Hydrophilic super-macroporous polystyrene composite microsphere loaded with inorganic / organic hybrid cascade catalytic system and preparation method of hydrophilic super-macroporous polystyrene composite microsphere

Abstract

The invention discloses hydrophilic super-macroporous polystyrene (HGPS (at) MAF-7 (at) FDH) composite microspheres loaded with an inorganic / organic hybrid cascade catalytic system and a preparation method of the hydrophilic super-macroporous polystyrene (HGPS (at) MAF-7 (at) FDH) composite microspheres. The method comprises the following steps: coupling HGPS microspheres with an organic ligand triazole through surface epoxy modification, and reacting the HGPS microspheres with a metal ion solution to obtain a metal ion-loaded microsphere skeleton; then dispersing the framework material in a buffer solution, adding an enzyme solution and an organic ligand triazole, and carrying out a one-pot reaction to obtain the HGPS-coated MAF-7-coated FDH composite microsphere which is used as an inorganic / organic hybrid cascade catalytic system to catalyze CO2 conversion to generate formic acid. According to the method, the defects that a nano material is easy to aggregate and collapse and difficult to recover are effectively overcome, and the material has high specific surface area, excellent mass transfer performance, biocompatibility and mechanical stability. The characteristics of the MAF-7 organic ligand and the controllable porous structure are beneficial to adsorption of CO2 gas, and the MAF-7 not only can be used as a nano-enzyme for replacing carbonic anhydrase to catalyze a CO2 hydration reaction, but also can be used as a carrier for immobilizing formate dehydrogenase. According to the HGPS and MAF-7 and FDH composite microspheres, the complexity of a multi-enzyme cascade reaction is simplified, the stability and robustness of enzymes are improved, a substrate channel is shortened, the overall reaction efficiency is improved, and the HGPS and MAF-7 and FDH composite microspheres have excellent popularization prospects in the fields of CO2 bioconversion and wider cascade reaction system biocatalysis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immobilized enzyme, and relates to a hydrophilic supermacroporous polystyrene composite microsphere (HGPS@MAF@FDH) loaded with an inorganic / organic hybrid cascade catalytic system and a preparation method thereof. The composite microsphere takes a hydrophilic supermacroporous polystyrene microsphere (HGPS) as an organic carrier skeleton, realizes synchronous CO2 capture and formic acid conversion by in-situ growth of a metal organic framework (MAF-7) and immobilization of formate dehydrogenase (FDH), and solves the problems of easy deactivation of a cascade enzyme system, poor reusability and difficult recovery of a nano-immobilized enzyme material. BACKGROUND

[0002] Although greenhouse gases are necessary conditions for maintaining the habitable temperature of the earth, with the acceleration of industrialization and the large-scale use of fossil energy, the concentration of greenhouse gases in the atmosphere continues to rise, exceeding the self-regulation threshold of the natural environment, causing the heat capture capacity to abnormally increase, and further causing a series of serious global climate and environmental problems, such as global warming, sea level rise, ocean acidification, and ecological system destruction. Therefore, the emission of greenhouse gases is one of the most challenging environmental problems at present, and CO2 is the largest source of greenhouse gases, accounting for 75% of the total amount of greenhouse gas emissions. At present, the technical path for controlling and reducing the content of CO2 in the atmosphere mainly includes two categories: carbon capture and storage technology (CCS) and carbon conversion technology. Carbon conversion technology converts CO2 into valuable chemicals or fuels through chemical and biological means, which can achieve the dual goals of "emission reduction" and "resource utilization", and is more in line with the concept of sustainable development. In the carbon conversion technology system, enzyme catalytic conversion technology has more sustainability than traditional chemical catalysis due to its mild reaction, high selectivity and low energy consumption, and can convert CO2 into useful substances such as methanol, formic acid and low-carbon olefins.

[0003] The enzymatic conversion of CO2 involves a cascade of enzymes, and the carbonic anhydrase (CA) and formate dehydrogenase (FDH) coordinate the cascade to catalyze the synthesis of CO2 into formic acid, which is the first step and the key limiting step of CO2 sequestration and high-value utilization. However, the free enzymes in the cascade reaction have problems such as low stability, difficult recovery, and long mass transfer distance. Immobilization technology is an effective means to improve the stability and reusability of enzymes. The main methods of enzyme immobilization include covalent binding, cross-linking, adsorption and embedding. Among them, embedding method, which embeds enzymes in the three-dimensional network structure of polymer materials, not only maintains the possibility of enzyme molecules contacting with substrates, but also effectively prevents the loss of enzymes. Moreover, it is easy to operate and can maximize the retention of enzyme activity, becoming the preferred solution for the immobilization of enzyme cascade reaction system. In the multi-enzyme cascade reaction, the order of enzyme immobilization, the combination method of enzyme and immobilized material, the activity of enzyme and the mass transfer efficiency of substrate and intermediate product all affect the final conversion efficiency. At present, there is no satisfactory immobilized enzyme carrier to provide ideal and repeatable conversion conditions for the cascade reaction of CO2 catalytic conversion.

