Preparation and application of iron-based nitrogen-doped carbonaceous materials with carbonic anhydrase-like activity

Abstract

This invention discloses the preparation and application of an iron-based nitrogen-doped carbonaceous material with carbonic anhydrase-like activity. The invention employs a co-precipitation method and a high-temperature carbonization method to prepare the material Fe10@CN-Mg, in which most of the iron is uniformly distributed in the form of single atoms on the nitrogen-doped carbon, wherein FeN... x The synergistic catalytic effect of the active site and Mg(OH)2 nanorods exhibits carbonic anhydrase-like activity. This material has a low metal content and low preparation cost, yet it still possesses kinetic constants comparable to other reported CA-like nanozymes and an excellent CaCO3 formation rate. It also demonstrates excellent stability even under extreme pH, high temperature, organic solvents, and high ionic strength conditions, maintaining high activity even after long-term storage and multiple recycling. Therefore, it can be applied to catalyze the conversion of CO2 to cyclic carbonates with biological and pharmaceutical applications.

Description

Technical Field

[0001] This invention pertains to iron-based nitrogen-doped carbonaceous materials with carbonic anhydrase-like activity, specifically relating to the preparation and application of such materials. Background Technology

[0002] With increasing human demand for energy and fossil fuels, carbon dioxide (CO2) emissions have gradually become a serious climate problem. While methods such as physical adsorption and chemical sequestration have long been used to address the CO2 crisis, they generally suffer from drawbacks such as high cost, demanding equipment regeneration requirements, and secondary pollution. In contrast, carbonic anhydrase (CA)-catalyzed CO2 hydration has become an effective tool for CO2 fixation due to its high catalytic activity and selectivity under mild conditions. However, the inherent defects of CA, including instability, sensitivity, and high cost, severely limit its application in CO2 bioconversion. Nanozymes are a class of nanomaterials with natural enzyme activity, which can be prepared from noble metal oxides, non-noble metal oxides, and carbon materials. Due to their advantages of high cost-effectiveness, good stability, durability, recyclability, and tunable activity, nanozymes have seen rapid development in recent years. To address these issues, some CA-like nanozymes prepared from supramolecular, nanomaterial, MOF, and polymer materials have been extensively explored. Due to the inherent active center of natural hydroxylamine (CA), most CA-like nanozymes are prepared by coordinating zinc ions with azole derivatives, such as histidine Bola amphiphilic molecules (His-C7), aza-macrocyclic ligands, benzothiazolyl Schiff base ligands, triazoles, and some hydrophilic and hydrophobic tripod ligands, etc.

[0003] Furthermore, nanozymes with other metals as active sites and exhibiting CA-like activity have also been explored. For example, cobalt was used as the active center and loaded onto a porous organic polymer (Co-BBP) to form hybrid matrix membranes (MMMs), thereby achieving efficient CO2 capture. For instance, a molybdenum oxide nanocapsule was prepared to mimic CA activity, where empty coordination sites reserved for CO2 molecules and hydrogen bonds formed between CO2 molecules and water within the catalyst ensured successful CO2 hydration. For example, Ni NPs were prepared that can generate Ni(OH). x This increases the alkalinity of the reaction solution, thereby breaking the pH dependence during hydration.

[0004] However, the cumbersome preparation process, the use of expensive ligands, and the high metal loading requirements limit their application in sustainable industrial production. Therefore, the rational design of CA-like nanozymes with high catalytic efficiency and economic benefits remains worthy of exploration. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, this invention provides an iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity, which belongs to an iron-based nanoenzyme. The Fe10@CN-Mg prepared by this invention has excellent carbonic anhydrase-like activity, while solving the problems of cumbersome preparation process, high cost and secondary pollution in the prior art.

[0006] Another object of the present invention is to provide a method for preparing the iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity.

[0007] The third objective of this invention is to provide the application of the iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity in simulating carbonic anhydrase activity and carbon dioxide hydration and conversion.

[0008] Technical solution: In order to achieve the above objectives, the present invention provides an iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity. In the material, most of the iron is uniformly distributed on the nitrogen-doped carbon in the form of single atoms, and almost no or very small amount of iron exists in the form of particles. In addition, magnesium hydroxide exists in the form of nanorods and is distributed around the nitrogen-doped carbon.

[0009] FeN in the material x The synergistic catalytic effect of the active site and Mg(OH)2 nanorods resulted in excellent carbonic anhydrase-like activity.

[0010] The (Fe10@CN-Mg) is mainly prepared by co-precipitation and high-temperature carbonization.

[0011] The method for preparing iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity according to the present invention includes the following steps:

[0012] (1) Mix aniline, 2-methylimidazole and water and stir to obtain a homogeneous solution;

[0013] (2) Mix zinc nitrate hexahydrate, iron source and water evenly and then add them to the homogeneous solution in step (1);

[0014] (3) Stir the homogeneous mixture from step (2) to react;

[0015] (4) After centrifuging the reaction system of step (3), take the precipitate, wash and dry the precipitate;

[0016] (5) Take the dried precipitate and magnesium oxide from step (4) and stir them in an organic solvent to obtain a mixed solution. Then, evaporate the mixed solution to obtain a dried precursor powder.

[0017] (6) The dried precursor powder in step (5) is heated at high temperature in the absence of air to obtain black powder.

[0018] (7) The black powder in step (6) is etched, centrifuged to obtain the precipitate, the precipitate is washed and dried, and finally iron-based nitrogen-doped carbon-based material (Fe10@CN-Mg) with Mg(OH)2 nanorods is obtained.

[0019] In the preparation method, stirring is carried out at room temperature.

