Application of magnetic separation-based amino modified metal organic framework immobilized ethanol dehydrogenase in screening of ethanol dehydrogenase inhibitor in traditional Chinese medicine
By magnetically separating amino-modified metal-organic framework material Fe3O4@NH2-MIL-101(Fe) to immobilize alcohol dehydrogenase, the problems of enzyme activity loss and separation difficulties were solved, enabling efficient screening of alcohol dehydrogenase inhibitors from traditional Chinese medicine. This provides a safe and stable enzyme preparation method and an efficient screening platform.
Patent Information
- Application Number
- CN202511335075.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-18
- Publication Date
- 2025-12-12
AI Technical Summary
Existing chemically synthesized or microbially derived alcohol dehydrogenase inhibitors are not safe or stable enough and have side effects. Traditional immobilization methods are prone to enzyme activity loss and separation difficulties, making it difficult to efficiently screen alcohol dehydrogenase inhibitors from traditional Chinese medicine.
Using Fe3O4@NH2-MIL-101(Fe), an amino-modified metal-organic framework material with magnetic separation, as a carrier, alcohol dehydrogenase was immobilized through covalent cross-linking and adsorption. Combined with magnetic separation technology, immobilized enzymes with high enzyme activity and stability were prepared for screening traditional Chinese medicine.
It improves enzyme stability and reusability, simplifies the separation process, reduces production costs, increases screening throughput and detection accuracy, and provides an efficient screening platform for alcohol dehydrogenase inhibitors from traditional Chinese medicine.
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Figure CN121109547A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of enzyme engineering, and particularly relates to application of ethanol dehydrogenase immobilized by magnetic separation amino-modified metal organic framework in screening of ethanol dehydrogenase inhibitors in traditional Chinese medicines. BACKGROUND
[0002] Alcoholic allergy and alcoholic liver injury have become an important problem threatening public health worldwide. The skin contains about one-third of the exogenous enzymes present in the liver, among which ethanol dehydrogenase (ADH) plays a key role in alcohol metabolism. When the human body ingests alcohol, ethanol is rapidly converted to acetaldehyde under the catalysis of ethanol dehydrogenase. Acetaldehyde, as a toxic metabolite, not only causes oxidative stress damage to liver cells, leading to hepatocyte inflammation, fatty degeneration and even cirrhosis, but also binds with proteins in skin tissue to form a hapten, triggering an immune response and causing skin redness, itching, edema and other allergic symptoms. According to statistics from the World Health Organization, the number of deaths caused by alcoholic liver disease worldwide continues to rise each year, and complications such as skin allergy and nervous system damage caused by alcohol metabolism also bring great pain to patients. Therefore, finding effective substances that can inhibit the activity of ethanol dehydrogenase and block the toxicity of ethanol metabolism has become a key direction for the prevention and treatment of alcohol-related diseases.
[0003] Immobilized enzyme technology, which combines free enzyme with carriers, significantly improves the stability and reusability of enzymes, and shows great potential in biocatalysis and drug screening. MIL-101(Fe) in metal-organic frameworks (MOFs) has become an ideal carrier for enzyme immobilization due to its ultra-high specific surface area, rich pore structure and good chemical stability. However, the unmodified MIL-101(Fe) has limited active sites on the surface and weak binding force with enzymes, and the immobilization process easily leads to loss of enzyme activity. After modification of MIL-101(Fe) with amino (-NH2) to form NH2-MIL-101(Fe), a large number of amino functional groups are introduced on the surface of the carrier, which can not only form a stable covalent bond network through high-efficiency cross-linking reaction with glutaraldehyde, enhancing the binding strength of the carrier and the enzyme, but also optimize the spatial orientation of the enzyme through electrostatic interaction and hydrogen bonding between the amino group and the enzyme molecule, reducing the loss of activity during the immobilization process, significantly improving the enzyme loading capacity and stability. Magnetic nanomaterials such as Fe3O4 have unique magnetic response characteristics and can be quickly separated under an external magnetic field, but the specific surface area of single magnetic material is small and the enzyme loading efficiency is low. The combination of Fe3O4 and NH2-MIL-101(Fe) to form Fe3O4@NH2-MIL-101(Fe) not only retains the high-efficiency immobilization capacity of NH2-MIL-101(Fe) for enzymes, but also endows the material with magnetic separation function, so that the immobilized enzyme can be quickly recovered by simple magnetic separation after the reaction is completed, greatly improving the separation efficiency and avoiding the damage to the enzyme structure caused by traditional separation methods such as centrifugation and filtration.
