Immobilized amine oxidase as well as preparation method and application thereof
By adsorbing amine oxidase on Fe3O4@Tb-mesoMOF crystals, forming an immobilized catalyst, the problems of poor stability and reusability of amine oxidase are solved, and efficient bioamine degradation effect is achieved.
Patent Information
- Application Number
- CN202411692094.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-03-28
Smart Images
Figure BDA0005151224660000061
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of amine oxidase, and in particular to an immobilized amine oxidase and a preparation method and application thereof. Background Art
[0002] Biogenic amines are low molecular weight nitrogen-containing organic bases, mainly formed by the decarboxylation of amino acids. Biogenic amines are widely distributed in nature and can be produced by the metabolism of microorganisms, plants and animals, and can be ingested into the human body through food. Appropriate amounts of biogenic amines have positive effects on the human body, such as improving human immunity, enhancing vascular activity, and regulating mental activities. However, when biogenic amines accumulate in the human body in large quantities, they can cause different toxic effects, such as headaches, hypotension, palpitations and vomiting, and in severe cases, they can be life-threatening. The biogenic amines in food mainly come from the food raw materials themselves and the food processing and storage process. Among them, fruits, vegetables, grains and other food raw materials contain a small amount of biogenic amines, and more biogenic amines are produced by the growth and metabolism of microorganisms during food processing and storage. The content of biogenic amines in fermented foods is generally high. Therefore, it is of great significance to reasonably control the content of biogenic amines in fermented foods.
[0003] Current research shows that amine oxidase is the main biogenic amine degrading enzyme. It is an important enzyme that can catalyze the oxidation of amines to generate aldehyde compounds and ammonia and release hydrogen peroxide. It is widely used in the fields of food industry, biosensor, environmental testing, etc. The amine oxidase used in the prior art is generally in a free state, but the free amine oxidase usually has the following disadvantages:
[0004] 1. Poor stability: easily affected by temperature, pH value and organic solvents;
[0005] 2. Poor reusability: cannot be recycled, increasing costs;
[0006] 3. Reduced enzyme activity: It is easy to be inactivated during operation.
[0007] This results in a low degradation rate of biogenic amines. Summary of the invention
[0008] In view of the deficiencies in the prior art, the present invention provides an immobilized amine oxidase and a preparation method and application thereof, which solve the problems in the prior art that the amine oxidase has poor performance and low degradation rate of biogenic amines.
[0009] A method for preparing immobilized amine oxidase comprises the following steps:
[0010] S1. Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O were used as raw materials to obtain Fe3O4@Tb-mesoMOF crystals by solvothermal synthesis reaction in a mixed solvent of DMA, methanol and water;
[0011] S2. The Fe3O4@Tb-mesoMOF crystals prepared in S1 were immersed in an AOs solution containing HEPES buffer for adsorption, and AOs-Fe3O4@Tb-mesoMOF was obtained after sufficient adsorption.
[0012] Preferably, in step S1, the mass ratio of Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O is 0.5-3.0:0.3-1.8:1.0-5.0.
[0013] Preferably, in step S1, the mass ratio of DMA, methanol and water is 2.0-4.0:0.5-1.5:0.1-0.5.
[0014] Preferably, in step S1, the added mass of Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O raw materials accounts for 4.5% to 5.5% of the volume of the mixed solvent of DMA, methanol and water.
[0015] Preferably, in step S1, the reaction temperature of the solvent thermal synthesis reaction is 102° C., and the reaction time is 2 days.
[0016] Preferably, in step S2, the AOs is a mixture of MAO, DAO and PAO, all of which have enzyme activities ≥1000 U / g, and the mass ratio of the three is 1.0-4.0:2.0-9.0:0.0-2.0.
[0017] Preferably, in step S2, the mass of Fe3O4@Tb-mesoMOF crystals added is 5% of the volume of the AOs solution containing HEPES buffer.
[0018] Preferably, in step S2, AOs-Fe3O4@Tb-mesoMOF needs to be rinsed multiple times with phosphate buffer, and the pH value of the phosphate buffer is 7.2.
[0019] The invention also provides an immobilized amine oxidase prepared by the preparation method.
[0020] The invention also provides an application of the immobilized amine oxidase in the degradation of biogenic amines.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] 1. Metal ion Tb of the MOF material prepared by the present invention 3+and ligand TATB to form a zeolite network structure. 3+ Ions and TATB ligands form the skeleton of the material through coordination, and a regular hexagonal pore structure with a diameter of 3.5-5.0nm is formed inside, namely mesoporous MOF (mesoMOF), thus forming a new enzyme catalytic platform with excellent properties such as large specific surface area, regular mesoporous structure, and abundant adsorption sites. It is suitable for the adsorption and fixation of spherical protein molecules with a molecular weight of 50K-300K, and has broad prospects for the application of enzyme, protein and other biological molecules fixation.
