Active Nanointerfaces Based on Enzyme Carbonic Anhydrase and Metal–Organic Framework for Carbon Dioxide Reduction

Carbonic anhydrases are enzymes capable of transforming carbon dioxide into bicarbonate to maintain functionality of biological systems. Synthetic isolation and implementation of carbonic anhydrases into membrane have recently raised hopes for emerging and efficient strategies that could reduce greenhouse emission and the footprint of anthropogenic activities. However, implementation of such enzymes is currently challenged by the resulting membrane’s wetting capability, overall membrane performance for gas sensing, adsorption and transformation, and by the low solubility of carbon dioxide in water, the required medium for enzyme functionality. We developed the next generation of enzyme-based interfaces capable to efficiently adsorb and reduce carbon dioxide at room temperature. For this, we integrated carbonic anhydrase with a hydrophilic, user-synthesized metal–organic framework; we showed how the framework’s porosity and controlled morphology contribute to viable enzyme binding to create functional surfaces for the adsorption and reduction of carbon dioxide. Our analysis based on electron and atomic microscopy, infrared spectroscopy, and colorimetric assays demonstrated the functionality of such interfaces, while Brunauer–Emmett–Teller analysis and gas chromatography analysis allowed additional evaluation of the efficiency of carbon dioxide adsorption and reduction. Our study is expected to impact the design and development of active interfaces based on enzymes to be used as green approaches for carbon dioxide transformation and mitigation of global anthropogenic activities.

Carbonic anhydrases (CAs; EC 4.2.1.1) are a family of metalloenzymes known to catalyze the reversible conversion of CO 2 into bicarbonate in the human body, all with ultrahigh turnover of up to 10 5 s −1 [25]. Isolated CAs used in synthetic assays were shown to allow evaluation of a range of disorders from edema, to glaucoma, cancer, and osteoporosis [26][27][28][29], just to name a few. CAs were also proved to play critical roles in the proliferation, survival, and differentiation of pathogens such as protozoa, fungi, and bacteria [30,31], with differences in the infected host relative to the uninfected habitat being evaluated for the potential development of novel therapeutic agents [32]. Furthermore, due to their involvement in reversible hydration/dehydration and pH homeostasis gluconeogenesis, lipogenesis, and ureagenesis [33,34], respectively, CAs have been considered as candidates for biosensors applications [35][36][37][38]. Moreover, their capability to enhance CO 2 capture and sequestration has been recently explored for the development of industrial applications based on membranes [39][40][41] to be used for contactors [39,40] and for supports of ionic liquid membranes [41]. Current active technologies rely on gas adsorption membrane (GAM) units [39,40] where the gas and liquid phases are spatially separated by a hydrophobic entity, with CA being immobilized on the liquid phase side. In such systems, the wetting relationship between the membrane and liquid solvent has proved critical for defining the overall membrane's performance for CO 2 adsorption [42,43]. However, low CO 2 solubility in water, the active solvent maintaining enzyme's functionality, continues to lead to intricate restrictions when large scale implementation of such CA-based systems [40,44] is considered. Moreover, challenges exist when aiming to identify any mass transfer limitations at its interface to thus relate the overall membrane's transformation ability to its scale-up and operational stability [45].
We hypothesized that nanoporous interfaces with hydrolytic stability could not only allow for user-controlled CA immobilization but could also ensure enzyme's activity and its stability retention for subsequent CO 2 adsorption, all at ambient conditions. To test our hypothesis, we synthesized and used metal-organic frameworks (MOFs), a type of porous crystal materials constructed by linking metal node (metal ions or clusters) and organic linkers together through bond coordination, as scaffolds for both enzyme immobilization and interfaces functionality demonstration. Studies in our group and others have previously showed that such frameworks are suitable for enzyme surface immobilization because of their customizable synthesis [46], large surface area which allows for increased enzyme loading [47,48], and tunable surface features which permit hydrophilic or hydrophobic residues implementation [48][49][50]. We and others have also showed that resulting MOF-enzyme conjugates [46] preserved enzymes' functionality to a larger extent than conjugates formed upon enzymes immobilized on other materials such as polyurethane foam [51], magnetic [52] and silica-based materials [53], or zeolites [54], respectively. Our proof-of-principle demonstration integrated in a lab-built bioreactor provides a functional interface for efficient CA functionality at ambient pressures and temperatures while allowing the study of kinetics efficiency, as well as system's operational stability in user-set-up conditions. Lastly, our demonstration provides a customizable interface for large-scale implementation with functionality determined by both the enzyme and MOF distribution, respectively.