[0020] The molar ratio of aniline, 2-methylimidazole, zinc nitrate hexahydrate, and iron source is 40-50:40-50:10-15:1-2.

[0021] Preferably, the molar ratio of aniline, 2-methylimidazole, zinc nitrate hexahydrate, and iron source is 40:40:10:1.

[0022] Preferably, in step (1), the molar ratio of aniline to dimethylimidazole is 1:1 to 1:2, preferably 1:1; and the amount of water used is 80-100 mL, preferably 80 mL.

[0023] The iron source in step (2) is ferrous sulfate heptahydrate.

[0024] In step (3), the homogeneous mixture is stirred and reacted at room temperature for 3-6 hours.

[0025] Preferably, in step (3), the homogeneous mixture is stirred and reacted at room temperature for 4 hours.

[0026] In step (5), 2-3g of dried precipitate and 2-3g of magnesium oxide are stirred in 50-100mL of anhydrous ethanol at room temperature for 12-15h.

[0027] As a preferred embodiment, in step (5), 2g of the dried precipitate and 3g of magnesium oxide are stirred in 50mL of anhydrous ethanol at room temperature for 12h.

[0028] In step (6), the temperature is increased to 900-1000℃ at a rate of 5-10℃ / min and maintained at this temperature for 1-3 hours.

[0029] Preferably, in step (6), the temperature is increased to 900°C at a rate of 5°C / min and maintained at that temperature for 2 hours.

[0030] In step (7), the black powder is etched in HNO3 solution for 1-3 hours.

[0031] Preferably, the HNO3 in step (7) is 80 mL of HNO3 (1 M) solution, and the etching is performed for 2 hours.

[0032] In steps (4) and (7), the constant temperature drying temperature of the precipitate is 40-60℃, preferably 60℃.

[0033] The present invention relates to the application of the iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity in simulating carbonic anhydrase activity.

[0034] The iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg, which exhibits carbonic anhydrase-like activity, was used to evaluate its carbonic anhydrase-like activity. The process included the following steps: Based on the ester hydrolysis ability of carbonic anhydrase, the carbonic anhydrase-like activity of Fe10@CN-Mg was evaluated by colorimetric determination according to the colorimetric reaction of p-NPA (acetyl-p-nitrophenyl ester). After hydrolysis of colorless p-NPA, the absorption wavelength of the yellow product p-NP (p-nitrophenol) could be monitored at UV 402 nm. Finally, the concentration of the obtained p-NP was evaluated using the formula y = 0.0166x + 0.0226 obtained from the calibration curve of standard p-NP (where x represents the absorbance of p-NP and y represents the concentration of p-NP).

[0035] The present invention relates to the application of the iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity in carbon dioxide hydration and carbon dioxide value-added conversion.

[0036] The method applied to carbon dioxide hydration includes the following steps: carbon dioxide, Fe10@CN-Mg, and (50mM pH=8) PBS buffer solution are reacted with shaking at room temperature for 30 min. After the reaction, the supernatant is collected by centrifugation. An excess of saturated CaCl2 solution is introduced into the supernatant, and the mixture is stirred at room temperature for 1 h, then allowed to stand for 2 h. The precipitate is collected by centrifugation, dried, and weighed.

[0037] One method applied to carbon dioxide conversion includes the following steps: different epoxides are reacted with Fe10@CN-Mg, tetrabutylammonium bromide (TBAB), and toluene at 80°C with shaking for 8 hours. After the reaction, the yield is determined by GC-MS, and the products are then separated by column chromatography.

[0038] This invention uses ferrous sulfate heptahydrate as the iron source and prepares a dry precipitate at room temperature using a co-precipitation method. The dried precipitate is then reacted with magnesium oxide in anhydrous ethanol at room temperature for 12 hours to obtain a mixed solution. The mixed solution is then evaporated to dryness using a rotary evaporator to obtain a precursor powder. The precursor powder is pyrolyzed at high temperature to obtain a black powder. Finally, the black powder is etched in a certain amount of HNO3 solution, centrifuged to collect the precipitate, washed, and dried at a constant temperature to finally obtain an iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with Mg(OH)2 nanorods. In the catalytic hydrolysis reaction of acetyl-p-nitrobenzene (p-NPA), the hydrolysis product catalyzed by Fe10@CN-Mg has a high absorption peak at 402 nm, indicating that the Fe10@CN-Mg prepared in this invention has excellent carbonic anhydride-like activity. Its application in carbon dioxide hydration and conversion reactions showed that, under optimal stoichiometric conditions, Fe10@CN-Mg exhibited a Michaelis constant and maximum reaction rate comparable to previously reported superior carbonic anhydrase-like nanozymes. Furthermore, Fe10@CN-Mg also demonstrated the optimal calcium carbonate precipitation rate. These results indicate the superior catalytic efficiency of Fe10@CN-Mg and reveal extremely promising prospects for industrial applications.

[0039] The nanomaterials prepared in this invention have a low metal (iron) content (2.4141 wt.%) and low preparation cost (0.93 RMB / g), yet they still possess kinetic constants (K0.05) comparable to other reported CA-like nanozymes. m 6.37mM, V max It exhibits a high efficiency (30.74 mM / min) and an excellent CaCO3 formation rate (618 mg / h). This material demonstrates excellent stability even under extreme pH, high temperature, organic solvent, and high ionic strength conditions, maintaining high activity even after long-term storage (2 months) and 7 recycling cycles. Furthermore, Fe10@CN-Mg has been successfully applied to catalyze the conversion of CO2 to cyclic carbonates with biological and pharmaceutical applications.

