Precipitation-Based Nanoscale Enzyme Reactor with Improved

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Research Article Cite This: ACS Catal. 2018, 8, 6526−6536

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Precipitation-Based Nanoscale Enzyme Reactor with Improved Loading, Stability, and Mass Transfer for Enzymatic CO2 Conversion and Utilization Han Sol Kim,† Sung-Gil Hong,† Kie Moon Woo,† Vanesa Teijeiro Seijas,† Seongbeen Kim,‡ Jinwoo Lee,‡ and Jungbae Kim*,† †

Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic of Korea Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Kyungbuk 37673, Republic of Korea

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‡

S Supporting Information *

ABSTRACT: Enzymatic CO2 conversion has gathered a growing attention due to its fast kinetics in converting CO2 to bicarbonate, but the carbonic anhydrase enzymes easily lose their activities in CO2 conversion processes. Here, we propose a “precipitationbased nanoscale enzyme reactor (p-NER)” approach, which stabilizes the activity of carbonic anhydrase, prepared via the two steps of enzyme adsorption into magnetic mesoporous silica and simultaneous enzyme precipitation/cross-linking. The simple addition of enzyme precipitation during cross-linking step resulted in the formation of cross-linked enzyme aggregates (CLEAs) not only inside the mesopores but also on the surface of mesoporous silica. External CLEAs of p-NER contributed to the improvement of enzyme loading (32.9% (w/w)) and mass transfer (KM = 3.68 mM) compared to those of NER (20.1% (w/w) and 4.29 mM, respectively), prepared without enzyme precipitation step and showing no external CLEAs. p-NER was stable under vigorous shaking (200 rpm) with no activity decrease for 160 days after the inactivation of 25% labile enzyme population at the initial stage of incubation. It suggests that external CLEAs were tightly bound on the surface of mesoporous silica by having roots of CLEAs in the internal mesopores. p-NER of carbonic anhydrase was used to convert CO2 to bicarbonate, and the resulting bicarbonate was further utilized for the generation of calcium carbonate. The addition of p-NER into the CO2 bubbling reactor resulted in 6.5-fold higher production of calcium carbonate than the control with no enzyme, revealing the accelerated kinetics of CO2 conversion in the presence of p-NER. p-NER can be easily recycled via magnetic separation, and retained 89% of initial activity after 10 recycled uses. This study has demonstrated great potential of p-NER not only for enzymatic CO2 conversion but also in various other applications where the short lifetimes of enzymes hamper their practical applications. KEYWORDS: Carbonic anhydrase, Precipitation-based nanoscale enzyme reactor, CO2 conversion, CO2 utilization, Calcium carbonate

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INTRODUCTION Global warming has fueled a great interest in carbon dioxide (CO2) capture and utilization (CCU) for the reduction of atmospheric CO2.1−3 Especially, the use of environmentally friendly biocatalysts has been gathering growing attention as a greener solution to CCU.4,5 As an example, carbonic anhydrase (CA) has been proposed for the expedition of CO2 capture6,7 because CA can catalyze the hydration of CO2 to bicarbonate at a high turnover rate up to 106 s−1.8 This superfast CAcatalyzed CO2 hydration can convert CO2 from the flue gas to bicarbonate, while the product bicarbonate can be subsequently utilized as a C1 feedstock for the synthesis of various chemicals.9−12 Even though CA accelerates the CO2 conversion at a high turnover rate, soluble form of enzymes can be easily denatured and lose their biocatalytic activities by the shear stress under vigorous gas bubbling condition.13,14 At the © XXXX American Chemical Society

same time, once CA enzymes are employed as a promotor for the sorbent-based CO2 capture processes, they are exposed to harsh reaction conditions, such as high temperature (40−60 °C) and alkaline environments (pH > 9),15,16 which can also induce the enzyme denaturation and inactivation. Therefore, the successful application of CA enzymes for CO2 conversion requires the stabilization of CA activity for their uses for an extended period of time. Many strategies have been proposed for the stabilization of CA activity, such as the discovery of thermo-stable or alkalistable CA from extremophiles,17−19 modifying the CA structure via protein engineering,20,21 and the immobilization Received: February 12, 2018 Revised: May 13, 2018

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in the first reactor, and then the bicarbonate solution from the first reactor was used to produce calcium carbonate (CaCO3) in the second reactor containing calcium ions. The accelerated kinetics of CO2 conversion in the presence of p-NER and its stability under iterative uses for CO2 conversion were also estimated.

