Preparation method and application of magnetic nano lignin
By constructing the Lignin-NH2@Fe3O4 vector, the thermal stability and recycling difficulties of ω-transaminase carrier were solved, and the cost of carriers and the recovery rate of enzyme activity was reduced, which was efficiently immobilized ω-transaminase for industrial applications.
Patent Information
- Application Number
- CN202510614685.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-14
- Publication Date
- 2025-07-25
AI Technical Summary
The existing immobilized ω-transaminase carriers have problems such as low thermal stability, difficulty in recycling and short catalytic life. Traditional carriers have high cost, low enzyme load, large enzyme activity loss, and difficulty in magnetic separation.
The Lignin-NH2@Fe3O4 carrier was constructed through amination-magnetic modification, and combined with the response surface optimization, magnetic nanolignin was prepared for immobilizing ω-transaminase, which solved the technical bottlenecks of low thermal stability, difficulty in recycling and short catalytic life of the carrier.
The cost of carrier is reduced by 40%, the recovery rate of enzyme activity is increased by more than 20%, and the magnetic separation efficiency is improved. It can operate stably under acidic and high temperature conditions, expanding industrial application scenarios.
Smart Images
Figure CN120365580A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of biocatalysis, and particularly relates to a preparation method and application of magnetic nano-lignin. Background Art
[0002] ω-transaminase is a key catalyst for synthesizing chiral amines, but its free form has defects such as substrate inhibition, poor thermal stability, and difficulty in reuse. Existing immobilization technologies mostly use silica gel, macroporous resin, or metal-organic frameworks, but there are problems such as high carrier cost, low enzyme loading, large enzyme activity loss, and difficulty in magnetic separation. Lignin, as a renewable biomass resource, has natural porosity (porosity ≈ 40%) and abundant hydroxyl functional groups (1.5 - 2.5 mmol / g), and theoretically is suitable for enzyme immobilization. However, natural lignin has problems such as surface inertness, lack of directional modification, lack of reaction sites such as amino groups on the unmodified lignin surface, difficulty in stabilizing enzyme molecules through covalent coupling, lack of magnetic responsiveness, and inability to meet the requirements of continuous flow reactors. Summary of the Invention
[0003] The present invention provides a preparation method and application of magnetic nano-lignin. In this preparation method, the Lignin-NH2@Fe3O4 carrier is constructed through amination-magnetic modification, and an efficient immobilized enzyme is obtained by combining response surface optimization, solving the technical bottlenecks of low thermal stability, difficult recovery, and short catalytic life of traditional carriers.
[0004] The purpose of the present invention is achieved through the following technical solutions: A preparation method of magnetic nano-lignin, the preparation method comprising the following steps: (1) Preparation of Lignin-NH2 Take lignin and disperse it in ultrapure water. After mechanical stirring, add polyethyleneimine, adjust the pH to 10, then add formaldehyde, react in a water bath at 60 °C for 5 h, stop the reaction, collect the product by suction filtration, wash it once with pure water, and dry it at 50 - 60 °C to obtain Lignin-NH2.
[0005] (2) Preparation of Lignin-NH2@Fe3O4 Pre-dissolve ammonium ferrous sulfate hexahydrate and ammonium ferric sulfate dodecahydrate in deionized water to prepare a solution for standby.
[0006] The aminated lignin was added to the pre-prepared solution. After ultrasonic stirring, the pH was adjusted to 10 with 6 mol / L ammonia water. After the pH was stabilized, it was mechanically stirred in a 50 °C constant temperature water bath for 1 h. After the reaction was completed, it was cooled to room temperature. After the solution was layered, magnetic aminated lignin was obtained by suction filtration. After washing with water multiple times until neutral, it was dried in vacuo at 60 °C for 24 h until constant weight, and magnetic nano-lignin Lignin-NH2@Fe3O4 was obtained.
[0007] Preferably, in step (1), the mass ratio of lignin to polyethyleneimine is 5:4.
[0008] Preferably, in step (2), the mass ratio of ammonium ferrous sulfate hexahydrate to ammonium ferric sulfate dodecahydrate is 0.65.
[0009] Preferably, in step (2), the specific surface area of the Lignin-NH2@Fe3O4 is 29.8 m² / g, the saturation magnetization intensity is 35 - 40 emu / g, and the nano-particle size is ≤100 nm.
