2. The thermal stability of all forms of the entrapped SazCA was quite similar; at 50 °C and 60 °C, the activity remained almost unchanged after 1 h of incubation. However, the activity of free SazCA decreased to 78 and 60% after 1 h of incubation at 50 and 60 °C, respectively. At 70 °C, the residual activity of free SazCA further dropped to 49%, whereas all of the entrapped SazCA maintained at least 61% of the original activity. The improvement in thermal stability by the silica matrix is evident; such improvement could be partly explained by the crystal structure of SazCA [8]. The physical entrapment imposed by the silica matrix should prevent the homodimers from dissociation and losing the Zn²⁺ cofactor. The silica network may also help to maintain the correct secondary and tertiary structure at elevated temperatures. As the temperature increased further to 70, 80, and 90 °C, the improvement by the silica matrix is less evident. Improved thermal stability is consistent with findings for D-amino acid oxidase immobilized using 15% MTMS in TMOS or solely TMOS [21]. Enhanced thermal stability was also reported for alcalase entrapped in silica using a mixture of DMDMS/TMOS at a molar ratio of 1:1 [24].

2.5. pH Stability

The pH stability is shown in Figure 3. The free SazCA was most stable at pH 7; SazCA entrapped with only TMOS and 10% MTMS was relatively stable in the pH range of 3–8, with at least 80% of activity remaining, and SazCA entrapped with 20% DMDMS was most stable at pH 8. For the free SazCA, the stability decreased rapidly when the pH became acidic, and the relative activity dropped to 31% at pH 3. The entrapment of SazCA significantly improved its stability under acidic pH, especially when only TMOS or 10% MTMS was employed. The acidic pH disrupts hydrogen bonds and electrostatic interactions, which are essential in maintaining the correct secondary, tertiary, and quaternary structure of a protein. The silica matrix may act like a rigid cage for the enzyme molecules and prevent distortion of its structure, thus maintaining the catalytically active conformation. In the alkaline pH, all of the entrapped SazCA were also more stable when compared with the free enzyme, especially when the silica was formed using 20% DMDMS. A similar improvement in stability in the pH range of 3–11 was reported for lipase immobilized on SiO₂ nanoparticles, followed by entrapment with sol–gel formed using a MTMS/TMOS molar ratio of five [25]. β-D-galactopyranosidase entrapped within sol–gel formed with 10% DMDMS in tetraethoxysilane also showed improved stability in the pH range of 8–11 [26].

2.6. Storage Stability

When stored at 4 °C, the activity of free SazCA decreased to 28% after 3 d. In contrast, after 42 d of storage, the activity of SazCA entrapped with only TMOS, 10% MTMS, and 20% DMDMS retained 83, 81, and 87% of the initial activity, respectively. At room temperature, as shown in Figure 4, the activity of the free enzyme decreased to 14% after 2 d; however, the enzyme entrapped with only TMOS, 10% MTMS, and 20% DMDMS retained 89, 89, and 79% of the initial activity after 42 d of storage, respectively. Our data indicate that the entrapment of SazCA in all three types of silica matrices greatly enhanced its storage stability. The rapid inactivation of the free SazCA is unexpected for an enzyme originating from a thermophile. We suspected that the poor storage stability of the free enzyme was caused by its low concentration (2.5 μg/mL), which was selected so that the concentrations of the free form and the entrapped forms were the same. The storage stability of an enzyme is affected by its concentration; for instance, the residual activity of 0.1 mg/mL alcohol oxidase was twofold higher than that of 0.01 mg/mL when stored at 37 °C for 7 h [27]. One possible mechanism of the poor storage stability associated with low protein concentration is inactivation by surface adsorption, which is much less evident at higher protein concentrations because the available adsorption sites are limited. We re-examined the storage stability of the free SazCA at a much higher concentration of 0.7 mg/mL, and the residual activity was 80% and 60% after six weeks of storage at 4 °C and room temperature, respectively. The data clearly show that the rapid inactivation of the free SazCA is caused by its low concentration; the entrapment of SazCA with biomimetic silica effectively prevents inactivation, potentially resulting from surface adsorption.

