CN116640757A - Construction method and application of immobilized enzyme system based on artificial antibody-antigen - Google Patents

Abstract

The invention belongs to the technical field of immobilized enzymes, and particularly relates to a construction method and application of an immobilized enzyme system based on an artificial antibody-antigen. The method comprises the following steps: (1) By Fe ₃ O ₄ As a magnetic core, the magnetic core is modified by tetraethoxysilane; (2) grafting double bonds on the surface thereof by using MPS; (3) Constructing an artificial antibody material by taking catechol as a template molecule; (4) Preparing a catechol-enzyme complex by reacting an enzyme molecule with catechol through Schiff base; (5) And adding the catechol-enzyme complex solution into an artificial antibody material to immobilize the enzyme. The method is simple and convenient, can reduce the cost, improve the stability of the enzyme and has higher immobilization rate while realizing the specific directional immobilization of the enzyme.

Description

Construction method and application of immobilized enzyme system based on artificial antibody-antigen

technical field

The invention belongs to the technical field of immobilized enzymes, in particular to a construction method and application of an artificial antibody-antigen-based immobilized enzyme system.

Background technique

As a catalyst, enzyme has the characteristics of mild reaction, no pollution and high catalytic efficiency. However, free enzymes have the disadvantages of being sensitive to the external environment, poor in stability, and easily inactivated. Immobilizing the enzyme on the matrix material can make up for this shortcoming (CesarMateo, Jose Palomo, Gloria Fernandez-Lorente, et al.Improvement of enzymeactivity, stability and selectivity via immobilization techniques[J].Enzyme and Microbial Technology,2007,40:1451 –1463). Conventional enzyme immobilization methods include physical adsorption, entrapment and encapsulation (María Fernández-Fernández, Sanromá Angeles, Moldes Diego.Recent developments and applications of immobilized laccase[J].Biotechnology Advances,2013,31:1808–1825) and covalent Crosslinking (Júlio César dos Santos, Patrícia Daniela Mijone, Gisel e Fátima Morais Nunes, et al. Covalent attachment of Candida rugosalipase on chemically modified hybrid matrix of polysiloxane–polyvinyl alcohol with different activating compounds[ J]. Journal of Chromatography B, 2007, 61:229–236). These traditional immobilized enzyme methods have improved the stability of the enzyme and realized the reuse of the enzyme, and at the same time realized the quantitative immobilization of the enzyme. However, the traditional immobilized enzyme method has disadvantages such as low specificity, poor biocompatibility, and poor reusable effect. In order to solve the above problems, inspired by DNA double helix base pairing, antigen-antibody recognition, etc., natural molecules were introduced into immobilized enzyme systems to solve specific recognition problems. However, biomolecular materials such as natural antibodies or DNA chains have the disadvantages of high cost, poor stability, and difficult preservation ( Renberg, Kae Sato, KazumaMawatari, et al.Serial DNA immobilization in micro-and extended nanospacechannels[J].Lab on a Chip,2009,9:1517–1523; Yun Liu,Huixiang Wang,JingyuHuang,Jie Yang,Baohong Liu, Pengyuan Yang, Microchip-based ELISA strategy for the detection of low-level disease biomarker in serum, An alytica ChimicaActa, 650 (2009) 77–82).

Contents of the invention

The purpose of the present invention is to provide a construction method and application of an immobilized enzyme system based on an artificial antibody-antigen, by covalently modifying the enzyme as an antigen, and through specific recognition with the artificial antibody material, the specificity of the enzyme can be achieved At the same time of directional immobilization, the cost is reduced, the stability of the enzyme is improved, and the enzyme loading capacity is high.

In order to achieve the above object, the present invention adopts the following technical solutions:

One aspect of the present invention provides a method for constructing an immobilized enzyme system based on an artificial antibody-antigen, the method comprising the following steps:

(1) Disperse Fe ₃ O ₄ nanoparticles in an ethanol-water mixed solvent system, and mix them with tetraethyl orthosilicate in an alkaline environment to obtain Fe ₃ O ₄ @SiO ₂ ;

(2) Disperse the Fe ₃ O ₄ @SiO ₂ prepared in step (1) in toluene, add triethylamine, α-3-(trimethoxysilyl)propyl methacrylate (MPS), and Reflux and mix under protection to obtain Fe ₃ O ₄ @SiO ₂ @MPS;

(3) Using catechol as a template molecule, combine the Fe ₃ O ₄ @SiO ₂ @MPS prepared in step (2) with catechol, α-methacrylic acid, ethylene glycol dimethacrylate, azo Diisobutyronitrile is polymerized in a toluene-acetonitrile mixed solution, and then the catechol is eluted with an eluent to prepare an artificial antibody material;

(4) performing a Schiff base reaction on the enzyme molecule and catechin aldehyde in a buffer solution to obtain a catechin aldehyde-enzyme complex solution;

(5) Add the catechin aldehyde-enzyme complex solution obtained in step (4) to the artificial antibody material prepared in step (3), mix well, and immobilize the enzyme.