Preparation of MIL-160/Al 2 O 3 Hybrids
FDCA-modified Al 2 O 3 filters were used for synthesis of MIL-160/Al 2 O 3 hybrids. For this, 10 mL of 5 mM FDCA solution in deionized water was first dosed onto the Al 2 O 3 filters contained in a Teflon-lined stainless steel autoclave and subsequently heated at 50 • C for 24 h. Secondly, such modified filters were subsequently dosed in a Teflonlined stainless steel autoclave in a solution of FDCA, aluminum chloride hexahydrate, and sodium hydroxide dissolved in deionized water (final concentrations of 55, 55, and 100 mM, respectively) at 100 • C for 12 h. After cooling to room temperature, the functionalized filter was taken out, washed extensively with deionized water to remove any of the unreacted species, dried overnight and at room temperature, and stored for further usage.

Characterization of MOFs and Resulting Hybrids
The morphologies of the Al 2 O 3 , FDCA functionalized Al 2 O 3 (FDCA/Al 2 O 3 ) filters, and MIL-160/Al 2 O 3 hybrids were investigated by field emission scanning electron microscopy (FESEM) performed on a S-4700 (Hitachi, Santa Clara, CA) with a cold field emission gun operating at 4 kV and 10 JA. Energy-dispersive X-ray spectroscopy (EDS) mapping was conducted for evaluating the elemental composition of the samples. Phase purity and crystallinity of the synthesized MOFs were confirmed by X-ray diffraction (XRD), with powders of MOFs being analyzed using a Bruker D8 ADVANCE X-ray diffractometer (Westborough, MA, USA) with CuKa radiation operating at 40 kV and 40 mA. XRD patterns were recorded in reflection mode, at room temperature, and under ambient conditions. Chemical composition of the Al 2 O 3 and functionalized Al 2 O 3 filters were evaluated in dried conditions by Fourier-transform infrared spectroscopy (FTIR) on a Digilab FTS 7000 (Digilab, Inc., Hopkinton, MA, USA) equipped with a diamond attenuated total reflection (ATR). Scans were collected in the range of 400-4000 cm −1 at a resolution of 4 cm −1 ; a total of 128 scans were co-added to form the final spectrum of each sample investigated. Experiments were performed in triplicates. The surface areas of Al 2 O 3 filter and MIL-160/Al 2 O 3 hybrids were determined from N 2 adsorption analysis conducted on a Micromeritics Instrument sorption analyzer (ASAP 2020, Micromeritics Instrument Corporation, Norcross, GA, USA) using liquid nitrogen at 77 K. An atomic force microscope (AFM, Asylum Research, Goleta, CA, USA) and Si tips (AC240TS, 50 kHz, Asylum Research, Goleta, CA, USA) were used to investigate the morphology of the samples in AC mode. Scans of 20 µm × 20 µm area or lower were acquired in air; order 3 image flattening was applied. Height evaluation were performed using the Asylum software (Asylum Research, Goleta, CA, USA), with subsequent MATLAB analysis.

CA/MIL-160/Al 2 O 3 Membrane Preparation
The CA/MIL-160/Al 2 O 3 membrane was prepared by loading CA onto the MIL-160/Al 2 O 3 hybrids. For this, the hybrids were immersed in a 2 mL solution containing different enzyme concentrations (1.0, 0.5, and 0.1 mg/mL, respectively) in Tris buffer (50 mM and pH 7.5) and in a covered glass vials (Fisher Scientific, Ward Hill, MA, USA), with the mixture being shaken at room temperature and 100 rpm. After 2 h, the resulting (now) membrane was removed and used for the activity assay and CO 2 -related tests, respectively. Control experiments have also been performed using Al 2 O 3 filters and incubating them with the above known representative concentrations of enzyme.

Enzyme Loading Assessment
Standard BCA assay was performed to evaluate enzyme loading or the amount of enzyme loaded per the membrane. Briefly, 1 mL of the working reagent containing 50 parts of reagent A with 1 part of reagent B (reagents were provided stock with the BCA assay kit) was mixed with 50 µL of the remaining solution, i.e., solution used for the membrane or filter and containing the different enzyme concentrations (see above), and subsequently incubated at 37 • C for 30 min. Absorbance at 562 nm was recorded for each sample using an UV−VIS spectrophotometer (UV2600 Shimadzu) and compared to a calibration curve of known concentrations of the CA suspended in the working reagent. Every experiment was repeated at least 3 times. Loadings were calculated using Equation (1): where C 0 and V 0 were the initial concentration and volume of CA solution, and C 1 and V 1 were the concentration and volume of CA solution after the immobilization process, respectively.

