Preparation method of modified ferroferric oxide and method for improving CO2 capture rate under illumination
By immobilizing formate dehydrogenase on a modified iron oxide composite carrier, the high cost and temperature mismatch issues of immobilized FDH technology were resolved, achieving low-cost and efficient CO2 capture and conversion, and meeting the carbon conversion requirements of room temperature environments.
Patent Information
- Application Number
- CN202511698047.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-19
- Publication Date
- 2026-03-17
AI Technical Summary
Existing immobilized formate dehydrogenase (FDH) technology suffers from high cost, temperature mismatch, and high energy consumption, which makes it difficult to fully realize its catalytic efficiency at room temperature. Furthermore, the immobilization process is complex, increasing the cost of carbon conversion.
A modified iron oxide composite carrier was used to construct a photothermal conversion carrier by loading Ni ions through a combination of Fe3O4 nanocores, a polydopamine photothermal conversion layer and a polyglutamic acid hydrophilic brush layer. This carrier was used to immobilize formate dehydrogenase and achieve photothermal synergy and room temperature catalysis of the enzyme.
It significantly reduces the cost of enzyme separation, purification, and energy consumption, improves the catalytic activity and stability of the enzyme, and achieves efficient CO2 capture and conversion under light irradiation, adapting to the carbon conversion requirements of room temperature environments.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] This invention belongs to the field of carbon conversion technology, specifically relating to a ferric oxide composite carrier, its preparation method, and a method for measuring CO2 capture rate under light irradiation. Background Technology
[0002] With escalating industrial emissions, atmospheric CO2 concentrations continue to rise, pushing natural carbon sequestration systems close to their ecological carrying capacity. Developing efficient artificial carbon sequestration technologies has become an urgent need to maintain carbon cycle balance. Among numerous artificial carbon sequestration technologies, enzymatic methods are highly promising due to their high efficiency, specificity, environmental friendliness, and mechanism of action closely resembling plant photosynthesis. Formate dehydrogenase (FDH), in particular, can efficiently catalyze the reversible conversion of CO2 and formic acid, and possesses advantages such as high substrate specificity and good environmental compatibility, making it one of the key enzymes in the field of enzymatic carbon conversion.
[0003] Although immobilization technology effectively addresses the core limitations of free FDH, such as its difficulty in recovery and poor reusability, it still faces three key challenges in large-scale biocatalytic carbon fixation applications. First, current immobilized FDH research primarily uses high-purity free enzymes as raw materials. However, the preparation and purification of high-purity enzymes are costly, significantly increasing the overall application cost of the immobilized system. This problem can be alleviated by researching the use of low-cost crude enzymes to replace high-purity enzymes, but the interference of impurities in crude enzymes on immobilization efficiency and catalytic activity still needs further optimization. Second, regarding temperature adaptability, the optimal catalytic temperature of existing immobilized FDH is often equal to or higher than that of free FDH. However, conventional applications of biocatalytic carbon fixation (such as industrial waste gas treatment and environmental CO2 conversion) are mostly at room temperature, below the optimal temperature of immobilized enzymes. This results in the immobilized system failing to fully exert its catalytic efficiency in practical applications, creating a core contradiction of "mismatch between optimal temperature and application temperature." More importantly, if high-temperature reaction conditions are used to meet the optimal temperature for immobilized FDH, a large amount of additional heat energy is required to maintain the temperature of the industrial system. Driving the carbon fixation reaction with the heat energy generated by carbon emissions essentially creates a paradox of "coexistence of carbon fixation and carbon emissions," which not only violates the low-carbon core goal of biocatalytic carbon fixation but also weakens the environmental benefits of the technology.
[0004] Therefore, developing a synchronous enzymatic carbon conversion system is key to promoting the large-scale application of enzymatic carbon conversion technology. The core advantages of this system are as follows: First, by constructing a composite carrier using iron oxide and polydopamine (PDA), the synergistic effect between the two can significantly improve the photothermal conversion efficiency. Its photothermal effect is more advantageous than that of iron oxide or PDA alone. Under light conditions, it can efficiently provide energy for formate dehydrogenase (FDH), thus providing specific support for the efficient conduct of enzymatic carbon conversion reactions. Second, precise control of enzyme activity can be achieved based on room temperature, without the need for complex temperature control equipment or additional high-energy consumption to maintain the reaction conditions. This can significantly reduce the energy consumption cost during system operation, further meeting the economic needs of large-scale application of enzymatic carbon conversion technology. Summary of the Invention
[0005] To overcome the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing modified iron oxide and a method for determining the CO2 capture rate under light irradiation using immobilized formate dehydrogenase, which has the characteristics of low carbon conversion cost and significantly improved CO2 conversion efficiency.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0007] Modified iron oxide composite carrier, including:
[0008] Magnetic Fe3O4 nanonuclei;
[0009] A polydopamine photothermal conversion layer, with a thickness of 50-100 nm, is coated around the Fe3O4 nanocore.
