Construction methods and applications of immobilized enzyme systems based on artificial antibody-antigen

Abstract

This invention belongs to the field of immobilized enzyme technology, specifically relating to a method for constructing and applying an immobilized enzyme system based on an artificial antibody-antigen. The method includes the following steps: (1) using Fe3O4 as a magnetic core and modifying it with tetraethyl orthosilicate; (2) grafting double bonds onto its surface using MPS; (3) constructing an artificial antibody material using catechol as a template molecule; (4) preparing a catechol-enzyme complex by reacting the enzyme molecule with catechol via a Schiff base reaction; and (5) adding the catechol-enzyme complex solution to the artificial antibody material to immobilize the enzyme. This invention provides a simple and convenient method that achieves specific and directional immobilization of the enzyme while reducing costs, improving enzyme stability, and exhibiting a high immobilization rate.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme technology, specifically relating to a method for constructing and applying an immobilized enzyme system based on artificial antibody-antigen. Background Technology

[0002] Enzymes, as catalysts, are characterized by mild reactions, no pollution, and high catalytic efficiency. However, free enzymes suffer from drawbacks such as sensitivity to the external environment, poor stability, and easy inactivation. Immobilizing enzymes on a matrix material can overcome these shortcomings (CesarMateo, Jose Palomo, Gloria Fernandez-Lorente, et al. Improvement of enzyme activity, stability and selectivity via immobilization techniques[J]. Enzyme and Microbial Technology, 2007, 40:1451–1463). Conventional methods for immobilizing enzymes include physical adsorption, encapsulation (María Fernández-Fernández, Sanromá Angeles, Moldes Diego. Recent developments and applications of immobilized laccase[J]. Biotechnology Advances, 2013, 31:1808–1825) and covalent cross-linking (Júlio César dos Santos, Patrícia Daniela Mijone, Gisele Fátima Morais Nunes, et al. Covalent attachment of Candida rugosaLipase on a chemically modified hybrid matrix of polysiloxane–polyvinyl alcohol with different activating compounds [J]. Journal of Chromatography B, 2007, 61:229–236). These traditional enzyme immobilization methods have improved enzyme stability, enabled enzyme reusability, and achieved quantitative enzyme immobilization. However, traditional enzyme immobilization methods suffer from drawbacks such as low specificity, poor biocompatibility, and poor reusability. To address these issues, inspired by the complementary base pairing of DNA double helixes and antigen-antibody recognition, natural molecules have been introduced into immobilized enzyme systems to solve the problem of specific recognition. However, biomolecular materials such as natural antibodies or DNA strands have disadvantages such as high cost, poor stability, and difficulty in preservation (Björn Renberg, Kae Sato, Kazuma Mawatari, et al. Serial DNA immobilization in micro- and extended nanospace channels[J]. Lab on a Chip, 2009, 9:1517–1523; Yun Liu, Huixiang Wang, Jingyu Huang, Jie Yang, Baohong Liu, Pengyuan Yang, Microchip-based ELISA strategy for the detection of low-level disease biomarker in serum, Analytica Chimica Acta, 650 (2009) 77–82). Summary of the Invention

[0003] The purpose of this invention is to provide a method for constructing and applying an immobilized enzyme system based on artificial antibody-antigen. By covalently modifying the enzyme as an antigen, it achieves specific and directional immobilization of the enzyme through specific recognition with artificial antibody materials, while reducing costs, improving enzyme stability, and achieving a high enzyme loading capacity.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for constructing an immobilized enzyme system based on an artificial antibody-antigen, the method comprising the following steps: (1) Fe3O4 nanoparticles were dispersed in an ethanol-water mixed solvent system and mixed with tetraethyl orthosilicate under alkaline conditions to obtain Fe3O4@SiO2; (2) Disperse the Fe3O4@SiO2 obtained in step (1) in toluene, add triethylamine and 3-(trimethoxysilyl)propyl α-methacrylate (MPS), and reflux and mix under nitrogen protection to obtain Fe3O4@SiO2@MPS; (3) Using catechol as a template molecule, Fe3O4@SiO2@MPS obtained in step (2) was polymerized with catechol, α-methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile in a toluene-acetonitrile mixed solution. Then, the catechol was eluted with an elution buffer to obtain the artificial antibody material. (4) The enzyme molecule and catechin are reacted with a Schiff base in a buffer solution to obtain a catechin-enzyme complex solution; (5) Add the catechin-enzyme complex solution obtained in step (4) to the artificial antibody material prepared in step (3), mix well, and immobilize the enzyme.