[0023] 2. The prepared AOs-Fe3O4@Tb-mesoMOF catalyst can adjust the ratio of monoamine oxidase (MAO), diamine oxidase (DAO) and polyamine oxidase (PAO) in amine oxidase (AOs) according to the types of major biogenic amines in food.
[0024] 3. After being reused 5 times, the degradation efficiency of biogenic amines of AOs-Fe3O4@Tb-mesoMOF prepared by the present invention can still reach more than 70%, and it has good reusability and degradation efficiency.
[0025] 4. The AOs-Fe3O4@Tb-mesoMOF prepared by the present invention exhibits higher enzyme catalytic performance. The regular mesoporous structure of this MOF material provides a specific attachment site for each enzyme molecule, effectively preventing the free enzymes from combining or even agglomerating through hydrogen bonds, ionic bonds, van der Waals forces, etc., so that the amine oxidase in Tb-mesoMOF exhibits high catalytic activity and solvent adaptability.
[0026] 5. The immobilized amine oxidase prepared by the present invention has high efficiency in catalyzing the degradation of biogenic amines, is easy to operate, and can be recycled and reused. It can solve the problem of excessively high biogenic amine content in fermented foods without changing the production process and conditions. DETAILED DESCRIPTION
[0027] In order to make the technical means, creative features, objectives and functions of the present invention clearer and easier to understand, the present invention is further described below in conjunction with specific embodiments:
[0028] Example 1
[0029] Preparation of immobilized amine oxidase:
[0030] S1. 0.45 g of Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O in a mass ratio of 0.5:0.3:1 were mixed in 10 ml of a mixed solvent of DMA, methanol and water in a mass ratio of 4:1.5:0.5, added to a hydrothermal reactor, sealed and reacted at 105°C for 60 h, and then magnetically separated, washed with water and dried to obtain Fe3O4@Tb-mesoMOF crystals.
[0031] S2. Immerse 2.5 g of Fe3O4@Tb-mesoMOF crystals prepared in S1 into 50 ml of AOs solution containing HEPES buffer, in which the mass ratio of MAO, DAO and PAO is 1:2:0. After fully adsorbing for 60 h at 37°C, rinse with phosphate buffer for multiple times to prepare AOs-Fe3O4@Tb-mesoMOF.
[0032] Biogenic amine degradation detection:
[0033] S3. Take 100 mL of a certain brand of rice wine sample on the market, add 0.1 g of AOs-Fe3O4@Tb-mesoMOF prepared in S2, and place it in a constant temperature box at 37°C for 6 hours. During this period, the magnet is pulled up and down every 5 minutes, and the changes in the total amine content before and after the rice wine treatment and the degradation rate of biogenic amines are measured.
[0034] S4. Use a magnetic field to adsorb and recover AOs-Fe3O4@Tb-mesoMOF, wash it with phosphate buffer, freeze-dry it, and perform the operation again according to S3.
[0035] S5. Repeat the operation of S4 1 to 5 times, and detect the degradation rate of biogenic amines in rice wine after recycling AOs-Fe3O4@Tb-mesoMOF 1 to 5 times.
[0036] Example 2
[0037] Preparation of immobilized amine oxidase:
[0038] S1. 0.55 g of Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O in a mass ratio of 1.5:1:3 were mixed in 10 ml of a mixed solvent of DMA, methanol and water in a mass ratio of 3:1:0.3, added to a hydrothermal reactor, sealed and reacted at 105°C for 60 h, and then magnetically separated, washed with water and dried to obtain Fe3O4@Tb-mesoMOF crystals.
[0039] S2. Immerse 2.5 g of Fe3O4@Tb-mesoMOF crystals prepared in S1 into 50 ml of AOs solution containing HEPES buffer, in which the mass ratio of MAO, DAO and PAO is 2:5:1. After fully adsorbed for 60 h at 37°C, rinse with phosphate buffer for multiple times to prepare AOs-Fe3O4@Tb-mesoMOF.
[0040] Biogenic amine degradation detection:
[0041] S3. Take 100 mL of a certain brand of rice wine sample on the market, add 0.5 g of AOs-Fe3O4@Tb-mesoMOF prepared in S2, and place it in a constant temperature box at 37°C for 6 hours. During this period, the magnet is pulled up and down every 5 minutes, and the changes in the total amine content before and after the rice wine treatment and the degradation rate of biogenic amines are measured.
[0042] S4. Use a magnetic field to adsorb and recover AOs-Fe3O4@Tb-mesoMOF, wash it with phosphate buffer, freeze-dry it, and perform the operation again according to S3.