The principle of the present invention is: take Fe ₃ O ₄ as the magnetic core, modify it with tetraethyl orthosilicate, wrap a layer of SiO ₂ on its surface, use silane coupling agent MPS to graft bis bond, using catechol as the "antigen" template molecule to construct the catechol molecularly imprinted polymer, that is, the artificial antibody material; then select the catechin aldehyde similar to the "antigen" template molecular structure, and use the aldehyde group at the para-position of the phenolic hydroxyl group Combine with the amine group on the enzyme molecule through Schiff base reaction to form a catechin-enzyme complex; then use the specific recognition between the phenolic hydroxyl end and the catechol molecularly imprinted polymer to link the complex to the artificial antibody material, Enzyme immobilization is achieved.

In the above technical solution, further, in step (1), in step (1), the particle diameter of the _Fe3O4 nanoparticles is _100-400nm ;

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 the ammonia water to tetraethyl orthosilicate is 2:1;

The molar ratio of Fe ₃ O ₄ to ethyl orthosilicate is 1:5-1:10.

In the above technical solution, further, in step (2), the volume ratio of the triethylamine to 3-(trimethoxysilyl)propyl α-methacrylate (MPS) is 1:1-1:3 , the ratio of the mass of Fe ₃ O ₄ @SiO ₂ to the volume of 3-(trimethoxysilyl)propyl α-methacrylate (MPS) is 0.1-0.5g:1mL.

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

In the above technical scheme, further, in step (3), the ratio of the molar mass of catechol to the mass of Fe ₃ O ₄ @Si O ₂ @MPS is 1-3mmol:1g, and the ratio of catechol to α- The molar ratio of methacrylic acid (MAA) is 1:2-1:6, and the molar ratio of catechol to ethylene glycol dimethacrylate (EGDMA) is 1:5-1:20;

The polymerization reaction temperature is 60-80°C, and the time is 12-24h;

The eluent is an acetic acid-ethanol mixed solution with a volume ratio of acetic acid and ethanol of 9:1.

In the above-mentioned technical scheme, further, in step (4), the buffer solution is a 0.01M phosphate buffer solution; the enzyme molecule is any one of lipase, α-amylase, and α-glucosidase ;

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, and the activity of the α-glucosidase is comparable to The mass ratio of catechin aldehyde is 200-1500U: 1μg;

The reaction temperature of the reaction between the catechin aldehyde and the enzyme molecule Schiff base is 20-60°C, the reaction pH is 6-8, and the reaction time is 1-6h.

In the above technical scheme, further, in step (4), the reaction parameters between the catechin and the enzyme molecule Schiff base need to be optimized according to the different types of enzymes:

The optimal conditions for the reaction of lipase and catechin aldehyde Schiff base are as follows: the reaction temperature is preferably 40-52°C, and the pH is preferably 7.2-7.6;

The optimal conditions for the reaction between α-amylase and catechin aldehyde Schiff base are as follows: the reaction temperature is preferably 20-45° C., and the pH is preferably 6.5-7.6.

The optimal conditions for the reaction between α-glucosidase and catechin aldehyde Schiff base are as follows: the reaction temperature is preferably 35-55° C., and the pH is preferably 6.5-7.6.

In the above technical solution, further, in step (5), the dosage relationship between the artificial antibody polymer and the catechin aldehyde-enzyme complex is as follows: 5-20 mg of artificial antibody material is added to every 1 mL of the catechin aldehyde-enzyme complex ;

The mixing speed is 100-300rpm;

The immobilization time of the enzyme is 1-24h, and the immobilization temperature is 10-60°C.

Another aspect of the present invention provides an application of the above construction method in the field of biocatalysis.