Enzyme Activity at the Designed Interfaces
The specific activity of CA (i.e., activity of the free, immobilized onto the membrane, or the control filters, respectively) was assessed by monitoring the hydrolysis of p-NPA at 400 nm [47]. For the free enzyme, 0.1 mL of free CA was mixed with 0. The absorbance was monitored every 2 min over a total period of 10 min. Additional blank experiments were conducted to estimate the self-dissociation of p-NPA in each of the solutions being used. Every experiment was repeated at least 3 times.

Assessment of Enzyme-Based Conjugates
Chemical composition of the CA-based samples was evaluated by FTIR. For this, all of the CA-based samples were prepared by suspending the supports (Al 2 O 3 and MIL-160/Al 2 O 3 ) in CA-Tris (pH 7.5) solutions with the concentration of 0.1 mg/mL for 2 h under 100 rpm shaking conditions and at room temperature. After drying the samples at room conditions, they were tested using FTIR as described above. Controls of dried CA was also used. Scans were collected in the range of 400-4000 cm −1 at a resolution of 4 cm −1 ; a total of 128 scans were co-added to form the final spectrum of each sample. Experiments were performed in triplicates. AFM using Si tips were used to investigate the morphology of the samples containing immobilized enzyme, all in AC mode. Scans of 20 µm × 20 µm area or lower were acquired in air with subsequent order 3 image flattening. Height evaluation were performed using the Asylum software with subsequent MATLab analysis.

Performance of the CA/MIL-160/Al 2 O 3 Membrane or CA/Al 2 O 3 Filters
Performance of as prepared CA/MIL-160/Al 2 O 3 membrane or CA/Al 2 O 3 filters used as controls for CO 2 adsorption was evaluated using a modified method reported by Hou et al. [41]. Briefly, an in-house platform (Supplementary Materials Scheme S1) composed of a membrane module, a gas chromatography unit (GC; Inficon, Micro GC Fusion, East Syracuse, NY, USA), mass flow meters, and pipelines was used. The membrane module was assembled from one right-angle flow rectangular manifold, three sets of stainless steel vacuum coupling O-ring (VCO) and stainless steel female nuts, and two stainless steel Swagelok tube fittings (Swagelok, Columbus, OH, USA). The GC unit was used for monitoring the CO 2 concentration of the output gases, while the flow meters were used for controlling the content of the feeding gas. Herein, the feeding gas contained a mixture of 5% CO 2 and 95% N 2 , with a controlled constant flow rate of 10 mL/min. This gas balance was chosen to mimic conditions in which the target molecule, i.e., CO 2 , corresponds to physical scenarios that are representative of current challenges in gas separation technologies [55,56], with carbon capture and storage (CCS) processes [55,[57][58][59][60][61][62], for instance, targeting uptake of CO 2 at dilute concentrations in power plants and chemical refineries [56,58,[63][64][65]. The self-made membrane module also comprised a gas chamber for gas flow and liquid chamber for removing any product being released. The synthesized membrane was mounted in the self-built module with the CA/MIL-160 side in direct contact with the gas phase and the Al 2 O 3 substrate side facing the liquid chamber. The overall and the specific CO 2 adsorption rate were determined by the difference of the CO 2 concentrations between the feeding and the output gas as shown in Equations (2) and (3), respectively: where C in, co 2 was the concentration of the feeding gas and C out, co 2 was the concentration of the output gas, while m CA was the effective mass of enzyme used. Control experiments using free CA (0.1 mg/mL) were also performed. For this, 3 mL of 0.1 mg/mL CA solution was suspended into a glass reactor (Supplementary Materials Scheme S2); the reactor was subsequently connected to the feeding gas and the rest of the configuration described above and used for evaluating the CO 2 concentration changes. As above, the flow rate was maintained at 10 mL/min; experiments were performed in triplicates.