of CA.22−24 In particular, enzyme immobilization not only stabilizes the activities of enzymes by preventing their structural denaturation, but also potentially improves enzyme activity, selectivity, specificity, and/or resistance to inhibitors or chemicals,25−27 while facilitating the separation and recycling of enzymes in the form of heterogeneous biocatalysts.28,29 As one of enzyme immobilization strategies, the approach of cross-linked enzyme aggregates (CLEAs) was proposed whereby enzymes in aqueous solution are precipitated by the addition of salting-out salts, water-miscible organic solvents, or nonionic polymers, and subsequently cross-linked with bifunctional cross-linking reagents.30,31 However, such carrier-free CLEAs are mechanically fragile for many industrial applications and requires tedious and timeconsuming manipulations for their recovery from the reaction mixture.32,33 At the other end of the spectrum in enzyme immobilization, enzymes have been immobilized within mesoporous materials.34 Mesoporous materials are good candidates as support materials for enzyme immobilization due to their highly ordered, robust, and tunable structure with large pore volume allowing for high enzyme loading.35 Enzymes in the confined microenvironment of mesoporous materials can be stabilized by macromolecular crowding effect,36−38 and enzymes inside mesoporous materials can be protected from harsh external conditions such as vigorous shaking or gas bubbling.14 Enzymes have been immobilized into mesoporous materials via conventional methods of simple adsorption and covalent attachment, but these approaches showed only a marginal improvement of enzyme stability due to vigorous enzyme leaching and denaturation.35 However, the simple addition of enzyme cross-linking step right after the enzyme adsorption into the mesoporous silica, called the approach of “nanoscale enzyme reactors (NER)”, could stabilize the activities of various enzymes by effectively preventing both denaturation and leaching of enzymes.39,40 The prevention of enzyme leaching is based on the ship-in-a-bottle mechanism, in which the larger-sized CLEAs cannot leach out through smaller bottleneck mesopores. However, immobilizing enzymes in porous materials uses only their internal pore volume, placing a potential problem with serious mass transfer limitation of substrate and product, as well as limiting the enzyme loading up to the pore volume of mesoporous materials.41 In the present study, we propose an approach of “precipitation-based nanoscale enzyme reactor (p-NER)”, which was prepared via a two-step process of (1) enzyme adsorption and (2) simultaneous precipitation/cross-linking. Magnetically separable spherical mesocellular silica foam (Mag-S-MCF) was used to prepare p-NER of CA. Simultaneous precipitation/cross-linking was carried out by adding ammonium sulfate (AS) as an enzyme precipitant during the step of enzyme cross-linking using glutaraldehyde (GA). We investigated the morphology, activity, stability, and kinetics of p-NER together with two control samples of enzyme adsorption (ADS) and nanoscale enzyme reactor (NER) with no enzyme precipitation step. Simultaneous enzyme precipitation/cross-linking of p-NER approach allowed for the formation of CLEAs on the surface of mesoporous silica, which led to the improvements of enzyme loading and substrate transfer while achieving long-term enzyme stability. p-NER of CA was used for the demonstration of biocatalytic CO2 conversion and utilization in a two reactor system: CO2 was converted to bicarbonate under the catalytic action of p-NER

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RESULTS AND DISCUSSION Immobilization of Carbonic Anhydrase in Magnetically Separable Spherical Mesocellular Silica Foam. Magnetically separable spherical mesocellular silica foam (Mag-S-MCF), having 40-nm mesocellular pores connected by 12-nm window mesopores (Figure S1), was used to immobilize monomeric CA from bovine erythrocytes via three different approaches: enzyme adsorption (ADS), nanoscale enzyme reactor (NER), and precipitation-based nanoscale enzyme reactor (p-NER) (Figure 1). p-NER was prepared via

Figure 1. Schematic illustrations for the immobilization of carbonic anhydrase in Mag-S-MCF via the approaches of enzyme adsorption (ADS), nanoscale enzyme reactor (NER), and NER with ammonium sulfate precipitation (p-NER).

a two-step process: (1) enzyme adsorption and (2) simultaneous enzyme precipitation/cross-linking, while NER was a control sample with no enzyme precipitation. Field emission−scanning electron microscope (FE-SEM) images of p-NER revealed the cross-linked enzyme aggregates (CLEAs) on the external surface of Mag-S-MCF, which were not observed in the FE-SEM images of Mag-S-MCF, ADS, and NER (Figures 2 and S2). This suggests that the addition of

Figure 2. FE-SEM images of Mag-S-MCF (a), ADS (b), NER (c), and p-NER (d). Insets are more magnified FE-SEM images of corresponding samples. 6527

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Figure 3. STEM images (left) and EDX elemental mapping (middle and right) of Mag-S-MCF (a), ADS (b), NER (c), and p-NER (d). Blue, red, and yellow dots in EDX elemental mapping represent Si-, Fe-, and N atoms, respectively. Upper and lower images are obtained at 7k and 115k magnifications, respectively.

amounts in solution before and right after the enzyme adsorption step, the enzyme loading of ADS before any washing was estimated to be 31.8% (w/w) (Figure S3a). Because the theoretical maximum loading of CA in Mag-SMCF is 32.2% (w/w) (see the Supporting Information for calculation), the initial ADS loading of 31.8% (w/w) suggests that most of the available mesopores are occupied by the adsorbed enzyme molecules right after enzyme adsorption. However, the final enzyme loading of ADS was reduced to 5.7% (w/w) after vigorous washings (Figure S3b), which reveals a serious leaching of adsorbed enzymes from the mesopores of Mag-S-MCF during the washing step. In particular, after the first 1 h washing under 150 rpm shaking, the enzyme loading of ADS was reduced from 31.8% (w/w) to 18.2% (w/w). This 1 h washing corresponds to the crosslinking or precipitation/cross-linking step of NER or p-NER, respectively. For the cases of NER and p-NER, their final enzyme loadings were estimated as 20.1% (w/w) and 32.9% (w/w), respectively, via elemental analysis. Marginally higher enzyme loading of NER (20.1% (w/w)) than that of ADS after the first 1 h washing (18.2% (w/w)) suggests that only GA with no AS does not exhibit significant effect on the fixation of leaching enzyme during the GA treatment, while it was effective in the cross-linking of enzymes retained inside Mag-SMCF. The 32.9% (w/w) enzyme loading of p-NER implies that the leaching of initially loaded enzymes (31.8% (w/w)) was effectively prevented during precipitation/cross-linking step. The leaching enzymes can be aggregated upon the ASinduced precipitation, probably mainly near the pore entrance of Mag-S-MCF due to the bottleneck effect, and simultaneously cross-linked by GA during the synthesis of p-NER in the form of external CLEAs. To confirm the contribution of enzymes outside Mag-SMCF to the development of external CLEAs for p-NER, we