[0010] The present invention also provides an application of the magnetic nano-lignin in the preparation of immobilized ω-transaminase, and the application specifically includes the following steps: S01. Inoculate the L118T strain into an LB medium containing 50 μg / mL, and culture it at 37 °C, 180 - 200 r / min for 8 - 10 hours; S02. Transfer the bacterial solution to an LB medium containing 50 μg / mL kanamycin, and continue to culture for 2 - 4 h; S03. When the OD value reaches 0.6 - 0.8, add 0.8 mM IPTG (inducer, isopropyl-β-D-thiogalactoside), and culture at 25 °C, 150 r / min for 18 - 20 h; S04. Collect the cells by centrifugation at 4 °C, 6000 rpm for 6 minutes, resuspend them in a 20 mM imidazole solution, break the cells by high-pressure homogenization, and then centrifuge at 4 °C, 8000 r / min for 50 minutes to collect the supernatant as the crude enzyme solution; S05. After the crude enzyme solution is purified, it is incubated with Lignin-NH2@Fe3O4 at a mass ratio of 46.3 mg / g in a Tris-HCl buffer solution at 25.2 °C, pH 9.0 for 51.2 min. After magnetic separation, immobilized ω-transaminase is obtained, denoted as AtATA@Lignin-NH2@Fe3O4.
[0011] Preferably, between step S04 and step S05, the following steps are further included, specifically: S04.1. After the crude enzyme solution is filtered through a 0.45 μm filter membrane, it is purified by passing through a Ni-NTA affinity chromatography column. S04.2. The chromatography column was equilibrated successively with 20% ethanol, deionized water, and 20 mM imidazole buffer. After sample loading, it was washed with 20 mM and 50 mM imidazole buffers, and finally eluted with 250 mM imidazole buffer to collect the enzyme solution. S04.3. The chromatography column was finally washed with 250 mM imidazole buffer, 20 mM imidazole buffer, deionized water, and 20% ethanol, and stored in a refrigerator at 4°C to obtain ω-transaminase.
[0012] Preferably, in step S05, the pH of the enzyme immobilization buffer is 9.0, the incubation temperature is 25.2°C ± 2°C, and the incubation time is 50 - 60 min.
[0013] Preferably, the immobilized ω-transaminase prepared in step S05 had a residual activity ≥ 57% after incubation at 50°C for 50 min, an activity retention ≥ 78% after 10 cycles of use, and an activity retention ≥ 93% after storage for 120 h.
[0014] Compared with the prior art, the advantages or beneficial effects of the technical solution of this application include: 1. The carrier cost was reduced by 40%, and the enzyme activity recovery rate was increased by more than 20%; 2. The magnetic separation efficiency was improved, reducing enzyme loss; 3. It can be stably operated under acidic (pH 4.0) and high-temperature (50°C) conditions, broadening the industrial application scenarios. Description of the Drawings
[0015] Figure 1 It is the transmission electron microscope image of Lignin-NH2@Fe3O4; Figure 2 It is the result graph of the effect of the mass ratio of enzyme to carrier on the immobilization efficiency; Figure 3 It is the result graph of the effect of immobilization time on the immobilization efficiency; Figure 4 It is the result graph of the effect of immobilization pH on the immobilization efficiency; Figure 5 It is the result graph of the effect of immobilization temperature on the immobilization efficiency. Detailed Embodiments
[0016] The following will combine the drawings and embodiments to detail the implementation manners of this application, so as to fully understand how this application uses technical means to solve technical problems and the implementation process of achieving corresponding technical effects and implement accordingly. The embodiments of this application and each feature in the embodiments can be combined with each other on the premise of not conflicting, and the formed technical solutions are all within the protection scope of this application.
[0017] It should be clear that the embodiments described below are only a part of the embodiments of the present application, rather than all of them. All other embodiments obtained by those skilled in the art based on the embodiments in the present application without creative efforts belong to the scope protected by the present application.
[0018] Example 1: Preparation of Lignin-NH2@Fe3O4 (1)Preparation of Lignin-NH2 Take 5 g of lignin and disperse it in 250 mL of ultrapure water. After mechanical stirring, add 4 g of polyethyleneimine, adjust the pH to 10, then add 4 g of formaldehyde, and react in a water bath at 60 °C for 5 h. Stop the reaction, collect the product by suction filtration, wash it once with pure water, and dry it at 50 - 60 °C.
