Lysine-modulated synthesis of enzyme-embedded hydrogen-bonded organic frameworks for efficient carbon dioxide fixation

Carbonic anhydrase (CA) is an important carbon fixation enzyme. Immobilization of CA can expand its application in the realm of adsorption, catalysis, and so on. As a typical metal-free framework, hydrogen-bonded organic frameworks (HOFs) featuring mild synthesis process, exquisite framework structure and good enzyme compatibility have been used for enzyme embedding. However, the catalytic performance of CA-embedded HOFs (CA@HOFs) is limited by the micropore size of HOFs and the slow adsorption of CO 2 . Herein, CA@Lys-HOF-1 was synthesized by introducing lysine (Lys), a basic amino acid, during the coprecipitation of CA and HOFs for CO 2 fixation. The addition of Lys enlarged the average pore size of HOF-1 from 1.8 to 3.2 nm, whereas the introduced - NH 2 groups increased the initial adsorption of CO 2 from 0.55 to 1.21 cm 3 g -1 . Compared to CA@HOF-1, the activity of CA@Lys-HOF-1 was enhanced by 71.25%, and the corresponding production of CaCO 3 was enhanced by 12.7%. After eight reaction cycles, CA@Lys-HOF-1 still maintained an output of 9.97 mg of CaCO 3 every 5 min, 83.7% of the initial production. It is hoped that the CA@Lys-HOF-1 reported offers a platform for efficient and continuous fixation of CO 2 .


INTRODUCTION
Carbon dioxide (CO 2 ) capture and utilization (CCU) is one of the ever-increasing research topics which can contribute to addressing environmental and ecological issues [1][2][3][4] .Enzymes such as formate dehydrogenase (FDH), ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO), and carbonic anhydrase (CA) can accurately activate CO 2 to lower the reaction energy barrier, which have received great attention as a green and feasible solution to CCU [5][6][7] .CA exhibits the highest catalytic rate among all carbon-fixation enzymes, and thus has good potential for the selective transformation of CO 2 to HCO 3 - [8] .However, enzymes that leave the bodies of organisms are easy to inactivate and difficult to reuse [9][10][11] .One frequently used method to address the above issues is to immobilize enzymes inside carrier materials [12] .
Herein, HOF-1 composed of two units of Tetrakis(4-amidiniumphenyl)methane and tetrakis (4-carboxyphenyl) methane was selected for CA embedding [37] .The structure of HOF-1 was modulated by introducing basic amino acids during the synthesis process.Briefly, carbonic anhydrase@HOF-1 (CA@HOF-1) was prepared by a coprecipitation method, in which CA was in situ embedded.The structure of CA@HOF-1 was modulated by altering the species and amount of amino acid.We found that basic amino acids, especially lysine (Lys), can interact with the carboxyl monomer of HOF-1 to occupy some of the hydrogen bond formation sites, thus causing defects of HOF-1 to promote mass transfer.Meanwhile, the introduced -NH 2 groups also facilitated the initial adsorption of CO 2 .Compared to CA@HOF-1, CA@Lys-HOF-1 showed a 12.7% enhancement in CO 2 fixation efficiency.When CO 2 was introduced at a flow rate of 25 mL min -1 , CaCO 3 precipitation reached 12.26 mg after 5 min of ventilation.After 8 cycles, CA@Lys-HOF-1 maintained an output of 9.97 mg CaCO 3 every 5 min.It is believed that Lys-HOF-1 is an ideal framework material for CO 2 -converting enzyme embedding.

Synthesis of HOF-1
Tetrakis(4-amidiniumphenyl)methane tetrahydrochloride (10 mg) was dissolved in H 2 O (2.5 mL) to form solution A. Tetrakis(4-carboxyphenyl)methane (7.5 mg) was dispersed in H 2 O (2375 μL), followed by the addition of aqueous ammonium hydroxide solution (1% v/v, 125 μL) to form solution B. Thereafter, solution B was added to solution A under stirring conditions at room temperature.Precipitation occurred immediately upon mixing.The reaction mixture was left to stir gently in the dark for 1 h.The HOF-1 material was then recovered by centrifugation, washed, dispersed, and centrifuged three times in H 2 O to remove any unreacted precursors.

