阅读说明：本技术 一种固定化酶载体、固定化酶以及制备方法 (Immobilized enzyme carrier, immobilized enzyme and preparation method ) 是由 陈峻青 汤玉琪 于 2021-09-01 设计创作，主要内容包括：本发明公开了一种固定化酶载体、固定化酶及制备方法。本发明通过壳聚糖的大分子颗粒附着在鸡蛋壳膜的表面并通过共沉淀的方式将磁性Fe-(3)O-(4)均匀沉淀分布在蛋壳膜上,制备出磁性Fe-(3)O-(4)/CS/ESMP作为固定化β-葡萄糖苷酶的酶载体,增加了酶结构的刚性以及耐受性,具有良好的操作稳定性；此外,制备的Fe-(3)O-(4)/CS/ESMP@BG可以重复使用,操作简便。Fe-(3)O-(4)/CS/ESMP@BG在磁场的作用下易于分离收集从而重复回收利用,提高了酶的使用效率,制备的Fe-(3)O-(4)/CS/ESMP@BG可应用于京尼平的生物合成。(The invention discloses an immobilized enzyme carrier, an immobilized enzyme and a preparation method. The invention attaches the macromolecular particles of chitosan on the surface of the eggshell membrane and makes the magnetic Fe in a coprecipitation way 3 O 4 Uniformly depositing and distributing on the eggshell membrane to prepare magnetic Fe 3 O 4 the/CS/ESMP is used as an enzyme carrier of the immobilized beta-glucosidase, so that the rigidity and the tolerance of an enzyme structure are increased, and the operation stability is good; in addition, Fe produced 3 O 4 the/CS/ESMP @ BG can be repeatedly used and is simple and convenient to operate. Fe 3 O 4 the/CS/ESMP @ BG is easy to separate and collect under the action of a magnetic field so as to be recycled, the use efficiency of enzyme is improved, and the prepared Fe 3 O 4 the/CS/ESMP @ BG can be applied to the biosynthesis of genipin.)

1. An immobilized enzyme carrier, which is prepared by the following method:

(1a) preparing a chitosan solution;

(1b) preparation of a catalyst containing Fe²⁺And Fe³⁺Adding the chitosan solution of step (1a) to the solution of (2)Adding into solution containing iron ions to obtain mixed solution, wherein the concentration of chitosan in the mixed solution is 0.015-0.025 g/mL; adding ESMP into the obtained mixed solution, stirring for dissolving, wherein the mass ratio of the chitosan to the ESMP is 1-3:1-3, uniformly stirring, adding ammonia water for primary precipitation, and adding a sodium hydroxide solution for secondary precipitation to obtain a granular carrier;

(1c) and (2) washing the carrier obtained in the step (1b), and adding the washed carrier into a genipin solution for infiltration to obtain the immobilized enzyme carrier.

2. The immobilized enzyme carrier according to claim 1, wherein in step (1c), the concentration of the genipin solution is 10-50 mmol/L.

3. The immobilized enzyme carrier according to claim 1, wherein in step (1b), the Fe is²⁺The concentration of Fe in the mixed solution is 0.1-0.2mol/L³⁺The concentration in the mixed solution is 0.2-0.4 mol/L.

4. The immobilized enzyme carrier according to claim 4, wherein the Fe is²⁺With Fe³⁺In a molar ratio of 2: 1.

5. The immobilized enzyme carrier according to claim 1, wherein in step (1b), the mass ratio of chitosan to ESMP is 1: 2.

6. the immobilized enzyme carrier according to claim 1, wherein in step (1b), the temperature of the preliminary precipitation is controlled at 75-85 ℃.

7. The process for producing an immobilized enzyme carrier according to claim 1, comprising the steps of:

(2a) preparing a chitosan solution;

(2b) preparation of a catalyst containing Fe²⁺And Fe³⁺Adding the chitosan solution obtained in the step (2a) into a solution containing iron ions to obtain the solutionAdding chitosan into the mixed solution, wherein the concentration of the chitosan in the mixed solution is 0.015-0.025 g/mL; then adding ESMP into the obtained mixed solution, wherein the mass ratio of the chitosan to the ESMP is 1-3:1-3, uniformly stirring, adding ammonia water for primary precipitation, and adding a sodium hydroxide solution for secondary precipitation to obtain a granular carrier;

(2c) and (3) washing the carrier obtained in the step (2b), and adding the washed carrier into a genipin solution for infiltration to obtain the immobilized enzyme carrier.