2.7. Reusability by Hydratase Activity

As shown in Figure 5, the activity of all of the entrapped SazCA demonstrated a gradual decrease, and about half of the original activity was retained after ten cycles of reuse. The profile of the activity change for all three types of entrapped SazCA was quite similar. To determine the cause of the activity loss during reuse, the weight change of all three types of silica particles was examined after ten reuse cycles, and about 55% of the initial weight remained. The loss of silica particles coincides with the loss of activity after ten reuse cycles, indicating that the decrease in activity after reuse is likely due to the loss of enzyme particles during the recovery and washing steps. The loss of activity after repeated use was reported for cellulase entrapped in sol–gel formed with an MTMS: TMOS molar ratio of 3:1; only 20% of the initial activity remained [28]. The reusability is not too different from that of CA entrapped in biomimetic silica formed using R5 peptide [20] or diethylenetriamine [29].

2.8. Carbon Sequestration

The onset of CaCO₃ precipitation is defined as the time when the average rate of increase in turbidity is greater than 0.001 per second (ΔA₆₀₀ > 0.001/s); the decrease in onset time is an indication of the rate enhancement in carbon sequestration [20]. When 0.125 μg/mL of SazCA entrapped with only TMOS, 10% MTMS, and 20% DMDMS was used in carbon sequestration, the onset time was 24.0 ± 0.5, 13.4 ± 1.1, and 13.8 ± 1.7 s, respectively. The negative control without enzyme had an onset time of 39.3 ± 2.5 s; adding SazCA entrapped with only TMOS, 10% MTMS, and 20% DMDMS shortened the onset time by 39, 66, and 65%, respectively. These results demonstrated that the entrapped SazCA effectively enhances the carbon sequestration rate, and the enhancement degree agrees with the activity recovery data described in Section 2.3.

The solid precipitates formed during CO₂ sequestration were analyzed with XRD and SEM. From the XRD patterns (Figure 6), all of the precipitates were rhombohedral calcites, and the diffraction peaks for spherical vaterite were barely visible. The XRD patterns of the CaCO₃ formed using entrapped SazCA (Figure 6, traces 4–6) were almost identical to those formed using their blank counterparts (silica particles without SazCA, Figure 6, traces 1–3), indicating that the presence of SazCA does not alter the structure of CaCO₃. The SEM images (Figure 7) also revealed that all of the precipitates were mainly composed of rhombohedral calcite.

All three types of entrapped SazCA were reused for carbon sequestration, and the results are presented in Figure 8. The amount of CaCO₃ produced was nearly identical after ten cycles of reuse, regardless of the type of entrapped SazCA used, suggesting that all forms of entrapped SazCA exhibit great reusability in carbon sequestration.

3. Materials and Methods

3.1. Plasmid Construction and Propagation

The DNA sequence of SazCA (NC_012438.1) was obtained from the RefSeq database of the NCBI website. The DNA sequence was synthesized and inserted into a pET-28a(+) plasmid using the restriction sites of NdeI and XhoI by Protech Technology (Taipei, Taiwan). This plasmid is referred to as pET-28a(+)-SazCA. To propagate the plasmid, the pET-28a(+)-SazCA was transformed into E. coli DH5α competent cells (Yeastern Biotech, New Taipei City, Taiwan) according to the manufacturer’s protocol. A single colony of E. coli DH5α transformant was inoculated into 10 mL of LB medium containing 30 μg/mL of kanamycin and incubated overnight at 37 °C with a shaker speed of 180 rpm. The cells were harvested through centrifugation at 12,800× g for 5 min, and then the plasmid was extracted using the Mini Plus plasmid DNA extraction system (Viogene Biotek, New Taipei City, Taiwan).