[0040] The preparation of the material in this invention breaks through the design limitations of traditional carbonic anhydrase mimics that use metallic zinc as the active site, achieving for the first time the use of non-precious metal iron as the active center, thus exhibiting excellent carbonic anhydrase-like activity. Furthermore, the preparation cost of Fe10@CN-Mg is only 0.93 RMB / g, demonstrating excellent industrial application value, and solving the problems of cumbersome preparation processes, high costs, and secondary pollution in existing technologies.

[0041] This invention uses FeN xThe site and MgO are combined in a material to exert excellent CA-like activity. Effective CA-like nanozymes should contain both Lewis acidic sites and basic sites, and their excellent catalytic activity is demonstrated by simulating carbonic anhydrase-like activity.

[0042] This invention breaks away from the traditional design concept of using zinc as the catalytic center. For the first time, it uses iron, a non-precious metal, as the metal center and combines it with the strong theoretical adsorption capacity of MgO for CO2 to design an iron-based nanozyme with carbonic anhydrase-like activity.

[0043] This invention primarily aims to achieve efficient hydration and conversion of carbon dioxide by designing iron-based nanozymes with carbonic anhydrase-like activity. This invention overcomes the inherent limitations of natural carbonic anhydrases and provides new possibilities for exploring more types of carbonic anhydrase-like nanozymes with metal centers and low metal content.

[0044] The iron-based nanozyme prepared in this invention contains FeN x The site and Mg(OH)2 nanorods played a synergistic catalytic role. Although the entire material has a low metal content and low preparation cost, it still has kinetic constants and excellent CaCO3 formation rate comparable to other reported CA-like nanozymes.

[0045] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0046] (1) This invention prepares an iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity, in which iron single atoms anchor FeN in nitrogen-doped carbon. x The structure is similar to the active site of natural enzymes in terms of electronic, geometric, and chemical structure. Compared with previously reported superior carbonic anhydrase-like nanozymes, Fe10@CN-Mg exhibits excellent K... m (Mi constant) and V max (Maximum rate) indicates that Fe10@CN-Mg has superior carbonic anhydrase-like activity.

[0047] (2) The catalytic stability of Fe10@CN-Mg is better than that of natural carbonic anhydrase. It can function well under extreme pH values, high temperatures, high ionic strength, different organic solvents, different concentrations of ethanol solutions and long-term storage conditions, and can be reused at least 7 times.

[0048] (3) The preparation process of this invention is simple, does not require expensive ligands, and has a high metal loading but low loading, which can ensure its application in sustainable industrial production. The Fe10@CN-Mg of this invention can be applied to gram-scale reactions of carbon dioxide hydration and conversion, and exhibits excellent hydration capacity and conversion efficiency, with very broad prospects for industrial application. Attached Figure Description

[0049] Figure 1 This is a summary diagram of the present invention;

[0050] Figure 2 The flowchart shows the preparation process of Fe10@CN-Mg.

[0051] Figure 3 The standard curve for reference standard p-NP;

[0052] Figure 4 The graph shows the test results for simulating CA activity for different catalysts;

[0053] Figure 5 The graph shows the catalytic kinetics performance evaluation of Fe10@CN-Mg.

[0054] Figure 6 The graph shows the CA-like activity of the prepared Fe10@CN-Mg under different parameters;

[0055] Figure 7 Images showing the application of the prepared Fe10@CN-Mg to carbon dioxide hydration;

[0056] Figure 8 Images showing the application of the prepared Fe10@CN-Mg in carbon dioxide conversion;

[0057] Figure 9 It is a cyclic carbonate 3a 1 H NMR spectrum;

[0058] Figure 10 3b is a cyclic carbonate 1 H NMR spectrum;

[0059] Figure 11 Stability assessment of Fe10@CN-Mg and carbonic anhydrase under different test conditions.

[0060] Figure 12 The X-ray diffraction (XRD) pattern of the prepared Fe10@CN-Mg;

[0061] Figure 13 High-resolution transmission electron microscopy (HRTEM) images and mapping images of the prepared Fe10@CN-Mg;

[0062] Figure 14 Transmission electron microscopy (TEM) image of the prepared Fe10@CN-Mg;

[0063] Figure 15 The specific surface area (BET) diagram of the prepared Fe10@CN-Mg is shown.

[0064] Figure 16 Comparison of CO2 temperature-programmed desorption (CO2-TPD) between the prepared Fe10@CN-Mg and Fe10@CN;

[0065] Figure 17 The image shows the XPS analysis results of the prepared Fe10@CN-Mg. Detailed Implementation

[0066] The technical solution of the present invention will be further described below with reference to embodiments. Before introducing specific embodiments, the situation of some materials and experimental devices used in the present invention will be briefly described below.

[0067] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer. Ferrous sulfate heptahydrate and zinc nitrate hexahydrate (purchased from Sinopharm Chemical Reagent Co., Ltd., China); sodium chloride and anhydrous ethanol (purchased from Aladdin Reagent Co., Ltd., China); aniline (purchased from Sigma-Aldrich, Shanghai, China); 2-methylimidazole, magnesium oxide (MgO), tetrabutylammonium bromide (TBAB), acetyl-p-nitrophenyl ester (p-NPA) and p-nitrophenol (p-NP) (purchased from Maclean's Biochemical Co., Ltd., China). Carbonic anhydrase (CA) (purchased from Shanghai Yuanye Biotechnology Co., Ltd., 100U, China), and carbon dioxide (CO2) (purchased from Nanjing Yehao Gas Co., Ltd., China). All chemical reagents used in this study were analytical grade.