Synthesis and Characterization of the Components and the BioMembrane
We hypothesized that hydrophilic metal-organic Framework (MOF) MIL-160 can be used as a porous scaffold to increase functionality of carbonic anhydrase (CA) enzyme for its subsequent usage in carbon dioxide (CO 2 ) gas adsorption. MIL-160s were selected based on their demonstrated capability to serve as flexible and hydrated "bridges" that ensure proton transfer to maintain enzyme functionality [46].
To demonstrate our hypothesis, we first synthesized the MIL-160/Al 2 O 3 hybrids using a user-controlled hydrothermal method. Briefly, Al 2 O 3 filters with uniform pore sizes (20 nm in diameter) were chosen as supports for MIL-160 "decorates", with Al 2 O 3 filters expected to allow coordination with 2,5-furandicarboxylic acid (FDCA), the known MIL-160 linker [46,66], through its Al metal ions [67,68]. It was expected that direct modification of the Al 2 O 3 filter with FDCA would lead to controlled formation of MIL-160 onto the filters interface [69,70]. The hypothesis is supported by previous studies demonstrating that metal oxides can be used as metal sources for ZIF-8 formation [71], while ZrO 2 can be used for UiO-66 [72] synthesis, as well as other reports [67,73] that showed the ability of porous Al 2 O 3 filters to be modified with organic chemicals such as dopamine through a hydroxide-induced covalent reaction [67]. Furthermore, previous studies showed that a filter's well-developed porosity and relatively high surface area, large pore volume, and abundant weak Lewis acid sites are essential to promote direct attachment of active molecules [74,75] (here, the FDCA). Such active molecules were further able to incorporate and disperse well into the individual filters' channels to thus serve as active catalytic species [76]; such species are all needed for the MIL-160 formation.
Surface modification was confirmed by scanning electron (SEM) [77] and atomic force (AFM) [78] microscopy analysis. For the first, changes in Al 2 O 3 thickness after FDCA functionalization were confirmed via cross-sectional SEM (Figure 1a), all relative to Al 2 O 3 control (Figure 1b). For the second, morphology evaluation showed that FDCA functionalization led to changes in the 3D profiles of the Al 2 O 3 filter surfaces (inserts, Figure 1a,b), as well as changes in the overall height profile distributions, with analysis revealing average height increases of about 4.7 times after filters modification with FDCA (Supplementary Materials Figure S1). ened relative to those of the FDCA used as control. Furthermore, energy-dispersive X-ray spectroscopy (EDS) analysis revealed concentration changes of the C, O, and Al elements for the functionalized FDCA/Al2O3 filters relative to the bare ones (Supplementary Materials Table S1), with C and O increasing from 8.59 ± 0.36% to 12.45 ± 0.08% and 37.48 ± 0.19 % to 44.45 ± 0.11%, while the concentration of Al decreased from 53.93 ± 0.21% to 43.11 ± 0.09%, respectively. FDCA/Al2O3 functionalized filter was subsequently used as support for the growth of MIL-160 under user-controlled hydrothermal method [46]. Formation of MIL-160 particles was confirmed by SEM analysis, with Figure 2a showing a representative image of the hybrids displaying clear edges and uniform distribution onto the filter. Control MIL-160 grown on Al2O3 filter (i.e., without any FDCA pretreatment) were edgeless and smaller in size and had poor uniformity (Figure 2a insert). The cross-section view of the hybrid indicated a continuous monolayer coverage with edge distribution resulting in an overall thickness changes of 13% to 15% relative to the thickness of the FDCA functionalized filter. Moreover, SEM analysis allowed particle size quantification, with the MIL-160 size in the hybrids being about 14.8 ± 0.9 µm while onto the filters, i.e., without FDCA pretreatment, being about 2.5 times smaller. AFM analysis also confirmed the edge distribution of the hybrids (Figure 2b). EDS mapping (Figure 2c) further demonstrated the formation of MIL-160/Al2O3 hybrids with layered distributions of C and O elements in the upper and Al in lower layer configuration, respectively. X-ray diffraction (XRD) confirmed the monophasic crystal nature of synthesized MOFs (Supplementary Materials Figure S3), with characteristic peaks recorded at 8.4° 9.4°, 11.98°, 15.2°, 18.8°, 22.8°, and 27°. Peaks were assigned to the planes (020), (011), (220), (031), (022), (051), and (502), respectively [80]. Lastly, the N2 adsorption analysis (Figure 2d) revealed Brunauer-Emmett-Teller (BET) and Langmuir surface area of MIL-160/Al2O3 hybrids of 214.58 and 264.70 m 2 /g, respectively, significantly larger than that of Al2O3 filters, which were 7.40 and 9.3 m 2 /g.  Figure S2). Peaks shifts were also visible and confirmed the coordination reaction. Indeed, previous analysis [46] showed that the peaks at 1674 and 1275 cm −1 associated with C=O and C−O bonds of FDCA disappeared in the spectra of MOFs, while the characteristic peaks at 1581 and 1414 cm −1 associated with the C=C bond of the furan ring and peaks at 962 and 825 cm −1 assigned to the C−H bond, respectively, were weakened relative to those of the FDCA used as control. Furthermore, energy-dispersive X-ray spectroscopy (EDS) analysis revealed concentration changes of the C, O, and Al elements for the functionalized FDCA/Al 2 O 3 filters relative to the bare ones (Supplementary Materials Table S1), with C and O increasing from 8.59 ± 0.36% to 12.45 ± 0.08% and 37.48 ± 0.19 % to 44.45 ± 0.11%, while the concentration of Al decreased from 53.93 ± 0.21% to 43.11 ± 0.09%, respectively.
FDCA/Al 2 O 3 functionalized filter was subsequently used as support for the growth of MIL-160 under user-controlled hydrothermal method [46]. Formation of MIL-160 particles was confirmed by SEM analysis, with Figure 2a showing a representative image of the hybrids displaying clear edges and uniform distribution onto the filter. Control MIL-160 grown on Al 2 O 3 filter (i.e., without any FDCA pretreatment) were edgeless and smaller in size and had poor uniformity (Figure 2a insert). The cross-section view of the hybrid indicated a continuous monolayer coverage with edge distribution resulting in an overall thickness changes of 13% to 15% relative to the thickness of the FDCA functionalized filter. Moreover, SEM analysis allowed particle size quantification, with the MIL-160 size in the hybrids being about 14.8 ± 0.9 µm while onto the filters, i.e., without FDCA pretreatment, being about 2.5 times smaller. AFM analysis also confirmed the edge distribution of the hybrids (Figure 2b