[0004] The existing inhibitors of ethanol dehydrogenase from chemical synthesis or microbial sources have the problems of insufficient safety and stability and some side effects, so it is an important direction to find ethanol dehydrogenase inhibitors with safety, stability and low side effects for the treatment and prevention of alcoholic liver damage and alcohol allergy. By using immobilized ethanol dehydrogenase, combining with modern analysis techniques such as chromatography-mass spectrometry, and high-throughput screening of ethanol dehydrogenase inhibitors from traditional Chinese medicines and natural products, important technical support can be provided for the drug research and development of alcohol allergy, alcoholic liver damage and related diseases. SUMMARY
[0005] The purpose of the present application is to provide a method for immobilizing ethanol dehydrogenase by covalent cross-linking and adsorption using magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe) as a carrier, to obtain immobilized ethanol dehydrogenase with high enzyme activity and stability and reusability, and to apply it to the screening of ethanol dehydrogenase inhibitors in traditional Chinese medicines.
[0006] In order to achieve the above object, the application adopts the following technical scheme: application of immobilized ethanol dehydrogenase based on magnetic separation amino-modified metal organic framework in screening of ethanol dehydrogenase inhibitors in traditional Chinese medicines, wherein the immobilized ethanol dehydrogenase is prepared by covalent cross-linking and adsorption of ethanol dehydrogenase with the magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe) as a carrier.
[0007] The application includes the following steps: activating and cross-linking the magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe) in a glutaraldehyde solution, magnetically separating, washing and drying the glutaraldehyde, mixing the activated and cross-linked magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe) with an ethanol dehydrogenase solution, magnetically separating after covalent cross-linking and adsorption, washing, drying, and obtaining the immobilized ethanol dehydrogenase.
[0008] The application has a mass ratio of ethanol dehydrogenase: Fe3O4@NH2-MIL-101(Fe)=(2-8):1.
[0009] The application has a mass concentration of the glutaraldehyde solution of 1%-6%, and the activation and cross-linking time is 1-2h.
[0010] The application has a preparation method of the magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe), which includes the following steps:
[0011] Iron chloride trihydrate is added to N,N-dimethylformamide and is dissolved by ultrasonic, then 2-amino terephthalic acid is added to N,N-dimethylformamide and is dissolved by ultrasonic, the above-mentioned solutions are mixed, then ferric oxide is added and is dispersed uniformly by ultrasonic, is transferred to a sealed reaction kettle for hydrothermal reaction, is cooled to room temperature after the reaction, is washed several times, is collected by a magnet, and is dried in vacuum to obtain Fe3O4@NH2-MIL-101(Fe).
[0012] The application has a reaction at 110℃ for 20h.
[0013] The application has an ethanol dehydrogenase solution prepared by dissolving ethanol dehydrogenase in a PBS buffer solution with a pH of 7-9.
[0014] The application has covalent cross-linking and adsorption at a rotation speed of 150-200 r / min, a temperature of 15-55℃, for 10min-4h.
[0015] The application of the above, the magnetic metal organic framework material Fe3O4@NH2-MIL-101 (Fe) is activated and cross-linked with 2% glutaraldehyde solution for 1h, is separated by magnetic attraction, is washed, is mixed with an ethanol dehydrogenase solution, is covalently cross-linked and adsorbed at a rotating speed of 150~200 r / min and 25 DEG C for 0.5h, is separated by magnetic attraction, is washed, is dried, and immobilized ethanol dehydrogenase is obtained.