[0019] (2)Preparation of Lignin-NH2@Fe3O4 Pre-dissolve 6.8 g of ammonium ferrous sulfate hexahydrate and 10.4 g of ferric ammonium sulfate dodecahydrate in 500 mL of deionized water to prepare a solution for standby. Take 4.0 g of aminated lignin and add it to the pre-prepared solution. After ultrasonic stirring for 0.5 h, adjust the pH to 10 with 6 mol / L ammonia water. After the pH stabilizes, mechanically stir in a constant temperature water bath at 50 °C for 1 h. After the reaction is completed, cool to room temperature. After the solution is layered, suction filter to obtain magnetic aminated lignin. Wash it with water multiple times until it is neutral, and then dry it in a vacuum at 60 °C for 24 h until it reaches a constant weight.
[0020] Example 2: Preparation and Purification of ω-Transaminase The ω-transaminase used in this study was obtained by transforming recombinant Escherichia coli containing the pET-28a-ω-TA gene. To improve the catalytic activity and stability, the strain with the L118T mutation site was selected. First, the L118T strain was inoculated into LB medium containing 50 μg / mL and cultured at 37 °C with 180 - 200 r / min for 8 - 10 hours. Then, the bacterial solution was transferred to LB medium containing 50 μg / mL kanamycin and cultured for another 2 - 4 h. When the OD value reached 0.6 - 0.8, 0.8 mM IPTG (inducer, isopropyl-β-D-thiogalactoside) was added, and the culture was continued at 25 °C with 150 r / min for 18 - 20 h. The cells were collected by centrifugation at 4 °C and 6000 rpm for 6 minutes, resuspended in 20 mM imidazole solution, and the cells were disrupted by high-pressure homogenization. After centrifugation at 4 °C and 8000 r / min for 50 minutes, the supernatant was collected as the crude enzyme solution. After the crude enzyme solution was filtered through a 0.45 μm filter membrane, it was purified by passing through a Ni-NTA affinity chromatography column. The chromatography column was equilibrated successively with 20% ethanol, deionized water, and 20 mM imidazole buffer. After sample loading, it was washed with 20 mM and 50 mM imidazole buffer, and finally eluted with 250 mM imidazole buffer to collect the enzyme solution. The chromatography column was finally washed with 250 mM imidazole buffer, 20 mM imidazole buffer, deionized water, and 20% ethanol and stored in a refrigerator at 4 °C. The obtained ω-transaminase was incubated with Lignin-NH2@Fe3O4 at a mass ratio of 46.3 mg / g in Tris-HCl buffer at 25.2 °C and pH 9.0 for 51.2 min. After magnetic separation, the immobilized ω-transaminase, denoted as AtATA@Lignin-NH2@Fe3O4, was obtained.
[0021] Example 3: Industrial-scale amplification preparation of Lignin-NH2@Fe3O4 carrier (1)Raw material pretreatment: Take 5 kg of alkali lignin (a company in Zhejiang, ash content 0.3%), crush it and pass through a 200-mesh sieve; (2)Amination reaction: Add 25 kg of deionized water, 5 kg of lignin, and 4 kg of PEI to a reaction kettle (100 L), and adjust the pH to 10.0 with 20% NaOH; Dropwise add 3.2 kg of formaldehyde (37%), stir and react at 60 °C for 5 h, perform centrifugal separation, and dry in hot air at 60 °C until the water content ≤ 5%; (3)Magnetic loading process: Dissolve 6.8 kg of Fe(NH4)2(SO4)2·6H2O and 10.4 kg of Fe(NH4)(SO4)2·12H2O in 500 L of deionized water; Add 4 kg of Lignin-NH2, disperse it by ultrasound (20 kHz, 1 h), adjust the pH to 10.0 with ammonia water, and react with cyclic stirring at 50 °C for 1 h; Separate the particles by a magnetic separator (magnetic field strength 0.5 T), wash them three times with ethanol, and dry them in a fluidized bed to obtain 4.3 kg of Lignin-NH2@Fe3O4.
[0022] Example 4: Continuous catalysis of immobilized enzyme in a 500 L reactor (1) Immobilized enzyme filling: Mix 2 kg of Lignin-NH2@Fe3O4 with 50 L of ω-transaminase solution (1.2 mg / mL, pH 9.0) at 25 °C; Stir constantly (150 rpm) for 55 min, and obtain 2.1 kg of AtATA@Lignin-NH2@Fe3O4 (enzyme loading 48.6 mg / g) after magnetic separation.