8. An immobilized enzyme obtained by crosslinking the immobilized enzyme carrier according to claim 1 with a solution of a free enzyme.

9. The immobilized enzyme according to claim 8, wherein the free enzyme is β -glucosidase.

10. A preparation method of immobilized enzyme is characterized by comprising the following steps:

(3a) preparing a chitosan solution;

(3b) preparation of a catalyst containing Fe²⁺And Fe³⁺Adding the chitosan solution obtained in the step (3a) into a solution containing iron ions to obtain a mixed solution, wherein the concentration of the chitosan in the mixed solution is 0.015-0.025 g/mL; then adding ESMP into the obtained mixed solution, wherein the mass ratio of the chitosan to the ESMP is 1-3:1-3, uniformly stirring, adding ammonia water for primary precipitation, and adding a sodium hydroxide solution for secondary precipitation to obtain a granular carrier;

(3c) washing the carrier obtained in the step (3b), and adding the washed carrier into a genipin solution for infiltration to obtain an immobilized enzyme carrier;

(3d) and (4) incubating the immobilized enzyme carrier obtained in the step (3c) with a beta-glucosidase solution to obtain immobilized beta-glucosidase.

Technical Field

The invention belongs to the technical field of immobilized enzymes, and particularly relates to an immobilized enzyme carrier, an immobilized enzyme and a preparation method.

Background

Genipin is an aglycone of geniposide, and is found in plants such as Gardenia jasminoides Ellis, Pelargonium graveolens, and eucommia ulmoides belonging to Rubiaceae. Genipin is reported to have the effects of resisting oxidation, inflammation, tumor, depression, fungi and reverse blood vessel hyperplasia, diabetes and immunosuppressant, is used for treating jaundice, edema and hypertension, and can inhibit the diffusion of some cancer cells, including leukemia, breast cancer, prostate cancer and hepatocellular carcinoma, so that genipin is widely applied to the biomedical field. Some studies have also reported the enormous ability of genipin to produce cross-links between proteins and polymers such as collagen, gelatin, chitosan, etc. In addition, genipin is safer to apply to food processing due to its lower cytotoxicity.

In the fields of modern biotechnology and biomedicine, enzymes play an important role due to the characteristics of high efficiency, strong specificity and the like. However, from an industrial point of view, free enzymes also have drawbacks such as unsatisfactory reusability, short catalytic life, low operational stability to temperature, pH and certain environmental factors, difficulty in recycling and reuse, and serious impediment to their large-scale application on a large industrial scale. In order to solve this technical problem, it is necessary to study the properties of the immobilized enzyme. Enzyme immobilization is a powerful means to overcome this limitation and provides the enzymatic properties of the biocatalyst. However, efficient and simple immobilization methods require further investigation.

Chitosan is the second most abundant natural polymer, deacetylated by chitin through alkaline treatment. The use of chitosan is commercially attractive due to the production of chitin and chitosan from shells of crabs, shrimps, prawns, lobsters, crawfish, krill and insects. Because of the non-toxicity, biocompatibility, good biodegradability, high affinity to protein and physiological inertia, the chitosan becomes an ideal enzyme immobilization carrier material.

The eggshell membrane is a large amount of waste in the food industry, is a promising reinforcing agent candidate material in the polymer industry, and has good toughness and impact strength. Approximately 60% of the protein in the organic matrix of eggshell membrane is distributed among collagen (35%), glucosamine (10%), chondroitin (9%) and hyaluronic acid (5%), while the inorganic fraction contains small amounts of Ca, Mg, Si, Zn and large amounts of nitrogen. Thus, ESM proteins stabilize them against acids, basic compounds and proteases. Furthermore, ESM is insoluble in water because it contains a large number of cysteines and lysines, which form molecular bridges between protein molecules. In particular, it has excellent permeability to substrates and products, making it an ideal support system for enzyme immobilization. Research results show that the application of the enzyme immobilization technology to household garbage (such as ESM) is not only a promising enzyme immobilization material, but also a promising biotechnology application platform.