3.2. Expression and Purification of SazCA

The extracted plasmid was transformed into E. coli BL21(DE3) competent cells (also from Yeastern Biotech) according to the standard protocol. For the production of SazCA, the cell stock of E. coli BL21(DE3) harboring the pET-28a(+)-SazCA plasmid was streaked on an LB agar plate and incubated overnight at 37 °C. A single colony was then selected and inoculated into 3 mL of LB medium containing 30 μg/mL of kanamycin. Then, the inoculated medium was incubated at 37 °C for 6 h at a shaker speed of 180 rpm. Subsequently, 2.5 mL of the culture was transferred into 100 mL of TB medium containing 30 μg/mL of kanamycin. Then, the inoculated medium was incubated at 37 °C for 6 h at a shaker speed of 180 rpm. When the OD₆₀₀ reached between 0.6 and 0.8, 1 mM of IPTG was added to induce protein expression, along with 0.5 mM ZnSO₄, because SazCA requires Zn²⁺ as a cofactor. The culture was further incubated at 28 °C for 16 h at the same shaker speed. The cells were harvested through centrifugation at 3250× g for 30 min. The supernatant was discarded, and the wet weight of the cell pellet was measured. The cell pellet was then stored at −20 °C for subsequent purification.

To purify SazCA, the cell pellet was resuspended in the storage buffer (20 mM Tris-HCl, pH 8.3); for every gram of cell pellet, 5 mL of the storage buffer was added. The cells were disrupted in an ice bath using a Q125 dismembrator (QSONICA, Newtown, CT, USA); the setting was 30 min at 40% amplitude (3 s pulse on and 12 s pulse off). The lysed cells were centrifuged at 3250× g for 30 min, and then the supernatant was collected and kept on ice for purification. The His-tagged protein was purified using Ni-NTA His-bind resin (Merck, Darmstadt, Germany) according to the standard protocol. The eluted enzyme was desalted against the storage buffer using a PD-10 column (Merck, Darmstadt, Germany) and then aliquoted and stored at −20 °C. The enzyme concentration was determined through Bradford assay using BSA standards; the purity and molecular weight of the enzyme were analyzed with SDS-PAGE.

3.3. Activity Assay

The hydratase activity assay is a modification of the procedure described by others [30]; the assay monitors the decrease in pH using bromothymol blue, a pH indicator, as hydrogen ions are released during the reaction. CO₂-saturated water was prepared by bubbling CO₂ at a flow rate of 2 L/min into 100 mL of deionized water placed in an ice bath for 1 h. The indicator solution was prepared by adding 1 mL of 0.1% bromothymol blue (in ca. 50% ethanol) to 49 mL of the storage buffer, and the solution was kept on ice until use. In a 2 mL microcentrifuge tube, 10–200 μL of the enzyme solution or suspension was mixed with 1 mL of the indicator solution. One milliliter of CO₂-saturated water was added to initiate the reaction, and a stopwatch was started immediately. The time required for the solution to change from blue (pH 8.3) to yellow (pH 6.3) was recorded. To improve the precision of identifying pH change, color references were prepared by mixing 1 mL of the indicator solution and 1 mL of deionized water, and the pH was adjusted to 8.3 and 6.3. The enzyme activity was then calculated and expressed in Wilbur–Anderson Unit (WAU), defined as follows:

WAU = (T₀ − T)/T

(2)

T₀ represents the time required for the pH change of the control (the enzyme solution or suspension was replaced with the storage buffer), and T represents the time required for the pH change of enzyme catalysis. The assay was performed in triplicate.

3.4. Entrapment of SazCA

The silicic acid was prepared by mixing 152 μL of TMOS with 848 μL of 1 mM HCl, and hydrolysis was performed at room temperature on a rotary shaker at 50 rpm for 15 min. The resulting silicic acid concentration was 1 M. In a 15 mL centrifuge tube, 8 mL of SazCA solution, 1 mL of freshly prepared 0.5 M silicic acid, 1 mL of 2 mM PAA, and 5 mL of 0.1 M KH₂PO₄ with 0.1 N NaOH (pH 8) were mixed; the reaction was carried out at room temperature for 5 min. The silica nanoparticles were collected through centrifugation at 6000× g for 5 min and then washed with 10 mL of the storage buffer twice. The supernatant and the washing fractions were examined for their hydratase activity. The silica particles were resuspended in 14 mL of the storage buffer and stored at 4 °C for later use. Immobilization efficiency and activity recovery were calculated based on the following definitions:

• Immobilization efficiency = (amount of entrapped SazCA/amount of added SazCA) × 100%.
• Activity recovery = (specific activity of entrapped SazCA/specific activity of free SazCA) × 100%.