[0004] Among the many immobilized enzyme materials, metal-organic frameworks (MOFs) have become a new star of immobilized enzyme carriers due to their high specific surface area, adjustable pore size and stable chemical structure. MOFs materials are self-assembled by metal ions and organic ligands, and can immobilize enzymes through in-situ encapsulation, physical adsorption and cross-linking, showing excellent performance in maintaining enzyme activity and stability. MOFs materials can also act as nanoscale enzymes, combining the characteristics of nanomaterials and catalytic function. Among them, zinc-based imidazole MOFs have carbonic anhydrase (CA) activity and can be used as nanoscale enzyme to assist CO2 adsorption and hydration. MAF-7 material composed of zinc salt and triazole has better hydrophilicity and biocompatibility, good water phase dispersibility and controllable affinity with proteins. The nitrogen-containing base of MAF-7 can also promote the forward progress of CO2 hydration reaction, ensuring the efficiency of cascade catalysis, and is a nanoscale enzyme carrier and micro-reaction platform with CA enzyme activity and immobilization efficiency. However, nanomaterials themselves have problems such as easy aggregation and low mechanical properties, and are difficult to recycle, which limits their use as immobilized enzyme carriers.

[0005] Polystyrene materials have been widely used in the field of immobilized enzymes. In our previous work, we designed a hydrophilic and macroporous polystyrene (HGPS) microsphere (ZL 201310475774.5; Macromolecules, 2018, 51, 4085-4093) with a particle size of 10-600 μm and a macroporous pore size of 200-7000 nm. The porosity is 45-85%, and the specific surface area is 20-300 m 2HGPS microspheres, with their high hydrophilicity, good biocompatibility, rich hydroxyl content for easy derivatization, high mechanical strength, high mass transfer efficiency, and ease of recovery and separation, possess the advantages of being an excellent carrier for immobilized enzymes. In the preparation of HGPS microspheres, the self-made amphiphilic diblock glycosylated macromolecular polymer exhibits directional alignment. Hydrophobic segments chemically bond with the polystyrene backbone of the microspheres through polymerization, stably embedding within the backbone. Hydrophilic segments spontaneously align towards the water channels, ultimately positioning themselves on the outer surface of the microsphere pores. This provides modifiable groups for subsequent functionalization grafting, construction of micro / nanocarriers, and enzyme immobilization. For multi-enzyme reaction systems, the separation and orderly positioning of different enzymes to achieve substrate channel effects and significantly improve the final reaction yield is crucial. Direct immobilization of multiple enzymes using HGPS microspheres cannot achieve this requirement. Summary of the Invention

[0006] To address the existing problems in current research, this invention designs a novel inorganic / organic hybrid cascade catalytic immobilized enzyme composite microsphere system. The nanozyme MAF-7 and formate dehydrogenase FDH are used in a cascade to convert CO2 to formic acid, and the mixture is immobilized on HGPS microspheres. MAF-7 serves as both a CO2 hydration enzyme and a carrier for FDH. This design cleverly utilizes the characteristics of MAF-7: on the one hand, the nanozyme replaces the biological enzyme, exhibiting higher robustness and stability; on the other hand, the simplified cascade system optimizes substrate channels, shortens substrate transport distances, and reduces intermediate product inhibition. Immobilizing MAF-7 on HGPS microspheres ensures efficient mass transfer while overcoming the problems of easy aggregation, poor dispersion, and difficult recovery of nanomaterials, maintaining enzyme stability and reproducibility, and ultimately achieving higher catalytic efficiency.

[0007] The technical solution adopted in the invention is:

[0008] A hydrophilic, ultraporous polystyrene (HGPS@MAF@FDH) composite microsphere supported on an inorganic / organic hybrid cascade catalytic system and its preparation method, comprising the following steps:

[0009] (1) HGPS microspheres were prepared according to the methods described in the patents and articles previously published by the research group (ZL 201310475774.5; Macromolecules, 2018, 51, 4085-4093).

[0010] (2) The hydroxyl groups on the surface of hydrophilic macroporous polystyrene microspheres were epoxy activated using the Williamson etherification reaction (Colloids Surf. B ​​Biointerfaces, 2015, 129, 206-210.). 1 g of HGPS microspheres, 20 mL of dimethyl sulfoxide (DMSO), a certain amount of epichlorohydrin, and 10 mL of 0.1 M NaOH solution were added to a three-necked flask equipped with a mechanical stirrer and reacted at 500 rpm for 3 h in a 60 ºC water bath. After the reaction, the mixture was filtered and washed to remove residual NaOH, followed by washing several times with methanol to remove excess epichlorohydrin. Finally, the microspheres were dried under vacuum to obtain epoxy-activated A-HGPS microspheres.

[0011] (3) The A-HGPS microspheres from step (2) were modified with triazole functionalizing reagent. A-HGPS and 50 mL of 0.5 M triazole functionalizing reagent in methanol were added to a three-necked flask and reacted at 300 rpm for 3 h in a 60 ºC water bath. The mixture was washed with methanol until the pH was neutral and then dried under vacuum at room temperature for 24 h to obtain HGPS microspheres with triazole groups.

[0012] (4) The HGPS microspheres with triazole groups obtained in step (3) were ultrasonically dispersed in a metal salt solution for 30-100 min. Then, formate dehydrogenase (FDH) and triazole solution were added under stirring at room temperature to encapsulate the enzyme in situ in the crystal MAF-7@FDH. The crystal was then in situ heterogeneous nucleation and growth initiated by the triazole groups on the surface of the HGPS microspheres. After the reaction was completed, the microspheres were filtered, washed, and freeze-dried to obtain hydrophilic macroporous HGPS@MAF-7@FDH composite microspheres.

[0013] (5) Pour 150 mL of deionized water into a conical flask (250 mL), place it in an ice-water bath, and continuously bubble it into the conical flask through a CO2 gas bottle for 30 min. When the rate of escaping gas is basically the same as the rate of gas introduced, a saturated CO2 solution can be obtained. Under stirring conditions, add the HGPS@MAF-7@FDH composite microspheres obtained in step (4) into the conical flask containing the saturated CO2 solution, add coenzyme NADH and mix, and incubate in an air shaker at 25 ºC for 30-120 min at a speed of 100-200 rpm to obtain the formic acid product.