[0023] (2) Continuous flow reaction system: Reactor: 500 L stirred tank (316L stainless steel), equipped with an internal magnetic grid separation unit (magnetic field strength 0.3 T); Substrate feeding: 1-(R)-PEA (20 mM), pyruvic acid (25 mM), PLP (0.1 mM) dissolved in PBS at pH 8.0, flow rate 10 L / h; Operating conditions: 25 °C, dissolved oxygen ≤ 5%, pressure 0.1 MPa; Results: Continuously operate for 120 h (10 batches), the average conversion rate of acetophenone is 78.4%, and the enzyme activity decay rate ≤ 2.1% / batch.
[0024] Control example group: Performance comparison of traditional Fe3O4@SiO2 carriers Carrier preparation: Synthesize Fe3O4@SiO2 by the Stöber method (SiO2 shell thickness ≈ 15 nm); Immobilization conditions: The same enzyme loading, pH, and temperature; Test results: Enzyme activity recovery rate: 58.3% (vs 74.09% of the present invention); Recyclability: The activity drops to 42.1% after the 5th batch (vs 78.4% in the 10th batch of the present invention).
[0025] Example 5: In this study, a variety of analytical techniques were comprehensively used to fully characterize the structure and properties of the materials.
[0026] First, a scanning electron microscope (SEM, model SU1510, Hitachi, Japan) and a transmission electron microscope (TEM, model JEM-2100, JEOL, Japan) were used to observe the morphological characteristics of the samples. Subsequently, a Fourier transform infrared spectrometer (FTIR, model Vertex 70, Molecular Devices, USA) was used to perform infrared spectroscopy detection on the materials, and the scanning range was set to 4000 - 400 cm -1 , in order to analyze the chemical bond and functional group information of the materials. To deeply explore the elemental composition and chemical state of the material surface, an X-ray photoelectron spectrometer (XPS, model EscaLab 250Xi, Thermo Fisher Scientific, USA) was used for analysis. In addition, an X-ray diffractometer (XRD, model XRD-7000, Sailwaven, Suzhou) was used to analyze the crystal structure of the samples. After the samples were prepared into XRD films, they were detected, the scanning range was 10° - 80°, and the scanning speed was 5° / min. To evaluate the thermal stability of the materials, a thermogravimetric analyzer (TG209F3, Netzsch, Germany) was used to perform thermogravimetric analysis (TG), the test temperature range was 50 K - 800 K, and the heating rate was 10 K / min. The specific surface area analysis (BET) was completed by a specific surface area analyzer (ASAP2460, Micromeritics, USA) to reveal the pore structure and specific surface area of the materials and provide a basis for evaluating the adsorption performance of the materials. Finally, a vibrating sample magnetometer (VSM, model 4HF, ADE, USA) was used to evaluate the saturation magnetization intensity of the samples. At 300 K, the magnetic field intensity was set in the range of ±10000 Oe, and the magnetization curve was plotted to comprehensively evaluate the magnetic properties of the materials. The characterization results are as Figure 1 shown.
[0027] Example 6: Determination of the Activity of Immobilized Enzyme In a 1.5 mL centrifuge tube, an equal amount of transaminase and immobilized enzyme were mixed with a reaction substrate containing 2.5 mM pyruvate, 1-(R)-PEA, 0.1 mM PLP, and 50 mL PBS. After incubation in a metal bath at 25 °C and 800 rpm for 3 min, the supernatant was placed in a boiling water bath for 10 min to terminate the transamination reaction, and then the content of acetophenone in the solution was measured by HPLC. The relative activity of the enzyme was calculated based on the obtained data, and the data with the highest activity of the free enzyme and immobilized enzyme were defined as 100%. The recovery rate of the immobilized enzyme was calculated according to the following formula: (1-1) In the formula: Ai: The activity of the immobilized enzyme (U) Af: The activity of the free enzyme equivalent to the immobilized enzyme loading (U) Example 7: Optimization of Reaction Conditions for Immobilized ω-Transaminase The enzyme loading and the activity of immobilized enzyme are two key parameters to measure the performance of immobilized enzyme. In order to prepare an immobilized enzyme with higher catalytic activity and larger enzyme loading, we optimized the conditions of the immobilization process. By optimizing these immobilization conditions, the overall performance of the immobilized enzyme was improved.
[0028] (1)Optimization of Enzyme-to-Carrier Mass Ratio Take 1 mL of transaminase enzyme solution with concentrations of 0.7, 0.8, 0.9, 1.0, and 1.1 mg / mL, add 0.02 g of magnetic nano-lignin (i.e., the enzyme-to-carrier mass ratio is 35, 40, 45, 50, 55 mg / g), incubate in a constant temperature oscillator at 25 °C and 800 rpm for a period of time, then perform magnetic separation, and wash twice with PBS (50 mM, pH 8.0) to obtain AtATA@Lignin-NH2@Fe3O4. Conduct 3 parallel experiments and calculate the relative activity and enzyme loading of the immobilized enzyme respectively. The results of the effect of enzyme-to-carrier mass ratio on the immobilization efficiency are as Figure 2 shown.