[0016] The beneficial effects of the application are as follows:
[0017] 1、MIL-101(Fe) in metal organic framework material (MOFs) has ultra-high specific surface area, rich pore structure and good chemical stability, and becomes an ideal enzyme immobilization carrier. However, the unmodified MIL-101(Fe) has limited active sites on the surface, and is prone to cause loss of enzyme activity in the immobilization process. After the MIL-101(Fe) is modified into NH2-MIL-101(Fe) by amino (-NH2), a large number of amino functional groups are introduced on the surface of the carrier, which can not only have a high cross-linking reaction with glutaraldehyde to form a stable covalent bond network and enhance the binding strength of the carrier and the enzyme, but also can optimize the spatial orientation of the enzyme through the electrostatic interaction and hydrogen bond interaction between the amino group and the enzyme molecule, reduce the activity loss in the immobilization process, and significantly improve the enzyme loading capacity and stability. After the NH2-MIL-101(Fe) is combined with magnetic nanoparticles to form Fe3O4@NH2-MIL-101(Fe), not only the high enzyme loading capacity of NH2-MIL-101(Fe) is retained, but also the problem of difficult recovery of traditional MOFs materials is overcome, so that the immobilized enzyme can be quickly separated from the reaction mixture through magnetic attraction, avoiding the tedious centrifugal process of traditional separation, and reducing the loss of immobilized enzyme in the separation process, and the separation efficiency is greatly improved.
[0018] 2、The preparation method of the immobilized ethanol dehydrogenase of the application adopts a two-step cross-linking method, glutaraldehyde is used as a cross-linking agent, the magnetic metal organic framework material is activated and cross-linked in the glutaraldehyde, and after being washed and dried, the enzyme solution is cross-linked and adsorbed, so that the toxicity of high-concentration glutaraldehyde solution to the enzyme can be avoided. The immobilization process has mild reaction conditions, avoiding the damage of high temperature, strong acid / alkali and other harsh conditions to the enzyme activity. The immobilized enzyme not only improves the thermal stability of ethanol dehydrogenase, but also improves the recyclability, helps to prolong the shelf life and service life of the enzyme, and enables the ethanol dehydrogenase to be used repeatedly. At the same time, the process does not require complex equipment, the raw materials are cheap and easy to obtain, and the production cost and resource consumption are significantly reduced.
[0019] 3、The prepared immobilized ethanol dehydrogenase is combined with liquid chromatography-mass spectrometry (LC-MS) technology to provide an efficient platform for screening of ethanol dehydrogenase inhibitors in traditional Chinese medicines. Compared with the traditional free enzyme screening method, the technology has the following advantages: short analysis time, greatly improved screening throughput, reuse of immobilized enzyme to greatly reduce the cost of single run. At the same time, magnetic separation avoids complex sample pretreatment steps, reduces impurity interference, and improves detection sensitivity and accuracy. This technology system can quickly and accurately screen active ingredients that inhibit the activity of ethanol dehydrogenase from traditional Chinese medicine compounds, providing key technical support for the drug research and development of diseases such as alcoholic liver damage and skin alcohol allergy, and is expected to accelerate the discovery of new alcoholism drugs and liver protection drugs. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 is the IR graph of Fe3O4@NH2-MIL-101 (Fe) prepared in the application.
[0021] Figure 2 is the XRD graph of Fe3O4@NH2-MIL-101 (Fe) prepared in the application.
[0022] Figure 3 is the effect of glutaraldehyde solution concentration on Fe3O4@NH2-MIL-101 (Fe) immobilized ethanol dehydrogenase.
[0023] Figure 4 is the effect of carrier material mass on Fe3O4@NH2-MIL-101 (Fe) immobilized ethanol dehydrogenase.
[0024] Figure 5 is the effect of immobilized ethanol dehydrogenase prepared in the application on relative enzyme activity at different temperatures.
[0025] Figure 6 is the effect of immobilized ethanol dehydrogenase prepared in the application on relative enzyme activity at different pH values.
[0026] Figure 7 is the relative enzyme activity of immobilized ethanol dehydrogenase prepared in the application after 7 times of repeated operation. DETAILED DESCRIPTION
[0027] The application will be further described in detail below in combination with examples and drawings. The specific examples described below are not used to limit the application, but only to explain the application.
[0028] In the following examples, the ethanol dehydrogenase powder was purchased from Shanghai Yuanye Biological Technology Co., Ltd.; for various reagents and operations used in the examples, unless otherwise specified, they are conventional reagents and operations in the art.