Functionality of the CA Enzyme at the BioMembrane Interface
Synthesized MIL-160/Al2O3 hybrids were subsequently used as supports for enzyme surface immobilization [46]. Immobilization was confirmed by FTIR and standard colorimetric assay. For the first, CA immobilized at the membrane interface showed broader peaks (1458-1735 cm −1 ) relative to the peaks of free enzyme, i.e., 1625 and 1523 cm −1 (Supplementary Materials Figure S4), presumably due to C=N stretching and N-H bending vibration [46,81] known to occur upon enzyme immobilization at interfaces. Additionally, control CA immobilized onto Al2O3 displayed narrower peaks relative to free enzyme in solution, most likely due to the enzyme's deformation at such a filter interface [46]. The colorimetric assay confirmed enzyme loading at about 0.101 ± 0.006 mg per membrane, which was lower than that for the control filter that showed an average loading of 0.124 ± 0.006 mg, values recorded when both were offered 0.2 mg of enzyme during interfaces' preparation. The higher enzyme loading observed for the CA/Al2O3 relative to CA/MIL-160/Al2O3 was presumable due to the porous structure of the Al2O3 filter; specifically, filter pore sizes of 25 nm could potentially allow entrapment of CAs (which are known to have individual diameters of ~4.5 nm [46]). Indeed, previous studies showed entrapment of enzymes in spaces as small as 3.0 and 4.1 nm when, for instance, microperoxidase-11 (MP-11) was interfaced with Tb-TATB [82], or in 9 and 15 nm in a periodic mesoporous organosilicas (PMOs) used for lipase immobilization [83], respectively. Considering that the porosity of MIL-160 is only 0.5 nm [84], such small pores are expected to provide additional space for gas adsorption analysis (as supported by BET evaluations) but, however, should not be prone for confinement of immobilized enzymes.
Activity evaluations (Figure 3a) and Michaelis-Menten kinetics of the CA/MIL-160/Al2O3 and CA/Al2O3 control were performed using the hydrolysis of para-nitrophenol acetate (p-NPA). Analysis showed that the enzyme immobilized at the membrane inter-