[0014] Preferably, in the epoxy activation reaction described in step (2), epichlorohydrin can be replaced with halogenated monoepoxy reagents such as epibromopropane, 1-chloro-2,3-epoxypropane, 3-chloro-1,2-epoxybutane, and 2,3-epoxy-1-iodopropane to achieve epoxy activation. Alternatively, epoxy reagents containing multiple epoxy groups such as 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, glycerol triglycidyl ether, and isocyanuric acid triglycidyl ester can be used to bind with the hydroxyl groups on the surface of the microspheres for epoxy activation.

[0015] Preferably, the triazole functionalizing agent described in step (3) is selected from at least one of 3-amino-1,2,4-triazole, 5-amino-1H-1,2,3-triazole, 4-amino-1,2,4-triazole, 1-(3-aminopropyl)-1H-1,2,3-triazole, 3-hydroxy-1,2,4-triazole, (3-aminopropyl)-4H-1,2,4-triazole, 5-mercapto-1H-1,2,3-triazole, 1-phenyl-5-amino-1H-1,2,3-triazole, and 4-(1H-1,2,3-triazole-1-yl)aniline.

[0016] Preferably, in step (3), the molar concentration of the triazole functionalizing reagent in the reaction system is 1-10 times the molar amount of epoxy groups on the surface of the microspheres. Different triazole functionalizing reagents react with HGPS microspheres under different conditions: for reagents containing amino groups, they can be reacted directly in a weakly alkaline system with pH 8-10 at a temperature of 40-80 °C for 1-6 h; for reagents containing hydroxyl groups, they need to be reacted in a polar aprotic solvent with strong base (NaOH, KOH, etc.) to catalyze the ring opening of the epoxy groups before reacting, at a temperature of 60-100 °C for 4-12 h; for reagents containing thiol groups, they can be reacted directly in a neutral to weakly alkaline system with pH 7-9 at a temperature of 30-60 °C for 0.5-3 h, and nitrogen gas is used for protection to prevent thiol group oxidation.

[0017] Preferably, the metal salt in step (4) is a zinc salt, selected from zinc nitrate, hydrochloride, sulfate and phosphate.

[0018] More preferably, the metal salt is zinc nitrate hexahydrate with a concentration range of 5-30 mM, the triazole functionalizing agent is 3-methyl-1,2,4-triazole with a concentration range of 10-60 mM, the molar ratio of the two is 1:(2-4), and the reaction temperature is 4-25 ℃.

[0019] Preferably, the ultrasonic power in step (4) is 300-600 W, the ultrasonic time is 10-40 min, the stirring temperature is 4 ℃ to room temperature, the stirring speed is 300-500 rpm, and the stirring time is 12h-48h.

[0020] The beneficial effects of this invention are as follows:

[0021] This invention effectively solves the problems of low loading rate, poor dispersibility, low mass transfer efficiency, and poor recyclability of existing enzyme immobilization carriers for CO2 bioconversion cascade reactions by designing HGPS@MAF-7@FDH composite microspheres to support an inorganic / organic hybrid cascade catalytic system. The hydrophilic, large-porous polystyrene microspheres ensure the mass transfer efficiency of the system and provide sufficient loading space, dispersibility, and recyclability for the MAF-7 nanomaterials. MAF-7 serves both as a carrier for immobilized FDH enzymes, ensuring high enzyme activity and reproducibility, and as a nanozyme mimicking carbonic anhydrase to perform CO2 hydration. The entire system simplifies the complexity of the cascade reaction, improves stability and robustness, shortens the substrate channel, and increases overall reaction efficiency. This invention offers a simple and mild technical method, providing a novel approach to the immobilization of cascade reaction systems and has broad application prospects in CO2 bioconversion and the wider field of biomanufacturing. Attached Figure Description

[0022] Figure 1 The image shows the SEM morphology of the HGPS@MAF@FDH composite microspheres in Example 1.

[0023] Figure 2 This is a comparison chart of pH stability of free FDH and immobilized FDH in Example 1;

[0024] Figure 3 This is a comparison chart of the temperature stability of free FDH and immobilized FDH in Example 1;

[0025] Figure 4 This is a comparison diagram of the mechanical shear stability of free FDH and immobilized FDH in Example 1;

[0026] Figure 5 The number of cycles and remaining activity of the immobilized FDH in Example 1;

[0027] Figure 6 This is a comparison of the CO2 conversion efficiency of the systems in Example 1 and the comparative example. Detailed Implementation

[0028] Example 1:

[0029] (1) HGPS microspheres (1.0 g) and 20 mL DMSO were added to a 250 mL three-necked flask equipped with a mechanical stirrer. The mechanical stirrer was turned on (500 rpm) to fully disperse the microspheres. The mixture was stirred at room temperature for 10 min. Under the mechanical stirring condition, epichlorohydrin (2 mL) and NaOH (0.1 M, 10 mL) were added sequentially. The mixture was heated to 60 °C and reacted at 500 rpm for 3 h. After filtration, the microspheres were washed with methanol until neutral, washed three times with water, and dried under vacuum at 60 °C to obtain epoxy-activated A-HGPS microspheres with an epoxy group content of 0.91-1.58 mmol / g.