Iron oxide nanoparticles are the most studied magnetic nanoparticles in the industrial and biotechnological field, and since iron particles can be of the nanoscale, they become superparamagnetic, thus avoiding self-agglomeration. The immobilized enzyme prepared by using the magnetic nano material as the enzyme carrier has improved stability, and can be quickly separated and recovered by adsorbing the magnetic material through a magnetic field, so that the aim of repeated use is fulfilled.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides an immobilized enzyme carrier, which is made of Fe₃O₄Composition of/CS/ESMP with genipin as cross-linking agent and Fe in advance₃O₄the/CS/ESMP infiltration ensures that the prepared immobilized enzyme carrier is more convenient to use. The invention further provides an immobilized enzyme immobilized with beta-glucosidase immobilized on magnetic Fe₃O₄The enzyme rigidity and tolerance are improved on/CS/ESMP. The invention also provides an immobilized enzyme carrier and a preparation method of the immobilized enzyme.

The technical scheme is as follows: the immobilized enzyme carrier is prepared by the following method:

(1a) preparing a chitosan solution;

(1b) preparation of a catalyst containing Fe²⁺And Fe³⁺Adding the chitosan solution obtained in the step (1a) into a solution containing iron ions to obtain a mixed solution, wherein the concentration of the chitosan in the mixed solution is 0.015-0.025 g/mL; then adding ESMP into the obtained mixed solution, wherein the mass ratio of the chitosan to the ESMP is 1-3:1-3, uniformly stirring, adding ammonia water for primary precipitation, and adding a sodium hydroxide solution for secondary precipitation to obtain a granular carrier;

(1c) and (2) washing the carrier obtained in the step (1b), and adding the washed carrier into a genipin solution for infiltration to obtain the immobilized enzyme carrier.

Preferably, in the step (1a), the chitosan has a deacetylation degree of 85% or more and a viscosity of 50-400 mPa.s.

Preferably, in the step (1c), the concentration of the genipin solution is 10-50 mmol/L.

Preferably, in step (1b), the Fe²⁺The concentration of Fe in the mixed solution is 0.1-0.2mol/L³⁺The concentration in the mixed solution is 0.2-0.4 mol/L.

Preferably, the Fe²⁺With Fe³⁺In a molar ratio of 2: 1.

Preferably, in the step (1b), the mass ratio of the chitosan to the ESMP is 1: 2.

preferably, in the step (1b), the solution is heated to 75-85 ℃, ammonia water is added as a precipitating agent, and primary precipitation is carried out, so that the temperature of the primary precipitation is controlled at 75-85 ℃.

Preferably, the mass percentage concentration of the ammonia water is 25-28%.

Preferably, in the step (1b), the concentration of the sodium hydroxide solution is 8-12% by mass.

Preferably, the solvent of the sodium hydroxide solution consists of ethanol to water in a volume ratio of 1: 3.

Preferably, in the step (1c), the crosslinking reaction of the carrier is carried out in a genipin solution of 15-25mmol/L at 45-55 ℃, and the crosslinking time is preferably 4-6 h; subsequently, the mixture was rinsed with deionized water and stored at 4 ℃.

The preparation method of the immobilized enzyme carrier comprises the following steps:

(2a) preparing a chitosan solution;

(2b) preparation of a catalyst containing Fe²⁺And Fe³⁺Adding the chitosan solution obtained in the step (2a) into a solution containing iron ions to obtain a mixed solution, wherein the mass of the chitosan in the mixed solution is 0.015-0.025 g/mL; then adding ESMP into the obtained mixed solution, wherein the mass ratio of the chitosan to the ESMP is 1-3:1-3, uniformly stirring, adding ammonia water for primary precipitation, and adding a sodium hydroxide solution for secondary precipitation to obtain a granular carrier;

(2c) and (3) washing the carrier obtained in the step (2b), and adding the washed carrier into a genipin solution for infiltration to obtain the immobilized enzyme carrier.

The parameter limitation in the preparation method of the immobilized enzyme carrier is the same as the parameter limitation in the preparation of the immobilized enzyme carrier.

The invention further provides an immobilized enzyme, which is formed by crosslinking a free enzyme solution and the immobilized enzyme carrier.

The free enzyme used herein may be selected from a wide variety of enzymes, and further, the free enzyme is β -glucosidase.