Functionality of the CA Enzyme at the BioMembrane Interface
Synthesized MIL-160/Al 2 O 3 hybrids were subsequently used as supports for enzyme surface immobilization [46]. Immobilization was confirmed by FTIR and standard colorimetric assay. For the first, CA immobilized at the membrane interface showed broader peaks (1458-1735 cm −1 ) relative to the peaks of free enzyme, i.e., 1625 and 1523 cm −1 (Supplementary Materials Figure S4), presumably due to C=N stretching and N-H bending vibration [46,81] known to occur upon enzyme immobilization at interfaces. Additionally, control CA immobilized onto Al 2 O 3 displayed narrower peaks relative to free enzyme in solution, most likely due to the enzyme's deformation at such a filter interface [46]. The colorimetric assay confirmed enzyme loading at about 0.101 ± 0.006 mg per membrane, which was lower than that for the control filter that showed an average loading of 0.124 ± 0.006 mg, values recorded when both were offered 0.2 mg of enzyme during interfaces' preparation. The higher enzyme loading observed for the CA/Al 2 O 3 relative to CA/MIL-160/Al 2 O 3 was presumable due to the porous structure of the Al 2 O 3 filter; specifically, filter pore sizes of 25 nm could potentially allow entrapment of CAs (which are known to have individual diameters of~4.5 nm [46]). Indeed, previous studies showed entrapment of enzymes in spaces as small as 3.0 and 4.1 nm when, for instance, microperoxidase-11 (MP-11) was interfaced with Tb-TATB [82], or in 9 and 15 nm in a periodic mesoporous organosilicas (PMOs) used for lipase immobilization [83], respectively. Considering that the porosity of MIL-160 is only 0.5 nm [84], such small pores are expected to provide additional space for gas adsorption analysis (as supported by BET evaluations) but, however, should not be prone for confinement of immobilized enzymes.
Activity evaluations (Figure 3a) and Michaelis-Menten kinetics of the CA/MIL-160/Al 2 O 3 and CA/Al 2 O 3 control were performed using the hydrolysis of para-nitrophenol acetate (p-NPA). Analysis showed that the enzyme immobilized at the membrane interface had a higher activity, higher V max (5.66 ± 0.57 mM/mg enzyme/min.), and lower K m (31.98 ± 4.46 mM) relative to the enzyme immobilized directly onto the filter (V max of 4.16 ± 0.27 mM/mg enzyme/min and K m of 39.97 ± 4.09 mM, respectively). The recorded surface-dependent activity and kinetic behavior was presumably due to enzyme deformation or enzyme entrapment (Figure 3b) at the two interfaces. In particular, the sharp edges and higher curvatures of MIL-160 framework could had led to a lower enzyme deformation and higher retained activity than the lower curvature of the Al 2 O 3 used as control. This hypothesis is supported by previous work that showed that MIL-160 hydrophilicity does not only allow for a higher activity of immobilized enzymes, but further, their flexible and hydrated nature provide "bridges" to ensure proton transfer at their interfaces and lead to improved kinetic behavior [46]. Previous studies [85][86][87][88] also showed that supports with higher local curvatures were more beneficial for retaining enzyme activity, with Tadepalli et al. [85] demonstrating, for instance, higher activity for horseradish peroxidase absorbed on gold nanoparticles known to have higher surface curvature. Campbell et al. [87] also reported that the power density of an enzymatic biofuel cell could be 10-fold greater when glucose oxidase and bilirubin oxidase used to form enzymatically active anodes and cathodes, respectively, were immobilized onto graphene-coated single-wall carbon nanotube gels known to possess higher curvature than controls gold/multiwall carbon nanotube fiber paddles. In addition, our previous work [86] confirmed this principle by revealing a higher activity for soybean peroxidase immobilized onto carbon nanotube as opposite to the enzyme immobilized onto graphene oxide sheets. Lastly, complementary previous analysis showed that the functionality of the enzymes entrapped in porous matrices/supports was affected significantly by the substrate diffusion limitation to enzyme's active site [89,90], with authors indicating that such diffusion limitations were due to the steric hindrances that the confined spaces imposed.  Observed changes in the CO2 concentration at the CA/MIL-160/Al2O3 membrane interface relative to both free CA and CA/Al2O3 controls, respectively, were endowed by the design of the nanoscale hierarchical structure based on the MIL-160 and enzyme adsorption at such interfaces, respectively. Specifically, framework flexibility and intrinsic regular porosity not only provided a favorable microenvironment for both enzyme loading and functionality, but they also allowed for an active and viable transfer for both the reactant and the product to occur at the immobilized enzyme's active site. These are supported by our previous study and our herein included BET and microscopy analysis showing that MIL-160 has high hydrophilicity and highly porous crystalline structure with defined square-shape sinusoidal channels of ~5 Å in diameter [84]. Moreover, water CO 2 adsorption and reduction at the CA/MIL-160/Al 2 O 3 membrane (i.e., changes in the CO 2 concentration upon gas interaction with the user-formed membrane as well as changes in operational stability of the membrane upon extended gas exposure) were evaluated using an "in-house" platform (Supplementary Materials Scheme S1). The CA/MIL-160/Al 2 O 3 membrane was sealed in a tunnel-like chamber, with the CA-loaded side towards the feed gas and the other side being maintained in direct contact with stirred water. A membrane contactor was aimed to allow direct contact of the target gas (CO 2 ) with the enzyme, while the MIL-160 porosity provided a hydrophilic microenvironment to retain immobilized enzyme's activity. This lab-designed platform was thus hypothesized to directly assist with membrane hydration while also ensuring CO 2 direct adsorption at the CA active site and eliminating the CO 2 solvation [91] limitations. The mechanism of CO 2 adsorption at the CA active site was previously described, with report showing that CO 2 binds near the Zn ions [92] to lead to a Zn-coordinated transformation of bound CO 2 into bicarbonate to be followed by the direct release of such product into the media (Supplementary Materials Equations (S1)-(S3)) [25]. CO 2 concentration changes at the membrane interface were evaluated by monitoring the concentration of CO 2 in the output gas (i.e., after its exposure to the CA interface) through the gas chromatograph (GC); CO 2 concentration in the feeding gas was maintained constant. The CO 2 changes in concentration are shown in Figure 3c; specifically, analysis revealed about 5% change on average in the output CO 2 concentration relative to the concentration used during membrane's feeding. The concentration plateaued after about 54 ± 7.8 min of continuous membrane operation in the CO 2 flow. In contrast, the changes at the CA-Al 2 O 3 filter interface decreased drastically after only 50 min operation from an initial maximum of 1.4% to 0.5% and then subsequently plateaued after about 76.3 ± 3.  Figure S5c). Such patterns were presumably triggered by the Brownian motion and the molecular diffusion of the individual CA molecules suspended in water and their subsequent collision with CO 2 at the gas-liquid phases.