[0005] The principle of this invention is as follows: Fe3O4 is used as a magnetic core, modified with tetraethyl orthosilicate, and coated with a layer of SiO2. Double bonds are grafted onto its surface using the silane coupling agent MPS. Catechol is used as the "antigen" template molecule to construct a catechol molecularly imprinted polymer, i.e., an artificial antibody material. Then, catechol aldehyde, which has a similar structure to the "antigen" template molecule, is selected. The aldehyde group at the para position of the phenolic hydroxyl group combines with the amino group on the enzyme molecule through a Schiff base reaction to form a catechol aldehyde-enzyme complex. Then, the complex is linked to the artificial antibody material by utilizing the specific recognition between the phenolic hydroxyl end and the catechol molecularly imprinted polymer, thereby achieving enzyme immobilization.

[0006] In the above technical solution, further, in step (1), the particle size of the Fe3O4 nanoparticles is 100-400 nm; In the ethanol-water mixed solvent system, the volume ratio of ethanol to water is 4:1; The alkaline environment is provided by ammonia water, and the volume ratio of ammonia water to tetraethyl orthosilicate is 2:1; The molar ratio of Fe3O4 to tetraethyl orthosilicate is 1:5 to 1:10.

[0007] In the above technical solution, further, in step (2), the volume ratio of triethylamine to 3-(trimethoxysilyl)propyl methacrylate (MPS) is 1:1-1:3, and the mass ratio of Fe3O4@SiO2 to the volume ratio of 3-(trimethoxysilyl)propyl methacrylate (MPS) is 0.1-0.5 g:1 mL.

[0008] In the above technical solution, the volume ratio of toluene to acetonitrile in the toluene-acetonitrile mixed solution is 1:1 to 1:5.

[0009] In the above technical solution, further, in step (3), the ratio of the amount of catechin to the mass of Fe3O4@SiO2@MPS is 1-3 mmol:1g, the molar ratio of catechin to α-methacrylic acid (MAA) is 1:2-1:6, and the molar ratio of catechin to ethylene glycol dimethacrylate (EGDMA) is 1:5-1:20; The polymerization reaction temperature is 60-80℃, and the time is 12-24 h; The eluent is an acetic acid-ethanol mixture with a volume ratio of 9:1 (acetic acid to ethanol).

[0010] In the above technical solution, further, in step (4), the buffer solution is a 0.01 M phosphate buffer solution; the enzyme molecule is any one of lipase, α-amylase, and α-glucosidase; The mass ratio of catechin aldehyde to lipase is 1:50-1:250, the mass ratio of catechin aldehyde to α-amylase is 1:10-1:40, and the ratio of α-glucosidase activity to catechin aldehyde mass is 200-1500U:1μg. The reaction temperature of the catechin with the Schiff base of the enzyme molecule is 20-60℃, the pH is 6-8, and the reaction time is 1-6 h.

[0011] In the above technical solution, further, in step (4), the reaction parameters of the catechin with the Schiff base of the enzyme molecule need to be optimized according to different types of enzymes: The optimized reaction conditions for lipase and catechol Schiff base are: the preferred reaction temperature is 40-52 ℃, and the preferred pH is 7.2-7.6. The optimized reaction conditions for α-amylase and catechin Schiff base are: the preferred reaction temperature is 20-45 ℃, and the preferred pH is 6.5-7.6.

[0012] The optimized conditions for the reaction between α-glucosidase and catechol Schiff base are: a reaction temperature preferably of 35-55 ℃ and a pH preferably of 6.5-7.6.

[0013] In the above technical solution, further, in step (5), the dosage relationship between the artificial antibody polymer and the catechin-enzyme complex is: 5-20 mg of artificial antibody material is added to each 1 mL of catechin-enzyme complex; The mixing speed is 100-300 rpm; The enzyme was immobilized for 1-24 h at a temperature of 10-60 °C.

[0014] Another aspect of the present invention provides an application of the above-described construction method in the field of biocatalysis.