The present invention has the following beneficial effects:

1. The method of the present invention is simple and convenient. While realizing the specific and directional immobilization of the enzyme, it can reduce the cost, improve the stability of the enzyme, and has a high immobilization rate, which can reach more than 60%, breaking through the traditional method. Immobilized enzyme materials can only achieve quantitative immobilization, and the disadvantages of using biomolecular materials for directional immobilization of enzymes are expensive, creating conditions for simple, convenient and affordable specific directional immobilization.

2. The invention uses artificial antibody materials to immobilize enzymes, which not only has a good immobilization effect, but also has high enzyme activity;

3. The present invention uses Fe ₃ O ₄ as the magnetic core, so that the immobilized enzyme can be easily separated from the solution system and can be reused.

Description of drawings

Fig. 1 is a schematic diagram of the construction process of the artificial antibody-antigen immobilized enzyme system of the present invention.

Detailed ways

The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention, and should not be regarded as specific limitations to the present invention.

Example 1

This embodiment carries out the construction of immobilized enzyme system to lipase, and construction method comprises the following steps:

(1) Disperse 0.6g of Fe ₃ O ₄ nanoparticles in 200mL of ethanol-water mixed solvent system with a volume ratio of ethanol and water of 4:1, add 8mL of ammonia water and 4mL of ethyl orthosilicate, and mix at 35°C for 6h , to get Fe ₃ O ₄ @SiO ₂ ;

(2) Disperse 0.5 g of Fe ₃ O ₄ @SiO ₂ prepared in step (1) in 50 mL of toluene, add 1 mL of triethylamine, 2 mL of α-3-(trimethoxysilyl)propyl methacrylate (MPS ), reflux and mix under the protection of nitrogen to obtain Fe ₃ O ₄ @SiO ₂ @MPS;

(3) Take 0.2g of Fe ₃ O ₄ @SiO ₂ @MPS prepared in step (2) and 0.25mmol catechol, 0.5mmol α-methacrylic acid, 2.5mmol ethylene glycol dimethacrylate, 20mg azo Diisobutyronitrile was polymerized at 60°C in a toluene-acetonitrile mixed solvent system with a volume ratio of 4:1 for 24 hours, and then the catechol was eluted with an acetic acid-ethanol mixed solution with a volume ratio of acetic acid and ethanol of 9:1 drop, to produce artificial antibody materials;

(4) Perform Schiff base reaction of lipase and catechin aldehyde in a phosphate buffer solution with a concentration of 0.01M to obtain a catechin aldehyde-enzyme complex solution; the reaction conditions are: the concentration of catechin aldehyde solution is 25 μg/mL , The concentration of α-amylase is 4mg/mL, the reaction temperature is 50°C, the pH is 7.5, and the reaction time is 4h;

(5) Add 1 mL of the catechin aldehyde-enzyme complex solution obtained in step (4) to 5 mg of the artificial antibody material prepared in step (3), and mix with a mixer with a rotation speed of 300 rpm to immobilize the enzyme The immobilization time was 11 hours, and the immobilization temperature was 20° C. to obtain immobilized lipase.

Example 2

In this embodiment, the construction of an immobilized enzyme system for α-amylase is carried out. The construction method includes the following steps:

(1) Disperse 0.6g of Fe ₃ O ₄ nanoparticles in 200mL of ethanol-water mixed solvent system with a volume ratio of ethanol and water of 4:1, add 8mL of ammonia water and 4mL of ethyl orthosilicate, and mix at 35°C for 6h , to get Fe ₃ O ₄ @SiO ₂ ;

(2) Disperse 0.5 g of Fe ₃ O ₄ @SiO ₂ prepared in step (1) in 50 mL of toluene, add 1 mL of triethylamine, 2 mL of α-3-(trimethoxysilyl)propyl methacrylate (MPS ), reflux and mix under the protection of nitrogen to obtain Fe ₃ O ₄ @SiO ₂ @MPS;

(3) Take 0.2g of Fe ₃ O ₄ @SiO ₂ @MPS prepared in step (2) and 0.25mmol catechol, 0.5mmol α-methacrylic acid, 2.5mmol ethylene glycol dimethacrylate, 20mg azo Diisobutyronitrile was polymerized at 60°C in a toluene-acetonitrile mixed solvent system with a volume ratio of 4:1 for 24 hours, and then the catechol was eluted with an acetic acid-ethanol mixed solution with a volume ratio of acetic acid and ethanol of 9:1 drop, to produce artificial antibody materials;