[0030] (2) 50 mL of DMSO solution (pH=9) containing 3-amino-1,2,4-triazole (25 mmol / L, molar amount 3 times that of epoxy groups on the surface of microspheres) was added to a three-necked flask. A-HGPS microspheres (0.5 g) prepared in step (1) were taken and stirred at 60 °C and 120 rpm for 4 h using a constant temperature water bath and a top-mounted mechanical stirrer. After that, the mixture was filtered by Buchner funnel and washed three times with methanol solution. The washed microspheres were placed in a vacuum drying oven and dried for 24 h to obtain triazole-modified HGPS microspheres.

[0031] (3) Add 0.3 g of the triazole-modified HGPS microspheres obtained in step (2) to a three-necked flask, then add 30 mL of zinc nitrate hexahydrate solution (15 mM), sonicate at 300 W for 20 min, and then stir magnetically at 4 ℃ for 30 min; add formate dehydrogenase FDH (100 mg) and 30 mL of 3-methyl-1,2,4-triazole solution (30 mM) (Zn 2+ The ligand ratio was 1:2. The mixture was magnetically stirred at 400 rpm for 24 h at room temperature. After filtration, the microspheres were washed three times with water and freeze-dried for 12 h to obtain HGPS@MAF-7@FDH composite microspheres. The resulting composite microspheres had an average particle size of 480 μm, an average pore size of 1200 nm, a porosity of 60.7%, and a specific surface area of ​​55.6 m². 2 / g, scanning electron microscope image see Figure 1 .

[0032] (4) Add 150 mL of ultrapure water to a 250 mL Erlenmeyer flask, and purge with CO2 for 30 min to obtain a CO2-saturated aqueous solution. Mix 0.1 g of HGPS@MAF-7@FDH composite microspheres and 100 μL of coenzyme NADH (3 mg / mL), add CO2-saturated aqueous solution to 2 mL, adjust the pH of the system to 6.5, and incubate at 25 ℃ and 150 rpm for 60 min with shaking. Detect the immobilized enzyme activity and formic acid yield. The loading of FDH to the microspheres was 40.75 mg / g. The immobilized FDH content remained above 75% within the pH range of 5-9. See [link to relevant documentation]. Figure 2 The temperature stability of immobilized FDH after incubation at 50-70℃ for 30 min was more than 25% higher than that of free FDH. (See...) Figure 3 .

[0033] Example 2:

[0034] (1) HGPS microspheres (1.0 g) and 20 mL DMSO were added to a 250 mL three-necked flask equipped with a mechanical stirrer. The mechanical stirrer was turned on (500 rpm) to fully disperse the microspheres. The mixture was stirred at room temperature for 10 min. Then, under mechanical stirring, 2 mL of epoxypropane and 10 mL of NaOH (0.1 M) were added sequentially. The mixture was heated to 60 °C and reacted at 500 rpm for 3 h. After filtration, the microspheres were washed with methanol until neutral, washed three times with water, and dried under vacuum at 60 °C to obtain epoxy-activated A-HGPS microspheres. The epoxy group content of the obtained epoxy microspheres was 0.25-0.6 mmol / g.

[0035] (2) 50 mL of DMSO solution (pH=8.5) containing 5-amino-1H-1,2,3-triazole (25 mmol / L) was added to a three-necked flask. The A-HGPS microspheres (0.5 g) prepared in step (1) were taken and stirred at 60 °C and 120 rpm for 4 h using a constant temperature water bath and a top-mounted mechanical stirrer. The remaining liquid was then filtered through a Buchner funnel and washed three times with methanol solution. The washed microspheres were then placed in a vacuum drying oven and dried for 24 h to obtain triazole-modified HGPS microspheres.

[0036] (3) Add 0.3 g of the triazole-modified HGPS microspheres obtained in step (2) to a three-necked flask, then add 30 mL of 10 mM zinc nitrate hexahydrate solution, sonicate at 300 W for 20 min, and then pre-treat with magnetic stirring at 4 ℃ for 30 min; add formate dehydrogenase FDH (120 mg) and 30 mL of 5-amino-1H-1,2,3-triazole solution (30 mM) (Zn 2+The ligand ratio was 1:3. The mixture was magnetically stirred at 400 rpm for 24 h at room temperature. After filtration, the microspheres were washed three times with water and freeze-dried for 12 h to obtain composite microspheres. The resulting composite microspheres had an average particle size of 450 μm, an average pore size of 920 nm, a porosity of 57.7%, and a specific surface area of ​​45.8 m². 2 / g.

[0037] (4) Add 150 mL of ultrapure water to a 250 mL Erlenmeyer flask, and purge with CO2 for 30 min to obtain a CO2-saturated aqueous solution. Mix 0.1 g of HGPS@MAF-7@FDH composite microspheres and 100 μL of coenzyme NADH (3 mg / mL), add CO2-saturated aqueous solution to 2 mL, adjust the pH of the system to 6.8, and incubate at 25℃ and 150 rpm for 60 min with shaking. Detect the immobilized enzyme activity and formic acid production. The loading of FDH to the microspheres was 25.36 mg / g. The immobilized FDH maintained above 74% in the pH range of 5-9. After incubation at 50℃-70℃ for 30 min, the temperature stability of the immobilized FDH was more than 20% higher than that of free FDH.

[0038] Example 3:

[0039] (1) HGPS microspheres (1.0 g) and DMSO solution (20 mL) were added to a 250 mL three-necked flask equipped with a mechanical stirrer. The mechanical stirrer was turned on (500 rpm) to fully disperse the microspheres. The mixture was stirred at room temperature for 10 min. Under the mechanical stirring condition, 3-chloro-1,2-epoxybutane (2.2 mL) and NaOH (0.1 M, 10 mL) were added sequentially. The mixture was heated to 60 °C and reacted at 500 rpm for 3 h. After filtration, the microspheres were washed with methanol until neutral, washed three times with water, and dried under vacuum at 60 °C to obtain epoxy-activated A-HGPS microspheres. The epoxy group content of the obtained epoxy microspheres was 0.95-1.26 mmol / g.