ammonium sulfate (AS) as an enzyme precipitant during the glutaraldehyde (GA)-induced enzyme cross-linking step of NER allows for the formation of CLEAs on the surface of MagS-MCF. Because each sample was excessively washed with deionized water via magnetic separation prior to the FE-SEM analysis, only tightly bound CLEAs on the surface of Mag-SMCF could be observed in the FE-SEM images. Without any amine groups on the surface of Mag-S-MCF, the images of CLEAs on the surface of Mag-S-MCF suggest the chemical cross-linking between CLEAs on the surface of Mag-S-MCF and CLEAs in the mesopores of Mag-S-MCF. In other words, CLEAs were formed on the surface of Mag-S-MCF by having roots of CLEAs in the internal mesopores of Mag-S-MCF via effective chemical cross-linking. We have also performed scanning transmission electron microscopy (STEM) analyses (Figure 3). After the fixation of samples in epoxy resin, samples were microtome sectioned and their cross sections were analyzed by high-angle annular darkfield (HAADF) STEM imaging and energy dispersive X-ray (EDX) elemental mapping. The HAADF images clearly revealed magnetic nanoparticles (white particles) embedded within the mesopore structure of Mag-S-MCF. The HAADF images of p-NER showed dense packing of cross-linked enzymes within Mag-S-MCF, and EDX elemental mapping of p-NER also revealed densely distributed N atoms from enzymes. The zoomed-out images of p-NER confirmed the formation of external CLEAs, which could not be observed in other samples. The formation of external CLEAs of p-NER can be explained by two hypothetical events: (1) CLEAs can be protruded from the mesoporous materials during their formation inside Mag-S-MCF, or (2) CLEAs outside of Mag-S-MCF can be recruited and linked with those formed inside the mesoporous materials. By measuring the enzyme 6528

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Figure 4. (a) Esterase activities of ADS, NER, and p-NER. One unit is defined by the enzyme amount for the hydrolysis of 1 μmol 4-NPA per 1 min in 100 mM Tris-HCl buffer (pH 7.6). (b) Stabilities of free carbonic anhydrase (×), ADS (Δ), NER (blue ■), and p-NER (red ■) at room temperature under shaking (200 rpm).

NER, and p-NER could be calculated to be 0.91, 0.63, and 0.55 units/mg CA, respectively, while that of free CA was 1.10 units/mg CA. The lower specific enzyme activities of NER and p-NER than ADS can be explained by the enzyme crosslinking, which can inactivate enzymes via chemically driven enzyme denaturation or reduction of enzyme flexibility due to multipoint chemical bonds on each enzyme molecule.42 The stabilities of free CA, ADS, NER, and p-NER were investigated by measuring the enzyme activities time dependently while each sample was incubated in aqueous buffer (100 mM Tris-HCl, pH 7.6) at room temperature under vigorous shaking (200 rpm, 45° tilted) (Figure 4b). The relative activity represents the ratio of residual activity at each time point to the initial activity of each sample. Free CA showed the rapid drop of enzyme activity, which can be explained by the denaturation of free CA under the shear stress of shaking. According to the first-order inactivation kinetics, the half-life of free CA was estimated to be 3.66 days. On the other hand, ADS showed a marginal stabilization of CA activity due to the protection of enzyme molecules from denaturation by the molecular crowding in the confined space of Mag-S-MCF mesopores.43 However, the stabilization effect was marginal with a half-life of 6.14 days, which can be explained by the enzyme leaching from the mesopores followed by the enzyme denaturation under shaking. Both NER and p-NER showed a biphasic enzyme inactivation with rapid enzyme inactivation at the initial phase and better-stabilized enzyme activity at the later phase (Figures 4b and S5). During the initial phase up to 4 days, NER and pNER lost 23.1% and 24.7% of their initial activities, respectively, and the remaining 76.9% and 75.3% activities of NER and p-NER were stably maintained during the following 155 days of incubation, respectively (Figures 4b and S5). This biphasic enzyme inactivation can be explained by the generation of two different populations of cross-linked enzymes upon chemical cross-linking: (1) labile form and (2) stable form.44 The initial activity loss of both NER and pNER can be explained by the rapid inactivation of labile form, while the stabilization of both samples at the later phase can be explained by the cross-linked enzyme aggregates (CLEAs) with enough number of chemical linkages on the surface of enzyme molecules to stabilize the activity of CLEAs. The estimated half-lives of NER and p-NER at the initial phase up to 4 days