The preparation method of the immobilized enzyme comprises the following steps:

(3a) preparing a chitosan solution;

(3b) preparation of a catalyst containing Fe²⁺And Fe³⁺Adding the chitosan solution obtained in the step (3a) into a solution containing iron ions to obtain a mixed solution, wherein the concentration of the chitosan in the mixed solution is 0.015-0.025 g/mL; then adding ESMP into the obtained mixed solution, wherein the mass ratio of the chitosan to the ESMP is 1-3:1-3, uniformly stirring, adding ammonia water for primary precipitation, and adding a sodium hydroxide solution for secondary precipitation to obtain a granular carrier;

(3c) washing the carrier obtained in the step (3b), and adding the washed carrier into a genipin solution for infiltration to obtain an immobilized enzyme carrier;

(3d) and (4) incubating the immobilized enzyme carrier obtained in the step (3c) with a beta-glucosidase solution to obtain immobilized beta-glucosidase.

The parameter limitation in the preparation method of the immobilized enzyme in the invention is the same as the parameter limitation in the preparation of the immobilized enzyme carrier.

Preferably, in the step (3d), the incubation conditions of the immobilized enzyme carrier and the beta-glucosidase solution are as follows: mixing the following components in a mass ratio of 6-8: 1 and beta-glucosidase at 4 ℃ for 3-5h, shaking slightly, fixing the beta-glucosidase on the immobilized enzyme carrier, washing with sodium phosphate buffer solution with pH 7.0 until no protein is detected, and collecting Fe under the action of magnetic field₃O₄/CS/[email protected]。

The preferable preparation method of the immobilized enzyme comprises the following steps: (a) firstly, preparing a chitosan solution;

(b) FeCl is added₂·4H₂O、FeCl₃·6H₂Mixing O according to the molar ratio of 1: 2, adding water for dissolving, adding the chitosan solution into the solution containing iron ions in the same volume to obtain a mixed solution, wherein the concentration of chitosan in the mixed solution is 0.017-0.018g/mL, and Fe²⁺The concentration of Fe in the mixed solution is 0.1-0.15mol/L³⁺The concentration in the mixed solution is 0.2-0.3 mol/L; and then adding ESMP into the obtained mixed solution, wherein the mass ratio of the chitosan to the ESMP is 1: 2, after uniformly stirring, heating to 75-85 ℃, adding 25-28% ammonia water, stirring for 1-2h, carrying out primary precipitation, then adding 8-12% sodium hydroxide solution (the volume ratio of ethanol to water is 1:3) at room temperature for secondary precipitation to obtain a granular carrier, and washing the granular carrier with deionized water, ethanol solution (the volume ratio of ethanol to water is 1:1) and deionized water in sequence until the carrier is neutral;

(c) washing the carrier obtained in the step (b), adding the washed carrier into a genipin solution of 15-25mmol/L, heating to 45-55 ℃, and infiltrating for 4-6h to obtain an immobilized enzyme carrier;

(d) mixing the following components in a mass ratio of 6-8: 1 and beta-glucosidase (the beta-glucosidase is dissolved in PBS buffer solution with pH 7.0), incubating for 3-5h at 4 ℃, washing all samples with sodium phosphate buffer solution with pH 7.0 until no protein is detected, and collecting Fe under the action of magnetic field₃O₄/CS/[email protected]。

In the present invention, "%" is a mass percent concentration unless otherwise specified.

Has the advantages that: (1) the invention prepares magnetic Fe by coprecipitation crosslinking method₃O₄the/CS/ESMP is used as an enzyme carrier for immobilizing beta-glucosidase (BG), and the beta-glucosidase is immobilized on magnetic Fe by using genipin as a cross-linking agent₃O₄/CS/ESMP, which increases the structural rigidity of the enzyme; (2) compared with free BG, Fe3O4/CS/ESMP @ BG prepared by the invention is Fe₃O₄the/CS/ESMP @ BG has strong tolerance to temperature, pH change and organic solvent, and has good operation stability; (3) the immobilized enzyme has good enzyme loading capacity and activity recovery rate, and Fe₃O₄the/CS/ESMP @ BG can be repeatedly used, the operation is simple and convenient, the immobilized enzyme is easy to separate and collect under the action of a magnetic field so as to be repeatedly recycled, and the use efficiency of the enzyme is improved.