[0010] A hydrophilic brush layer (PGA) grafted onto the polydopamine photothermal conversion layer has a molecular weight of 600,000-800,000 Da.
[0011] The Ni ion loading of the coordination-modified polyglutamic acid hydrophilic brush layer is 1.5-3 mg / mg.
[0012] A method for preparing a modified iron oxide composite carrier, characterized by comprising the following steps:
[0013] Step 1: Weigh 50-200 mg of Fe3O4 and 0.15-0.35 g of dopamine hydrochloride (DA) and add them to Tris-HCl buffer. After reacting for 3-5 hours, centrifuge, wash 3-4 times with deionized water, and vacuum dry to obtain Fe3O4@PDA. Fe3O4@PDA is a composite material with iron(III) oxide core and polydopamine shell, abbreviated as FP.
[0014] Step 2: Take 100-300 mg of Fe3O4@PDA from Step 1, add 20-60 mg of N-hydroxysuccinimide (NHS) and 10-40 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and then add 20-60 mg of polyglutamic acid (PGA). React at room temperature for 12-15 hours. After the reaction is complete, centrifuge, wash with deionized water 3-4 times, and vacuum dry to obtain Fe3O4@PDA-PGA. Fe3O4@PDA-PGA is a core-shell-brush structure composite material with iron(III) oxide as the core, polydopamine as the coating shell, and polyglutamic acid as the grafted brush chain.
[0015] Step 3: Take 100-300 mg of Fe3O4@PDA-PGA from Step 2, add 100-150 mL of Tris-HCl buffer, then add 0.2-0.4 g of NaCl and 0.9-2.4 g of nickel chloride hexahydrate (NiCl2·6H2O), and react at room temperature for 2-5 h. After the reaction is complete, centrifuge, wash with deionized water 2-3 times, and store under vacuum at 4℃ to obtain Fe3O4@PDA-PGA-Ni composite material, i.e. modified iron(III) oxide. Fe3O4@PDA-PGA-Ni composite material is a core-shell-brush structure composite functional material with iron(III) oxide as the core, polydopamine as the coating shell, polyglutamic acid as the grafted brush chain, and further functionalized nickel (Ni) on the surface.
[0016] A method for determining CO2 capture rate of modified iron oxide-fixed formate dehydrogenase under light irradiation, characterized by comprising the following steps:
[0017] Step a: Inoculate a single colony of recombinant Escherichia coli carrying the formate dehydrogenase gene into 4-7 mL of LB medium containing 50-100 mg / mL kanamycin and culture for 12-14 h; transfer to 100-300 mL of fresh LB medium at a 2% inoculation rate and continue culturing for 2.5-4 h, then add 1 mol / L IPTG (isopropyl-β-D-thiogalactopyranoside) to make the system concentration 1-10 mmol / L and react for 20-24 h to obtain bacterial suspension; collect bacterial cells by centrifugation at room temperature for 5-10 min, resuspend in 10-30 mL of lysis buffer, and sonicate to disrupt the cells; centrifuge the lysis buffer for 20-30 min and use the supernatant for subsequent fixation and purification; obtain crude FDH enzyme solution for later use.
[0018] Step b: Add 25-50 mg of Fe3O4@PDA-PGA-Ni to 1-3 mL of crude FDH enzyme solution obtained in step 1, and shake for 30-40 min. After the reaction, collect the complex by magnetic separation, wash 2-3 times with PBS buffer to obtain the Fe3O4@PDA-PGA-Ni complex with immobilized FDH. (The Fe3O4@PDA-PGA-Ni composite material can bind to the histidine (His) tag on the formate dehydrogenase (FDH) molecule in the crude enzyme solution through specific interaction, thereby mediating the formation of a complex system between Fe3O4@PDA-PGA-Ni and FDH.)
[0019] Step c: Place 25-70 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex into a 50 mL centrifuge tube containing 10-20 mL of PBS buffer, and incubate in a 36 °C water bath for 10-15 min. Adjust the visible light intensity to raise the temperature of the immobilized FDH Fe3O4@PDA-PGA-Ni complex to a constant 45 °C and maintain it for 10-15 min. Add NADH and mix thoroughly. Introduce a mixture of CO2 and N2 gas at a flow rate of 20-100 mL / min, with a CO2 to N2 volume ratio of 0.15:0.85, and react for 30-90 min. Measure the outlet CO2 concentration using a CO2 infrared analyzer and calculate the CO2 capture efficiency. Measure the formic acid concentration in the reaction solution using a high-performance liquid chromatograph and calculate the CO2 conversion efficiency.
[0020] In step c, the method for determining 45℃ is as follows:
[0021] 25 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was added to 3 mL of PBS buffer and incubated in a 35 °C water bath for 1 min. Then, the immobilized FDH Fe3O4@PDA-PGA-Ni complex solution was heated to 35 °C, 40 °C, 45 °C and 50 °C by adjusting the visible light intensity. Finally, the enzyme activity was detected according to the enzyme activity assay method.