prepared p-NER variations with modified protocols. In the standard protocol of p-NER synthesis, after the enzyme adsorption into Mag-S-MCF, all the enzyme solution is decanted and enzyme-adsorbed Mag-S-MCF is incubated in the mixture of AS and GA solutions. We intentionally retained certain volumes of enzyme solution (1 or 2 mL) to check the effect of external enzymes on the morphologies of external CLEAs during the p-NER synthesis (p-NER-R1 or p-NER-R2, respectively). Then, AS and GA was added to the same final concentration of standard p-NER protocol. Although SEM analysis revealed that there were no significant differences among the morphologies of p-NER, p-NER-R1, and p-NER-R2 (Figure S4), elemental analysis showed increased enzyme loadings of p-NER-R1 (36.3% (w/w)) and p-NER-R2 (37.4% (w/w)) compared to that of standard p-NER (32.9% (w/w)). These results suggest that tight conjugation between the CLEAs in bulk solution and those inside mesopores can be formed. At the same time, higher enzyme loading of p-NER (32.9% (w/w)) over the theoretical maximum loading (32.2% (w/w)) implies the recruitment of external enzymes to the surface of Mag-S-MCF in the form of external CLEAs. In other words, during the p-NER synthesis, imperfectly removed enzymes after the enzyme adsorption step can be precipitated/ cross-linked in the form of CLEAs, and conjugated with those inside the mesoporous materials. Activity and Stability of Immobilized Carbonic Anhydrase. The esterase activity of CA was measured by the hydrolysis of 4-nitrophenyl acetate (4-NPA) in 100 mM Tris-HCl (pH 7.6). The apparent activities of ADS, NER, and p-NER were 0.055, 0.158, and 0.271 units/mg Mag-S-MCF, respectively (Figure 4a). The activity of p-NER was 4.9 and 1.7 times higher than those of ADS and NER, respectively. The leaching of adsorbed enzymes under washings can explain the low activity of ADS. On the other hand, the higher NER activity than ADS could be achieved by the effective prevention of enzyme leaching via the ship-in-a-bottle mechanism, under which the cross-linked enzymes in large mesocellular pores (40 nm) cannot leach out through smaller window mesopores (12 nm).39,40 The 1.7-fold higher activity of p-NER than NER can be explained by the CLEAs on the surface of Mag-S-MCF as shown in the FE-SEM images (Figures 2 and S2). The specific enzyme activities of ADS, 6529

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ACS Catalysis were 10.6 and 9.8 days, respectively, while those at the second phase were 1470 and 2270 days, respectively. These highly stabilized CLEAs of NER and p-NER at the second phase starting from the 4-day incubation reveals that the simple addition of enzyme cross-linking right after the enzyme adsorption into the Mag-S-MCF is very effective in stabilizing the CA activity in the form of CLEAs both in and on Mag-SMCF. Much longer half-lives of NER and p-NER than free CA and ADS suggest that the enzyme cross-linking not only prevents the structural denaturation of immobilized enzymes by forming multipoint chemical linkages among enzyme molecules, but also inhibits the leaching of enzymes via the ship-in-a-bottle mechanism.39,40 The half-life of p-NER at the second phase was 1.54 times longer than that of NER even though p-NER had the CLEAs on the external surface of Mag-S-MCF, where more shear stress would be imposed under shaking. One possible explanation for this result is that ammonium sulfate precipitation plays an important role for effective enzyme cross-linking by allowing closer contact among the precipitated enzyme molecules. Enzyme precipitation leads to the formation of insoluble aggregates of enzymes,45,46 and the intermolecular proximity of aggregated molecules is close compared to that of pseudohomogeneously dissolved molecules.47 Upon cross-linking, the CLEAs of p-NER would be more resistant against enzyme denaturation by forming a higher number of chemical linkages per unit enzyme molecule, and thus effectively inhibiting the enzyme denaturation and leaching.11 High stability of p-NER also reveals the tight crosslinking between external and internal CLEAs, which would be the only driving force for the survival of external CLEAs under vigorous shaking incubation and washings via magnetic separation of Mag-S-MCF, which has no amine groups on its surface. It would be worthwhile to note that p-NER achieved higher enzyme loading/activity with even improved enzyme stability when compared to NER. As a control of p-NER, we also prepared p′-NER by performing the three-step immobilization of (1) enzyme adsorption, (2) precipitation, and (3) cross-linking, rather than the two-step (1) enzyme adsorption and (2) simultaneous enzyme precipitation/cross-linking of p-NER (Figure S6). The activity of p′-NER was 0.207 units/mg of Mag-S-MCF, which is 0.8 times lower and 1.4 times higher than those of p-NER and NER, respectively. The FE-SEM images showed that p′NER had less population of external CLEAs on Mag-S-MCF than those of p-NER (Figure S7). These results suggest that the simultaneous enzyme precipitation and cross-linking for pNER is more effective in developing more CLEAs on the surface of Mag-S-MCF compared to the sequential treatment of precipitation and cross-linking. Enzyme Kinetics for Immobilized Carbonic Anhydrase. The kinetic parameters of free CA, ADS, NER, and pNER were estimated by analyzing the measured activities at various 4-NPA concentrations, ranging from 0.5 to 5 mM (Table 1). The Michaelis−Menten constants (KM) of free CA, ADS, NER, and p-NER were 2.21, 4.03, 4.29, and 3.68 mM, respectively. Once CA was immobilized in the form of ADS, NER, or p-NER, the KM values were increased compared to free CA due to the increased diffusional limitation of substrate through the pore of Mag-S-MCF. NER showed higher KM value than ADS, which can be explained by more diffusional limitation imposed by the higher enzyme loading of NER in the pore of Mag-S-MCF. Interestingly, p-NER resulted in

Table 1. Enzyme Kinetics of the Immobilized Carbonic Anhydrase in Mag-S-MCFa sample free CA ADS NER p-NER

KM (mM) 2.21 4.03 4.29 3.68

± ± ± ±

0.74 0.02 0.18 0.08

kcat (min−1) 33.1 6.5 13.0 9.8

± ± ± ±

9.5 0.2 0.1 1.3

kcat/KM (mM−1 min−1) 15.09 1.62 3.04 2.66

± ± ± ±

0.77 0.03 0.15 0.40

a

The activity of CA was measured by the hydrolysis of 4-NPA (0.5−5 mM) in 100 mM Tris-HCl (pH 7.6). Kinetic constants were obtained by Lineweaver−Burk Plot. Enzyme loadings of immobilized samples were estimated by either BCA protein assay (ADS) or elemental analysis (NER and p-NER).