(4) Carry out Schiff base reaction with the catechin aldehyde of α-amylase and the phosphate buffer solution that concentration is 0.01M, obtain catechin aldehyde-enzyme complex solution; Reaction condition is: the concentration of catechin aldehyde solution is 30μg/mL, the concentration of α-amylase is 1mg/mL, the reaction temperature is 36°C, the pH is 6.8, and the reaction time is 5h;

(5) Add 1 mL of the catechin aldehyde-enzyme complex solution obtained in step (4) to 5 mg of the artificial antibody material prepared in step (3), and mix with a mixer with a rotation speed of 300 rpm to immobilize the enzyme The immobilization time was 11 h, and the immobilization temperature was 20° C. to obtain immobilized lipase.

Example 3

This embodiment carries out the construction of immobilized enzyme system to α-glucosidase, and construction method comprises the following steps:

(1) Disperse 0.6g of Fe ₃ O ₄ nanoparticles in 200 mL of ethanol-water mixed solvent system with a volume ratio of ethanol and water of 4:1, add 8 mL of ammonia water, 4 mL of tetraethyl orthosilicate, and mix at 35°C for 6 hours. Get Fe ₃ O ₄ @SiO ₂ ;

(2) Disperse 0.5 g of Fe ₃ O ₄ @SiO ₂ prepared in step (1) in 50 mL of toluene, add 1 mL of triethylamine, 2 mL of α-3-(trimethoxysilyl)propyl methacrylate (MPS ), reflux and mix under the protection of nitrogen to obtain Fe ₃ O ₄ @SiO ₂ @MPS;

(3) Take 0.2g of Fe ₃ O ₄ @SiO ₂ @MPS prepared in step (2) and 0.25mmol catechol, 0.5mmol α-methacrylic acid, 2.5mmol ethylene glycol dimethacrylate, 20mg azo Diisobutyronitrile was polymerized at 60°C in a toluene-acetonitrile mixed solvent system with a volume ratio of 4:1 for 24 hours, and then the catechol was eluted with an acetic acid-ethanol mixed solution with a volume ratio of acetic acid and ethanol of 9:1 drop, to produce artificial antibody materials;

(4) Carry out Schiff base reaction with α-glucosidase and catechin aldehyde in the phosphate buffer solution of 0.01M, obtain catechin aldehyde-enzyme complex solution; Reaction condition is: catechin aldehyde solution concentration 28 μg/mL, the volume ratio of α-glucosidase to catechin aldehyde solution is 2.5%, the reaction temperature is 45°C, the pH is 7.3, and the reaction time is 5h;

(5) Add 1 mL of the catechin aldehyde-enzyme complex solution obtained in step (4) to 5 mg of the artificial antibody material prepared in step (3), and mix with a mixer with a rotation speed of 300 rpm to immobilize the enzyme The immobilization time was 11 h, and the immobilization temperature was 20° C. to obtain immobilized lipase.

Comparative example 1

The difference from Example 1 is that the matrix material is a non-artificial antibody material, and other processes are completely consistent with Example 1;

The preparation method of the non-artificial antibody material is:

(1) Disperse 0.6g of Fe ₃ O ₄ nanoparticles in 200mL of ethanol-water mixed solvent system with a volume ratio of ethanol and water of 4:1, add 8mL of ammonia water and 4mL of ethyl orthosilicate, and mix at 35°C for 6h , to get Fe ₃ O ₄ @SiO ₂ ;

(2) Disperse 0.5 g of Fe ₃ O ₄ @SiO ₂ prepared in step (1) in 50 mL of toluene, add 1 mL of triethylamine, 2 mL of α-3-(trimethoxysilyl)propyl methacrylate (MPS ), reflux and mix under the protection of nitrogen to obtain Fe ₃ O ₄ @SiO ₂ @MPS;

(3) Take 0.2g of Fe ₃ O ₄ @SiO ₂ @MPS prepared in step (2) and 0.5mmol α-methacrylic acid, 2.5mmol ethylene glycol dimethacrylate, 20mg azobisisobutyronitrile at 60 Polymerize in a mixed solvent system of toluene-acetonitrile with a volume ratio of 4:1 at ℃ for 24 hours, then elute catechol with a mixed solution of acetic acid-ethanol with a volume ratio of acetic acid and ethanol of 9:1 to obtain non-artificial Antibody material.