[0040] (2) 50 mL of DMSO solution (pH=9) containing 1-(3-aminopropyl)-1H-1,2,3-triazole (30 mmol / L) was added to a three-necked flask. A-HGPS (0.5 g) prepared in step (1) was taken and stirred at 70 °C and 120 rpm for 4 h using a constant temperature water bath and a top-mounted mechanical stirrer. The remaining liquid was then filtered through a Buchner funnel and washed three times with methanol solution. The washed microspheres were then placed in a vacuum drying oven and dried for 24 h to obtain triazole-modified HGPS microspheres.

[0041] (3) Add 0.3 g of the triazole-modified HGPS obtained in step (2) to a three-necked flask, then add 30 mL of zinc nitrate hexahydrate solution (10 mM), sonicate at 300 W for 20 min, and then pretreat with magnetic stirring at 4 ℃ for 30 min; add formate dehydrogenase FDH (180 mg) and 30 mL of 3-ethyl-1,2,4-triazole solution (30 mM) (Zn 2+ The ligand ratio was 1:3, and the mixture was magnetically stirred at 400 rpm for 24 h at room temperature. After filtration, the microspheres were washed three times with water and freeze-dried for 12 h to obtain composite microspheres. The resulting composite microspheres had an average particle size of 450 μm, an average pore size of 1070 nm, a porosity of 65.8%, and a specific surface area of ​​50.3 m². 2 / g.

[0042] (4) Add 150 mL of ultrapure water (solvent) to a 250 mL Erlenmeyer flask, and purge with CO2 for 30 min to obtain a CO2-saturated aqueous solution. Mix 0.1 g of HGPS@MAF-7@FDH composite microspheres and 100 μL of coenzyme NADH (3 mg / mL), add CO2-saturated aqueous solution to 2 mL, adjust the pH of the system to 7.0, and incubate at 25 ℃ and 150 rpm for 60 min with shaking. Detect the immobilized enzyme activity and formic acid product yield. The loading of FDH to the microspheres was 38.69 mg / g. The immobilized FDH maintained above 70% in the pH range of 5-9. After incubation at 50℃-70℃ for 30 min, the temperature stability of the immobilized FDH was more than 21% higher than that of free FDH.

[0043] Example 4:

[0044] (1) HGPS microspheres (1.0 g) and 20 mL DMSO were added to a 250 mL three-necked flask equipped with a mechanical stirrer. The mechanical stirrer was turned on (500 rpm) to fully disperse the microspheres. The mixture was stirred at room temperature for 10 min. Under the mechanical stirring condition, 2,3-epoxy-1-iodopropane (1.8 mL) and NaOH (0.1 M, 10 mL) were added sequentially. The mixture was heated to 60 °C and reacted at 500 rpm for 3 h. After filtration, the microspheres were washed with methanol until neutral, washed three times with water, and dried under vacuum at 60 °C to obtain epoxy-activated A-HGPS microspheres. The epoxy group content of the obtained epoxy microspheres was 0.52-0.83 mmol / g.

[0045] (2) 50 mL of DMSO solution containing 3-hydroxy-1,2,4-triazole (40 mmol / L), 0.1 M NaOH, and A-HGPS (0.5 g) prepared in step (1) were added to a three-necked flask. The mixture was stirred at 80 °C and 120 rpm for 4 h using a constant temperature water bath and a top-mounted mechanical stirrer. The remaining liquid was then filtered through a Buchner funnel and washed three times with methanol solution. The washed microspheres were then placed in a vacuum drying oven and dried for 24 h to obtain triazole-modified HGPS microspheres.

[0046] (3) Add 0.3 g of the triazole-modified HGPS obtained in step (2) to a three-necked flask, then add 30 mL of zinc nitrate hexahydrate solution (10 mM), sonicate at 300 W for 20 min, and then pretreat by magnetic stirring at 4 ℃ for 30 min; add formate dehydrogenase FDH (240 mg) and 30 mL of 1-methyl-1,2,4-triazole (35 mM) (Zn 2+ The ligand ratio was 1:3.5, and the mixture was magnetically stirred at 400 rpm for 24 h at room temperature. After filtration, the microspheres were washed three times with water and freeze-dried for 12 h to obtain HGPS@MAF-7@FDH composite microspheres. The obtained composite microspheres had an average particle size of 500 μm, an average pore size of 955 nm, a porosity of 61.2%, and a specific surface area of ​​52.2 m². 2 / g.

[0047] (4) Add 150 mL of ultrapure water to a 250 mL Erlenmeyer flask and purge with CO2 for 30 min to obtain a CO2-saturated aqueous solution. Mix 0.1 g of HGPS@MAF-7@FDH composite microspheres and 100 μL of coenzyme NADH (3 mg / mL), and add CO2-saturated aqueous solution to 2 mL. Adjust the pH of the system to 7.0, and incubate at 25 ℃ and 150 rpm for 60 min with shaking. Detect the immobilized enzyme activity and formic acid product yield. The loading of FDH to the microspheres was 36.85 mg / g. The immobilized FDH content remained above 70% within the pH range of 5-9. After incubation at 50℃-70 ℃ for 30 min, the temperature stability of the immobilized FDH was more than 19% higher than that of free FDH.