[0029] As Figure 2 shown, the effects of different enzyme molecule-to-carrier mass ratios on the relative activity and enzyme loading of AtATA@Lignin-NH2@Fe3O4 were investigated. When the enzyme-to-carrier mass ratio was 45 mg / g, the relative enzyme activity of AtATA@Lignin-NH2@Fe3O4 reached the peak, and the enzyme loading at this time was 22.60 mg / g. After increasing the enzyme-to-carrier mass ratio, the relative activity of the immobilized enzyme gradually decreased. This may be because the active sites on the surface of the Lignin-NH2@Fe3O4 carrier reached saturation, and continuing to increase the addition amount of enzyme molecules would lead to a decrease in enzyme activity.
[0030] (2)Optimization of Immobilization Time Take 1 mL of enzyme solution, add 0.02 g of magnetic nano-lignin, incubate in a constant temperature oscillator at 25 °C and 800 rpm for 10, 20, 30, 40, 50, 60, 70 min, wash twice with PBS (50 mM, pH 8.0), perform magnetic separation, and obtain AtATA@Lignin-NH2@Fe3O4. Conduct 3 parallel experiments and calculate the relative activity and enzyme loading of the immobilized enzyme respectively. The results of the effect of immobilization time on the immobilization efficiency are as Figure 3 shown.
[0031] As Figure 3As shown in the figure, the effects of different immobilization times on the relative activity and enzyme loading of the immobilized enzyme were studied. It can be seen that when the immobilization reaction was 50 min, both the relative enzyme activity and enzyme loading (22.74 mg / g) of AtATA@Lignin-NH2@Fe3O4 reached their peaks. After that, as the immobilization time increased, the relative enzyme activity of the immobilized enzyme decreased instead. This may be because when the immobilization time is too long, some enzyme molecules will fall off the carrier under excessive oscillation, leading to a decrease in the stability of the enzyme.
[0032] (3)Optimization of immobilization pH Buffers with pH values of 8.0, 8.5, 9.0, 9.5, and 10.0 (50 mM Tris-HCl) were prepared. 1 mL of the enzyme solution under different buffers was taken, and 0.02 g of magnetic nano-lignin was added. The mixture was incubated in a constant temperature oscillator at 25 °C and 800 rpm for 50 min, then separated by magnetic force and washed twice with PBS (50 mM, pH 8.0) to obtain AtATA@Lignin-NH2@Fe3O4. Three parallel experiments were carried out, and the relative activity and enzyme loading of the immobilized enzyme were calculated respectively. The results of the effect of immobilization pH on the immobilization efficiency are as Figure 4 shown.
[0033] As Figure 4 shown, the effects of buffers with different pH values on the immobilization reaction were further studied. The experimental data showed that when the enzyme was immobilized under the condition of pH 9.0, both the relative enzyme activity and enzyme loading (23.02 mg / g) of AtATA@Lignin-NH2@Fe3O4 reached their maximum values. This is mainly because the spatial structure of the enzyme may change in an overly acidic or alkaline solution environment, resulting in irreversible inactivation of ω-transaminase, thus affecting the results of immobilization.
[0034] (4)Optimization of immobilization temperature 1 mL of the enzyme solution was taken, and 0.02 g of magnetic nano-lignin was added. The mixture was incubated in a constant temperature oscillator at 10, 15, 20, 25, and 30 °C at 800 rpm for 50 min, then washed twice with PBS (50 mM, pH 8.0), and separated by magnetic force to obtain AtATA@Lignin-NH2@Fe3O4. Three parallel experiments were carried out, and the relative activity and enzyme loading of the immobilized enzyme were calculated respectively. The results of the effect of immobilization temperature on the immobilization efficiency are as Figure 5 shown.
[0035] As Figure 5As shown, the effects of different immobilization temperatures on the immobilization of transaminase were investigated. When the immobilization temperature was 25 °C, both the relative enzyme activity and the enzyme loading (23.12 mg / g) reached their peaks. Further increasing the temperature affected the enzyme activity, which gradually decreased. This may be because temperature not only affects the stability and activity of the enzyme itself but also acts on the binding of the enzyme to the carrier, and a reasonable temperature promotes the effective binding of the carrier to the enzyme molecules.