Comparative example 2

The difference from Example 2 is that the matrix material is different, and the non-artificial antibody material in Comparative Example 1 is used, and other processes are completely consistent with Example 2.

Comparative example 3

The difference from Example 3 is that the matrix material is different, and the non-artificial antibody material in Comparative Example 1 is used, and other processes are completely consistent with Example 3.

Performance Testing:

Carry out performance test to the immobilized enzyme material that embodiment 1, embodiment 2, embodiment 3 and comparative example 1, comparative example 2, comparative example 3 make, method is as follows:

(1) Enzyme load:

The absorbance of the supernatant enzyme solution before and after immobilization was measured by the Coomassie brilliant blue method at UV 595nm, substituted into the BSA bovine serum albumin standard curve, and the protein concentration before and after immobilization was determined to calculate the immobilization rate and immobilization amount.

The formula of loading rate: Yield(%)=(C ₀ -C _t )/C ₀ ×100%

Solid loading formula: solid loading = (C ₀ -C _t )×V×1000/M

Where C ₀ is the protein concentration before immobilization, C _t is the protein concentration after immobilization, V is the volume of the solution used for immobilization, and M is the mass of the matrix.

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

(3) Reusability of enzymes

The enzyme activity measured for the first time was defined as 100%, and after the immobilized enzyme was separated from the solution system using a magnet, the activity was repeatedly measured, and the ratio of the repeated measurement and the first measurement was compared.

(4) Comparison of temperature adaptability between immobilized enzyme and free enzyme

The activities of immobilized enzymes and free enzymes were measured under different temperature conditions, and the best one was selected as 100%, and the remaining temperature activity was compared with it.

(5) Comparison of pH adaptability between immobilized enzyme and free enzyme

The activities of immobilized enzymes and free enzymes were measured under different pH conditions, and the best one was selected as 100%, and the remaining pH activity was compared with it.

The performance measurement results are as follows:

Table 1 shows the immobilization capacity, enzyme activity and repeated enzyme activity determination results of Examples 1-3 and Comparative Examples 1-3.

Table 1

The artificial antibody-antigen immobilized enzyme system of Example 1 has a lipase immobilization capacity of 13.22mg _protein /g _support , an initial enzyme activity of 104.66U/g _support , an immobilization rate _of 73.58%, and an enzyme activity of It was 104.30U/g _support ; the non-artificial antibody material in Comparative Example 1 had an immobilization capacity of lipase of 0.77 mg _protein /g _support , which was much different from the immobilized lipase effect of the artificial antibody material, and had a certain initial activity. 75.87U/g _support , but _only 5.17U/g _support left when reused.

The α-amylase of Example 2 has an immobilization capacity of 14.78mg _protein /g _support , an initial enzyme activity of 199.33u/g _support , an immobilization rate of 68.29%, and an enzyme activity of 199.33U/g _support during repeated use; The non-artificial antibody material in ratio 2 has an immobilized capacity of 0.20mg _protein /g _support for α-amylase, and an initial enzyme activity of 114.30U/g _support . At the same time, the activity recovery effect decreases rapidly when it is reused, leaving only 29.74U/g _support .

The α-glucosidase of Example 3 has an immobilization capacity of 25.09mg _protein /g _support , an initial enzyme activity of 143.25U/g _support , an immobilization rate of 62.84%, and an enzyme activity of 137.70U/g _support during repeated use; The non-artificial antibody material in Comparative Example 2 has an immobilization capacity of α-glucosidase of 5.91mg _protein /g _support , an initial enzyme activity of 70.29U/g _support , and only 29.46U/g _support of enzyme activity after repeated use.

At the same time, the artificial antibody-immobilized lipase, α-amylase, and α-glucosidase materials prepared in Examples 1-3 of the present invention were 62.79% and 47.48% respectively relative to the initial enzyme activity when they were reused for the 15th time. %, 39.85%. Facts have proved that using artificial antibody materials to immobilize enzymes not only has a good immobilization effect, but also has higher enzyme activity, and has higher stability than non-artificial antibodies when repeated use.