[0048] Example 5:

[0049] (1) HGPS microspheres (1.0 g) and 20 mL DMSO were added to a 250 mL three-necked flask equipped with a mechanical stirrer. The mechanical stirrer was turned on (500 rpm) to fully disperse the microspheres. The mixture was stirred at room temperature for 10 min. Under the mechanical stirring condition, 1-chloro-2,3-epoxypropane (2 mL) and NaOH (0.1 M, 10 mL) were added sequentially. The mixture was heated to 60 °C and reacted at 500 rpm for 3 h. After filtration, the microspheres were washed with methanol until neutral, washed three times with water, and dried under vacuum at 60 °C to obtain epoxy-activated A-HGPS microspheres. The epoxy group content of the obtained epoxy microspheres was 0.59-1.18 mmol / g.

[0050] (2) The three-necked flask reaction vessel was protected with nitrogen for 30 min. Then, 50 mL of DMSO solution (pH=8) containing 5-mercapto-1H-1,2,3-triazole (25 mmol / L) was added to the three-necked flask. A-HGPS (0.5 g) prepared in step (1) was taken and stirred at 50 ℃ and 120 rpm for 4 h using a constant temperature water bath and a top-mounted mechanical stirrer. The remaining liquid was then filtered with a Buchner funnel and washed three times with methanol solution. The washed microspheres were then placed in a vacuum drying oven and dried for 24 h to obtain triazole-modified HGPS microspheres.

[0051] (3) Add 0.3 g of the triazole-modified HGPS obtained in step (2) to a three-necked flask, then add 20 mL of zinc nitrate hexahydrate solution (10 mM), sonicate at 300 W for 20 min, and then pre-treat with magnetic stirring at 4 ℃ for 30 min; add formate dehydrogenase FDH (300 mg) and 30 mL of 5-amino-3-methyl-1,2,4-triazole (60 mM) (Zn 2+ The ligand ratio was 1:4. The mixture was mechanically stirred at 400 rpm for 24 h at room temperature. After filtration, the microspheres were washed three times with water and freeze-dried for 12 h to obtain composite microspheres. The resulting composite microspheres had an average particle size of 460 μm, an average pore size of 1130 nm, a porosity of 59.7%, and a specific surface area of ​​35.6 m². 2 / g.

[0052] (4) Add 150 mL of ultrapure water to a 250 mL Erlenmeyer flask and purge with CO2 for 30 min to obtain a CO2-saturated aqueous solution. Mix 0.1 g of HGPS@MAF-7@FDH composite microspheres and 100 μL of coenzyme NADH (3 mg / mL), and add CO2-saturated aqueous solution to 2 mL. Adjust the pH of the system to 8.0, and incubate at 25 ℃ and 150 rpm for 60 min with shaking. Detect the immobilized enzyme activity and formic acid product yield. The loading of FDH to the microspheres was 35.52 mg / g. The immobilized FDH content remained above 68% within the pH range of 5-9. After incubation at 50℃-70℃ for 30 min, the temperature stability of the immobilized FDH was more than 17% higher than that of free FDH.

[0053] Example 6:

[0054] The specific detection and characterization methods of this invention are as follows:

[0055] (1) Morphology and structure of the HGPS@MAF-7@FDH microsphere system:

[0056] We used SEM to characterize the morphology and structure of the immobilized enzyme vector HGPS@MAF-7@FDH, and the results are as follows: Figure 1 As shown, the well-formed spherical surface of HGPS@MAF-7@FDH exhibits the crystal structure of MAF-7, and pores can be observed on its surface, indicating that HGPS@MAF-7@FDH was successfully synthesized.

[0057] (2) Determination of immobilized enzyme loading:

[0058] The obtained FDH immobilized enzyme mixture was centrifuged at 4 ℃ and 8000 rpm for 10 min. The supernatant was used as the test solution, and the amount of immobilized enzyme was determined by the Coomassie Brilliant Blue method. This method measures the optical adsorption of the supernatant (free enzyme) at 595 nm, and the amount of enzyme is calculated using bovine serum albumin as a standard. The FDH immobilization efficiency is calculated by the following formula:

[0059]

[0060] (3) FDH enzyme activity assay:

[0061] Take 1.3 mL (10 mg / mL) of NaHCO3 (dissolved in PBS buffer, pH 6), add 100 mg of immobilized enzyme and 100 μL of NADH, react at 25 °C for 30 min, and measure the change in absorbance at 340 nm to reflect the amount of NADH consumed.

[0062] The formula for calculating FDH enzyme activity is as follows:

[0063]

[0064] Where ΔAbs represents the change in absorbance, Δt represents the reaction time (min), V represents the reaction volume (mL), Ɛ is the molar extinction coefficient, and d is the grating diameter.

[0065] (4) Determination of pH stability of immobilized enzymes:

[0066] Different buffer solutions with pH gradients of 3, 4, 5, 6, 7, 8, 9, 10, and 11 were prepared, with a NaHCO3 substrate concentration of 10 mg / mL. Based on these buffers, the enzyme activities of free FDH and immobilized FDH at different pH gradients were measured. The ratio of the activity to the highest activity was used as the relative enzyme activity, expressed as a percentage. Results were obtained from... Figure 2 As shown, the optimal pH for both free and immobilized FDH is 6. The activity of the free enzyme rapidly decreases to below 80% when the pH of the reaction system deviates from the optimal pH value, while the immobilized enzyme can maintain above 75% in the pH range of 5-9, and is particularly stable under alkaline conditions. This indicates that the immobilized FDH is more stable and suitable for CO2 absorption scenarios.