[0029] Example 1
[0030] The preparation method of the immobilized ethanol dehydrogenase enzyme comprises the following steps:
[0031] 1. Preparation of magnetic organic framework material Fe3O4@NH2-MIL-101(Fe)
[0032] Dissolve 3.38 g of iron trichloride hexahydrate in 20 mL of N,N-dimethylformamide, and ultrasonic for 10 min; then take 1.08 g of 2-amino terephthalic acid and add it to 30 mL of N,N-dimethylformamide, and ultrasonic for 10 min; mix the above solutions, and then add 300 mg of Fe3O4, and ultrasonic for 30 min to make it uniformly dispersed; place the obtained mixture in a polytetrafluoroethylene reaction kettle, and react at 110°C for 20 h; magnetically collect the product, then sequentially wash with N,N-dimethylformamide and anhydrous methanol for three times, and vacuum dry at 60°C for 10-12 h until anhydrous, to obtain the magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe), which is labeled and stored for use.
[0033] 2. Preparation of ethanol dehydrogenase solution
[0034] Take 5.0 mg of ethanol dehydrogenase, dissolve it with PBS buffer solution at pH 7.4, and constant volume to 50 mL, to obtain an ethanol dehydrogenase solution with a concentration of 0.1 mg / mL, which is stored at 4°C for use.
[0035] 3. Preparation of immobilized ethanol dehydrogenase
[0036] Take 10-50 mg of magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe), add 2 mL of 2% glutaraldehyde solution, and activate and crosslink at 25°C and 150-200 r / min for 1 h; then wash with PBS buffer solution at pH 7.4 for three times to remove unreacted glutaraldehyde solution; then mix with 1 mL of 0.1 mg / mL ethanol dehydrogenase solution, and covalently crosslink and adsorb in a constant temperature oscillator at a temperature of 15-55°C and a rotation speed of 150-200 r / min for 10 min-4 h; magnetically separate, wash with PBS buffer solution at pH 7.4 for three times, and dry, to obtain immobilized ethanol dehydrogenase.
[0037] Example 2: Effect of glutaraldehyde solution concentration on Fe3O4@NH2-MIL-101(Fe) immobilized ethanol dehydrogenase
[0038] 1. Preparation of magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe)
[0039] 3.38 g of ferric chloride hexahydrate was dissolved in 20 mL of N,N-dimethylformamide and sonicated for 10 min to dissolve. Then, 1.08 g of 2-aminoterephthalic acid was added to 30 mL of N,N-dimethylformamide and sonicated for 10 min to dissolve. The solutions were mixed, and then 300 mg of Fe3O4 was added and sonicated for 30 min to disperse the mixture evenly. The resulting mixture was placed in a polytetrafluoroethylene reactor and reacted at 110 °C for 20 h. The product was collected by magnetic absorption and then washed three times each with N,N-dimethylformamide and anhydrous methanol. The product was then vacuum dried at 60 °C for 12 h until anhydrous to obtain the magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe), which was labeled and stored for later use. Figure 1 This is the IR spectrum of the prepared Fe3O4@NH2-MIL-101(Fe). (From...) Figure 1 The presence of characteristic peaks indicates the successful synthesis of the composite material. Figure 2 This is the XRD pattern of the prepared Fe3O4@NH2-MIL-101(Fe), from... Figure 2 It is evident that the composite material exhibits the same characteristic peaks as NH2-MIL-101(Fe) and Fe3O4, confirming the formation of highly crystalline NH2-MIL-101(Fe) on the magnetic nanoparticles.
[0040] 2. Preparation of alcohol dehydrogenase solution
[0041] Weigh 5.0 mg of alcohol dehydrogenase, dissolve it in PBS buffer solution at pH 7.4 and bring the volume to 50 mL to obtain an alcohol dehydrogenase solution with a concentration of 0.1 mg / mL. Store the solution at 4 °C for later use.
[0042] 3. Preparation of immobilized alcohol dehydrogenase
[0043] Take 30 mg of magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe), add 2 mL of 1%, 2%, 3%, 4%, 5%, and 6% glutaraldehyde solution respectively, and set up 3 parallel samples for each concentration. Activate and crosslink at 25℃ and 200 r / min for 1 h. Then wash 3 times with PBS buffer solution at pH 7.4 to remove unreacted glutaraldehyde solution. Then mix with 1 mL of 0.1 mg / mL alcohol dehydrogenase solution, and covalently crosslink and adsorb for 0.5 h in a constant temperature shaker at 25℃ and 200 r / min. Separate by magnetic adsorption, wash 3 times with PBS buffer solution at pH 7.4, and dry to obtain immobilized alcohol dehydrogenase.