Observed changes in the CO 2 concentration at the CA/MIL-160/Al 2 O 3 membrane interface relative to both free CA and CA/Al 2 O 3 controls, respectively, were endowed by the design of the nanoscale hierarchical structure based on the MIL-160 and enzyme adsorption at such interfaces, respectively. Specifically, framework flexibility and intrinsic regular porosity not only provided a favorable microenvironment for both enzyme loading and functionality, but they also allowed for an active and viable transfer for both the reactant and the product to occur at the immobilized enzyme's active site. These are supported by our previous study and our herein included BET and microscopy analysis showing that MIL-160 has high hydrophilicity and highly porous crystalline structure with defined square-shape sinusoidal channels of~5 Å in diameter [84]. Moreover, water molecules played critical roles in maintaining CA activity since the framework, through its endowed hydrophilic and porous characteristics, served as a "bridge" for proton transfer for the enzyme regeneration step (Supplementary Materials Equations (S1)-(S3)) to ensure CO 2 hydration [46].
We also varied the concentration of CA offered during the immobilization process; analysis showed that even though enzyme loading increased from 0.115 to 0.403 mg/membrane when the CA amount in the feeding solution increased by 10 times for instance, the catalytic performance of the membrane did not change (Figure 3d). We thus hypothesize that what dictates efficiency is the enzyme packing/arrangement at the MOF interface. Indeed, the specific CO 2 change in adsorption (Figure 3e) at the CA/MIL-160/Al 2 O 3 membrane prepared using a CA concentration of 0.1 mg/mL reached the highest value of 1.26 mmol/mg CA/min. This was 1.6 and 5.0 times that of CA/MIL-160/Al 2 O 3 membranes prepared under the CA concentrations of 0.5 and 1.0 mg/mL with effective enzyme loading being 0.042 ± 0.0003 and 0.117 ± 0.024 mg/membrane, respectively. Herein, the effective enzyme loading is referred to the amount of the accessible enzyme for CO 2 adsorption as dictated by the enzyme-support specific interface. Such reduced enzyme efficiency was probably due to the intramolecular interaction of multilayered immobilized enzymes shown previously to cause activity losses at given interfaces [46,92]. Moreover, a larger enzyme loading could also result in mass transfer limitations due to the multilayer coverage to again lead to reduced overall efficiency of the immobilized enzymes [93,94].
Our results are superior to previous results from Hou et al. [95] or Wang et al. [96] (Supplementary Materials Table S2), with the higher CO 2 adsorption achieved by the design CA/MIL-160/Al 2 O 3 membrane being presumably due to the advantages of the design itself which allows for both direct contact of CO 2 with the enzyme as well as hydrophilic wetting through MIL-160. Specifically, Hou et al. [95] reported CO 2 changes at a CA integrated Janus membrane (i.e., carbonic anhydrase (CA)-1H,1H,2H,2H-peruorododecyltrichlorosilane (FTCS)-carbon nanotubes (CNTs)-polyvinylidene fluoride (PVDF)) with authors studying the CO 2 hydration performance when the enzyme was integrated into the hydrophobic polypropylene (PP) hollow fiber membranes. They showed a CO 2 hydration rate of about 62% of the one achieved in this study while their effective membrane area was over 67 times larger than the one used herein [40]. Furthermore, Wang's group achieved high efficiency of CO 2 absorption flux of 2.5 × 10 −3 mol·m −2 ·s −1 , which is considerably reduced when compared to the one achieved in our study. Moreover, in their study the authors immobilized CA onto a polydopamine (PDA)/polyethylenimine (PEI)-polyvinylidene fluoride (PVDF) composite hollow fiber membrane through a crossing linking method [96], which provides limited scalable capabilities.
Our results demonstrated the potential of a user-designed membrane to be used as a bioreactor to provide a viable platform for efficient CO 2 adsorption and reduction specifically considering that our previous analysis [46] that showed that thermal stability (investigated upon applying heat treatment to the conjugates, under various temperatures, for 30 min) is enhanced relative to the thermal stability of the free enzyme stored under the same conditions. In particular, we have previously shown [46] that CA-MIL-160 conjugates retained all of their original activity after heating treatment at 60 • C and only lost about 7% of their original activity after heating treatment at 70 • C. Our analysis demonstrated that the flexibility of the MOF interface protects the enzyme from rigid multiattachment and leads to cooperative enhancement of water affinity [97] with the excellent heat storage capacity of MIL-160 [80,84] further enhancing the high temperature tolerance of the immobilized CA relative to its free forms. Furthermore, our results indicate that such a bioreactor could possibly be used to evaluate CO 2 hydration, with such process to be controlled by the functionality of the immobilized enzyme. Moreover, our study hints at the ability of the membrane to allow for evaluation of CO 2 benign transformation, through direct measurement of bicarbonate product. Lastly, our analysis allows for complete integration and extension of the CO 2 adsorption and transformation system through a parallel configuration of the membrane bioreactor design to thus allow for flow control and transformation in a modular geometry.