[0022] The PBS buffer has a pH of 7.0 and a concentration of 0.05 M.
[0023] The beneficial effects of this invention are:
[0024] This invention utilizes the photothermal conversion carrier prepared by the above method to immobilize formate dehydrogenase, achieving cost reduction in three aspects: Firstly, since the photothermal conversion carrier directly completes the one-step purification and immobilization of the enzyme, eliminating the need for chromatography reagents, centrifugation equipment, etc., required by traditional multi-step processes, the separation and purification cost is only 2‰ of that of free enzyme purification processes, significantly reducing the economic investment in the initial preparation stage, thus having the advantage of significantly reducing separation and purification costs. Secondly, due to the use of a magnetically responsive photothermal conversion carrier, the immobilized enzyme can be efficiently recovered through magnetic separation. Experiments show that after continuous reuse for 10 times, the enzyme catalytic activity still maintains more than 50%, reducing the amount of enzyme required for a single reaction, thus having the advantage of reducing unit cycle cost. Thirdly, due to the use of a carrier with excellent photothermal conversion performance, the local micro-thermal field can be precisely controlled to the optimal temperature for enzyme catalysis (35-40℃) under light irradiation, matching the enzyme catalytic requirements without additional heating equipment and energy input, thus effectively reducing energy consumption costs. Fourthly, this system can efficiently and synergistically participate in the carbon conversion of formate dehydrogenase, improving the problem of insufficient adaptability of reaction conditions while reducing costs throughout the entire process, providing an economical solution for the large-scale application of enzymatic carbon conversion technology. Attached Figure Description
[0025] Figure 1 This is a schematic diagram of the carbon conversion process for preparing artificial chloroplasts according to the present invention.
[0026] Figure 2 This is a Fourier transform infrared diagram of the preparation of Fe3O4@PDA-PGA according to the present invention.
[0027] Figure 3 This is a schematic diagram showing the Ni ion loading in Fe3O4@PDA-PGA-Ni prepared according to the present invention.
[0028] Figure 4 This is a schematic diagram of the laser confocal microscopy used in the preparation of the Fe3O4@PDA-PGA-Ni composite with immobilized FDH according to the present invention. Detailed Implementation
[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0030] See Figure 1 , Figure 1This paper demonstrates the preparation and biomimetic carbon fixation process of the Fe3O4@PDA-PGA-Ni complex with immobilized FDH: First, using Fe3O4 as a substrate, dopamine (DA) and polyglutamic acid (PGA) are sequentially modified and nickel ions are combined to construct a modified iron oxide support. Then, crude enzyme is loaded onto the support in a one-step method to form the Fe3O4@PDA-PGA-Ni complex with immobilized FDH. Subsequently, the Fe3O4@PDA-PGA-Ni complex with immobilized FDH mimics the function of natural chloroplasts, using visible light as the sole energy source to activate the immobilized formate dehydrogenase (FDH), catalyzing the conversion of carbon dioxide into formic acid. At the same time, the enzyme can be recycled and reused. This not only replicates the light-driven carbon conversion logic of natural chloroplasts, but also solves the problems of enzyme stability and cost through artificial carriers.
[0031] Modified iron oxide composite carrier, including:
[0032] Magnetic Fe3O4 nanonuclei;
[0033] A polydopamine photothermal conversion layer, with a thickness of 50-100 nm, is coated around the Fe3O4 nanocore.
[0034] A hydrophilic brush layer (PGA) grafted onto the polydopamine photothermal conversion layer has a molecular weight of 600,000-800,000 Da.
[0035] The Ni ion loading of the coordination-modified polyglutamic acid hydrophilic brush layer is 1.5-3 mg / mg.
[0036] A method for preparing a modified iron oxide composite carrier, characterized by comprising the following steps:
[0037] Step 1: Weigh 50-200 mg of Fe3O4 and 0.15-0.35 g of dopamine hydrochloride (DA) and add them to Tris-HCl buffer. After reacting for 3-5 h, centrifuge, wash 3-4 times with deionized water, and vacuum dry to obtain Fe3O4@PDA. Fe3O4@PDA is a composite material with iron(III) oxide core and polydopamine shell.
[0038] Step 2: Take 100-300 mg of Fe3O4@PDA from Step 1, add 20-60 mg of N-hydroxysuccinimide (NHS) and 10-40 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and then add 20-60 mg of polyglutamic acid (PGA). React at room temperature for 12-15 hours. After the reaction is complete, centrifuge, wash with deionized water 3-4 times, and vacuum dry to obtain Fe3O4@PDA-PGA. Fe3O4@PDA-PGA is a core-shell-brush structure composite material with iron(III) oxide as the core, polydopamine as the coating shell, and polyglutamic acid as the grafted brush chain.