[0015] The present invention has the following beneficial effects: 1. The method of the present invention is simple and convenient. While achieving specific and directional immobilization of enzymes, it can reduce costs, improve enzyme stability, and has a high immobilization rate of over 60%. It overcomes the shortcomings of traditional immobilized enzyme materials, which can only achieve quantitative immobilization, and the high cost of using biomolecular materials for directional immobilization of enzymes. This invention creates conditions for simple, convenient and affordable specific and directional immobilization.

[0016] 2. The enzyme immobilization using artificial antibody materials in this invention not only has a good immobilization effect, but also has high enzyme activity; 3. This invention uses Fe3O4 as a magnetic core, which makes it easy to separate the immobilized enzyme from the solution system and allows for repeated use. Attached Figure Description

[0017] Picture 1 This is a schematic diagram illustrating the construction process of the artificial antibody-antigen immobilized enzyme system of the present invention. Detailed Implementation

[0018] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely for the purpose of helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0019] Example 1 This embodiment describes the construction of an immobilized lipase enzyme system. The construction method includes the following steps: (1) 0.6 g Fe3O4 nanoparticles were dispersed in 200 mL of ethanol-water mixed solvent system with a volume ratio of 4:1, 8 mL of ammonia and 4 mL of tetraethyl orthosilicate were added, and the mixture was stirred at 35 °C for 6 h to obtain Fe3O4@SiO2. (2) Take 0.5 g of Fe3O4@SiO2 obtained in step (1) and disperse it in 50 mL of toluene. Add 1 mL of triethylamine and 2 mL of 3-(trimethoxysilyl)propyl α-methacrylate (MPS). Mix under nitrogen protection by reflux to obtain Fe3O4@SiO2@MPS. (3) Take 0.2 g of Fe3O4@SiO2@MPS obtained in step (2) and 0.25 mmol of catechol, 0.5 mmol of α-methacrylic acid, 2.5 mmol of ethylene glycol dimethacrylate and 20 mg of azobisisobutyronitrile and polymerize them in a toluene-acetonitrile mixed solvent system with a volume ratio of 4:1 at 60℃ for 24 h. Then, the catechol is eluted with an acetic acid-ethanol mixed solution with a volume ratio of 9:1 to obtain the artificial antibody material. (4) Lipase and catechin were subjected to Schiff base reaction in a 0.01 M phosphate buffer solution to obtain a catechin-enzyme complex solution. The reaction conditions were: catechin solution concentration of 25 μg / mL, lipase concentration of 4 mg / mL, reaction temperature of 50℃, pH of 7.5, and reaction time of 4h. (5) Add 1 mL of the catechin-enzyme complex solution obtained in step (4) to 5 mg of the artificial antibody material prepared in step (3), mix with a mixer at 300 rpm, and immobilize the enzyme for 11 hours at 20°C to obtain immobilized lipase.

[0020] Example 2 This embodiment describes the construction of an immobilized enzyme system for α-amylase. The construction method includes the following steps: (1) 0.6 g Fe3O4 nanoparticles were dispersed in 200 mL of ethanol-water mixed solvent system with a volume ratio of 4:1, 8 mL of ammonia and 4 mL of tetraethyl orthosilicate were added, and the mixture was stirred at 35 °C for 6 h to obtain Fe3O4@SiO2. (2) Take 0.5 g of Fe3O4@SiO2 obtained in step (1) and disperse it in 50 mL of toluene. Add 1 mL of triethylamine and 2 mL of 3-(trimethoxysilyl)propyl α-methacrylate (MPS). Mix under nitrogen protection by reflux to obtain Fe3O4@SiO2@MPS. (3) Take 0.2 g of Fe3O4@SiO2@MPS obtained in step (2) and 0.25 mmol of catechol, 0.5 mmol of α-methacrylic acid, 2.5 mmol of ethylene glycol dimethacrylate and 20 mg of azobisisobutyronitrile and polymerize them in a toluene-acetonitrile mixed solvent system with a volume ratio of 4:1 at 60℃ for 24 h. Then, the catechol is eluted with an acetic acid-ethanol mixed solution with a volume ratio of 9:1 to obtain the artificial antibody material. (4) α-amylase and catechin were subjected to a Schiff base reaction in a 0.01 M phosphate buffer solution to obtain a catechin-enzyme complex solution. The reaction conditions were: catechin solution concentration of 30 μg / mL, α-amylase concentration of 1 mg / mL, reaction temperature of 36℃, pH of 6.8, and reaction time of 5 h. (5) Add 1 mL of the catechin-enzyme complex solution obtained in step (4) to 5 mg of the artificial antibody material prepared in step (3), mix it with a mixer at 300 rpm, and immobilize the enzyme for 11 h at 20 °C to obtain immobilized α-amylase.