Drawings

FIG. 1 shows the effect of immobilized enzyme vectors prepared under different conditions on enzyme activity;

FIG. 2 shows free BG and Fe₃O₄Screening the optimum temperature of/CS/ESMP @ BG;

FIG. 3 shows free BG and Fe₃O₄Screening the optimal pH value of/CS/ESMP @ BG;

FIG. 4 is Fe₃O₄a/CS/ESMP @ BG cyclic utilization result;

FIG. 5 shows ESMP and Fe₃O₄The scanning electron microscope images of/CS/ESMP, wherein, images A and B are the scanning electron microscope images of ESMP, and images C, D, E and F are Fe₃O₄a/CS/ESMP scanning electron microscope picture;

FIG. 6 is Fe₃O₄/CS/ESMP (A, C) and Fe₃O₄Confocal microscopy analysis of/CS/ESMP @ BG (B, D), where panels A and C are immobilized enzyme support Fe₃O₄the/CS/ESMP, the graph B and the graph D are immobilized enzyme Fe₃O₄/CS/[email protected]。

Detailed Description

Preparation of immobilized enzyme carrier

1.1 Effect of ESMP on the Performance of immobilized enzyme Carriers

0.5g of Fe₃O₄(preparation conditions refer to Fe in MCESM1₃O₄Preparation method) and 20mL of a filtered chitosan solution (mass concentration of 3.5%) were stirred at room temperature for 1 hour, and a 10% sodium hydroxide solution (25% ethanol: 75% water, v/v), and the resulting large particles were sequentially washed with deionized water, ethanol solution (50% ethanol: 50% water, v/v) and deionized water until neutral. Subjecting the obtained product toIncubating large particles in 20mmol/L genipin solution at 50 ℃, washing with deionized water after 5h, and mixing BG with Fe₃O₄The mass ratio of/CS is 1: 8 into a 10mL reactor, adding 1mL PBS buffer (pH 7.0) of beta-glucosidase, incubating at 4 deg.C for 3h, and collecting Fe under the action of magnetic field₃O₄/CS/ESMP @ BG. The enzyme activity was found to be 52.4%.

In the forming process of the chitosan-based hydrogel particles, ESMP is added to be used as a template part to be dissolved in a sodium hydroxide solution, so that the porous structure is improved. The morphology of the ESM presents a multi-layer interwoven fiber network structure. Due to its special shape, more chitosan chains can be attached by physical and chemical cross-linking. Therefore, ESMP can improve the connectivity of the internal structure of the large particles, which can lead to multiple lattice structures and increased porosity of the large particles. In addition, the abundant protein residues in the eggshell membrane can play a role of a multi-site sucker, and the capture capacity of the chitosan-based large particles on protein molecules is improved. Therefore, the addition of ESMP can increase the activity of the immobilized enzyme.

1.2 the influence of different preparation conditions on the morphology of large particles and the relative enzyme activity.

An immobilized enzyme carrier Fe prepared by the following different methods₃O₄the/CS/ESMP are named MCESM1, MCESM2, MCESM3, MCESM4, MCESM5, MCESM6, respectively.

MCESM 1: FeCl is added₂·4H₂O、FeCl₃·6H₂Mixing the two solutions according to the molar ratio of 1: 2, heating to 80 ℃, stirring for 60min, adding 28% ammonia water as a precipitator, filtering, washing and drying the mixed solution to obtain nanoscale Fe₃O₄。

Chitosan (high viscosity, viscosity > 400mPa. s) powder was dissolved in an aqueous solution containing 2% (w/v) acetic acid, stirred at 50 ℃ for 4h to give a chitosan solution of 0.035g/mL, and then filtered with gauze. Simultaneously, ESMP (particle size less than 80 microns) is dispersed in the aqueous solution, and stirring is continued to obtain an ESMP solution with the concentration of 0.07 g/mL.

0.5g of Fe₃O₄And 20mL of the filtered shellThe polysaccharide solution was stirred at room temperature for 1h, then mixed with 5mL of ESMP solution and stirred for 2h, and then a 10% sodium hydroxide solution (25% ethanol: 75% water, v/v) was added dropwise thereto. The resulting large particles were washed with deionized water, ethanol solution (50% ethanol: 50% water, v/v) and deionized water in this order until neutral.

And (3) incubating the obtained large particles in a 20mmol/L genipin solution at 50 ℃, washing with deionized water after 5 hours, and storing at 4 ℃.