[0068] The experimental instruments involved include:

[0069] X-ray diffractometer (XRD), AMATLAB9 from Japan;

[0070] Transmission electron microscope (TEM), FEI Tecnai G2 F20 S-Twin;

[0071] BET surface area and pore size measurement, Micromeritics ASAP 2020 instrument;

[0072] Ultraviolet spectroscopy measurement, UV-1800PC ultraviolet spectrophotometer;

[0073] CO2-TPD, AutoChem 1II 2920;

[0074] XPS, ESCALAB 250Xi spectrometer (Thermo Scientific, USA).

[0075] Example 1

[0076] This embodiment briefly describes the preparation process of iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity. First, it should be noted that Fe10@CN-Mg in this application is mainly prepared using co-precipitation and high-temperature carbonization methods. Co-precipitation refers to the process where, when a solution contains two or more cations, all cations exist homogeneously in the solution. With the addition of a precipitant, a uniform precipitate of various components can be obtained after the precipitation reaction. High-temperature carbonization refers to the reaction process of heating and decomposing a solid under air-isolated conditions. The combination of these two methods has the advantages of simple preparation process, short synthesis cycle, and easy operation. During the reaction, the addition of aniline is beneficial for forming precipitates with smaller particle sizes, thereby obtaining a higher specific surface area; in addition, aniline, as a nitrogen source, can improve the dispersion of iron atoms, resulting in a more uniform system. The addition of magnesium oxide not only helps the catalyst form a porous structure but also gives the catalyst more basic sites. The iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity prepared in this invention has significant application prospects in the context of the era of "carbon peaking and carbon neutralization". Figure 1 ).

[0077] The iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity prepared in this example was prepared using the following steps (e.g.) Figure 2 ):

[0078] (1) Aniline (32 mmol, 2.92 mL), 2-methylimidazole (32 mmol, 2.62 g) and water (80 mL) were mixed at room temperature and stirred at 500 rpm to obtain a homogeneous solution;

[0079] (2) Mix zinc nitrate hexahydrate (8 mmol, 2.37 g), ferrous sulfate heptahydrate (0.8 mmol, 222.4 mg) and 80 mL of water thoroughly and add them to the homogeneous solution in step (1);

[0080] (3) Stir the homogeneous mixture in step (2) at 500 rpm for 4 h at room temperature;

[0081] (4) Centrifuge the reaction system of step (3) to collect the precipitate, and wash the precipitate; after washing, dry the precipitate at a constant temperature of 60°C.

[0082] (5) Take the dried precipitate (2g) and magnesium oxide (3g) from step (4) and stir them in anhydrous ethanol (50mL) solution at room temperature at a speed of 500rpm for 12h to obtain a mixed solution. Then, evaporate the mixed solution by rotary evaporator to obtain the dried precursor powder.

[0083] (6) The dried precursor powder from step (5) is isolated from air and heated in a furnace at a heating rate of 5°C / min from room temperature to 900°C, and held at this temperature for 2 hours to obtain a black powder.

[0084] (7) Etch all the black powder obtained in step (6) in 80 mL of 1 M HNO3 solution for 2 h, centrifuge to collect the precipitate, wash the precipitate with water until neutral, and dry it at 60 °C overnight to finally obtain iron-based nitrogen-doped carbon material (Fe10@CN-Mg) with Mg(OH)2 nanorods, that is, the iron-based nitrogen-doped carbon material with carbonic anhydrase-like activity of the present invention.

[0085] Comparative Example 1:

[0086] Preparation of Fe3@CN-Mg

[0087] (1) Aniline (32 mmol, 2.92 mL), 2-methylimidazole (32 mmol, 2.62 g) and water were mixed at room temperature and stirred vigorously to obtain a homogeneous solution;

[0088] (2) Mix zinc nitrate hexahydrate (6 mmol, 1.78 g), ferrous sulfate heptahydrate (2 mmol, 556 mg) and 80 mL of water evenly and then add them to the homogeneous solution in step (1);

[0089] (3) Stir the homogeneous mixture from step (2) at room temperature for 4 hours;

[0090] (4) Centrifuge the reaction system of step (3) to collect the precipitate, and wash the precipitate; after washing, dry the precipitate at a constant temperature of 60°C.

[0091] (5) Take the dried precipitate (2g) and magnesium oxide (3g) from step (4) and stir in anhydrous ethanol (50mL) solution at room temperature for 12h to obtain a mixed solution. Then, evaporate the mixed solution by rotary evaporator to obtain the dried precursor powder.

[0092] (6) The dried precursor powder from step (5) is isolated from air and heated in a furnace at a heating rate of 5°C / min from room temperature to 900°C, and held at this temperature for 2 hours to obtain a black powder.

[0093] (7) Etch all the black powder obtained in step (6) in 80 mL of 1 M HNO3 solution for 2 h, centrifuge to collect the precipitate, wash the precipitate with water until neutral, and dry it at 60 °C overnight to finally obtain iron-based nitrogen-doped carbonaceous material (Fe3@CN-Mg) with Mg(OH)2 nanorods.

[0094] Comparative Example 2:

[0095] Preparation of Fe20@CN-Mg

[0096] (1) Aniline (32 mmol, 2.92 mL), 2-methylimidazole (32 mmol, 2.62 g) and water were mixed at room temperature and stirred vigorously to obtain a homogeneous solution;

[0097] (2) Mix zinc nitrate hexahydrate (8 mmol, 2.37 g), ferrous sulfate heptahydrate (0.4 mmol, 111.2 mg) and 80 mL of water thoroughly and add them to the homogeneous solution in step (1);

[0098] (3) Stir the homogeneous mixture from step (2) at room temperature for 4 hours;

[0099] (4) Centrifuge the reaction system of step (3) to collect the precipitate, and wash the precipitate; after washing, dry the precipitate at a constant temperature of 60°C.