[0021] Example 3 This embodiment describes the construction of an immobilized enzyme system for α-glucosidase. The construction method includes the following steps: (1) 0.6 g Fe3O4 nanoparticles were dispersed in 200 mL of ethanol-water mixed solvent system with a volume ratio of 4:1, 8 mL of ammonia and 4 mL of tetraethyl orthosilicate were added, and the mixture was stirred at 35 °C for 6 h to obtain Fe3O4@SiO2. (2) Take 0.5 g of Fe3O4@SiO2 obtained in step (1) and disperse it in 50 mL of toluene. Add 1 mL of triethylamine and 2 mL of 3-(trimethoxysilyl)propyl α-methacrylate (MPS). Mix under nitrogen protection by reflux to obtain Fe3O4@SiO2@MPS. (3) Take 0.2 g of Fe3O4@SiO2@MPS obtained in step (2) and 0.25 mmol of catechol, 0.5 mmol of α-methacrylic acid, 2.5 mmol of ethylene glycol dimethacrylate and 20 mg of azobisisobutyronitrile and polymerize them in a toluene-acetonitrile mixed solvent system with a volume ratio of 4:1 at 60℃ for 24 h. Then, the catechol is eluted with an acetic acid-ethanol mixed solution with a volume ratio of 9:1 to obtain the artificial antibody material. (4) α-glucosidase and catechin were subjected to a Schiff base reaction in a 0.01 M phosphate buffer solution to obtain a catechin-enzyme complex solution. The reaction conditions were: catechin solution concentration of 28 μg / mL, volume ratio of α-glucosidase to catechin solution of 2.5%, reaction temperature of 45℃, pH of 7.3, and reaction time of 5h. (5) Add 1 mL of the catechin-enzyme complex solution obtained in step (4) to 5 mg of the artificial antibody material prepared in step (3), mix it with a mixer at 300 rpm, and immobilize the enzyme for 11 h at 20 °C to obtain immobilized α-glucosidase.

[0022] Comparative Example 1 The difference from Example 1 is that the matrix material is different; it is a non-artificial antibody material. The other processes are completely the same as in Example 1. The preparation method of the non-artificial antibody material is as follows: (1) 0.6 g Fe3O4 nanoparticles were dispersed in 200 mL of ethanol-water mixed solvent system with a volume ratio of 4:1, 8 mL of ammonia and 4 mL of tetraethyl orthosilicate were added, and the mixture was stirred at 35 °C for 6 h to obtain Fe3O4@SiO2. (2) Take 0.5 g of Fe3O4@SiO2 obtained in step (1) and disperse it in 50 mL of toluene. Add 1 mL of triethylamine and 2 mL of 3-(trimethoxysilyl)propyl α-methacrylate (MPS). Mix under nitrogen protection by reflux to obtain Fe3O4@SiO2@MPS. (3) Take 0.2 g of Fe3O4@SiO2@MPS obtained in step (2) and 0.5 mmol of α-methacrylic acid, 2.5 mmol of ethylene glycol dimethacrylate and 20 mg of azobisisobutyronitrile and polymerize them in a toluene-acetonitrile mixed solvent system with a volume ratio of 4:1 at 60 °C for 24 h. Then, the catechol is eluted with an acetic acid-ethanol mixed solution with a volume ratio of 9:1 to obtain non-artificial antibody material.

[0023] Comparative Example 2 The difference from Example 2 is the matrix material; the non-artificial antibody material from Comparative Example 1 is used. All other processes are completely consistent with Example 2.

[0024] Comparative Example 3 The difference from Example 3 is the matrix material; the non-artificial antibody material from Comparative Example 1 is used. The other processes are completely consistent with Example 3.