Synthesis of CA@HOF-1
Tetrakis(4-amidiniumphenyl)methane tetrahydrochloride (10 mg) was dissolved in H 2 O (1.25 mL) to form solution A. An aqueous solution of CA (1.25 mL of 2 mg mL -1 stock solution) was added to solution A and stirred at room temperature for 10 min to form solution B. Tetrakis(4-carboxyphenyl)methane (7.5 mg) was dissolved in 2375 μL of H 2 O and 125 μL of 1% NH 4 OH to form solution C. Solution C was then added dropwise to solution B under stirring.The mixture was then left to stir gently for another 1 h to ensure the completion of the synthesis.Thereafter, CA@HOF-1 was collected by centrifugation and then washed, dispersed, and centrifuged three times in H 2 O to remove the unreacted precursors and loosely adsorbed CA.

Synthesis of CA@amino acid-HOF-1
Tetrakis (4-carboxyphenyl) methane (7.5 mg) was dissolved in 2375 μL of H 2 O and 125 μL of 1% NH 4 OH to form solution A. An aqueous solution of CA (0.625 mL of 4 mg mL -1 stock solution) and amino acid (0.625 mL of 1/10/20 mg mL -1 stock solution) was added to solution A and stirred at room temperature for 10 min to form solution B. Tetrakis(4-amidiniumphenyl)methane tetrahydrochloride (10 mg) was dissolved in H 2 O (1.25 mL) to form solution C. Solution C was then added dropwise to solution B under stirring.The mixture was then left to gently stir for another 1 h to ensure the completion of the synthesis.Thereafter, CA@amino acid-HOF-1 was collected by centrifugation and then washed, dispersed, and centrifuged three times in H 2 O to remove any unreacted precursors and loosely adsorbed CA.

Characterization
The sample morphologies were analyzed by scanning electron microscopy (SEM, S-4800, Hitachi) and transmission electron microscopy (TEM, JEM-100CX II, JEOL).The chemical compositions of the samples were identified by Fourier transform infrared (FTIR) spectrometer (Nicolet-6700, Nicolet).The crystal structures of the samples were identified by X-ray diffraction (XRD, X' Pert Pro), with 2-theta ranging from 10° to 90° by a step width of 0.033° with 15.24° min -1 speed at 40 mA and 40 kV.The specific surface area and pore size distribution of the samples were examined by Brunauer-Emmett-Teller (BET) method based on N 2 adsorption/desorption isotherms on an AUTOSORB-1 surface area and pore size analyzer (Quantachrome Instruments).

Conversion of CO 2
CA@HOF-1 and CA@Lys-HOF-1 were used for the conversion of CO 2 .In a typical experiment, nitrogen was first injected into the aqueous phase of the reaction device for 10 min to remove CO 2 gas in the solution.Then, CO 2 with a rate of 25 mL min -1 was injected into the system.A sample was taken every 5 min for follow-up reaction.Specifically, pH value of the reaction solution was detected by a pH meter and kept at 7.9, which was adjusted by adding 5 mol L -1 NaOH.Then, a certain amount of reaction solution was mixed with 670 mmol L -1 calcium chloride solution.The mixture was shaken at 200 r min -1 to form CaCO 3 precipitate.The precipitated CaCO 3 was filtered by a filter paper with an average pore diameter of 2.5 μm, which was then dried overnight and weighed to determine the relative yield of CaCO 3 .