[0039] Step 3: Take 100-300 mg of Fe3O4@PDA-PGA from Step 2, add 100-150 mL of Tris-HCl buffer, then add 0.2-0.4 g of NaCl and 0.9-2.4 g of nickel chloride hexahydrate (NiCl2·6H2O), and react at room temperature for 2-5 h. After the reaction is complete, centrifuge, wash with deionized water 2-3 times, and store under vacuum at 4℃ to obtain Fe3O4@PDA-PGA-Ni composite material, i.e. modified iron(III) oxide. Fe3O4@PDA-PGA-Ni composite material is a core-shell-brush structure composite functional material with iron(III) oxide as the core, polydopamine as the coating shell, polyglutamic acid as the grafted brush chain, and further functionalized nickel (Ni) on the surface.
[0040] A method for determining CO2 capture rate of modified iron oxide-fixed formate dehydrogenase under light irradiation, characterized by comprising the following steps:
[0041] Step a: Inoculate a single colony of recombinant Escherichia coli carrying the formate dehydrogenase gene into 4-7 mL of LB medium containing 50-100 mg / mL kanamycin and culture for 12-14 h; transfer to 100-300 mL of fresh LB medium at a 2% inoculation rate and continue culturing for 2.5-4 h, then add 1 mol / L IPTG (isopropyl-β-D-thiogalactopyranoside) to make the system concentration 1-10 mmol / L and react for 20-24 h to obtain bacterial suspension; collect bacterial cells by centrifugation at room temperature for 5-10 min, resuspend in 10-30 mL of lysis buffer, and sonicate to disrupt the cells; centrifuge the lysis buffer for 20-30 min and use the supernatant for subsequent fixation and purification; obtain crude FDH enzyme solution for later use.
[0042] Step b: Add 25-50 mg of Fe3O4@PDA-PGA-Ni to 1-3 mL of crude FDH enzyme solution obtained in step 1, and shake for 30-40 min. After the reaction, collect the complex by magnetic separation, wash 2-3 times with PBS buffer to obtain the Fe3O4@PDA-PGA-Ni complex with immobilized FDH. (The Fe3O4@PDA-PGA-Ni composite material can bind to the histidine (His) tag on the formate dehydrogenase (FDH) molecule in the crude enzyme solution through specific interaction, thereby mediating the formation of a complex system between Fe3O4@PDA-PGA-Ni and FDH.)
[0043] Step c: Place 25-70 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex into a 50 mL centrifuge tube containing 10-20 mL of PBS buffer, and incubate in a 36 °C water bath for 10-15 min. Adjust the visible light intensity to raise the temperature of the immobilized FDH Fe3O4@PDA-PGA-Ni complex to a constant 45 °C and maintain it for 10-15 min. Add NADH and mix thoroughly. Introduce a mixture of CO2 and N2 gas at a flow rate of 20-100 mL / min, with a CO2 to N2 volume ratio of 0.15:0.85, and react for 30-90 min. Measure the outlet CO2 concentration using a CO2 infrared analyzer and calculate the CO2 capture efficiency. Measure the formic acid concentration in the reaction solution using a high-performance liquid chromatograph and calculate the CO2 conversion efficiency.
[0044] In step c, the method for determining 45℃ is as follows:
[0045] 25 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was added to 3 mL of PBS buffer and incubated in a 35 °C water bath for 1 min. Then, the immobilized FDH Fe3O4@PDA-PGA-Ni complex solution was heated to 35 °C, 40 °C, 45 °C and 50 °C by adjusting the visible light intensity. Finally, the enzyme activity was detected according to the enzyme activity assay method.
[0046] The PBS buffer, pH 7.0, has a concentration of 0.05 M.
[0047] Example 1
[0048] Magnetic Fe3O4 nanonuclei;
[0049] A polydopamine photothermal conversion layer with a thickness of 50 nm is coated around the Fe3O4 nanocore.
[0050] A hydrophilic brush layer (PGA) grafted onto the polydopamine photothermal conversion layer has a molecular weight of 600,000 Da.
[0051] The Ni ion loading of the coordination-modified polyglutamic acid hydrophilic brush layer was 1.5 mg / mg.
[0052] Example 2
[0053] Magnetic Fe3O4 nanonuclei;
[0054] A polydopamine photothermal conversion layer, 100 nm thick, is coated around the Fe3O4 nanocore.
[0055] A hydrophilic brush layer (PGA) grafted onto the polydopamine photothermal conversion layer has a molecular weight of 800,000 Da.
[0056] The Ni ion loading of the coordination-modified polyglutamic acid hydrophilic brush layer was 3 mg / mg.
[0057] Example 3
[0058] Magnetic Fe3O4 nanonuclei;
[0059] A polydopamine photothermal conversion layer, 75 nm thick, is coated around the Fe3O4 nanocore.
[0060] A hydrophilic brush layer (PGA) grafted onto the polydopamine photothermal conversion layer has a molecular weight of 700,000 Da.
[0061] The Ni ion loading of the coordination-modified polyglutamic acid hydrophilic brush layer was 2.3 mg / mg.