[0100] (5) Take the dried precipitate (2g) and magnesium oxide (3g) from step (4) and stir in anhydrous ethanol (50mL) solution at room temperature for 12h to obtain a mixed solution. Then, evaporate the mixed solution by rotary evaporator to obtain the dried precursor powder.

[0101] (6) The dried precursor powder from step (5) is isolated from air and heated in a furnace at a heating rate of 5°C / min from room temperature to 900°C, and held at this temperature for 2 hours to obtain a black powder.

[0102] (7) Etch all the black powder obtained in step (6) in 80 mL of 1 M HNO3 solution for 2 h, centrifuge to collect the precipitate, wash the precipitate with water until neutral, and dry it at 60 °C overnight to finally obtain iron-based nitrogen-doped carbonaceous material (Fe20@CN-Mg) with Mg(OH)2 nanorods.

[0103] Comparative Example 3:

[0104] Preparation of Fe10@CN

[0105] (1) Aniline (32 mmol, 2.92 mL), 2-methylimidazole (32 mmol, 2.62 g) and water (80 mL) were mixed at room temperature and stirred vigorously to obtain a homogeneous solution;

[0106] (2) Mix zinc nitrate hexahydrate (8 mmol, 2.37 g), ferrous sulfate heptahydrate (0.8 mmol, 222.4 mg) and 80 mL of water thoroughly and add them to the homogeneous solution in step (1);

[0107] (3) Stir the homogeneous mixture from step (2) at room temperature for 4 hours;

[0108] (4) Centrifuge the reaction system of step (3) to collect the precipitate, and wash the precipitate; after washing, dry the precipitate at a constant temperature of 60°C.

[0109] (5) The dried precipitate from step (4) is isolated from air and heated in a furnace at a heating rate of 5°C / min from room temperature to 900°C, and held at this temperature for 2 hours to obtain a black powder.

[0110] (6) The black powder from step (5) was etched in 80 mL of 1 M HNO3 solution for 2 h, centrifuged to collect the precipitate, and the precipitate was washed with water until neutral and dried at 60 °C overnight to finally obtain the iron-based nitrogen-doped carbonaceous material (Fe10@CN).

[0111] Comparative Example 4:

[0112] Preparation of CN-Mg

[0113] (1) Aniline (32 mmol, 2.92 mL), 2-methylimidazole (32 mmol, 2.62 g) and water (80 mL) were mixed at room temperature and stirred vigorously to obtain a homogeneous solution;

[0114] (2) Mix zinc nitrate hexahydrate (8 mmol, 2.37 g) and 80 mL of water thoroughly and then add it to the homogeneous solution in step (1);

[0115] (3) Stir the homogeneous mixture from step (2) at room temperature for 4 hours;

[0116] (4) Centrifuge the reaction system of step (3) to collect the precipitate, and wash the precipitate; after washing, dry the precipitate at a constant temperature of 60°C.

[0117] (5) Take the dried precipitate (2g) and magnesium oxide (3g) from step (4) and stir in anhydrous ethanol (50mL) solution at room temperature for 12h to obtain a mixed solution. Then, evaporate the mixed solution by rotary evaporator to obtain the dried precursor powder.

[0118] (6) The dried precursor powder from step (5) is isolated from air and heated in a furnace at a heating rate of 5°C / min from room temperature to 900°C, and held at this temperature for 2 hours to obtain a black powder.

[0119] (7) Etch all the black powder obtained in step (6) in 80 mL of 1 M HNO3 solution for 2 h, centrifuge to collect the precipitate, wash the precipitate with water until neutral, and dry it at 60 °C overnight to finally obtain nitrogen-doped carbonaceous material (CN-Mg) with Mg(OH)2 nanorods.

[0120] Comparative Example 5:

[0121] CN preparation

[0122] (1) Aniline (32 mmol, 2.92 mL), 2-methylimidazole (32 mmol, 2.62 g) and water (80 mL) were mixed at room temperature and stirred vigorously to obtain a homogeneous solution;

[0123] (2) Mix zinc nitrate hexahydrate (8 mmol, 2.37 g) and 80 mL of water thoroughly and then add it to the homogeneous solution in step (1);

[0124] (3) Stir the homogeneous mixture from step (2) at room temperature for 4 hours;

[0125] (4) Centrifuge the reaction system of step (3) to collect the precipitate, and wash the precipitate; after washing, dry the precipitate at a constant temperature of 60°C.

[0126] (5) The dried precipitate from step (4) is isolated from air and heated in a furnace at a heating rate of 5°C / min from room temperature to 900°C, and held at this temperature for 2 hours to obtain a black powder.

[0127] (6) The black powder in step (5) was etched in 80 mL of 1 M HNO3 solution for 2 h, centrifuged to collect the precipitate, washed with water until neutral, and dried at 60 °C overnight to finally obtain nitrogen-doped carbonaceous material (CN).