RESULTS AND DISCUSSION
Preparation and Characterization of CA@HOF-1 and CA@Lys-HOF-1 A reported HOF material (HOF-1) was chosen for the embedding of CA [37] .The preparation and structure regulation of CA@HOF-1 are shown in Figure 1.Briefly, HOF-1 was prepared by mixing tetrakis (4-amidiniumphenyl)methane tetrahydrochloride (monomer 1) solution and tetrakis(4-carboxyphenyl) methane (monomer 2) solution under stirring at room temperature [Supplementary Figure 1].CA@Lys-HOF-1 was prepared by introducing CA and lysine (Lys) during the HOF-1 synthesis process.For control, CA@HOF-1 was also prepared by only adding CA in the HOF-1 synthesis.In advance of the discussion about CA@HOF-1 and CA@Lys-HOF-1, the chemical composition and physical structure of HOF-1 were characterized by FTIR, XRD and 13 C-NMR, which are shown in Supplementary Figures 2 and 3.
The topological structures of all samples were also examined by SEM and TEM.As shown in Figure 2 and Supplementary Figure 4, both CA@HOF-1 and CA@Lys-HOF-1 maintain the rod-like structure of HOF-1 with similar dimensions (around 300 nm wide).This indicates that the incorporation of CA and Lys did not alter the structure and size of HOF-1 during crystallization.As depicted in Figure 2C, some defects can be observed on the surface of CA@Lys-HOF-1, while CA@HOF-1 and HOF-1 remain intact.Figure 2D further shows that CA@HOF-1 and CA@Lys-HOF-1 well maintain the crystal structure of HOF-1 with the characteristic peaks at 2θ values of 8.6°, and 17.4°.The results of FTIR analysis of CA@HOF-1 and CA@Lys-HOF-1 are shown in Figure 2E.The absorption band centered at around 1030 cm -1 may be assigned to the N-H bending vibration of amide in Lys, indicating the introduction of Lys into HOF-1.To reveal the variation of surface area and pore size distribution of HOF-1 after modulation and enzyme embedding, the N 2 adsorption-desorption isotherms of HOF-1, CA@HOF-1, and CA@Lys-HOF-1 were examined [Supplementary Figure 5].As shown in Figure 2F and Table 1, CA@Lys-HOF-1 shows a larger pore size and broader size distribution, which may facilitate the transfer of CO 2 from the particle surface to CA. CA@Lys-HOF-1 also has a larger surface area due to the defects caused by Lys, which provides more sites for CO 2 conversion.Then, the CO 2 adsorption tests were performed and the results are shown in Figure 2G and Supplementary Figure 6.The CO 2 adsorption of CA@Lys-HOF-1 is higher when the CO 2 partial pressure is low, a result due to the relatively larger pore size.With an increase in CO 2 partial pressure, CA@Lys-HOF-1 reaches CO 2 adsorption saturation most quickly, mainly owing to the higher amount of -NH 2 with high CO 2 affinity on the particle surface.As the pressure continued to increase, the final amount of CO 2 adsorption of CA@Lys-HOF-1 was close to that of HOF-1 and CA@HOF-1.
Subsequently, EDS mapping was performed to further investigate whether enzymes were embedded in HOF-1.As shown in Figure 3, the distribution of C and N (the main elements in the two monomers of HOF-1) match the morphology of materials, which can also prove the successful synthesis of HOF-1.
Particularly, S (the characteristic element of CA) is uniformly dispersed in the two samples of CA@HOF-1 and CA@Lys-HOF-1, indicating the successful embedding of CA.