smaller KM value than ADS and NER. Improved mass transfer of p-NER than NER stems from the external CLEAs, which provide much easier accessibility of substrate to the enzymes. Internal CLEA population of p-NER would place more serious limitation of substrate transfer, but the overall mass transfer of p-NER was facilitated more than NER based on the beneficial effect of the external CLEAs of p-NER on mass transfer. pNER also exhibited better mass transfer than ADS, where enzymes are simply adsorbed in the pore of Mag-S-MCF. This reveals that the presence of external CLEAs is favorable in terms of mass transfer, which counterbalances the mass transfer limitation that arise from the close proximity of precipitated and cross-linked enzymes. Turnover number (kcat) was obtained by dividing the maximum velocity of enzyme reaction by total enzyme concentration. The kcat values of free CA, ADS, NER, and pNER were 33.1, 6.5, 13.0, and 9.8 min−1, respectively. All the immobilized CA samples showed decreased kcat values when compared to that of free CA. The flexibility of enzymes generally decreases upon the immobilization, which can result in the decrease of turnover number. Higher kcat value of NER than that of ADS can be explained by the prevention of enzyme denaturation, maintaining more enzyme population to be active. Decreased kcat value of p-NER than NER implies that the introduction of precipitation step during the enzyme crosslinking resulted in the formation of CLEAs with closely packed enzyme molecules and reduced the enzyme flexibility more than CLEAs without enzyme precipitation. The overall catalytic efficiencies, kcat/KM, of free CA, ADS, NER, and p-NER were 15.09, 1.62, 3.04, and 2.69 mM−1 min−1, respectively. Although all the immobilized CA samples exhibited lower catalytic efficiencies than free enzyme due to the increased mass transfer limitation and decreased enzyme flexibility, the immobilization of CA can improve the enzyme stability for the long-term and recycled uses of biocatalysts. The catalytic efficiency of p-NER was comparable with that of NER, while the enzyme loading and stability of p-NER were better than those of NER. More importantly, the external CLEAs of p-NER not only increased the enzyme loading, but also improved the overall mass transfer limitation. Pre-Loading of Bovine Serum Albumin (BSA). To confirm the hypothesis that the simultaneous enzyme precipitation/cross-linking of p-NER protocol forms the external CLEAs on Mag-S-MCF, and to quantify the effect of external CLEAs on the activity of p-NER, bovine serum albumin (BSA) was preadsorbed into Mag-S-MCF before the addition of CA enzymes for the synthesis of NER and p-NER (Figure 5a). BSA is a model protein with no biocatalytic activity, and the mesopores of Mag-S-MCF was prefilled with 6530

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Figure 5. (a) Schematic illustration of NER and p-NER with BSA preloading. (b) Activities of BSA-preloaded NER (blue ■) and p-NER (red ■). (c) Activity comparison of NER/p-NER and BSA-preloaded NER/p-NER when 100% (v/v) of pore volume was preloaded by BSA.

BSA from 50% to 100% (v/v) prior to the synthesis of NER and p-NER with additional CA (see the Supporting Information for the calculation of maximum BSA loading amount). Interestingly, the difference of CA activities between NER and p-NER with the same amount of preloaded BSA did not change much regardless of preloaded BSA amounts (Figure 5b). This result reveals that the activity difference between NER and p-NER, regardless of preloaded BSA amounts, represents the activity of CLEAs on the surface of Mag-S-MCF. The average of activity differences between NER and p-NER with the same amount of preloaded BSA was 0.106 units/mg Mag-S-MCF, representing 38.2% of p-NER activity when prepared with no BSA adsorption (0.278 units/mg MagS-MCF). p-NER activity after subtracting the activity of external CLEAs (0.106 units/mg Mag-S-MCF) was 0.172 units/mg Mag-S-MCF, which is marginally higher than that of NER (0.162 units/mg Mag-S-MCF). This activity difference between NER and internal CLEAs of p-NER suggests that enzyme precipitation of p-NER allows for more compact form of CLEAs in the mesopores of Mag-S-MCF. We also checked the hydratase activities of BSA preloaded NER and p-NER by using CO2 as substrate and measuring the amount of calcium carbonate (Figure S8). The trend of hydratase activities was similar to that of esterase activities, obtained by using 4-NPA as a substrate (Figure 5b). NER with 100% (v/v) BSA preloading exhibited activity of 0.018 units/mg Mag-S-MCF, which represents 12.6% of NER activity (Figure 5c). This result suggests that preadsorbed BSA molecules were partially exchanged with CA molecules during the follow-up CA adsorption and cross-linking steps. On the other hand, p-NER with 100% (v/v) BSA preloading retained 46.0% of p-NER activity, which mostly represents the activity of external CLEAs on the surface of Mag-S-MCF. The KM

value of p-NER with 100% (v/v) BSA preloading was estimated as 3.42 mM, which represents the improved mass transfer over p-NER with no BSA preloading (KM = 3.68 mM) by substituting internal CLEAs of p-NER with fairly cheap BSA molecules. The kcat value, and the kcat/KM, of p-NER with 100% (v/v) BSA preloading could not be determined because the CA was mixed with BSA and the elemental analysis cannot give information on each protein amount. For the comparative enzyme kinetics, p-NER of αchymotrypsin (CT) was also prepared. CT is a protease whose activity can be determined by the hydrolysis of Nsuccinyl-Ala-Ala-Pro-Phe p-nitroanilide (molecular weight 624.6 g mol−1), which is bigger than the substrate of CA (4NPA, molecular weight 181.2 g mol−1). The KM values of ADS, NER, and p-NER with CT were 3.0, 4.4, and 2.5 times higher than that of free CT (Table S1) while the KM values of ADS, NER, and p-NER with CA were 1.8, 2.0, and 1.7 times higher than that of free CA (Table 1). The larger differences of KM values between free enzyme and immobilization samples of CT than those of CA can be explained by the difference of substrate size. In other words, the larger substrate (N-succinylAla-Ala-Pro-Phe p-nitroanilide) of CT led to more serious mass transfer limitation than CA with small substrate (4-NPA). The KM value for 100% (v/v) BSA preloaded p-NER of CT was 84.2% of that of p-NER without preloaded BSA. With the CA enzyme, on the other hand, the KM value ratio of BSA preloaded p-NER to p-NER was 93.0%. The larger reduction of KM value ratio by replacing CA with CT can be explained by more serious transfer limitation of larger substrate (N-succinylAla-Ala-Pro-Phe p-nitroanilide) through the external CLEAs of p-NER with CT. Depending on the combination of enzyme and substrate, the NER approach can encounter a serious issue of mass transfer 6531