[0128] Example 2

[0129] Assessment of simulated carbonic anhydrase activity

[0130] To verify that the iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with Mg(OH)2 nanorods provided in this invention possesses optimal carbonic anhydrase-like activity, further comparative experiments were conducted using Fe10@CN-Mg prepared in Example 1 as a basis, and compared with Comparative Examples 1-4. The relevant experimental procedures are as follows. Based on the ester hydrolysis ability of carbonic anhydrase, the carbonic anhydrase-like activity of Fe10@CN-Mg was evaluated by colorimetric determination according to the colorimetric reaction of p-NPA (acetyl-p-nitrophenyl ester). After the colorless p-NPA hydrolysis, the absorption wavelength of the yellow product p-NP (p-nitrophenol) could be monitored at UV 402nm. In a typical experiment, 250 μL of Fe10@CN-Mg (1 mg / mL) aqueous solution, 100 μL of p-NPA (1 mM), and 2150 μL of PBS buffer solution (pH 7.4, 10 mM) were added sequentially to a 5 mL centrifuge tube. The above reaction solution was reacted at room temperature on a long-axis mixer for 30 minutes. Then, the supernatant was obtained by centrifugation (1000 rpm, 3 min), and further monitoring was performed at 402 nm. In addition, simulated enzyme activity experiments were conducted by replacing Fe10@CN-Mg with the products prepared in Comparative Examples 1-4. To facilitate the determination and evaluation of the activity of Fe10@CN-Mg simulating carbonic anhydrase, a linear relationship between p-NP absorbance and concentration was established using a spectrophotometer. Specifically, the absorbance values ​​of different concentrations of p-NP (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM) were measured at 402 nm, and a standard curve was plotted. Figure 3 Finally, the ability of the products of Fe10@CN-Mg and Comparative Examples 1-4 to catalyze the hydrolysis of p-NPA was evaluated using the formula y = 0.0166x + 0.0226 (where x represents the absorbance of p-NP and y represents the concentration of p-NP) obtained from the calibration curve of standard p-NP, thereby assessing the carbonic anhydrase activity of Fe10@CN-Mg and Comparative Examples 1-4. The results are as follows: Figure 4 As shown, Fe10@CN-Mg exhibits the best carbonic anhydrase-like activity.

[0131] Furthermore, the hydrolysis of p-NPA at different concentrations (0.025 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 3 mM) catalyzed by Fe10@CN-Mg prepared in Example 1 was investigated. According to the kinetic formula V = (V max ×[S]) / (K m +[S])(where [S] represents the concentration of the substrate, V represents the apparent initial reaction rate, V max K represents the maximum reaction rate. m The kinetic constant of Fe10@CN-Mg, calculated using the Michaelis constant, is V. max=30.74 mM / min, K m =6.37 mM. Compared to the best reported carbonic anhydrase mimic, Fe10@CN-Mg, although with a lower metal loading, still exhibits excellent kinetic constants ( Figure 5 ).

[0132] Example 3

[0133] Parameter experiments simulating carbonic anhydrase activity

[0134] To verify the effectiveness of the iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity provided by this invention in simulating carbonic anhydrase activity under different parameter considerations, further experimental verification was conducted based on the Fe10@CN-Mg prepared in Example 1. The relevant experimental procedures are as follows.

[0135] Based on the enzyme activity test in Example 2, the catalytic parameters of Fe10@CN-Mg were further investigated according to the method in Example 2, including the reaction time (0-30 min), the concentration of Fe10@CN-Mg (0.010, 0.050, 0.075, 0.100, 0.200 mg / mL), the concentration of substrate p-NPA (0.25, 0.5, 1.0, 2.0, 3.0 mM), and the effect of the UV absorption of p-NPA itself at 402 nm. Figure 6 The results showed that the hydrolysis of p-NPA by Fe10@CN-Mg was more complete over time. Furthermore, the higher the concentration of both the catalyst and the substrate, the faster the hydrolysis of p-NPA. The effect of the UV absorption of p-NPA itself at 402 nm was negligible.

[0136] Example 4

[0137] Hydration and transformation of carbon dioxide

[0138] To verify that the iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity provided by this invention has the best technical effect in carbon dioxide hydration compared with Comparative Examples 3-5, further experiments were conducted based on Fe10@CN-Mg prepared in Example 1 and Comparative Examples 3-5. The relevant experimental procedures are as follows.

[0139] To evaluate the CO2 hydration capacity of Fe10@CN-Mg and Comparative Example 3-5, CO2 gas was passed into 30 mL of PBS buffer solution (pH 8.0, 50 mM) containing 1 mg / mL Fe10@CN-Mg (or the product of Comparative Example 3-5) at a rate of 60 mL / min, and the reaction was continued at room temperature for 30 minutes. After the hydration process was completed, Fe10@CN-Mg was removed by centrifugation, and excess CaCl2 solution (4 M) was introduced into the supernatant. After stirring at room temperature for 1 hour and then standing for 2 hours, CaCO3 precipitation was observed. The obtained CaCO3 precipitate was collected by centrifugation and dried overnight under vacuum at 60 °C. The mass of CaCO3 was finally weighted and recorded. In addition, a blank control experiment without Fe10@CN-Mg and a Mg(OH)2 control experiment were also performed. The CaCO3 precipitates obtained under different catalytic conditions were statistically analyzed. Figure 7 The combined results of Fe10@CN-Mg and Comparative Examples 3-5 in the hydration of carbon dioxide further demonstrate that the Fe10@CN-Mg prepared in Example 1 plays a crucial role in catalyzing the hydration of CO2.

[0140] Furthermore, the conversion of CO2 was studied through the reaction of CO2 and epoxides, and the resulting cyclic carbonate compounds have wide applications in medicinal chemistry, biomedicine, and other fields. The specific experimental procedure was as follows: 0.1 mmol of epoxides (2-phenylethylene oxide, 2-(4-chlorophenyl)ethylene oxide, 2-(phenoxymethyl)ethylene oxide, or 2-[(2-propen-1-yloxy)methyl]ethylene oxide and 2-(butoxymethyl)ethylene oxide) were reacted with 10 mg of Fe10@CN-Mg (4.3 mmol% [Fe]) prepared in Example 1 and 0.5 equivalents of TBAB under a CO2 atmosphere at 80 °C for 8 hours. After completion, the reaction solution was diluted with ethyl acetate (6 mL) and filtered through a silica gel bed layered on diatomaceous earth (EtOAc: petroleum ether = 1:1). Then, volatiles were removed by rotary evaporation, and the yield of the crude product was determined by GC-MS. Figure 8 To further confirm the nature of the products, products 3a and 3b were selected for further 1H NMR characterization. Figure 9 and Figure 10 Furthermore, scaling up the above steps 100 times still yields a high yield (98%). These results demonstrate that Fe10@CN-Mg provides a feasible method for the conversion of CO2 into other important chemicals. Moreover, the success of the gram-scale experiments with epoxy compounds suggests the potential of Fe10@CN-Mg for industrial applications.