[0025] Performance testing: The performance of the immobilized enzyme materials prepared in Examples 1, 2, and 3, and Comparative Examples 1, 2, and 3 was tested using the following methods: (1) Enzyme loading: The supernatant enzyme solution absorbance before and after immobilization was measured using the Coomassie Brilliant Blue method at 595 nm UV. The protein concentrations before and after immobilization were then substituted into the BSA bovine serum albumin standard curve to determine the immobilization rate and amount.

[0026] Formula for yield ratio: Yield(%) = (C0 - C) t ) / C0×100% Fixed load formula: Fixed load = (C0 - C) t )×V×1000 / M Where C0 is the protein concentration before immobilization, C t V represents the protein concentration after immobilization, V represents the volume of the solution used for immobilization, and M represents the mass of the matrix.

[0027] (2) Enzyme activity is defined as the amount of enzyme consumed per minute to hydrolyze 1 μmol of substrate or produce 1 μmol of product, which is defined as 1 U.

[0028] (3) Reusability of enzymes The enzyme activity measured for the first time was defined as 100%. Subsequently, the immobilized enzyme was separated from the solution system using a magnet, and the activity was measured repeatedly to compare the ratio of the repeated measurements to the first measurement.

[0029] (4) Comparison of temperature adaptability between immobilized enzymes and free enzymes The activities of immobilized and free enzymes were measured under different temperature conditions, and the best value was defined as 100%. The remaining temperature activities were then compared with these values.

[0030] (5) Comparison of pH adaptability between immobilized enzymes and free enzymes The activities of immobilized and free enzymes were measured under different pH conditions, and the best value was defined as 100%. The remaining pH activities were then compared with these values.

[0031] The performance test results are as follows: The results of immobilization, enzyme activity, and repeated enzyme activity determination of Examples 1-3 and Comparative Examples 1-3 are shown in Table 1.

[0032] Table 1

[0033] The artificial antibody-antigen immobilized enzyme system in Example 1 had an immobilization loading of 13.22 mg of lipase. protein / g support The initial enzyme activity was 104.66 U / g support The immobilization rate was 73.58%, and the enzyme activity upon repeated use was 104.30 U / g. support The non-artificial antibody material in Comparative Example 1 had an immobilization capacity of 0.77 mg for lipase. protein / g support Compared to immobilized lipases using artificial antibody materials, the effect is significantly worse, although it exhibits some initial activity of 75.87 U / g. support However, only 5.17 U / g remained when reused. support .

[0034] The α-amylase loading in Example 2 was 14.78 mg. protein / g supportThe initial enzyme activity was 199.33 u / g. support The immobilization rate was 68.29%, and the enzyme activity upon repeated use was 199.33 U / g. support The non-artificial antibody material in Comparative Example 2 had an immobilization capacity of 0.20 mg for α-amylase. protein / g support It has 114.30 U / g support The initial enzyme activity decreased rapidly upon repeated use, with only 29.74 U / g remaining. support .

[0035] The α-glucosidase loading in Example 3 was 25.09 mg. protein / g support The initial enzyme activity was 143.25 U / g. support The immobilization rate was 62.84%, and the enzyme activity upon repeated use was 137.70 U / g. support The non-artificial antibody material in Comparative Example 2 had an immobilization capacity of 5.91 mg for α-glucosidase. protein / g support The initial enzyme activity was 70.29 U / g. support When reused, the enzyme activity is reduced to only 29.46 U / g. support .

[0036] Meanwhile, the artificial antibody immobilized lipase, α-amylase, and α-glucosidase materials prepared in Examples 1-3 of this invention showed that, after 15 repeated uses, their enzyme activities relative to the initial enzyme activities were 62.79%, 47.48%, and 39.85%, respectively. This demonstrates that immobilizing enzymes with artificial antibody materials not only has a good immobilization effect and high enzyme activity, but also exhibits higher stability compared to non-artificial antibodies during repeated uses.