MCESM 2: FeCl is added₂·4H₂O、FeCl₃·6H₂Mixing the two solutions according to the molar ratio of 1: 2, heating to 80 ℃, stirring for 60min, adding 28% ammonia water as a precipitator, filtering, washing and drying the mixed solution to obtain nanoscale Fe₃O₄。

Dissolving chitosan (deacetylation degree is more than or equal to 85% and viscosity is 50mPa.s) powder in 2% (w/v) acetic acid aqueous solution, stirring at 50 ℃ for 4h to obtain 0.035g/mL chitosan solution, and filtering with gauze. At the same time, ESMP was dispersed in the aqueous solution, and stirring was continued to obtain an ESMP-dispersed aqueous solution having a concentration of 0.07 g/mL. 0.5g of Fe₃O₄After stirring with 20mL of filtered chitosan at room temperature for 1 hour, followed by mixing with 5mL of ESMP solution and stirring for 2 hours, 10% sodium hydroxide solution (25% ethanol: 75% water, v/v) was added dropwise thereto. The resulting large particles were washed with deionized water, ethanol solution (50% ethanol: 50% water, v/v) and deionized water in this order until neutral. The crosslinking reaction of all large particles was carried out at 50 ℃ in a 20mmol/L genipin solution. Washing with deionized water after 5 hr, and storing at 4 deg.C.

MCESM 3: taking 1g of the nano-scale Fe prepared above₃O₄After 20mL of a filtered 2% (w/v) acetic acid aqueous solution of 0.035g/mL chitosan (degree of deacetylation: 85% or more, viscosity: 50mPa.s) was stirred at room temperature for 1 hour and 5mL of a 0.07g/mL ESMP solution were mixed and stirred for 2 hours, 10% sodium hydroxide solution (25% ethanol: 75% water, v/v) was added dropwise thereto, and the obtained large particles were washed with deionized water, an ethanol solution (50% ethanol: 50% water, v/v) and deionized water in this order until neutral. Cross-linking reaction of all Large particles 20mmol/L Jing at 50 deg.CIn nipalene solution. Washing with deionized water after 5 hr, and storing at 4 deg.C.

MCESM 4: in 20mL FeCl-containing solution₂·4H₂O、0.89g、FeCl₃·6H₂O2.42g of the aqueous solution, 20mL of a 2% (w/v) aqueous acetic acid solution of 0.035g/mL of chitosan (degree of deacetylation: 85% and viscosity: 50mPa.s) after filtration was added thereto, stirred at room temperature for 1 hour, mixed with 20mL of a 0.07g/mL ESMP solution and stirred for 2 hours, heated to 80 ℃, added with 28% aqueous ammonia as a precipitant and stirred for 1 hour, added dropwise with a 10% sodium hydroxide solution (25% ethanol: 75% water, v/v), and the resulting large particles were washed with deionized water, an ethanol solution (50% ethanol: 50% water, v/v) and deionized water in this order until neutral. The crosslinking reaction of all large particles was carried out at 50 ℃ in a 20mmol/L genipin solution. Washing with deionized water after 5 hr, and storing at 4 deg.C.

MCESM 5: in 20mL FeCl-containing solution₂·4H₂O 0.89g、FeCl₃·6H₂O2.42g of the aqueous solution, 20mL of a 2% (w/v) acetic acid aqueous solution of 0.035g/mL of chitosan (degree of deacetylation: 85% and viscosity: 50mPa.s) after filtration was added thereto, stirred at room temperature for 1 hour, 1.4g of ESMP was added thereto, mixed and stirred for 2 hours, then heated to 80 ℃, ammonia water was added thereto as a precipitant, stirred for 1 hour, 10% sodium hydroxide solution (25% ethanol: 75% water, v/v) was added dropwise thereto, and the obtained large particles were washed with deionized water, an ethanol solution (50% ethanol: 50% water, v/v)) and deionized water in this order until neutral. The crosslinking reaction of all large particles was carried out at 50 ℃ in a 20mmol/L genipin solution. Washing with deionized water after 5 hr, and storing at 4 deg.C.

MCESM 6: in 20mL FeCl-containing solution₂·4H₂O 0.89g、FeCl₃·6H₂O2.42g of the aqueous solution, 20mL of a 2% (w/v) aqueous acetic acid solution of 0.035g/mL of chitosan (degree of deacetylation: 85% or more) after filtration was added thereto, stirred at room temperature for 1 hour, 1.4g of ESMP was added thereto, mixed and stirred for 2 hours, then heated to 80 ℃ and 10% sodium hydroxide solution (25% ethanol: 75% water, v/v) was added dropwise thereto, and the obtained large particles were washed with deionized water, ethanol (50% ethanol: 50% water, v/v) and deionized water in this order until neutral. All ofThe crosslinking reaction of the large particles was carried out at 50 ℃ in a 20mmol/L genipin solution. Washing with deionized water after 5 hr, and storing at 4 deg.C.