[0044] 4. Determination of the relative activity of immobilized alcohol dehydrogenase
[0045] With the assistance of oxidized coenzyme I (NAD+), alcohol dehydrogenase catalyzes the dehydrogenation of ethanol to acetaldehyde, simultaneously generating reduced coenzyme (NADH), which exhibits characteristic absorption at 340 nm, while NAD+ shows no absorption at the same wavelength. This method is used to determine the enzyme activity of alcohol dehydrogenase. 2 mL of PBS, 0.5 mL of 1 mg / mL oxidized coenzyme I (NAD+), and 0.5 mL of anhydrous ethanol were added to the immobilized alcohol dehydrogenase prepared in step 3 above. The mixture was thoroughly mixed and reacted at room temperature for 5 min. The immobilized enzyme and reaction solution were separated by magnetic adsorption, and the absorbance of the reaction solution at 340 nm was measured using a UV spectrophotometer. The relative activity of the immobilized alcohol dehydrogenase at glutaraldehyde concentrations of 1%–6% was determined.
[0046] Depend on Figure 3 It can be seen that the relative activity of immobilized alcohol dehydrogenase reaches its highest value when the glutaraldehyde concentration is 2%. Subsequently, the relative enzyme activity decreases as the glutaraldehyde concentration increases. Therefore, the optimal cross-linking agent glutaraldehyde concentration for immobilized alcohol dehydrogenase is 2%.
[0047] Example 2: Effect of carrier material quality on Fe3O4@NH2-MIL-101(Fe) immobilized alcohol dehydrogenase
[0048] 1. Preparation of magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe)
[0049] 3.38 g of ferric chloride hexahydrate was dissolved in 20 mL of N,N-dimethylformamide and sonicated for 10 min to dissolve. Then, 1.08 g of 2-aminoterephthalic acid was added to 30 mL of N,N-dimethylformamide and sonicated for 10 min to dissolve. The solutions were mixed, and then 300 mg of Fe3O4 was added and sonicated for 30 min to disperse the mixture evenly. The resulting mixture was placed in a polytetrafluoroethylene reactor and reacted at 110 °C for 20 h. The product was collected by magnetic absorption and then washed three times each with N,N-dimethylformamide and anhydrous methanol. The product was then vacuum dried at 60 °C for 12 h until anhydrous to obtain the magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe), which was labeled and stored for later use.
[0050] 2. Preparation of alcohol dehydrogenase solution
[0051] Weigh 5.0 mg of alcohol dehydrogenase, dissolve it in PBS buffer solution at pH 7.4 and bring the volume to 50 mL to obtain an alcohol dehydrogenase solution with a concentration of 0.1 mg / mL. Store the solution at 4 °C for later use.
[0052] 3. Preparation of immobilized alcohol dehydrogenase
[0053] Respectively, 10, 20, 30, 40, 50 mg of magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe) was weighed, 2 mL of 2% glutaraldehyde solution was added to each, 3 parallel samples were set up for each carrier material mass, and crosslinking was activated at 25°C and 200 r / min for 1 h; then washed with 3 times of pH 7.4 PBS buffer solution to remove the unreacted glutaraldehyde solution; then mixed with 1 mL of 0.1 mg / mL ethanol dehydrogenase solution, covalently crosslinked and adsorbed in a constant temperature oscillator at a temperature of 25°C and a rotation speed of 200 r / min for 0.5 h, magnetically separated, washed with 3 times of pH 7.4 PBS buffer solution, and dried to obtain immobilized ethanol dehydrogenase.
[0054] 5、Immobilized ethanol dehydrogenase relative activity determination
[0055] Under the assistance of oxidized coenzyme I (NAD+), ethanol dehydrogenase catalyzes ethanol dehydrogenation into acetaldehyde, and reduced coenzyme (NADH) is generated at the same time, which has a characteristic absorption at 340 nm, while NAD+ has no absorption at the same wavelength. The enzyme activity of ethanol dehydrogenase was determined by this method. 2 mL of PBS, 0.5 mL of 1 mg / mL oxidized coenzyme I (NAD+) and 0.5 mL of anhydrous ethanol were added to the immobilized ethanol dehydrogenase prepared in step 3 above, mixed well, reacted at room temperature for 5 min, magnetically separated the immobilized enzyme and the reaction solution, and the absorbance value of the reaction solution at 340 nm was determined by ultraviolet spectrophotometry. The relative activity of ethanol dehydrogenase immobilized without mass carrier material was determined.