After the enzyme is immobilized, the optimal enzymatic reaction conditions may change. The initial activity of the free enzyme and the immobilized enzyme activity are respectively defined as 100%. It can be seen from the experimental results that the best enzyme activity of the three immobilized enzymes The reaction temperature was changed compared with the free enzyme. In Example 2, after the α-amylase was immobilized, the activity recovery effect was higher than that of the free enzyme no matter at high temperature or low temperature. The relative activity of the immobilized α-amylase was 77.21% at 25°C, and the free starch The enzyme content is 67.68%. The relative activity of immobilized α-amylase at 50°C is 95.31%, while the activity of free amylase is 79.76%. After the α-glucosidase is immobilized in Example 3, the activity recovery effect under low temperature conditions is close to that of the free enzyme, but it is superior to the free enzyme under high temperature conditions. The residual activity of the immobilized α-glucosidase at 80°C is 47.82%, the remaining activity of free glycosidase is 38.51%. In Example 1, the activity recovery effect of immobilized lipase is not as good as that of free enzyme at low temperature, but higher than that of free enzyme at high temperature. At 50°C, the recovery activity of immobilized lipase is 93.72%, while that of free lipase Only 87.35% left. In general, when using artificial antibody materials to immobilize enzymes, the adaptability of enzymes to higher extreme temperatures is improved.

Comparison of activity recovery effects of immobilized lipase, immobilized α-amylase, immobilized α-glucosidase and corresponding free enzymes at different pH values, α-glucosidase, lipase and α-amylase were The optimal pH of the enzymatic reaction varies after immobilized on the artificial antibody material. The activity recovery effect of the immobilized lipase is more excellent under alkaline conditions. When the pH=10.5, the recovery rate of the activity of the immobilized lipase is 77.27±0.25%, while the activity of the free lipase is only 39.65±0.33%. Although the activity recovery effect of α-glucosidase is not as good as that of free enzyme under acidic conditions after being immobilized, its activity recovery rate under alkaline conditions is higher than that of free enzyme. When pH=9, the activity recovery efficiency of free enzyme was 64.35%, while the activity recovery effect of the immobilized enzyme was 72.72%. After the α-amylase is immobilized, it is superior to the free enzyme no matter under the condition of peracid or overalkali. When the pH=10.5, the relative activity of the immobilized α-amylase is 82.37%, and the relative activity of the α-amylase in the free state It is 69.46%. When pH=5.5, the activity recovery rate of immobilized α-amylase is 89.55%, and the enzyme activity recovery rate of free state is then 81.04%. Activity retention effect in acid and over-alkali environment.

Application example 1

Use immobilized lipase to catalyze the synthesis of benzyl acetate. The synthesis conditions are: add 500 μL of vinyl acetate and 5 μL of benzyl alcohol to 50 mg of immobilized lipase material, react at 70 ° C, and the reaction reaches an equilibrium state after 6 hours of reaction; After reacting the mixture for 6 hours, the unreacted reactants were evaporated to dryness at 70° C., and the yield of the reaction product benzyl acetate was measured by liquid phase, and the yield was 88.72%. After the supernatant was separated from the immobilized lipase material with a magnet, vinyl acetate and benzyl alcohol were added continuously. After five consecutive injections, the yield of benzyl acetate obtained was 74.13% of the product obtained by the initial catalysis. This proves that the lipase immobilized by the method not only has good stability and reusability in aqueous solution, but also maintains good stability and reusability in organic solvents.

Application example 2

Use immobilized α-amylase to catalyze the synthesis of o-amino-p-cresol. The synthesis conditions are: add the starch solution to the immobilized α-amylase material and activate it at 37°C for 10 minutes, then suck out the supernatant and mix with 2-nitro -4-Methylphenol was reacted, and the product amount was basically stable when the reaction was carried out at 100° C. for 9 minutes, and the yield was 58.88% when detected by liquid phase at 275 nm. Next, the continuous sampling efficiency of the immobilized α-amylase material catalyzing the reaction of 2-amino-p-cresol was measured. After 5 times of repeated use, the catalytic efficiency was 80.86% of the initial use.

Application example 3

Using immobilized α-glucosidase to catalyze the synthesis of 4-methylumbelliferone, the synthesis condition is: add 4-methylumbelliferone-α-D-glucopyranoside solution to immobilized α-glucosidase The material was reacted at 37° C., and the yield was measured by high-performance liquid chromatography at 254 nm after reacting for 30 minutes. At this time, the reaction yield was 77.54%. Then, the continuous injection effect of the immobilized α-glucosidase catalyzing the conversion of 4-methylumbelliferone-α-D-glucopyranoside into 4-methylumbelliferone was determined. After 5 consecutive catalysis, the yield was 81.12% of the initial catalysis.