[0043] S5. Repeat the operation of S4 1 to 5 times, and detect the degradation rate of biogenic amines in rice wine after recycling AOs-Fe3O4@Tb-mesoMOF 1 to 5 times.
[0044] Example 3
[0045] Preparation of immobilized amine oxidase:
[0046] S1. 1.5 g of Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O in a mass ratio of 3:1.8:5 were mixed in 30 ml of a mixed solvent of DMA, methanol and water in a mass ratio of 2:0.5:0.1, added to a hydrothermal reactor, sealed and reacted at 105°C for 60 h, and then magnetically separated, washed with water and dried to obtain Fe3O4@Tb-mesoMOF crystals.
[0047] S2. 5 g of Fe3O4@Tb-mesoMOF crystals prepared in S1 were immersed in 100 ml of AOs solution containing HEPES buffer, in which the mass ratio of MAO, DAO and PAO was 2:9:2. After fully adsorbed for 60 h at 37°C, they were rinsed multiple times with phosphate buffer to prepare AOs-Fe3O4@Tb-mesoMOF.
[0048] Biogenic amine degradation detection:
[0049] S3. Take 100 mL of a certain brand of rice wine sample on the market, add 1 g of AOs-Fe3O4@Tb-mesoMOF prepared in S2, and place it in a constant temperature box at 37°C for 6 hours. During this period, the magnet is pulled up and down every 5 minutes, and the changes in the total amine content before and after the rice wine treatment and the degradation rate of biogenic amines are measured.
[0050] S4. Use a magnetic field to adsorb and recover AOs-Fe3O4@Tb-mesoMOF, wash it with phosphate buffer, freeze-dry it, and perform the operation again according to S3.
[0051] S5. Repeat the operation of S4 1 to 5 times, and detect the degradation rate of biogenic amines in rice wine after recycling AOs-Fe3O4@Tb-mesoMOF 1 to 5 times.
[0052] Example 4
[0053] Preparation of immobilized amine oxidase:
[0054] S1. 1.5 g of Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O in a mass ratio of 2:1:2 were mixed in 30 ml of a mixed solvent of DMA, methanol and water in a mass ratio of 3:1:0.2, added to a hydrothermal reactor, sealed and reacted at 105°C for 60 h, and then magnetically separated, washed with water and dried to obtain Fe3O4@Tb-mesoMOF crystals.
[0055] S2. 5 g of Fe3O4@Tb-mesoMOF crystals prepared in S1 were immersed in 100 ml of AOs solution containing HEPES buffer, in which the mass ratio of MAO, DAO and PAO was 1:2:1. After fully adsorbed for 60 h at 37°C, they were rinsed multiple times with phosphate buffer to prepare AOs-Fe3O4@Tb-mesoMOF.
[0056] Biogenic amine degradation detection:
[0057] S3. Take 100mL of a certain brand of commercially available light soy sauce sample, add 0.1g of AOs-Fe3O4@Tb-mesoMOF prepared in S2, and place it in a constant temperature box at 37℃ for 6h. During this period, the magnet is pulled up and down every 5 minutes, and the changes in the total amine content before and after rice wine treatment and the degradation rate of biogenic amines are measured.
[0058] S4. Use a magnetic field to adsorb and recover AOs-Fe3O4@Tb-mesoMOF, wash it with phosphate buffer, freeze-dry it, and perform the operation again according to S3.
[0059] S5. Repeat the operation of S4 1 to 5 times, and detect the degradation rate of biogenic amines in rice wine after recycling AOs-Fe3O4@Tb-mesoMOF 1 to 5 times.
[0060] Example 5
[0061] Preparation of immobilized amine oxidase:
[0062] S1. 1.5 g of Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O in a mass ratio of 2:1:2 were mixed in 30 ml of a mixed solvent of DMA, methanol and water in a mass ratio of 3:1:0.2, added to a hydrothermal reactor, sealed and reacted at 105°C for 60 h, and then magnetically separated, washed with water and dried to obtain Fe3O4@Tb-mesoMOF crystals.
[0063] S2. 5 g of Fe3O4@Tb-mesoMOF crystals prepared in S1 were immersed in 100 ml of AOs solution containing HEPES buffer, in which the mass ratio of MAO, DAO and PAO was 1:2:1. After fully adsorbed for 60 h at 37°C, they were rinsed multiple times with phosphate buffer to prepare AOs-Fe3O4@Tb-mesoMOF.
[0064] Biogenic amine degradation:
[0065] S3. Take 100 mL of a certain brand of commercially available wine, add 0.1 g of AOs-Fe3O4@Tb-mesoMOF prepared in S2, and place it in a constant temperature box at 37°C for 6 hours. During this period, the magnet is pulled up and down every 5 minutes, and the changes in the total amine content before and after the rice wine treatment and the degradation rate of biogenic amines are measured.