[0056] From Figure 4 It can be seen that the relative activity of immobilized ethanol dehydrogenase tends to be flat when the mass of magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe) is 30 mg. Therefore, considering the economic benefits, the optimal mass of magnetic metal organic framework material for immobilizing ethanol dehydrogenase is determined to be 30 mg.
[0057] Example 4 Effect of Fe3O4@NH2-MIL-101(Fe) immobilized ethanol dehydrogenase on relative enzyme activity at different temperatures
[0058] 1、Preparation of magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe)
[0059] Take 3.38 g of iron trichloride hexahydrate dissolved in 20 mL of N, N- dimethylformamide, ultrasonic 10 min dissolved; Take 1.08 g of 2-amino terephthalic acid into 30 mL of N, N-dimethylformamide, ultrasonic 10 min dissolved, mix the above solution, then add 300 mg of Fe3O4, ultrasonic 30 min to make it uniform dispersion; The resulting mixture was placed in a polytetrafluoroethylene reactor at 110°C for 20 h, and the product was collected by magnetic absorption, then washed with N, N-dimethylformamide and anhydrous methanol respectively for three times, and dried at 60°C under vacuum for 12 h to obtain the magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe), labeled and stored for standby.
[0060] 2. Preparation of ethanol dehydrogenase solution
[0061] Take 5.0 mg of ethanol dehydrogenase, dissolve and constant volume to 50 mL with PBS buffer solution of pH 7.4 to obtain ethanol dehydrogenase solution with a concentration of 0.1 mg / mL, and store at 4°C for standby.
[0062] 3. Preparation of immobilized ethanol dehydrogenase
[0063] Take 30 mg of magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe), add 2 mL of 2% glutaraldehyde solution respectively, activate and crosslink at 25°C and 200 r / min for 1 h; Then wash with PBS buffer solution of pH 7.4 for 3 times to remove unreacted glutaraldehyde solution; Then mix with 1 mL of 0.1 mg / mL ethanol dehydrogenase solution, covalently crosslink and adsorb in a constant temperature oscillator at 25°C and 200 r / min for 0.5 h, separate by magnetic absorption, wash with PBS buffer solution of pH 7.4 for 3 times, and dry to obtain immobilized ethanol dehydrogenase.
[0064] 4. Determination of relative activity of immobilized ethanol dehydrogenase
[0065] Under the assistance of oxidized coenzyme I (NAD+), ethanol dehydrogenase catalyzes ethanol dehydrogenation into acetaldehyde, while generating reduced coenzyme (NADH), which has a characteristic absorption at 340 nm, while NAD+ has no absorption at the same wavelength. The enzyme activity of ethanol dehydrogenase is determined by this method. The immobilized ethanol dehydrogenase prepared in step 3 above and the corresponding free enzyme are incubated at 5, 15, 25, 35, 45, 55, 60 and 65°C for 1h, and 3 parallel samples are set up at each temperature. Then 2mL PBS, 0.5mL 1mg / mL oxidized coenzyme I (NAD+) and 0.5mL anhydrous ethanol are added, mixed thoroughly, and reacted at room temperature for 5min. The immobilized enzyme and the reaction solution are separated by magnetic attraction, and the absorbance value of the reaction solution at 340nm is determined by ultraviolet spectrophotometry. The relative activity of the immobilized ethanol dehydrogenase and the free enzyme at different temperatures from 5 to 65°C is determined.
[0066] Figure 5 The results of the thermal stability of the immobilized ethanol dehydrogenase and the free enzyme are shown in the graph. As can be seen from the graph, compared with the free enzyme, the immobilized enzyme has temperature tolerance (temperature tolerance range: 5-65°C), so it is proved that the immobilized ethanol dehydrogenase has better thermal stability than the free enzyme.