The obtained vector was screened, and the results are shown in FIG. 1, and from the results shown in FIG. 1, CS, addition sequence and concentration all had an influence on the activity of the final immobilized enzyme, and finally the experimental conditions of MCESM5 were selected for further screening.

1.3 evaluation of results

Example 1: magnetic chitosan eggshell membrane (Fe)₃O₄CS/ESMP) comprising the steps of:

step 1: chitosan powder was dissolved in 2% (w/v) acetic acid aqueous solution, stirred at 50 ℃ for 4h to give 0.035g/mL chitosan solution, which was then filtered with gauze. In 20mL FeCl-containing solution₂·4H₂O 0.89g、FeCl₃·6H₂O2.42g of aqueous solution, 20mL of 2% (w/v) acetic acid aqueous solution of chitosan (deacetylation degree is more than or equal to 85% and viscosity is 50mPa.s) with mass percent concentration of 0.035g/mL after filtration is added thereto, the mixture is stirred at room temperature for 1h, 1.4g of ESMP is added thereto, the mixture is stirred for 2h, the temperature is raised to 80 ℃, ammonia water is added thereto as a precipitant, the mixture is stirred for 1h, subsequently, 10% sodium hydroxide solution (25% ethanol: 75% water, v/v) is added dropwise thereto at room temperature, and the obtained large particles are washed with deionized water, ethanol (50% ethanol: 50% water, v/v) and deionized water in sequence until neutral. All large particle crosslinking reactions were performed in a 20mmol/L genipin solution at 50 deg.C, rinsed with deionized water after 5h, and stored at 4 deg.C.

The scanning electron micrograph of the obtained immobilized enzyme carrier is shown in FIG. 5, the pictures A and B in FIG. 5 are the scanning electron micrographs of ESMP, and the pictures C, D, E and F are Fe₃O₄the/CS/ESMP scanning electron microscope image shows that the ESMP presents a multi-layer interwoven fiber network structure as shown in an A image and a B image in figure 5. The ESMP has a pulling effect on chitosan chains, so that the arrangement of chitosan chains is looser, the porous structure formed by stacking the chitosan chains is reduced, and the immobilization capacity of macro particles is improved. From the shapes and surface structures of the C, D, E and F diagrams in FIG. 5, general observation can be madeFe prepared by coprecipitation method₃O₄The uniform particle size loading on the ESMP material surface and the chitosan layer covering, maintains the basic ESMP interlaced rod-like surface morphology, which is consistent with the expected result, and can see that Fe₃O₄Success of the/CS/ESMP preparation.

Example 2: preparation of immobilized beta-glucosidase

BG prepared in example 1 and Fe₃O₄The mass ratio of/CS/ESMP is 1: 8 into a 10mL reactor, adding 1mL PBS buffer (pH 7.0) of beta-glucosidase, incubating at 4 deg.C for 3h, immobilizing beta-glucosidase on cross-linked large particles, washing all samples with pH 7.0 sodium phosphate buffer until no protein is detected, and collecting Fe under the action of magnetic field₃O₄/CS/[email protected]。

To further confirm the Fe prepared₃O₄Presence of the target beta-glucosidase on CS/ESMP, we are on Fe₃O₄/CS/ESMP and Fe₃O₄Cofocal Laser Scanning Microscope (CLSM) analysis of/CS/ESMP @ BG with fluorescein isothiocyanate labeled Fe₃O₄Confocal microscopy analysis of/CS/ESMP @ BG is shown in FIG. 6. From the morphological images of the samples shown in panels a and C in fig. 6, it is clear that no fluorescence signal is shown by BG loading. However, in panels B and D of FIG. 6, it can be observed that Fluorescein Isothiocyanate (FITC) -labeled β -glucosidase showed strong green fluorescence signal after immobilization, indicating that β -glucosidase had successfully cross-linked to Fe by genipin₃O₄Surface of/CS/ESMP.