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Figure 6. Schematic representation of reaction system for the conversion and utilization of CO2 to calcium carbonate based on immobilized carbonic anhydrase.

Figure 7. (a) Temporal formation profile of CaCO3 by the reaction between calcium ion and bicarbonate solution produced by blank (×), ADS (Δ), NER (blue ■), and p-NER (red ■). Stabilities of ADS (b), NER (c), and p-NER (d) under their iterative uses for the CO2 conversion based on magnetic separation.

limitation with a large substrate, which potentially leads to an economic burden due to the under-utilization of expensive enzymes in the mesoporous materials. The p-NER approach can give us an alternate solution by providing additional CLEAs on the external surface of mesoporous materials by simply adding the enzyme precipitants during the cross-linking step. The economics of p-NER application can be further improved by forming the CLEAs only on the surface of mesoporous materials by prefilling the internal mesopores with cheap proteins such as BSA. At the same time, the

multienzyme system can be potentially developed by adding one enzyme inside while forming the external CLEAs with another enzyme. We anticipate that this p-NER approach can be employed for various applications where either the poor enzyme stability or serious mass transfer limitation hampers the practical successes of enzyme applications. Enzymatic CO2 Conversion and Calcium Carbonate Formation. As a potential application of p-NER with CA, enzymatic CO2 conversion and utilization were demonstrated by using a two-reactor system (Figure 6). In the first reactor, 6532

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was successfully employed for the conversion of CO2 to bicarbonate, followed by the formation of calcium carbonate. The present success has demonstrated the potential of p-NER as stabilized enzyme system to be employed for the enzymatic CO2 conversion and utilization. Improved stability of p-NER, and its facile magnetic separation, will be greatly beneficial in its recycled uses for the other types of CO2 utilization protocols, such as the production of methanol10 and the growth of photosynthetic microorganisms.11 It is also anticipated that the p-NER approach can be employed not only for CO2 conversion and utilization, but also for various other applications where high enzyme loading and stability with relieved mass transfer limitation play a key role for their eventual successes.

CA-catalyzed CO2 hydration to bicarbonate was carried out by bubbling CO2 gas to the aqueous solution under the catalytic action of immobilized CA for 1 min. Immobilized CA was recovered via magnetic separation and the bicarbonate solution was transferred to the second reactor, containing calcium ions, for the generation of calcium carbonate (CaCO3) (Figure S9). The weight of CaCO3 is stoichiometrically proportional to the amount of bicarbonate from the first reactor, and thus represents the biocatalytic performance of CA. The weights of CaCO3 after 150 min reaction in the second reactor were 16, 32, 78, and 104 mg, when no enzyme (blank), ADS, NER, and p-NER, respectively, were used in the first reactor as biocatalysts (Figure 7a). p-NER produced 6.5, 3.3, and 1.3 times higher amounts of CaCO3 than blank, ADS, and NER, respectively. When the CaCO3 weight of blank was subtracted from those of samples, p-NER showed 5.5 and 1.4 times higher CaCO3 generation compared to ADS and NER, respectively. This result suggests that high CA loading and relieved mass transfer limitation of p-NER accelerated the kinetics of CO2 conversion in the first reactor and provided higher concentration of bicarbonate ion for the generation of CaCO3 in the second reactor compared to the other control samples. The CaCO3 production correlates well with the esterase activities, where p-NER showed 4.9 and 1.7 times higher activity than ADS and NER, respectively. This result indicates that the CaCO3 production is determined by the CA activity in the first reactor. We further investigated the stabilities of ADS, NER, and pNER under their recycled uses for CO2 conversion. After each cycle of CO2 conversion, immobilized CA samples were recovered from the reaction mixture via magnetic separation, washed, and reused for the iterative CO2 conversion. Relative CaCO3 production, defined by the ratio of CaCO3 amount at each cycle to that of the first cycle, was used to estimate the reuse stabilities of samples. After 10 times of recycled uses for CO2 conversion, the relative CaCO3 production of ADS, NER, and p-NER were 35%, 79% and 89%, respectively (Figure 7b− d). The decreased relative CaCO3 production of ADS can be explained by the leaching and denaturation of enzymes under CO2 bubbling. Once enzymes are leached from the Mag-SMCF, they can be denatured under the vigorous condition of CO2 bubbling. On the other hand, both NER and p-NER maintained significant biocatalytic performance under recycled uses due to their stabilization effect on enzyme activity, which resulted from the effective prevention of both enzyme leaching and denaturation.