[0067] (5) Determination of the heat resistance of immobilized enzymes:

[0068] Temperature gradients of 30, 40, 50, 60, and 70 °C were set, with a NaHCO3 substrate concentration of 10 mg / mL. Reactions were performed for 30 min at each temperature, and the activities of free and immobilized FDH were measured. The ratio of enzyme activity at different temperature gradients to the highest enzyme activity was expressed as relative enzyme activity as a percentage. Results are presented by... Figure 3 It can be seen that the optimal reaction temperature for both free FDH and immobilized FDH is 30 ℃. As the temperature increases, the activity of free FDH enzyme decreases rapidly, reaching only 20% at 70 ℃. In contrast, the activity of immobilized FDH is higher than that of the free enzyme at the same temperature, especially above 50 ℃, where it is more than 25% higher than the free enzyme, indicating that the immobilization system improves the enzyme's heat resistance.

[0069] (6) Determination of the mechanical stability of the microsphere system:

[0070] Considering that continuous bubbling is required when CO2 is used as a substrate in cascade catalysis, which generates shear force that affects microspheres and enzyme activity, potentially leading to microsphere breakage or enzyme inactivation, we used varying magnetic stirring speeds in the reaction system to generate shear force and measured enzyme activity at different stirring speeds (0 rpm, 500 rpm, 1000 rpm, 1500 rpm, and 2000 rpm). The ratio of each measured enzyme activity to the initial enzyme activity was used as the relative enzyme activity. The results are as follows: Figure 4The free enzyme rapidly loses its activity under increased shear force, retaining only 30% at 1500 rpm. However, the enzyme immobilized in the HGPS@MAF-7@FDH system has a certain mechanical strength and can withstand greater shear force, maintaining more than 80% of its activity at 2000 rpm.

[0071] (7) Determination of repeated use effect:

[0072] After activity measurement, HGPS@MAF-7@FDH was filtered and washed three times with deionized water. The activity of the immobilized FDH after recycling was measured. This cycle was repeated 10 times, and the ratio of the enzyme activity measured each time to the enzyme activity measured in the first cycle was taken as the relative enzyme activity. The results are presented by... Figure 5 As shown, the enzyme activity decreased relatively steadily in the first two cycles, and remained at around 80% until the fifth cycle, indicating that HGPS@MAF-7@FDH can be used stably and repeatedly, significantly improving the overall yield.

[0073] (8) Formic acid yield determination:

[0074] First, prepare standard solution A—trans-aconitine: dissolve 0.1 g of trans-aconitine and 2 g of sodium acetate in 20 mL of isopropanol to prepare solution A. Prepare standard solution B: dissolve 6 g of sodium acetate in 20 mL of deionized water to prepare solution B. Take 0.5 mL of formic acid standard working solutions with concentrations of 4 mM, 3 mM, 2 mM, and 1 mM, respectively, and add 1 mL of solution A, 40 μL of solution B, and 3.5 mL of acetic anhydride. Heat in a water bath at 50 ℃ for 30 min to allow for color development. Measure the absorbance at 515 nm for each formic acid concentration. Plot a formic acid standard curve with formic acid concentration (mM) on the x-axis and absorbance on the y-axis. Then, 0.5 mL of the reaction solution from the comparative and experimental groups after incubation for 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h were heated in a water bath at 50 °C for 30 min with 1 mL of solution A, 40 μL of solution B, and 3.5 mL of acetic anhydride to carry out the colorimetric reaction. The absorbance of each concentration of formic acid at 515 nm was then measured and substituted into the formic acid standard curve to calculate the formic acid content.

[0075] The results showed that the composite microsphere HGPS@MAF-7@FDH system could stably increase the formic acid content, and the formic acid yield gradually increased to 8 mM with the extension of reaction time. Figure 6 ).

[0076] Comparative example:

[0077] (1) 0.3 g of hydrophilic macroporous HGPS microspheres were added to 40 mL of formate dehydrogenase solution (FDH, 2.5 mg / mL, prepared with PBS buffer). The mixture was mechanically stirred at 500 rpm for 48 h at room temperature. After filtration, the microspheres were washed three times with water and freeze-dried for 12 h to obtain FDH-loaded HGPS microspheres HGPS@FDH. The obtained FDH-loaded microspheres had an average particle size of 460 μm, a pore size of 2200 nm, a macropore size of 1.5 μm, a porosity of 75.9%, and a specific surface area of ​​82.3 m². 2 .

[0078] (2) Add 150 mL of ultrapure water (solvent) to a 250 mL conical flask and purge with CO2 for 30 min until saturated. Then add 0.1 g of HGPS microspheres HGPS@FDH that adsorb FDH, 100 μL of coenzyme NADH (3 mg / mL, prepared with PBS buffer) and CO2 saturated aqueous solution to bring the reaction system to a final volume of 2 mL. Adjust the pH of the system to 7.0 and incubate at 25 °C and 150 rpm for 60 min. It was found that no MAF-7 was produced on the HGPS microspheres. After 6 h of reaction, less than 1 mM of formic acid was detected.

[0079] Effect Experiment:

[0080] To verify the catalytic effect of the HGPS@MAF-7@FDH system of the present invention on the production of formic acid from CO2, the catalytic effects of the HGPS@MAF-7@FDH prepared in Example 1 and the HGPS@FDH prepared in the comparative example were compared. Figure 6 It can be seen that with the extension of reaction time, the formic acid concentration of the composite microspheres HGPS@MAF-7@FDH gradually increased to 8 mM, while the formic acid concentration of the comparative HGPS@FDH microspheres was less than 1 mM. The experimental results indicate that HGPS microspheres immobilized with FDH alone cannot effectively catalyze CO2 due to the lack of calcium carbonate enzyme activity. The presence of MAF-7 nanozyme can both promote CO2 hydration and cascade formic acid dehydrogenase, thus catalyzing the conversion of bicarbonate ions to formic acid. The complete HGPS@MAF-7@FDH system can more efficiently catalyze the conversion of carbon dioxide to formic acid.