Example 3: fe₃O_4/Determination of the catalytic Properties of CS/ESMP @ BG

As shown in FIG. 1, the optimum temperature for free BG is 50 ℃ and Fe₃O₄the/CS/ESMP @ BG showed the greatest activity at 60 ℃. BG crosslinking of Fe by genipin₃O₄the/CS/ESMP @ BG enzyme protein forms covalent bonds, thereby greatly stabilizing the spatial conformation of the enzyme protein. At higher temperature, the molecular structure of the enzyme can still maintain stable structureHigh activity is maintained. The determination method comprises the following steps: and (3) activity determination: respectively adding free beta-glucosidase, Fe₃O₄adding/CS/ESMP @ BG into 1ml PBS phosphate buffer (100mmol/L, pH 7), and measuring enzyme activity of free beta-glucosidase, Fe3O4/CS/ESMP at 30-90 deg.C, respectively₃O₄The enzyme activity of the optimum temperature of/CS/ESMP @ BG is 100%, and the relative enzyme activities of free enzyme and immobilized enzyme under various temperature conditions are calculated. The results are shown in FIG. 2.

As shown in FIG. 3, free BG and Fe₃O₄The optimum pH value of/CS/ESMP @ BG is 6. And Fe₃O₄the/CS/ESMP @ BG is more adaptable to severe pH conditions than free BG. Fe₃O₄The excellent performance of/CS/ESMP @ BG under relatively basic conditions may highlight its potential for industrial applications. Typically, BG is fixed to Fe by cross-linking₃O₄On a/CS/ESMP support, Fe₃O₄the/CS/ESMP @ BG has better pH adaptability, and the performance of the/CS/ESMP @ BG is obviously superior to that of free BG.

The determination method comprises the following steps: respectively adding free beta-glucosidase, Fe3O4/CS/ESMP @ BG into 1ml PBS phosphate buffer (100mmol/L, pH 7), measuring the enzyme activities of the free beta-glucosidase, Fe3O4/CS/ESMP under the condition of pH 3-9, respectively calculating the relative enzyme activities of the free enzyme and the immobilized enzyme under each pH condition by taking the optimum pH enzyme activities of the free beta-glucosidase, Fe3O4/CS/ESMP @ BG as 100%.

As shown in FIG. 4, Fe₃O₄the/CS/ESMP @ BG has recyclability. Using Fe₃O₄The catalyst can be reused by using the/CS/ESMP @ BG as the catalyst. After each reaction, the materials can be easily separated under the action of a magnetic field. After 10 cycles of use, the recovery rate of enzyme activity can still reach 91.4%. The immobilized enzyme has the greatest advantage of being reusable to improve the economy of the enzyme. Thus, Fe is used₃O₄The catalyst is/CS/ESMP @ BG, so that the production cost can be reduced, and the catalyst has a greater industrial prospect.

The determination method comprises the following steps: with Fe₃O₄pNPG (1mM) catalyzed by/CS/ESMP as template reaction to study Fe₃O₄Operational stability of/CS/ESMP @ BG. Mixing Fe₃O₄the/CS/ESMP @ BG was added to a buffer solution containing pNPG (1mM, pH 6) to react under the action of a magnetic field, and the immobilized enzyme was separated and collected and washed 3 times with the buffer solution. To ensure that no product marks are detected. Thereafter, the reaction was continued with an equal amount of a buffer containing pNPG (1mM, pH 6) and 10 batches were repeated in this manner, with the activity retention data based on 100% of the enzyme activity of the first reaction.

Example 4: fe₃O₄Method for synthesizing genipin by/CS/ESMP @ BG biocatalysis

Taking 1g of the immobilized enzyme prepared in the embodiment 2, adding the immobilized enzyme into a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 6, quickly adding 20mg of geniposide, stirring for 5 hours at the temperature of 50 ℃ to completely react, and taking out the immobilized enzyme to obtain a genipin crude product aqueous solution. The genipin crude product water solution generated under the optimal condition is adsorbed by a macroporous tree to be saturated by adopting a method for purifying genipin by using macroporous resin (HPD 700). After adsorption, absolute ethyl alcohol is used for elution, and the eluent is evaporated in a rotary mode to obtain a product. Extracting with diethyl ether repeatedly, rotating the extractive solution, recrystallizing with acetone, and drying the crystal at low temperature.

According to the experimental result of the invention, genipin is synthesized by immobilized enzyme catalysis, the catalysis time is slightly longer than that of free beta-glucosidase, but the catalysis yield can reach 92.6%, and the catalyst can be used for multiple times, thereby realizing maximization of production benefit

The above embodiments are only some embodiments of the present invention, and are not intended to limit the scope of the present invention; all equivalent changes and modifications made according to the present disclosure are intended to be covered by the scope of the claims of the present invention.