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EXPERIMENTAL SECTION

Reagents and Materials. Carbonic anhydrase from bovine erythrocytes (CA), α-chymotrypsin from bovine pancreas (CT), bovine serum albumin (BSA), 4-nitrophenyl acetate (4-NPA), acetonitrile, calcium chloride, N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, N,N-dimethylformamide (DMF), glutaraldehyde (GA), ammonium sulfate (AS), sodium phosphate monobasic, sodium phosphate dibasic, tris(hydroxymethyl)aminomethane hydrochloride, tris(hydroxymethyl)aminomethane, hydrocholoric acid (HCl), sodium hydroxide (NaOH), Pluronic P123 ((EO)20(PO)70(EO)20), ethanol, 1,3,5-trimethylbenzene, tetraethyl orthosilicate (TEOS), and ammonium fluoride (NH4F) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Iron(III) nitrate nonahydrate (Fe(NO3)3·9H2O) and potassium chloride (KCl) were purchased from Samchun Chemicals (Seoul, Korea). Bicinchoninic acid (BCA) protein assay kit was purchased from Pierce Biotechnology (Rockford, IL, USA). Preparation of Magnetically Separable Spherical Mesocellular Silica Foam. Magnetically separable spherical mesocellular silica foam (Mag-S-MCF) was prepared by following the previous protocol of Woo et al.48 Detailed process for the synthesis is as follow. Acidic solution, consisting of 37% HCl (10 mL), ethanol (5 mL), and water (65 mL), was prepared. Pluronic P123 (4 g) and KCl (5 g) were added to the acidic solution and the mixture was stirred until the chemicals dissolved. 1,3,5-Trimethylbenzene (4 g) was added into the solution, and the mixture was incubated at 40 °C under vigorous stirring for 2 h. TEOS (9.2 mL) was added to the solution under stirring for 5 min. The reaction mixture was incubated at 40 °C for 20 h. After adding NH4F (46 mg) to the incubated solution, additional aging was carried out at 100 °C for 24 h. Suspended materials in the solution were filtered and dried at room temperature. The powder form of materials was calcined at 550 °C for 4 h to eliminate the Pluronic P123 template. The resulting materials are spherical mesocellular silica foam (S-MCF). To embed magnetic nanoparticles in S-MCF, S-MCF (1 g) was added to a Fe(NO3)3·9H2O solution, where Fe(NO3)3·9H2O (1.31 g) was dissolved in ethanol (40 mL). The mixture was stirred at room temperature until all the solvent was removed via evaporation. The resulting powder was calcined at 400 °C for 4 h under a mixed gas of argon (96%) and hydrogen (4%) to synthesize magnetically separable spherical mesocellular silica foam (Mag-S-MCF). Mag-S-MCF was excessively washed with deionized water (DI) and dried by using vacuum oven. Synthesis of ADS, NER, p-NER, and p′-NER. Two mL of 5 mg mL−1 CA solution in 100 mM sodium phosphate buffer (PB, pH 7.6) was added to 5 mg of Mag-S-MCF, and the mixture was shaken under 150 rpm for 1 h. CA-adsorbed Mag-S-MCF was recovered via magnetic capture, and the supernatant was decanted. Two mL of 0.5% (w/v) GA solution with and without 50% (w/v) AS was added to the CA-adsorbed Mag-S-MCF to prepare p-NER and NER samples, respectively, while 100 mM PB was added to CA-adsorbed Mag-SMCF for the preparation of adsorption (ADS) sample. For a comparative study, p′-NER was prepared by performing the sequential steps of enzyme precipitation and cross-linking, instead of simultaneous enzyme precipitation and cross-linking for p-NER. In

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CONCLUSIONS Carbonic anhydrase was immobilized and stabilized by using an approach of p-NER, consisting of enzyme adsorption and simultaneous precipitation/cross-linking. The addition of enzyme precipitation improved both enzyme loading and stability, which can be explained by effective enzyme crosslinking due to the close contact among the precipitated enzyme molecules upon adding the enzyme precipitant. Especially, the visualization of CLEAs on the surface of mesoporous silica has opened up a new potential that not only the internal mesopores but also the external surface of mesoporous materials can be used for the enzyme immobilization with no sacrifice of enzyme stability at all. Kinetic analysis confirmed that CLEAs on the surface of mesoporous silica contributed to the improved mass transfer of p-NER. Highly loaded and stabilized carbonic anhydrase in the form of p-NER 6533

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ACS Catalysis

The first reactor with immobilized CA samples in 100 mM Tris-HCl was bubbled with CO2 gas at a flow rate of 150 mL min−1 for 1 min by a CO2 flow meter (RK1600R, Kofloc, Kyoto, Japan). During the CA reaction, pH was maintained at 7.6 by the automatic addition of 5 M NaOH from a pH titrator (Mettler Toledo T70, Greifensee, Switzerland). After the bubbling of CO2 gas, immobilized CA was magnetically captured, and the reaction solution containing bicarbonate was mixed with 670 mM calcium chloride solution in the second reactor. The mixture was shaken under 200 rpm to form the precipitates of calcium carbonate (CaCO3). Precipitated CaCO3 was filtered by Whatman filter paper with average pore diameter of 2.5 μm (No. 42, GE Healthcare, Buckinghamshire, UK). CaCO3 was dried overnight and weighed. Immobilized CA samples were recovered via magnetic separation after each reaction in the first reactor, and reused for the repetitive conversion of CO2 to bicarbonate. The relative production of CaCO3 at each cycle, defined by the ratio of CaCO3 weight at each cycle to the CaCO3 weight at the first cycle, was estimated at the reaction time of 90 min in the second batch reactor. Preparation and Enzyme Kinetic Analysis of Immobilized αChymotrypsin in Mag-S-MCF. ADS, NER, p-NER, and BSApreloaded p-NER of CT were prepared by following the protocol for the immobilization of CA with the modified buffer pH, enzyme concentration, and GA concentration. PB and Tris buffer with pH 7.9 were used throughout the synthesis of immobilized CT. Two mg mL−1 CT was used for the adsorption of enzymes in Mag-S-MCF. GA solution (0.1% (w/v)) was used to prepare NER, while 0.1% (w/v) GA solution with 50% (w/v) AS was used to prepare p-NER and BSA-preloaded p-NER of CT. Kinetic parameters of free and immobilized CT samples were estimated by checking their activities with different N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide concentration, prepared in DMF, from 10 to 400 μM. The results were analyzed by Lineweaver−Burk plot.