[0067] Example 5 Effect of Fe3O4@NH2-MIL-101(Fe) immobilized ethanol dehydrogenase on relative enzyme activity at different pH values
[0068] 1. Preparation of magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe)
[0069] Take 3.38g of iron trichloride hexahydrate and dissolve it in 20mL of N,N-dimethylformamide, and ultrasonic for 10min; then take 1.08g of 2-amino terephthalic acid and add it to 30mL of N,N-dimethylformamide, and ultrasonic for 10min; mix the above solutions and then add 300mg of Fe3O4, and ultrasonic for 30min to make it uniformly dispersed; place the obtained mixture in a polytetrafluoroethylene reaction kettle and react at 110°C for 20h; collect the product by magnetic attraction, then wash it with N,N-dimethylformamide and anhydrous methanol respectively for three times, and vacuum dry at 60°C for 12h until it is anhydrous; thus the magnetic metal organic framework material Fe3O4@NH2-MIL-101(Fe) is obtained, which is labeled and stored for use.
[0070] 2. Preparation of ethanol dehydrogenase solution
[0071] Take 5.0mg of ethanol dehydrogenase, dissolve it with PBS buffer solution of pH 7.4 and dilute to 50mL to obtain an ethanol dehydrogenase solution with a concentration of 0.1mg / mL, which is stored at 4°C for use.
[0072] 3. Preparation of immobilized alcohol dehydrogenase
[0073] Take 30 mg of magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe), add 2 mL of 2% glutaraldehyde solution, and activate cross-linking at 25℃ and 200 r / min for 1 h; then wash 3 times with PBS buffer solution at pH 7.4 to remove unreacted glutaraldehyde solution; then mix with 1 mL of 0.1 mg / mL alcohol dehydrogenase solution, and covalently cross-link and adsorb for 0.5 h in a constant temperature shaker at 25℃ and 200 r / min, separate by magnetic adsorption, wash 3 times with PBS buffer solution at pH 7.4, and dry to obtain immobilized alcohol dehydrogenase.
[0074] 4. Determination of the relative activity of immobilized alcohol dehydrogenase
[0075] With the assistance of oxidized coenzyme I (NAD+), alcohol dehydrogenase catalyzes the dehydrogenation of ethanol to acetaldehyde, simultaneously generating reduced coenzyme (NADH), which exhibits characteristic absorption at 340 nm. NAD+, however, shows no absorption at the same wavelength. This method is used to determine the enzyme activity of alcohol dehydrogenase. The immobilized alcohol dehydrogenase and the corresponding free enzyme prepared in step 3 above were incubated for 1 h in PBS buffer solutions at pH values of 5, 5.5, 6, 6.5, 7, 7.4, 8, 8.5, 9, 9.5, and 10, respectively, with three replicates for each pH. Then, 2 mL of PBS, 0.5 mL of 1 mg / mL oxidized coenzyme I (NAD+), and 0.5 mL of anhydrous ethanol were added, thoroughly mixed, and reacted at room temperature for 5 min. The immobilized enzyme and reaction solution were separated by magnetic adsorption, and the absorbance of the reaction solution at 340 nm was measured using a UV spectrophotometer. The relative activities of immobilized alcohol dehydrogenase and free enzyme were determined at different pH values of 5, 5.5, 6, 6.5, 7, 7.4, 8, 8.5, 9, 9.5, and 10.
[0076] Figure 6 The figure shows the pH stability results for immobilized alcohol dehydrogenase and the free enzyme. As can be seen from the figure, the immobilized enzyme exhibits pH tolerance (pH tolerance range of 5-10). However, the figure also shows that the immobilized alcohol dehydrogenase has a relatively lower enzyme activity than the free enzyme under slightly acidic conditions. This may be because the amino groups (-NH2) on the surface of the NH2-MIL-101(Fe) support undergo protonation to form positively charged -NH3. +On the one hand, this change in charge may affect the electrostatic interaction and hydrogen bond network between the carrier and alcohol dehydrogenase, leading to unfavorable changes in the spatial conformation of the enzyme on the carrier surface. The structure of the active site will no longer be in its optimal catalytic state, thus reducing enzyme activity. On the other hand, protonated amino groups may repel certain acidic groups on the alcohol dehydrogenase molecule, weakening the binding force between them. This can cause some enzyme to detach from the carrier or shift, affecting the effective concentration and catalytic efficiency of the enzyme. Meanwhile, an alkaline environment has little effect on the structural stability of the NH2-MIL-101(Fe) carrier; the binding between the carrier and the enzyme remains strong, maintaining a relatively stable enzyme conformation, which is beneficial for the enzyme to exert its catalytic activity. Furthermore, alkaline conditions may also affect the form of the substrate, making it easier for it to bind to the enzyme's active site, indirectly improving the enzyme's catalytic efficiency and making the immobilized enzyme more alkali-tolerant.