[0062] Example 4
[0063] Step 1: Weigh 50 mg of Fe3O4 and 0.15 g of dopamine hydrochloride (DA) and add them to Tris-HCl buffer. After reacting for 3 h, centrifuge, wash 3-4 times with deionized water, and vacuum dry to obtain Fe3O4@PDA. Fe3O4@PDA is a composite material with iron oxide core and polydopamine shell.
[0064] Step 2: Take 100 mg of Fe3O4@PDA from Step 1, add 20 mg of N-hydroxysuccinimide (NHS) and 10 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and then add 20 mg of polyglutamic acid (PGA). React at room temperature for 12 h. After the reaction is complete, centrifuge, wash three times with deionized water, and vacuum dry to obtain Fe3O4@PDA-PGA. Fe3O4@PDA-PGA is a core-shell-brush structure composite material with iron(III) oxide as the core, polydopamine as the coating shell, and polyglutamic acid as the grafted brush chain.
[0065] Step 3: Take 100 mg of Fe3O4@PDA-PGA from Step 2, add 100 mL of Tris-HCl buffer, then add 0.2 g of NaCl and 0.9 g of nickel chloride hexahydrate (NiCl2·6H2O), and react at room temperature for 2 h. After the reaction is complete, centrifuge, wash twice with deionized water, and store under vacuum at 4 °C to obtain Fe3O4@PDA-PGA-Ni composite material, i.e. modified iron(III) oxide. Fe3O4@PDA-PGA-Ni composite material is a core-shell-brush structure composite functional material with iron(III) oxide as the core, polydopamine as the coating shell, polyglutamic acid as the grafted brush chain, and further functionalized nickel (Ni) on the surface.
[0066] Example 5
[0067] The preparation method of the modified iron oxide composite carrier includes the following steps:
[0068] Step 1: Weigh 200 mg of Fe3O4 and 0.35 g of dopamine hydrochloride (DA) and add them to Tris-HCl buffer. After reacting for 5 h, centrifuge, wash 4 times with deionized water, and vacuum dry to obtain Fe3O4@PDA. Fe3O4@PDA is a composite material with iron oxide core and polydopamine shell.
[0069] Step 2: Take 300 mg of Fe3O4@PDA from Step 1, add 60 mg of N-hydroxysuccinimide (NHS) and 40 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and then add 60 mg of polyglutamic acid (PGA). React at room temperature for 15 h. After the reaction is complete, centrifuge, wash with deionized water 4 times, and vacuum dry to obtain Fe3O4@PDA-PGA. Fe3O4@PDA-PGA is a core-shell-brush structure composite material with iron(III) oxide as the core, polydopamine as the coating shell, and polyglutamic acid as the grafted brush chain.
[0070] Step 3: Take 300 mg of Fe3O4@PDA-PGA from Step 2, add 150 mL of Tris-HCl buffer, then add 0.4 g of NaCl and 2.4 g of nickel chloride hexahydrate (NiCl2·6H2O), and react at room temperature for 5 h. After the reaction is complete, centrifuge, wash three times with deionized water, and store under vacuum at 4 °C to obtain Fe3O4@PDA-PGA-Ni composite material, namely modified iron(III) oxide. Fe3O4@PDA-PGA-Ni composite material is a core-shell-brush structure composite functional material with iron(III) oxide as the core, polydopamine as the coating shell, polyglutamic acid as the grafted brush chain, and further functionalized nickel (Ni) on the surface.
[0071] Example 6
[0072] Step 1: Weigh 150 mg of Fe3O4 and 0.25 g of dopamine hydrochloride (DA) and add them to Tris-HCl buffer. After reacting for 4 h, centrifuge, wash 4 times with deionized water, and vacuum dry to obtain Fe3O4@PDA. Fe3O4@PDA is a composite material with iron oxide core and polydopamine shell.
[0073] Step 2: Take 200 mg of Fe3O4@PDA from Step 1, add 35 mg of N-hydroxysuccinimide (NHS) and 25 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), then add 35 mg of polyglutamic acid (PGA), and react at room temperature for 14 h; after the reaction, centrifuge, wash four times with deionized water, and vacuum dry to obtain Fe3O4@PDA-PGA. Fe3O4@PDA-PGA is a core-shell-brush structure composite material with iron(III) oxide as the core, polydopamine as the coating shell, and polyglutamic acid as the grafted brush-like chains; see [link to relevant documentation]. Figure 2 The infrared spectra of Fe3O4, FP, and FPP are shown, with FP (red curve) at 1521 cm⁻¹. -1 (CN stretching vibration), 1281cm -1 (C=C stretching vibration) and 1620cm -1 Characteristic peaks appeared at the (benzene ring skeletal vibration) position, all of which correspond to the characteristic functional groups of PDA. These characteristic peaks were not observed in the spectrum of pure Fe3O4 samples, thus confirming that PDA had been successfully coated onto the Fe3O4 surface. Further observation of the FPP (black curve) revealed a peak at 1184 cm⁻¹. -1 (CN / NH vibration), 1730cm -1 (Amide I with C=O stretching vibration) and 3300 cm -1 The appearance of a new characteristic peak at the (broad peak) indicates that PGA has been successfully grafted onto the FP surface.