[0141] Example 5

[0142] Stability test

[0143] This invention investigates the stability of iron-anchored nitrogen-doped carbon material Fe10@CN-Mg to evaluate its performance under extreme conditions and compares it with carbonic anhydrase. Factors such as temperature, pH, organic solvent, ethanol concentration, ionic strength, storage time, and cycle number were investigated. Figure 11 Detailed enzyme activity measurement procedure: Before measuring enzyme activity, Fe10@CN-Mg and carbonic anhydrase were treated under different variable conditions before further use. For carbonic anhydrase, 100U of carbonic anhydrase was prepared by taking 1 mg of enzyme and adding it to 1 mL of ultrapure water.

[0144] In a 5 mL centrifuge tube, add 250 μL of Fe10@CN-Mg (1 mg / mL) or 250 μL of carbonic anhydrase solution (1 mg / mL) (both adjusted for the aforementioned variables of temperature, pH, organic solvent, ethanol concentration, ionic strength, storage time, and number of cycles), 100 μL of p-NPA (1 mM), and 2150 μL of buffer solution sequentially. Incubate the reaction solution at room temperature on a long-axis mixer for 30 minutes. Then, centrifuge (1000 rpm, 3 min) to obtain the supernatant and monitor it at 402 nm. The temperature was determined by incubating 250 μL of catalyst at different temperatures for two hours; the pH was determined by replacing the buffer solution with different pH buffer solutions; the organic solvent was determined by replacing the buffer solution with an organic solvent; and the ion concentration was determined by weighing out the corresponding NaCl solid and adding it to 250 μL of catalyst before incubation.

[0145] Temperature effect test:

[0146] Fe10@CN-Mg or carbonic anhydrase were incubated at different temperatures (37℃, 50℃, 80℃, 100℃, or 130℃) for 2 h, and then their catalytic activity was evaluated. Relative activity can be obtained by comparing with the activity at 37℃.

[0147] from Figure 11 (a) It can be seen that Fe10@CN-Mg maintains high activity at 130℃, while the activity of carbonic anhydrase (CA) begins to decrease significantly at 80℃, and even loses activity at 100℃. Therefore, Fe10@CN-Mg is an effective catalyst with better temperature tolerance than carbonic anhydrase.

[0148] Organic solvent effect test:

[0149] Fe10@CN-Mg or carbonic anhydrase were incubated at 37°C for 12 h in different organic solvents (methanol, DMF, or DMSO). Relative activities were determined by comparison with activities incubated in PBS buffer (pH 7.4).

[0150] from Figure 11 (b) It can be seen that after treatment with DMSO, DMF or MeOH, Fe10@CN-Mg still maintains high activity, while the activity of carbonic anhydrase CA is significantly reduced or even lost.

[0151] Ethanol concentration effect test:

[0152] Fe10@CN-Mg or carbonic anhydrase was incubated with different concentrations of ethanol (0%, 10%, 30%, 50%, or 60%) at room temperature for 2 hours. Relative activities were obtained by comparing the activities when incubated with 0% ethanol. Different ethanol volume ratios were added to reflect the effect of organic solvent content on the CA-like activity of carbonic anhydrase.

[0153] from Figure 11 (c) It can be seen that the activity of Fe10@CN-Mg increases significantly with the increase of ethanol concentration. However, high concentration of ethanol inhibits the activity of carbonic anhydrase, indicating that under the action of high concentration of ethanol, Fe10@CN-Mg has better stability and catalytic activity than carbonic anhydrase.

[0154] Ion strength effect test:

[0155] Fe10@CN-Mg or carbonic anhydrase were incubated at room temperature for 12 h in NaCl aqueous solutions of different concentrations (0 M, 0.1 M, 0.25 M, 0.5 M, or 1.0 M), and their catalytic activity was then evaluated. Relative activity was obtained by comparing the activity with that of NaCl aqueous solution at 0 M.

[0156] from Figure 11 (d) It can be seen that with the increase of NaCl concentration, the activity of carbonic anhydrase decreases rapidly or even disappears due to the influence of strong ionic strength on the charge distribution and spatial structure. However, with the increase of ion concentration, Fe10@CN-Mg can still maintain high catalytic activity and exhibit high ion tolerance.

[0157] Storage stability test:

[0158] Fe10@CN-Mg or carbonic anhydrase (1 mg / mL, 250 μL) was stored at room temperature, and its activity was measured every 7 / 10 / 20 days. Relative activity could be determined by comparison with the activity of fresh Fe10@CN-Mg or carbonic anhydrase.

[0159] from Figure 11 (e) It can be seen that Fe10@CN-Mg retained 89% of its activity after 2 months of storage, exhibiting more stable properties to water and oxygen. The activity of carbonic anhydrase decreased significantly over time and eventually lost all activity.

[0160] Loop testing:

[0161] Fe10@CN-Mg (1 mg / mL, 250 μL) and p-NPA (1 mM, 100 μL) were mixed with PBS buffer (pH 7.4, 10 mM, 2150 μL) and reacted at 25 °C for 30 min. After the reaction was complete, Fe10@CN-Mg was collected by centrifugation (10000 rpm, 5 min), washed three times with water, and then reused in the next reaction cycle.

[0162] from Figure 11 (f) It can be seen that Fe10@CN-Mg can still maintain 85% of its activity after being used seven times.