more details, 50% (w/v) AS solution was added to the CA-adsorbed Mag-S-MCF, and incubated at room temperature for 30 min. After AS treatment, the solution was replaced with 0.5% (w/v) GA solution. All the samples of ADS, NER, p-NER, and p′-NER were incubated under 150 rpm for 1 h and followed by washing with 100 mM PB three times. After the removal of 100 mM PB, 2 mL of 100 mM Tris-HCl (pH 7.6) was added to each sample. The samples were shaken at 200 rpm for 1 h to cap the unreacted aldehyde groups. After five times of washing with 100 mM Tris-HCl, all the samples were diluted by half with 100 mM Tris-HCl (pH 7.6) and stored at 4 °C until use. Synthesis of BSA Pre-Loaded NER and p-NER. Different amounts of BSA, which theoretically occupies 0−100% (v/v) pore volume of Mag-S-MCF, was adsorbed into Mag-S-MCF prior to the synthesis of NER and p-NER. The BSA solution with different concentrations was prepared and incubated with Mag-S-MCF under shaking at 150 rpm for 1 h. The BSA solution was removed after the magnet capture of BSA preadsorbed Mag-S-MCF. The protocols for the synthesis of NER and p-NER were followed as described above to prepare BSA preloaded NER and p-NER, respectively. Determination of Enzyme Loading by Protein Quantification. The amount of protein in the solution during the preparation of ADS sample was determined via BCA protein assay by following the protocol provided by Pierce Biotechnology.49 Enzyme loading was calculated from the difference of protein amount in the waste solution and in the initial stock solution, which represents the amount of protein loaded in the mesoporous silica. Elemental Analysis. Enzyme loadings of NER and p-NER cannot be obtained due to the generation of insoluble CLEAs because the conventional protein assays are based on the measurement of soluble enzymes in solution. As a bypass, we performed elemental analysis. Immobilized CA samples were prepared without 100 mM Tris-HCl (pH 7.6) treatment. Instead, excessive washing with DI water was carried out for the removal of GA or AS molecules. After DI washing, immobilized CA samples were freeze-dried. The composition of nitrogen atom in Mag-S-MCF, free CA, ADS, NER, and p-NER was analyzed by Flash 2000 (Thermo Scientific, Waltham, MA, USA). Field-Emission Scanning Electron Microscope Analysis. Field emission−scanning electron microscope (FE-SEM) images of Mag-S-MCF, ADS, NER, p-NER, and p′-NER were obtained using a Quanta 250 FEG microscope of FEI (Hillsboro, OR, USA). Samples were excessively washed with DI water and freeze-dried. The freezedried samples were coated with platinum and analyzed by FE-SEM at an accelerating voltage of 15 kV. Activity and Stability Measurement. The activities of CA samples were measured by the hydrolysis reaction of 3 mM 4-NPA in 100 mM Tris-HCl (pH 7.6).50 For the case of immobilized CA samples, 50-μL aliquots of immobilized CA samples were added to 3 mM 4-NPA in 100 mM Tris-HCl (pH 7.6). Immobilized CA samples were captured by a magnet at a certain time interval during their reaction. The supernatant of reaction mixture was aliquoted to check the absorbance at 348 nm with a spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan). For the case of free CA, enzyme solution and 3 mM 4-NPA were mixed in a cuvette, and the absorbance increase at 348 nm was monitored by the spectrophotometer. One unit is defined by the amount of enzyme that catalyzes the hydrolysis of 1 μmol 4-NPA in a minute. The stabilities of CA samples were determined by temporally checking activities during the storage of 2 mL of stock solution under 200 rpm shaking at room temperature. The relative activities of samples were calculated based on the ratio of residual activity at each time point to the initial activity of each sample. The half-life was obtained from the linear correlation of semilogarithm values of relative activities with time, which represents the first-order inactivation kinetics. Kinetic Analysis. Activities of free and immobilized CA samples were measured by using 4-NPA with different concentration from 0.5 to 5 mM. The results were analyzed by Lineweaver−Burk plot to determine the kinetic parameters of samples. Enzymatic CO2 Conversion and Calcium Carbonate Formation. A reaction system with two reactors was developed for the conversion of CO2 and utilization in the form of calcium carbonate.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.8b00606. Structural properties of Mag-S-MCF, theoretical background for the calculation of maximum loading, additional discussion and supporting figures for the immobilized CA and CT samples (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jinwoo Lee: 0000-0001-6347-0446 Jungbae Kim: 0000-0001-8280-7008 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (20142020200980). This work was also supported by the Global Research Laboratory Program (2014K1A1A2043032) and Nano·Material Technology Development Program (2014M3A7B4052193) through the National Research Foundation of Korea (NRF) grants funded by the Korea government Ministry of Science and ICT (MSIT). This study was also supported by the Korea CCS R&D Center (2018M1A8A1057172) grant funded by the Korea government Ministry of Science and ICT (MSIT). We thank Dr. 6534

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Hionsuck Baik (Seoul Center, Korea Basic Science Institute) for TEM analysis.

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