[0077] Example 6: Relative enzyme activity of Fe3O4@NH2-MIL-101(Fe) immobilized alcohol dehydrogenase after 7 repeated operations
[0078] Methods: Take 30 mg of immobilized alcohol dehydrogenase, add 2 mL of PBS buffer solution, 0.5 mL of 1 mg / mL oxidized coenzyme I (NAD+) and 0.5 mL of anhydrous ethanol, mix thoroughly, react at room temperature for 5 min, magnetically separate the immobilized enzyme and reaction solution, and measure the absorbance of the reaction solution at 340 nm using a UV spectrophotometer. Wash the immobilized alcohol dehydrogenase with PBS buffer solution, and then add the immobilized alcohol dehydrogenase back into the substrate solution to repeat the experiment.
[0079] The results are as follows Figure 7 As shown, after seven repetitions, the relative activity of the immobilized alcohol dehydrogenase remained above 88%, indicating that Fe3O4@NH2-MIL-101(Fe) is a good carrier for immobilizing alcohol dehydrogenase.
Claims
1. An application of an immobilized alcohol dehydrogenase based on magnetic separation of an amino-modified metal-organic framework in screening alcohol dehydrogenase inhibitors in traditional Chinese medicine, characterized in that, The immobilized alcohol dehydrogenase is prepared by covalent cross-linking and adsorption of alcohol dehydrogenase using the magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe) as a carrier.
2. The application according to claim 1, characterized in that, The process includes the following steps: activating and cross-linking the magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe) in glutaraldehyde solution, magnetically separating the material, washing away the glutaraldehyde, mixing the activated and cross-linked magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe) with an alcohol dehydrogenase solution, covalently cross-linking and adsorbing the material, then magnetically separating the adsorbed material, washing, and drying to obtain immobilized alcohol dehydrogenase.
3. The application according to claim 2, characterized in that, By mass ratio: alcohol dehydrogenase: Fe3O4@NH2-MIL-101(Fe) = (2~8):
1.
4. The application according to claim 2, characterized in that, The mass concentration of the glutaraldehyde solution is 1% to 6%, and the activation crosslinking time is 1 to 2 hours.
5. The application according to claim 2, characterized in that, The preparation method of the magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe) includes the following steps: Ferric chloride hexahydrate was added to N,N-dimethylformamide and sonicated to dissolve it. Then, 2-aminoterephthalic acid was added to N,N-dimethylformamide and sonicated to dissolve it. The above solutions were mixed and then iron(III) oxide was added and sonicated to disperse it evenly. The mixture was then transferred to a closed reactor for hydrothermal reaction. After the reaction was completed, the mixture was cooled to room temperature, washed several times, and the product was collected with a magnet. After vacuum drying, Fe3O4@NH2-MIL-101(Fe) was obtained.
6. The application according to claim 5, characterized in that, The reaction was carried out at 110°C for 20 hours.
7. The application according to claim 2, characterized in that, The alcohol dehydrogenase solution is prepared by dissolving alcohol dehydrogenase in PBS buffer solution with a pH of 7-9.
8. The application according to claim 2, characterized in that, The covalent crosslinking and adsorption were carried out at a rotation speed of 150-200 r / min and a temperature of 15-55℃ for 10 min to 4 h.
9. The application according to claim 2, characterized in that, The magnetic metal-organic framework material Fe3O4@NH2-MIL-101(Fe) was activated and crosslinked with 2% glutaraldehyde solution for 1 h, separated by magnetic adsorption, washed, and then mixed with alcohol dehydrogenase solution. The mixture was covalently crosslinked and adsorbed at 150~200 r / min and 25℃ for 0.5 h, separated by magnetic adsorption, washed, and dried to obtain immobilized alcohol dehydrogenase.