[0081] Experimental results show that the HGPS@MAF-7@FDH composite material constructed in this invention can simultaneously resolve the contradictions between carrier loading rate, dispersibility, mass transfer efficiency and recyclability, breaking through the limitations of traditional carrier materials and providing a new approach for the high-value utilization of CO2 and the construction of immobilized carriers for more multi-enzyme cascade reaction systems.

Claims

1. Hydrophilic superporous polystyrene composite microspheres (HGPS@MAF-7@FDH) loaded with inorganic / organic hybrid cascade catalytic system and a preparation method thereof, characterized in that Comprise the following steps: (1) using an epoxy reagent to activate the hydrophilic super macroporous polystyrene (HGPS) microspheres by Williamson etherification reaction, after the reaction, remove the residual NaOH by filtration and washing, then wash with methanol for several times, vacuum drying, to obtain epoxy-activated A-HGPS microspheres; the particle size of the HGPS microspheres ranges from 10 to 600 μm, the pore size is 200-7000 nm, the porosity is 45-85%, and the specific surface area is 20-300 m 2 / g. (2) The surface of the A-HGPS microspheres in step (1) is modified by using a triazole functionalization reagent, and the A-HGPS and the triazole functionalization reagent are reacted, then the pH value is neutralized by washing with methanol, and the mixture is vacuum dried at room temperature for 24 hours to obtain HGPS microspheres coupled with triazole groups; (3) The HGPS microspheres coupled with triazole organic ligands obtained in step (3) are ultrasonically dispersed in a metal ion solution, formic acid dehydrogenase (FDH) and a triazole solution are added, the FDH is in-situ encapsulated in the crystal MAF-7 to obtain MAF-7@FDH, and the MAF-7 crystals on the surface and in the pores of the HGPS microspheres are in-situ heterogeneously nucleated and grown under the initiation of the triazole groups, then the reaction is completed, the mixture is filtered, washed and freeze-dried to obtain HGPS@MAF-7@FDH composite microspheres loaded with inorganic / organic hybrid cascade catalytic systems.

2. The method for preparing HGPS@MAF-7@FDH composite microspheres according to claim 1, characterized in that: The epoxidation reagent in step (1) is at least one selected from the group consisting of epichlorohydrin, epibromohydrin, 1-chloro-2,3-epoxypropane, 3-chloro-1,2-epoxybutane, 2,3-epoxy-1-iodopropane, 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, glycerol triglycidyl ether and isocyanuric acid triglycidyl ester.

3. The method for preparing HGPS@MAF-7@FDH composite microspheres according to claim 1, characterized in that: The triazole functionalization reagent in step (2) is at least one selected from the group consisting of 3-amino-1,2,4-triazole, 5-amino-1H-1,2,3-triazole, 4-amino-1,2,4-triazole, 1-(3-aminopropyl)-1H-1,2,3-triazole, 3-hydroxy-1,2,4-triazole, (3-aminopropyl)-4H-1,2,4-triazole, 5-mercapto-1H-1,2,3-triazole, 1-phenyl-5-amino-1H-1,2,3-triazole and 4-(1H-1,2,3-triazol-1-yl)aniline.

4. The method for preparing HGPS@MAF-7@FDH composite microspheres according to claim 1, characterized in that, The molar concentration of the triazole functionalization reagent in the reaction system in step (2) ranges from 1 to 10 times the molar amount of the epoxy groups on the surface of the microspheres, and the reaction conditions of different triazole functionalization reagents with the HGPS microspheres are different: for the reagents containing amino groups, the reaction can be directly carried out in a weak alkaline system with pH 8-10 at a temperature of 40-80 ℃ for 1-6 h; for the reagents containing hydroxyl groups, the reaction needs to be carried out after the epoxy groups are ring-opened in a polar aprotic solvent with a strong base (NaOH, KOH, etc.) as a catalyst at a temperature of 60-100 ℃ for 4-12 h; for the reagents containing mercapto groups, the reaction can be directly carried out in a neutral to weak alkaline system with pH 7-9 at a temperature of 30-60 ℃ for 0.5-3 h, and nitrogen protection is needed to prevent the oxidation of mercapto groups.

5. The method for preparing HGPS@MAF-7@FDH composite microspheres according to claim 1, characterized in that, The metal ion in step (3) is zinc ion, and the metal salt is at least one selected from the group consisting of zinc nitrate, zinc chloride, zinc sulfate and zinc phosphate.

6. The method for preparing HGPS@MAF-7@FDH composite microspheres according to claim 1, characterized in that, The concentration of the metal ion in step (3) ranges from 5 to 30 mM, the concentration of the triazole ranges from 10 to 60 mM, and the molar ratio of the two is 1: (2-4).

7. The HGPS@MAF-7@FDH composite microspheres of claim 1, wherein The composite microspheres are micro-nano structures, the super-large pore HGPS is a micro-carrier, and the MAF-7 is a nano-carrier, formic acid dehydrogenase is encapsulated into the MAF-7 to be immobilized, and an inorganic / organic hybrid cascade catalytic system is formed; the loading amount of the composite microspheres FDH accounts for 20-60 mg / g of the microspheres, and the CO2 is effectively converted into formic acid.

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