[0066] S4. Use a magnetic field to adsorb and recover AOs-Fe3O4@Tb-mesoMOF, wash it with phosphate buffer, freeze-dry it, and perform the operation again according to S3.
[0067] S5. Repeat the operation of S4 1 to 5 times, and detect the degradation rate of biogenic amines in rice wine after recycling AOs-Fe3O4@Tb-mesoMOF 1 to 5 times.
[0068] Among them, H3TATB in Examples 1-5 is 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine, DMA is N,N-dimethylacetamide, HEPES buffer is 4-hydroxyethylpiperazineethanesulfonic acid buffer, AOs is amine oxidase, MAO is monoamine oxidase, DAO is diamine oxidase, PAO is polyamine oxidase, and the particle size of Fe3O4 nanoparticles is 50 to 200 nm. The finally prepared AOs-Fe3O4@Tb-mesoMOF is the immobilized amine oxidase.
[0069] In Example 1-5, the Fe3O4@Tb-mesoMOF crystal was reacted at 105°C for 60 h to fully adsorb AOs with a loading of 20 μmol / g. When the phosphate buffer was used for multiple rinses, the Cu in the supernatant needed to be continuously detected. 2+ , until there is no Cu 2+ Detected, Cu 2+ Contained in biogenic amines.
[0070] Moreover, in Examples 1-5, when performing biogenic amine degradation detection, the added mass of AOs-Fe3O4@Tb-mesoMOF is 1% to 10% of the sample volume.
[0071] The detection data of step S3 and step S4 in Example 1-5 are shown in the following table:
[0072]
[0073] It can be seen from the above table that the AOs-Fe3O4@Tb-mesoMOF prepared in the present invention has a good degradation effect on biogenic amines, and through the comparison of Examples 1-3, as the amount of AOs-Fe3O4@Tb-mesoMOF added increases, the degradation effect on biogenic amines is better and the degradation rate is higher.
[0074] At the same time, it can be seen from the table that in Examples 1-5, after 5 repeated uses, the degradation rate of AOs-Fe3O4@Tb-mesoMOF for biogenic amines can still reach more than 70%, and it still has a good biogenic amine degradation effect.
[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.
Claims
1. A method for preparing immobilized amine oxidase, characterized in that: The following steps are involved: S1. Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O were used as raw materials to obtain Fe3O4@Tb-mesoMOF crystals by solvothermal synthesis reaction in a mixed solvent of DMA, methanol and water; S2. The Fe3O4@Tb-mesoMOF crystals prepared in S1 were immersed in an AOs solution containing HEPES buffer for adsorption, and AOs-Fe3O4@Tb-mesoMOF was obtained after sufficient adsorption.
2. The method for preparing an immobilized amine oxidase according to claim 1, characterized in that: In step S1, the mass ratio of Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O is 0.5-3.0:0.3-1.8:1.0-5.
0.
3. The method for preparing an immobilized amine oxidase according to claim 1, characterized in that: In step S1, the mass ratio of DMA, methanol and water is 2.0-4.0:0.5-1.5:0.1-0.
5.
4. The method for preparing an immobilized amine oxidase according to claim 1, characterized in that: In step S1, the added mass of Fe3O4 nanoparticles, H3TATB and Tb(NO3)3·5H2O raw materials accounts for 4.5% to 5.5% of the volume of the mixed solvent of DMA, methanol and water.
5. The method for preparing an immobilized amine oxidase according to claim 1, characterized in that: In step S1, the reaction temperature of the solvent thermal synthesis reaction is 105° C. and the reaction time is 60 h.
6. The method for preparing an immobilized amine oxidase according to claim 5, characterized in that: In step S2, the AOs is a mixture of MAO, DAO and PAO, all of which have enzyme activities ≥1000 U / g, and the mass ratio of the three is 1.0-4.0:2.0-9.0:0.0-2.
0.
7. The method for preparing an immobilized amine oxidase according to claim 1, characterized in that: In step S2, the added mass of Fe3O4@Tb-mesoMOF crystals is 5% of the volume of the AOs solution containing HEPES buffer.
8. The method for preparing an immobilized amine oxidase according to claim 1, characterized in that: In step S2, AOs-Fe3O4@Tb-mesoMOF needs to be rinsed multiple times with phosphate buffer, and the pH value of the phosphate buffer is 7.
2.
9. An immobilized amine oxidase, characterized in that Prepared by the preparation method according to any one of claims 1 to 8.
10. Use of the immobilized amine oxidase according to claim 9 in the degradation of biogenic amines.