Claims
1. A preparation method of magnetic nano-lignin, characterized in that, It includes the following steps: (1) Preparation of Lignin-NH2 Disperse lignin into ultrapure water. After mechanical stirring, add polyethyleneimine, adjust the pH to 10, then add formaldehyde, react in a water bath at 60 °C for 5 h, stop the reaction, collect the product by suction filtration, wash it once with pure water, and dry it at 50 - 60 °C to obtain Lignin-NH2; (2) Preparation of Lignin-NH2@Fe3O4 Pre-dissolve ammonium ferrous sulfate hexahydrate and ammonium ferric sulfate dodecahydrate in deionized water to prepare a solution for standby; Add aminated lignin to the pre-prepared solution. After ultrasonic stirring, adjust the pH to 10 with 6 mol / L ammonia water. After the pH stabilizes, mechanically stir in a constant temperature water bath at 50 °C for 1 h. After the reaction ends, cool to room temperature. After the solution is layered, filter by suction to obtain magnetic aminated lignin. After washing it with water multiple times until it is neutral, dry it in a vacuum at 60 °C for 24 h until it reaches a constant weight to obtain magnetic nano-lignin Lignin-NH2@Fe3O4.
2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of lignin to polyethyleneimine is 5:
4.
3. The preparation method according to claim 1, wherein In step (2), the mass ratio of ammonium ferrous sulfate hexahydrate to ammonium ferric sulfate dodecahydrate is 0.
65.
4. The preparation method according to claim 1, characterized in that, In step (2), the specific surface area of the Lignin-NH2@Fe3O4 is 29.8 m² / g, the saturation magnetization intensity is 35 - 40 emu / g, and the nano-particle size ≤ 100 nm.
5. Application of magnetic nano-lignin in the preparation of immobilized ω-transaminase, characterized in that, It includes the following steps: S01: Inoculate the L118T strain into an LB medium containing 50 μg / mL, and culture it at 37 °C and 180 - 200 r / min for 8 - 10 hours; S02: Transfer the bacterial liquid to an LB medium containing 50 μg / mL kanamycin and continue to culture for 2 - 4 h; S03: When the OD value reaches 0.6 - 0.8, add 0.8 mM IPTG (inducer, isopropyl-β-D-thiogalactoside), and culture it at 25 °C and 150 r / min for 18 - 20 h; S04: Collect the cells by centrifugation at 4 °C and 6000 rpm for 6 minutes, resuspend them in a 20 mM imidazole solution, break the cells by high-pressure homogenization, and then centrifuge at 4 °C and 8000 r / min for 50 minutes to collect the supernatant as the crude enzyme solution; S05: After the crude enzyme solution is purified, incubate it with Lignin-NH2@Fe3O4 at a mass ratio of 46.3 mg / g in a Tris-HCl buffer solution at 25.2 °C and pH 9.0 for 51.2 min. After magnetic separation, the immobilized ω-transaminase, denoted as AtATA@Lignin-NH2@Fe3O4, is obtained.
6. Use of a magnetic nano-lignin according to claim 5 in the preparation of immobilized ω-transaminase, characterized in that Between step S04 and step S05, the following steps are also included, specifically: S04.1: Filter the crude enzyme solution through a 0.45 μm filter membrane and purify it through a Ni-NTA affinity chromatography column; S04.2: Equilibrate the chromatography column with 20% ethanol, deionized water, and 20 mM imidazole buffer solution in sequence. After loading the sample, wash it with 20 mM and 50 mM imidazole buffer solutions, and finally elute and collect the enzyme solution with 250 mM imidazole buffer solution; S04.
3. Finally, the chromatography column was washed with 250 mM imidazole buffer, 20 mM imidazole buffer, deionized water, and 20% ethanol and stored in a refrigerator at 4°C to obtain ω-transaminase.
7. Use of a magnetic nano-lignin according to claim 5 in the preparation of an immobilized ω-transaminase, characterized in that, In step S05, the pH of the enzyme immobilization buffer was 9.0, the incubation temperature was 25.2°C ± 2°C, and the incubation time was 50 - 60 min.
8. Use of a magnetic nano-lignin according to claim 5 in the preparation of an immobilized ω-transaminase, characterized in that, The immobilized ω-transaminase prepared in step S05 had a residual activity of ≥57% after incubation at 50°C for 50 min, an activity retention of ≥78% after 10 cycles of use, and an activity retention of ≥93% after storage for 120 h.