Conclusions
We developed an interface based on biocatalyst CA and a hydrophilic MOF, MIL-160, to be used for CO 2 adsorption in synthetic environment and at room temperature. Our study successfully showed that membrane's design and functionality depend on the materials characteristics, i.e., MIL-160 porosity and its ability to ensure a hydrophilic environment for the CA-driven reaction. We further demonstrated that enzyme's efficiency is function of the geometry and morphology of the interface itself; we envision extension of such CA-based technology to industrial conditions upon adopting reactor design and modular implementation.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/nano11041008/s1, Scheme S1: Schematic illustration of platform used for the membrane performance evaluation; Scheme S2: Schematic illustration of the reactor used for CO 2 hydration of free carbonic anhydrase; Figure S1: height profile of surface of the FDCA modified Al 2 O 3 (FDCA/Al 2 O 3 ) filter and Al 2 O 3 filter; Figure S2: FTIR spectra of FDCA; Figure S3: XRD spectra of the MOF; Table S1: elemental composition of Al 2 O 3 and FDCA/Al 2 O 3 membrane; Figure S4: FTIR analysis of membrane and controls; Equations (S1)-(S3): established mechanism for CO 2 transformation by the enzyme;

Data Availability Statement:
The data presented in this study are available on request from the first author.

Conflicts of Interest:
The authors declare no conflict of interest.