The test results of application examples 1-3 are shown in Table 2.

Table 2

The present invention mainly explores the feasibility of the artificial antibody-antigen immobilized enzyme system. After successfully constructing the single antibody immobilized enzyme system, its performance is measured. Compared with ordinary immobilized enzyme materials, artificial antibody immobilized enzyme materials The immobilization effect and stability are better, and finally the immobilized enzyme material has been successfully applied.

The applicant declares that the present invention illustrates the preparation method and application of the artificial antibody-antigen-immobilized enzyme system of the present invention through the above examples, but the present invention is not limited to the above steps, that is, it does not mean that the above process steps must be relied on for implementation. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of selected raw materials and the addition of auxiliary components in the present invention, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims (8)

1. A method for building an immobilized enzyme system based on artificial antibody-antigen, characterized in that, The method comprises the steps of: (1) Disperse Fe ₃ O ₄ nanoparticles in an ethanol-water mixed solvent system, and mix them with tetraethyl orthosilicate in an alkaline environment to obtain Fe ₃ O ₄ @SiO ₂ ; (2) Disperse the Fe ₃ O ₄ @SiO ₂ prepared in step (1) in toluene, add triethylamine, α-3-(trimethoxysilyl)propyl methacrylate (MPS), and Reflux and mix under protection to obtain Fe ₃ O ₄ @SiO ₂ @MPS; (3) Using catechol as a template molecule, combine the Fe ₃ O ₄ @SiO ₂ @MPS prepared in step (2) with catechol, α-methacrylic acid, ethylene glycol dimethacrylate, azo Diisobutyronitrile is polymerized in a toluene-acetonitrile mixed solution, and then the catechol is eluted with an eluent to prepare an artificial antibody material; (4) performing a Schiff base reaction on the enzyme molecule and catechin aldehyde in a buffer solution to obtain a catechin aldehyde-enzyme complex solution; (5) Add the catechin aldehyde-enzyme complex solution obtained in step (4) to the artificial antibody material prepared in step (3), mix well, and immobilize the enzyme. 2. The construction method according to claim 1, characterized in that, in step (1), the _Fe3O4 nanoparticles have _a particle diameter of 100-400nm; 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 the ammonia water to tetraethyl orthosilicate is 2:1; The molar ratio of Fe ₃ O ₄ to ethyl orthosilicate is 1:5-1:10. 3. The construction method according to claim 1, characterized in that, in step (2), the volume ratio of the triethylamine to α-3-(trimethoxysilyl)propyl methacrylate is 1: 1-1:3, the ratio of the mass of Fe ₃ O ₄ @SiO ₂ to the volume of 3-(trimethoxysilyl)propyl α-methacrylate is 0.1-0.5g:1mL. 4. The construction method according to claim 1, wherein the volume ratio of toluene to acetonitrile in the toluene-acetonitrile mixed solution is 1:1-1:5. 5. The construction method according to claim 1, characterized in that, in step (3), the ratio of the molar mass of catechol to the mass of Fe ₃ O ₄ @SiO ₂ @MPS is 1-3mmol:1g, The molar ratio of catechol to α-methacrylic acid is 1:2-1:6, and the molar ratio of catechol to ethylene glycol dimethacrylate is 1:5-1:20; The polymerization reaction temperature is 60-80°C, and the time is 12-24h; The eluent is an acetic acid-ethanol mixed solution with a volume ratio of acetic acid and ethanol of 9:1. 6. The construction method according to claim 1, characterized in that, in step (4), the buffer solution is a 0.01M 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 to α-amylase is 1:10-1:40; The ratio of the activity of the α-glucosidase to the mass of catechin aldehyde is 200-1500U: 1 μg; The reaction temperature of the reaction between the catechin aldehyde and the enzyme molecule Schiff base is 20-60°C, the reaction pH is 6-8, and the reaction time is 1-6h. 7. construction method according to claim 1, is characterized in that, in step (5), the consumption relation of described artificial antibody polymer and catechin aldehyde-enzyme complex is: every 1mL catechin aldehyde-enzyme complex Add 5-20mg of artificial antibody material to the The mixing speed is 100rpm-300rpm; The immobilization time of the enzyme is 1-24h, and the immobilization temperature is 10-60°C. 8. Application of the construction method described in any one of claims 1-7 in the field of biocatalysis.