[0074] Step 3: Take 200 mg of Fe3O4@PDA-PGA from Step 2, add 130 mL of Tris-HCl buffer, then add 0.3 g of NaCl and 1.6 g of nickel chloride hexahydrate (NiCl2·6H2O), and react at room temperature for 3.5 h. After the reaction, centrifuge, wash twice with deionized water, and store under vacuum at 4 °C to obtain the Fe3O4@PDA-PGA-Ni composite material, i.e., modified iron(III) oxide. The Fe3O4@PDA-PGA-Ni composite material is a core-shell-brush structured functional material with iron(III) oxide as the core, polydopamine as the coating shell, polyglutamic acid as the grafted brush-like chain, and further functionalized with nickel (Ni) on the surface. See also... Figure 3 This diagram illustrates the Ni ion loading in Fe3O4@PDA-PGA-Ni. The adsorption capacity of Fe3O4@PDA-PGA varies with the initial Ni concentration and time. The adsorption rate is fastest in the first 0-1 h, reaching its maximum after approximately 1 h. The adsorption capacity is highest at a solution ion concentration of 30 mg / mL, reaching 2.13 mg / mL. As the ion concentration in the solution increases, the adsorption capacity gradually decreases. This is because excessively high ion concentrations hinder the chelation between ions and the resin.
[0075] Example 7
[0076] Step a: A single colony of recombinant Escherichia coli carrying the formate dehydrogenase gene was inoculated into 4 mL of LB medium containing 50 mg / mL kanamycin and cultured for 12 h. The inoculum was then transferred to 100 mL of fresh LB medium at a 2% inoculation rate and cultured for another 2.5 h. 1 mol / L IPTG (isopropyl-β-D-thiogalactopyranoside) was added to bring the concentration to 1 mmol / L, and the reaction was allowed to proceed for 20 h to obtain the bacterial suspension. The bacterial suspension was centrifuged at room temperature for 5 min to collect the cells, resuspended in 10 mL of lysis buffer, and then sonicated. The lysis buffer was centrifuged for 20 min, and the supernatant was used for subsequent fixation and purification to obtain crude FDH enzyme solution for later use.
[0077] Step b: Add 25 mg of Fe3O4@PDA-PGA-Ni to 1 mL of the crude FDH enzyme solution obtained in step 1, and shake for 30 min. After the reaction, collect the complex by magnetic separation, wash twice with PBS buffer to obtain the Fe3O4@PDA-PGA-Ni complex with immobilized FDH. (The Fe3O4@PDA-PGA-Ni composite material can bind to the histidine (His) tag on the formate dehydrogenase (FDH) molecule in the crude enzyme solution through specific interactions, thereby mediating the formation of a complex system between Fe3O4@PDA-PGA-Ni and FDH; see [link to relevant documentation]). Figure 4 The distribution of FDH in FPP can be directly observed. FDH enzyme molecules were stained with the fluorescent dye FITC, and the material was then characterized using a laser confocal microscope (CLSM). The results showed that FPP exhibited a distinct green fluorescence field under CLSM. This phenomenon not only directly confirmed the successful presence of FDH in FPP, but also, based on the uniform distribution of the fluorescence signal, further indicated that FDH enzyme molecules were uniformly dispersed on and within the FPP material, without significant aggregation.
[0078] Step c: 25 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was placed in a 50 mL centrifuge tube containing 10 mL of PBS buffer and incubated in a 36 °C water bath for 10 min. The temperature of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was then raised to a constant 45 °C by adjusting the visible light intensity and maintained for 10 min. NADH was added and mixed thoroughly. A mixture of CO2 and N2 gas was introduced at a flow rate of 20 mL / min, with a CO2 to N2 volume ratio of 0.15:0.85, and the reaction was allowed to proceed for 30 min. The outlet CO2 concentration was measured using a CO2 infrared analyzer, and the CO2 capture efficiency was calculated. The formic acid concentration in the reaction solution was measured using a high-performance liquid chromatograph, and the CO2 conversion efficiency was calculated.
[0079] In step c, the method for determining 45℃ is as follows:
[0080] 25 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was added to 3 mL of PBS buffer and incubated in a 35 °C water bath for 1 min. Then, the immobilized FDH Fe3O4@PDA-PGA-Ni complex solution was heated to 35 °C, 40 °C, 45 °C and 50 °C by adjusting the visible light intensity. Finally, the enzyme activity was detected according to the enzyme activity assay method.
[0081] The PBS buffer, pH 7.0, has a concentration of 0.05 M.