The specific surface area and the pore size of the biomimetic silica were determined through N₂ adsorption/desorption analysis using a Micromeritics model ASAP 2010 analyzer (Norcross, GA, USA). Surface area was determined according to the Brunauer–Emmett–Teller (BET) method, and the pore size was determined using the Barrett–Joyner–Halenda (BJH) method with the desorption branch of the isotherm.

3.5. Thermal, pH, and Storage Stability

To investigate the thermal stability, free or entrapped SazCA at 2.5 μg/mL was incubated at 50, 60, 70, 80, and 90 °C for 1 h, and then the residual hydratase activity was determined as described in Section 3.3.

As for pH stability, a buffer system (150 mM glycine, 150 mM H₃PO₄, 150 mM Tris) was prepared and adjusted to pH values ranging from 3 to 12 using 1 N HCl or 1 N NaOH. Free SazCA at 100 μg/mL was mixed with this buffer system at a 1:9 (v/v) ratio. For entrapped SazCA, 2 mL of the enzyme suspension (2.5 μg/mL) was centrifuged at 6000× g for 5 min to remove the supernatant. Then, the entrapped enzyme was resuspended in the buffer system to a final volume of 0.5 mL. For both free and entrapped SazCA with its concentration adjusted to 10 μg/mL, the residual activities were determined after incubating at various pH levels for 30 min at room temperature.

As for storage stability, free or entrapped SazCA at 2.5 μg/mL was stored at 4 °C or room temperature, and then residual hydratase activity was measured daily for the free enzyme or weekly for the entrapped enzyme.

3.6. Determination of Reusability by Hydratase Activity

In a 2 mL microcentrifuge tube, 1 mL of 2.5 μg/mL entrapped SazCA was added, followed by the addition of 1 mL of CO₂-saturated water to initiate the hydration reaction; the reaction was allowed to proceed for 1 min, and the process is defined as one reaction cycle. To recover the entrapped enzyme, the mixture was centrifuged at 10,000× g for 5 min, and then the supernatant was removed, followed by washing the entrapped SazCA with the storage buffer twice. After the desired number of cycles was achieved, the activity of the entrapped SazCA was determined as described in Section 3.3.

3.7. Determination of Reusability by Carbon Sequestration

In a pre-weighed 1.5 mL microcentrifuge tube, 50 µL of 2.5 µg/mL entrapped SazCA was added, followed by 500 µL of CO₂-saturated water prepared as previously described, except at room temperature. The mixture was allowed to react for 3 min, followed by centrifugation at 13,370× g for 5 min, and then the supernatant was transferred to a pre-weighed 2 mL microcentrifuge tube. In the 2 mL microcentrifuge tube, 450 µL of 0.2 M CaCl₂ (prepared in 1 M Tris with pH adjusted to 11) was added to initiate the mineralization reaction, and the mixture was incubated for 3 min at room temperature. The CaCO₃ pellet was collected by centrifuging at 13,370× g for 5 min. After removing the supernatant, the pellet was dried in an oven at 55 °C for 24 h, and its weight was measured. The entire procedure was repeated using the remaining entrapped SazCA to assess its reusability. To characterize the CaCO₃ precipitate through XRD and SEM, the reaction was scaled up to a total volume of 50 mL to provide a sufficient sample.

To determine the onset of carbon sequestration, 50 µL of 2.5 µg/mL entrapped SazCA was added to 500 µL of CO₂-saturated water prepared at room temperature in a disposable cuvette. The mineralization reaction was initiated by adding 450 µL of 0.2 M CaCl₂, and then the reaction was carried out for 5 min at room temperature. The change in turbidity was monitored by measuring A₆₀₀ using a UV-VIS spectrophotometer (Jasco, Tokyo, Japan).

4. Conclusions

The addition of MTMS and DMDMS to TMOS greatly improves the activity of SazCA entrapped in biomimetic silica. The enhancement in activity through the addition of these methyl-substituted silanes does not compromise the stability of the entrapped enzyme; the thermal, pH, and storage stability were not significantly altered. When applied to carbon sequestration, after ten cycles of reuse, all three forms of entrapped SazCA showed little decrease in efficiency. The results reported in this work should provide insight to readers who are interested in developing reusable and robust immobilized enzymes.