[0037] The optimal enzymatic reaction conditions may change after enzyme immobilization. The initial activity of both the free and immobilized enzymes was defined as 100%. Experimental results showed that the optimal enzymatic reaction temperature for all three immobilized enzymes changed compared to the free enzyme. In Example 2, after immobilization, the activity recovery of α-amylase was higher than that of the free enzyme under both high and low temperature conditions. At 25°C, the relative activity of immobilized α-amylase was 77.21%, while that of free amylase was 67.68%; at 50°C, the relative activity of immobilized α-amylase was 95.31%, while that of free amylase was 79.76%. In Example 3, after immobilization, the activity recovery of α-glucosidase was similar to that of the free enzyme at low temperatures, but superior at high temperatures. At 80°C, the remaining activity of immobilized α-glucosidase was 47.82%, while that of free glycosidase was 38.51%. In Example 1, the immobilized lipase showed inferior activity recovery at low temperatures compared to the free enzyme, but significantly higher activity at high temperatures. At 50°C, the immobilized lipase recovered 93.72% of its activity, while the free lipase only retained 87.35%. In summary, immobilizing enzymes with artificial antibody materials improves their adaptability to higher extreme temperatures.

[0038] A comparison of the activity recovery efficiency of immobilized lipase, immobilized α-amylase, immobilized α-glucosidase, and their corresponding free enzymes under different pH conditions was conducted. The optimal pH for enzymatic reactions of α-glucosidase, lipase, and α-amylase all changed after immobilization on artificial antibody materials. Immobilized lipase showed superior activity recovery under alkaline conditions; at pH=10.5, the activity recovery rate of immobilized lipase was 77.27±0.25%, while the activity of free lipase was only 39.65±0.33%. Although the activity recovery efficiency of immobilized α-glucosidase under acidic conditions was not as good as that of free enzymes, its activity recovery rate under alkaline conditions was higher. At pH=9, the activity recovery efficiency of free enzymes was 64.35%, while that of immobilized enzymes was 72.72%. Immobilized α-amylase exhibits superior activity compared to free enzymes under both highly acidic and highly alkaline conditions. At pH 10.5, the relative activity of immobilized α-amylase is 82.37%, while that of free α-amylase is 69.46%. At pH 5.5, the activity recovery rate of immobilized α-amylase is 89.55%, while that of free enzyme is 81.04%. Overall, immobilizing enzymes with artificial antibody materials can improve the retention of enzyme activity under highly acidic and highly alkaline conditions.

[0039] Application Example 1 Benzyl acetate was synthesized using immobilized lipase catalysis. The synthesis conditions were as follows: 500 μL of vinyl acetate and 5 μL of benzyl alcohol were added to 50 mg of immobilized lipase material, and the reaction was carried out at 70 °C for 6 h until equilibrium was reached. After 6 h of reaction, the unreacted reactants in the mixture were evaporated to dryness at 70 °C, and the yield of benzyl acetate was determined by liquid chromatography, which showed a yield of 88.72%. After separating the supernatant from the immobilized lipase material with a magnet, vinyl acetate and benzyl alcohol were added again. After five consecutive injections, the yield of benzyl acetate obtained was 74.13% of the product obtained in the first catalysis. This demonstrates that the lipase immobilized by this method not only exhibits good stability and reusability in aqueous solutions but also maintains good stability and reusability in organic solvents.

[0040] Application Example 2 The immobilized α-amylase was used to synthesize o-amino-p-methylphenol. The synthesis conditions were as follows: starch solution was added to the immobilized α-amylase material and activated at 37°C for 10 min. The supernatant was then aspirated and reacted with 2-nitro-4-methylphenol. The product yield stabilized after reacting at 100°C for 9 min, and the yield was 58.88% at 275 nm using liquid chromatography. The continuous injection efficiency of the immobilized α-amylase material in the reaction of 2-amino-p-methylphenol was then measured. After five repeated uses, the catalytic efficiency was 80.86% of the initial efficiency.

[0041] Application Example 3 4-Methylumbelliferone was synthesized using immobilized α-glucosidase catalysis. The synthesis conditions were as follows: a solution of 4-methylumbelliferone-α-D-glucopyranoside was added to the immobilized α-glucosidase material, and the reaction was carried out at 37°C. After 30 min of reaction, the yield was determined by high-performance liquid chromatography at 254 nm, and the yield was 77.54%. Subsequently, the effect of continuous injection on the conversion of 4-methylumbelliferone-α-D-glucopyranoside to 4-methylumbelliferone catalyzed by immobilized α-glucosidase was determined. After 5 consecutive catalytic reactions, the yield was 81.12% of that after the first catalysis.

[0042] The test results for Application Example 1-3 are shown in Table 2.