[0082] Example 8
[0083] Step a: A single colony of recombinant Escherichia coli carrying the formate dehydrogenase gene was inoculated into 7 mL of LB medium containing 100 mg / mL kanamycin and cultured for 14 h. The inoculum was then transferred to 300 mL of fresh LB medium at a 2% inoculation rate and cultured for another 4 h. 1 mol / L IPTG (isopropyl-β-D-thiogalactopyranoside) was added to bring the concentration to 10 mmol / L, and the reaction was allowed to proceed for 24 h to obtain the bacterial suspension. The bacterial suspension was centrifuged at room temperature for 10 min to collect the cells, resuspended in 30 mL of lysis buffer, and then sonicated. The lysis buffer was centrifuged for 30 min, and the supernatant was used for subsequent fixation and purification to obtain crude FDH enzyme solution for later use.
[0084] Step b: Add 50 mg of Fe3O4@PDA-PGA-Ni to 3 mL of crude FDH enzyme solution obtained in step 1, and shake for 40 min. After the reaction, collect the complex by magnetic separation, wash three times with PBS buffer to obtain the Fe3O4@PDA-PGA-Ni complex with immobilized FDH. (The Fe3O4@PDA-PGA-Ni composite material can bind to the histidine (His) tag on the formate dehydrogenase (FDH) molecule in the crude enzyme solution through specific interaction, thereby mediating the formation of a complex system between Fe3O4@PDA-PGA-Ni and FDH.)
[0085] Step c: 70 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was placed in a 50 mL centrifuge tube containing 20 mL of PBS buffer and incubated in a 36 °C water bath for 15 min. The temperature of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was raised to a constant 45 °C by adjusting the visible light intensity and maintained for 15 min. NADH was added and mixed thoroughly. A mixture of CO2 and N2 gas was introduced at a flow rate of 100 mL / min, with a CO2 to N2 volume ratio of 0.15:0.85, and the reaction was carried out for 90 min. The outlet CO2 concentration was measured using a CO2 infrared analyzer, and the CO2 capture efficiency was calculated. The formic acid concentration in the reaction solution was measured using a high-performance liquid chromatograph, and the CO2 conversion efficiency was calculated.
[0086] In step c, the method for determining 45℃ is as follows:
[0087] 25 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was added to 3 mL of PBS buffer and incubated in a 35 °C water bath for 1 min. Then, the immobilized FDH Fe3O4@PDA-PGA-Ni complex solution was heated to 35 °C, 40 °C, 45 °C and 50 °C by adjusting the visible light intensity. Finally, the enzyme activity was detected according to the enzyme activity assay method.
[0088] The PBS buffer has a pH of 7.0 and a concentration of 0.05 M.
[0089] Example 9
[0090] Step a: A single colony of recombinant Escherichia coli carrying the formate dehydrogenase gene was inoculated into 6 mL of LB medium containing 75 mg / mL kanamycin and cultured for 13 h. The inoculum was then transferred to 200 mL of fresh LB medium at a 2% inoculation rate and cultured for another 3.5 h. 1 mol / L IPTG (isopropyl-β-D-thiogalactopyranoside) was added to bring the concentration to 5 mmol / L, and the reaction was allowed to proceed for 22 h to obtain the bacterial suspension. The bacterial suspension was centrifuged at room temperature for 8 min to collect the bacterial cells. The cells were resuspended in 20 mL of lysis buffer and sonicated. The lysis buffer was centrifuged for 25 min, and the supernatant was used for subsequent fixation and purification to obtain crude FDH enzyme solution for later use.
[0091] Step b: Add 35 mg of Fe3O4@PDA-PGA-Ni to 2 mL of crude FDH enzyme solution obtained in step 1, and shake for 35 min. After the reaction, collect the complex by magnetic separation, wash three times with PBS buffer to obtain the Fe3O4@PDA-PGA-Ni complex with immobilized FDH. (The Fe3O4@PDA-PGA-Ni composite material can bind to the histidine (His) tag on the formate dehydrogenase (FDH) molecule in the crude enzyme solution through specific interaction, thereby mediating the formation of a complex system between Fe3O4@PDA-PGA-Ni and FDH.)
[0092] Step c: 50 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was placed in a 50 mL centrifuge tube containing 15 mL of PBS buffer and incubated in a 36 °C water bath for 13 min. The temperature of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was raised to a constant 45 °C by adjusting the visible light intensity and maintained for 13 min. NADH was added and mixed thoroughly. A mixture of CO2 and N2 gas was introduced at a flow rate of 70 mL / min, with a CO2 to N2 volume ratio of 0.15:0.85, and the reaction was allowed to proceed for 60 min. The outlet CO2 concentration was measured using a CO2 infrared analyzer, and the CO2 capture efficiency was calculated. The formic acid concentration in the reaction solution was measured using a high-performance liquid chromatograph, and the CO2 conversion efficiency was calculated.