[0163] Example 6

[0164] Material characterization

[0165] The iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity prepared in Example 1 was characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM) morphology analysis, and specific surface area analysis (BET). Simultaneously, its simulated biological enzyme activity was analyzed. Figure 12 , Figure 13 and Figure 14 As can be seen from the figure, only a very small number of characteristic peaks of iron particles are present in the Fe10@CN-Mg material, indicating that the iron species in Fe10@CN-Mg may all exist in the form of single iron atoms. Furthermore, the distribution of Mg and O in HRTEM and mapping confirms that magnesium hydroxide exists in the form of nanorods and is distributed around nitrogen-doped carbon. Figure 15 As shown, Fe10@CN-Mg has a large specific surface area (140.9 m²). 2 / g). For example Figure 16 As shown in the figure, the carbon dioxide temperature-programmed desorption / resorption process of Fe10@CN-Mg and Fe10@CN indicates that Fe10@CN-Mg has more basic sites than Fe10@CN. For example... Figure 7 As shown, Fe10@CN-Mg exhibits excellent CO2 hydration capacity. For example... Figure 17 As shown, XPS peak segmentation confirms the presence of FeN in the material. x The existence of the site. For example... Figure 8 As shown, Fe10@CN-Mg exhibits excellent CO2-generating conversion capability. For example... Figure 11 As shown, Fe10@CN-Mg exhibits better stability compared to carbonic anhydrase.

[0166] Overall, the preparation process of the iron-based nitrogen-doped carbonaceous material (Fe10@CN-Mg) with carbonic anhydrase-like activity described in this invention is relatively mature and highly operable. Compared to natural carbonic anhydrase, Fe10@CN-Mg exhibits better stability because it can operate under extreme pH, high temperature, high ionic strength, organic solvents, different ethanol concentrations, and long storage times, and can be reused at least 7 times. Furthermore, it provides a new approach to solving the carbon dioxide crisis, demonstrating promising application prospects.

[0167] Example 7

[0168] Example 7 was prepared using the same method as Example 1, except that the ratio of aniline, 2-methylimidazole, zinc nitrate hexahydrate, and iron source was 50:50:15:2; the homogeneous mixture described in step (3) was stirred at 500 rpm for 3 hours at room temperature. In step (5), 3 g of dried precipitate and 2 g of magnesium oxide were stirred at 500 rpm for 15 hours at room temperature in 100 mL of anhydrous ethanol. In step (6), the temperature was increased to 1000°C at a rate of 10°C / min and maintained at that temperature for 1 hour.

[0169] Example 8

[0170] Example 8 was prepared using the same method as Example 1, except that the ratio of aniline, 2-methylimidazole, zinc nitrate hexahydrate, and iron source was 45:45:12:1.5; the homogeneous mixture described in step (3) was stirred at 500 rpm for 6 hours at room temperature. In step (5), 2.5 g of the dried precipitate and 2.5 g of magnesium oxide were stirred at 500 rpm for 14 hours at room temperature in 75 mL of anhydrous ethanol. In step (6), the temperature was increased to 950°C at a rate of 10°C / min and maintained at that temperature for 2 hours.

Claims

1. An iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity, characterized in that, Most of the iron in the material is uniformly distributed on the nitrogen-doped carbon in the form of single atoms, and a very small amount of iron exists in the form of particles. At the same time, magnesium hydroxide exists in the form of nanorods and is distributed around the nitrogen-doped carbon. The method for preparing the iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg includes the following steps: (1) Mix aniline, 2-methylimidazole and water and stir to obtain a homogeneous solution; (2) Mix zinc nitrate hexahydrate, iron source and water evenly and then add them to the homogeneous solution in step (1); (3) Stir the homogeneous mixture from step (2) to react; (4) Centrifuge the reaction system of step (3) to collect the precipitate, and wash and dry the precipitate; (5) Take the dried precipitate and magnesium oxide from step (4) and stir them in anhydrous ethanol to obtain a mixed solution. Rotate the mixed solution to dry the precursor powder. (6) The dried precursor powder in step (5) is heated at high temperature in the absence of air to obtain black powder. (7) The black powder in step (6) is etched, centrifuged to obtain the precipitate, the precipitate is washed and dried, and finally the iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity is obtained. The etching in step (7) is performed by etching in 80 mL of 1M HNO3 for 2 hours.

2. The iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity according to claim 1, characterized in that, In the preparation method described above, stirring is carried out at room temperature.

3. The iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity according to claim 1, characterized in that, The molar ratio of aniline, 2-methylimidazole, zinc nitrate hexahydrate, and iron source is 40-50:40-50:10-15:1-2.

4. The iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity according to claim 1, characterized in that, The iron source in step (2) is ferrous sulfate heptahydrate.

5. The iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity according to claim 1, characterized in that, The homogeneous mixture described in step (3) is stirred and reacted at room temperature for 3-6 h.

6. The iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity according to claim 1, characterized in that, In step (5), 2-3 g of dried precipitate and 2-3 g of magnesium oxide are stirred in 50-100 mL of anhydrous ethanol at room temperature for 12-15 h.

7. The iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity according to claim 1, characterized in that, In step (6), the high-temperature heating is carried out at a rate of 5-10 °C / min to 900-1000 °C, and the temperature is maintained at this temperature for 1-3 h.

8. The application of the iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity as described in claim 1 in simulating the activity of natural carbonic anhydrase.

9. The application of the iron-based nitrogen-doped carbonaceous material Fe10@CN-Mg with carbonic anhydrase-like activity as described in claim 1 in carbon dioxide hydration and carbon dioxide value-added conversion.

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