[0043] Table 2

[0044] This invention mainly explores the feasibility of artificial antibody-antigen immobilized enzyme system. After successfully constructing the monoclonal antibody immobilized enzyme system, its performance was measured. Compared with ordinary immobilized enzyme materials, the artificial antibody immobilized enzyme material has better immobilization effect and stability. Finally, the immobilized enzyme material was successfully applied.

[0045] The applicant declares that this invention illustrates the preparation method and application of the artificial antibody-antigen immobilized enzyme system through the above examples, but this invention is not limited to the above steps, that is, it does not mean that it must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials used in this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

Claims

1. A method for constructing an immobilized enzyme system based on artificial antibody-antigen, characterized in that, The method includes the following steps: (1) Fe3O4 nanoparticles were dispersed in an ethanol-water mixed solvent system and mixed with tetraethyl orthosilicate under alkaline conditions to obtain Fe3O4@SiO2; (2) Disperse the Fe3O4@SiO2 obtained in step (1) in toluene, add triethylamine and 3-(trimethoxysilyl)propyl α-methacrylate (MPS), and reflux and mix under nitrogen protection to obtain Fe3O4@SiO2@MPS; (3) Using catechol as a template molecule, Fe3O4@SiO2@MPS obtained in step (2) was polymerized with catechol, α-methacrylic acid, ethylene glycol dimethacrylate and azobisisobutyronitrile in a toluene-acetonitrile mixed solution. Then, the catechol was eluted with an elution buffer to obtain the artificial antibody material. (4) The enzyme molecule and catechin are reacted with a Schiff base in a buffer solution to obtain a catechin-enzyme complex solution; (5) Add the catechin-enzyme complex solution obtained in step (4) to the artificial antibody material prepared in step (3), mix well, and immobilize the enzyme. In step (4), the enzyme molecule is any one of lipase, α-amylase, or α-glucosidase.

2. The construction method according to claim 1, characterized in that, In step (1), the particle size of the Fe3O4 nanoparticles is 100-400 nm; In the ethanol-water mixed solvent system, the volume ratio of ethanol to water is 4:1; The alkaline environment is provided by ammonia water, and the volume ratio of ammonia water to tetraethyl orthosilicate is 2:1; The molar ratio of Fe3O4 to tetraethyl orthosilicate is 1:5 to 1:

10.

3. The construction method according to claim 1, characterized in that, In step (2), the volume ratio of triethylamine to 3-(trimethoxysilyl)propyl methacrylate is 1:1 to 1:3, and the mass ratio of Fe3O4@SiO2 to the volume ratio of 3-(trimethoxysilyl)propyl methacrylate is 0.1 to 0.5 g: 1 mL.

4. The construction method according to claim 1, characterized in that, The volume ratio of toluene to acetonitrile in the toluene-acetonitrile mixed solution is 1:1 to 1:

5.

5. The construction method according to claim 1, characterized in that, In step (3), the ratio of the amount of catechin to the mass of Fe3O4@SiO2@MPS is 1-3 mmol:1g, the molar ratio of catechin to α-methacrylic acid is 1:2-1:6, and the molar ratio of catechin to ethylene glycol dimethacrylate is 1:5-1:

20. The polymerization reaction temperature is 60-80℃, and the time is 12-24 h; The eluent is an acetic acid-ethanol mixture with a volume ratio of 9:1 (acetic acid to ethanol).

6. The construction method according to claim 1, characterized in that, In step (4), the buffer solution is a 0.01 M phosphate buffer solution; The mass ratio of catechin to lipase is 1:50-1:250; The mass ratio of catechin to α-amylase is 1:10-1:40; The ratio of α-glucosidase activity to catechin mass is 200-1500 U: 1 μg; The reaction temperature of the catechin with the Schiff base of the enzyme molecule is 20-60℃, the pH is 6-8, and the reaction time is 1-6 h.

7. The construction method according to claim 1, characterized in that, In step (5), the ratio of the amount of artificial antibody polymer to the amount of catechin-enzyme complex is as follows: 5-20 mg of artificial antibody material is added to each 1 mL of catechin-enzyme complex. The mixing speed is 100 rpm-300 rpm; The enzyme was immobilized for 1-24 h at a temperature of 10-60 °C.

8. The application of the construction method according to any one of claims 1-7 in the field of biocatalysis.

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