[0093] In step c, the method for determining 45℃ is as follows:
[0094] 25 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was added to 3 mL of PBS buffer and incubated in a 35 °C water bath for 1 min. Then, the immobilized FDH Fe3O4@PDA-PGA-Ni complex solution was heated to 35 °C, 40 °C, 45 °C and 50 °C by adjusting the visible light intensity. Finally, the enzyme activity was detected according to the enzyme activity assay method.
[0095] The PBS buffer has a pH of 7.0 and a concentration of 0.05 M.
Claims
1. A modified ferroferric oxide characterized in that, Comprise: Magnetic Fe3O4 nanometer core; Polydopamine light-heat conversion layer coated outside the Fe3O4 nanometer core, thickness is 50-100nm; Polyglutamic acid hydrophilic brush layer grafted outside the polydopamine light-heat conversion layer, molecular weight is 600-800 thousand Da; Ni ions modified on the polyglutamic acid hydrophilic brush layer, loading capacity is 1.5-3mg / mg.
2. A method for preparing modified ferroferric oxide, characterized by, Comprise the following steps: Step 1, weigh 50-200mg of Fe3O4, 0.15-0.35g of hydrochloric acid dopamine is added into Tris-HCl buffer solution, after reaction for 3-5h, centrifugal, deionized water washing 3-4 times, vacuum drying, to obtain Fe3O4@PDA; Step 2, take 100-300mg of Fe3O4@PDA in step 1, add 20-60mg of N-hydroxysuccinimide and 10-40mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, then add 20-60mg of polyglutamic acid, react at room temperature for 12h-15h; After the reaction is completed, centrifugal, deionized water washing 3-4 times, vacuum drying, to obtain Fe3O4@PDA-PGA; Step 3, take 100-300mg of Fe3O4@PDA-PGA in step 2, add 100-150mL of Tris-HCl buffer solution, then add 0.2-0.4g of NaCl and 0.9-2.4g of nickel chloride hexahydrate, react at room temperature for 2-5h; After the reaction is completed, centrifugal, deionized water washing 2-3 times, 4℃ vacuum preservation, to obtain Fe3O4@PDA-PGA-Ni composite material, that is, modified ferroferric oxide.
3. Method for measuring the CO2 capture rate under light of modified triiron tetraoxide immobilized formate dehydrogenase, characterized in that, Comprise the following steps: Step a, inoculate the recombinant escherichia coli carrying formate dehydrogenase gene single colony into 4-7mL LB medium containing 50-100mg / mL kanamycin, and culture for 12-14h; Transfer to 100-300mL fresh LB medium with 2% inoculation amount, continue to culture for 2.5-4h, then add 1mol / L IPTG (isopropyl-beta-D-thiogalactoside) to make the system concentration 1-10mmol / L, react for 20-24h, obtain bacterial liquid; Centrifugal the bacterial liquid at room temperature for 5-10min to collect bacterial body, resuspend with 10-30mL lysis buffer, then ultrasonic breakage, centrifugal the lysis solution for 20-30min, take the supernatant for subsequent immobilization and purification; Obtain FDH crude enzyme solution for standby; Step b, take 25-50mg of Fe3O4@PDA-PGA-Ni and add to 1-3mL of FDH crude enzyme solution obtained in step a, oscillation reaction for 30-40min; After the reaction is completed, collect the composite by magnetic separation, wash with PBS buffer solution for 2-3 times, to obtain Fe3O4@PDA-PGA-Ni composite of immobilized FDH; Step c, 25-70 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was placed in a 50 mL centrifuge tube containing 10-20 mL of PBS buffer and incubated in a 36 °C water bath for 10-15 min. The immobilized FDH Fe3O4@PDA-PGA-Ni complex was warmed to a constant 45 °C by adjusting the intensity of the visible light, and maintained for 10-15 min. NADH was added and mixed well. A mixture of CO2 and N2 gas was passed at a flow rate of 20-100 mL / min, with a CO2 to N2 volume ratio of 0.15:0.
85. The reaction was carried out for 30-90 min. The CO2 concentration at the outlet was determined by a CO2 infrared analyzer, and the CO2 capture efficiency was calculated. The formic acid concentration in the reaction solution was determined by high performance liquid chromatography, and the CO2 conversion efficiency was calculated.
4. The method for measuring the CO2 capture rate in formate dehydrogenase using modified magnetite according to claim 3, characterized by, In step c, the method for determining 45 °C is as follows: 25 mg of the immobilized FDH Fe3O4@PDA-PGA-Ni complex was added to 3 mL of PBS buffer and placed in a 35 °C water bath for 1 min. Then, the immobilized FDH Fe3O4@PDA-PGA-Ni complex solution at the same temperature was heated to 35 °C, 40 °C, 45 °C and 50 °C by adjusting the visible light. Finally, the enzyme activity was detected according to the enzyme activity determination method.
5. The method for measuring the CO2 capture rate of the modified ferroferric oxide immobilized formate dehydrogenase under light according to claim 3, characterized by, The PBS buffer has a pH of 7.0 and a concentration of 0.05 M.