Catalytic activity of CA@Lys-HOF-1
In this work, five different types of (poly) amino acids [Supplementary Figure 7] were adopted to regulate the structure and catalytic performance of CA@HOF-1.Specifically, the hydrogen bonds in HOF-1 are formed between -NH 2 and -COOH of the two monomers, while amino acids are organic compounds containing both -NH 2 and -COOH in the molecule.Amino acids may occupy part of the hydrogen bond formation site, and interfere with the synthesis of HOF-1 through competitive ligand interaction, thus altering the pore size of HOF-1 and influencing the mass transfer.
When γ-poly-l-glutamic acid (PLGA) was introduced, its -COOH groups can rapidly bind to monomer 1, inhibiting the formation of HOF-1.[Supplementary Figure 8].This was probably due to the preferential combination of -COOH in PLGA and -NH 2 in monomer.Among the other four types of amino acidmodulated CA@HOF-1, the CA@Lys-HOF-1 exerted the highest activity [Supplementary Figures 9 and 10].
The catalytic activity of HOF-1, Lys-HOF-1, CA@HOF-1, and CA@Lys-HOF-1 were tested by dispersing them individually in solution for CO 2 conversion.The activity was reflected by detecting the pH change in the solution after the ventilation of CO 2 .The solution without catalyst samples was chosen as control, the Δ pH was measured in real-time, and the value at the highest point was chosen to reflect the catalyst activity.Figure 4A and B shows that the activity of CA@Lys-HOF-1 was enhanced.The higher activity of CA@Lys-HOF-1 may be attributed to the defects resulting from Lys, which promoted mass transfer.The -NH 2 groups in Lys were also introduced into the material, which fortified the affinity between the materials and CO 2 [Supplementary Figure 11].Furthermore, we investigated the effect of Lys concentrations on the activity.As seen in Figure 4C and D, moderate concentration of Lys is favorable for HOF-1 modulation, thus acquiring the most active CA@Lys-HOF-1.It should be noted that the results of Arg modulation are shown in Supplementary Figure 12, which also shows the same result.

Stability and CO 2 fixation ability of CA@Lys-HOF-1
Subsequently, the stability, reusability, and CO 2 fixation ability of CA@Lys-HOF-1 were investigated.As shown in Figure 5, Lys-HOF-1 exhibits better protection of CA at different pH values.Regarding the thermal stability, the protective ability of Lys-HOF-1 for CA began to decrease when the temperature was higher than 60 °C.This may be owing to the weakened strength of hydrogen bonds within the carrier material after Lys modification, which made Lys-HOF-1 more prone to decomposition at higher temperatures.The reusability of CA@Lys-HOF-1 was also evaluated for its importance in industrial applications.As shown in Figure 5C, CA@Lys-HOF-1 shows excellent recyclability.After the 8th cycle of reaction, CA@Lys-HOF-1 still maintained 83.7% of its initial activity, and demonstrated unaltered morphological and crystal structure [Supplementary Figure 13].
Finally, to examine the CO 2 fixation ability of CA@Lys-HOF-1, CO 2 was introduced at a flow rate of 25 mL min -1 , and the CaCO 3 mineralization reaction was performed after 5 min of ventilation.In detail, the reaction rates of the four samples, including HOF-1, Lys-HOF-1, CA@HOF-1, and CA@ Lys-HOF-1, were assessed by measuring the amount of CaCO 3 precipitate produced.As shown in Figure 5D, the CaCO 3 precipitate amount of HOF-1, Lys-HOF-1, CA@HOF-1, and CA@Lys-HOF-1 is, respectively, 4.81 mg, 6.29 mg, 10.88 mg, 12.26 mg.The production of CaCO 3 enabled by CA@Lys-HOF-1 was the highest, again validating the superiority of CA@Lys-HOF-1 in fortifying CO 2 fixation processes.Moreover, a hot filtration [38] experiment was performed over CA@Lys-HOF-1 for CO 2 mineralization.As shown in Supplementary Figure 14, no more increment in the production of CaCO 3 is observed after the filtration process, suggesting the heterogeneous nature of our catalytic system.

CONCLUSIONS
In summary, HOF-1 modulated by amino acids was synthesized through a coprecipitation method for CA immobilization.By regulating the type and concentration of introduced amino acids, CA@Lys-HOF-1 with optimized activity and desirable stability was obtained.Compared with unmodulated CA@HOF-1, the activity of CA@Lys-HOF-1 was enhanced by 71.3%, whereas the CO 2 fixation efficiency reflected by CaCO 3 production was enhanced by 12.7%.This could be ascribed to the large pore size of CA@Lys-HOF-1 that facilitated CO 2 transfer as well as the abundant surface -NH 2 groups that promoted CO 2 adsorption.Moreover, CA@Lys-HOF-1 maintained over 80% of the initial activity after the 8th cycle reaction.Our findings may pave the way for the immobilization of CA and other CO 2 -fixation enzymes.