Preparation of heat-resistant magnetic chitosan immobilized enzyme

Abstract

The application discloses a heat-resistant magnetic chitosan immobilized enzyme, and relates to the technical field of immobilized enzymes. The immobilized enzyme carrier takes chitosan and sodium alginate as raw materials, utilizes the method of solubility under the freezing condition of an alkaline solution and hydrogen bond rearrangement, and prepares chitosan composite hydrogel which is excellent in mechanical properties and resistant to high temperature, and further prepares the immobilized heat-resistant enzyme by using a chemical crosslinking method. Based on this, the immobilized enzyme preparation method established in the present application is helpful to solve the problems that industrial enzymes are prone to inactivation under high-temperature conditions and are difficult to be continuously used.

Description

Technical Field

[0001] This invention relates to the technical field of immobilized enzymes, and more specifically to the preparation of a heat-resistant magnetic chitosan immobilized enzyme. Background Technology

[0002] Enzyme immobilization refers to a technique that uses physical adsorption or chemical bonding to fix enzymes onto a carrier or confine them within a specific space, thereby improving enzyme utilization through continuous or repeated use. Common immobilization methods include encapsulation, adsorption, covalent bonding, and cross-linking. Compared to free enzymes, immobilized enzymes have advantages such as high thermal stability, low cost, ease of control, and strong tolerance to denaturants. They can be used repeatedly or continuously and are easily separated from products, making them a research hotspot in the food, pharmaceutical, and chemical industries.

[0003] However, most current immobilized enzyme materials are suitable for immobilization reactions at room temperature. They struggle to maintain high enzyme activity at temperatures above 70°C, or begin to dissociate during the reaction, eventually leading to inactivation. For example, resins and sodium alginate, while exhibiting good biocompatibility and stability at room temperature, are often not heat-resistant and have limited mechanical strength, making them unsuitable for directly immobilizing heat-resistant enzymes. On the other hand, some carriers with high mechanical strength, such as nano-iron or glass, have poor biocompatibility and low operational stability. Chinese patent CN105695442A discloses a modified magnetic chitosan microsphere for enzyme immobilization. This patent uses emulsion polymerization to prepare the immobilized enzyme carrier, but still relies on acid dissolution of chitosan, thus failing to address the carrier's poor heat resistance and exhibiting drawbacks such as low mechanical strength and poor heat resistance. Chinese patent CN113652415A discloses chitosan iron oxide nanoparticles for enzyme immobilization. This patent uses a co-precipitation cross-linking method to prepare the immobilized enzyme carrier. However, as magnetic nanoparticles, iron oxide nanoparticles have limited industrial applicability. For substrates with slight viscosity, the immobilized enzyme may be unrecoverable. Therefore, it is of great significance to prepare an immobilized enzyme that can undergo continuous immobilization reactions at high temperatures and maintain high enzyme activity. Summary of the Invention

[0004] To address the drawbacks of enzyme inactivation and difficulty in continuous use under high industrial temperatures, this invention provides a method for preparing chitosan-immobilized enzymes. The method offers advantages such as good biocompatibility, high mechanical strength, and heat resistance, allowing for continuous use at high temperatures. Furthermore, the addition of magnetic Fe3O4 particles facilitates rapid separation of the enzyme from the substrate and enables repeated or continuous use of the enzyme. After eight repetitions at 80°C, the relative enzyme activity remains above 75%.

[0005] To achieve the above objectives, the present invention provides a method for preparing a heat-resistant magnetic chitosan-immobilized enzyme, the steps of which include:

[0006] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in water, then adjust the pH of the reaction system with an alkaline solution, heat and stir, filter, wash the filter residue, dry the filter residue, and obtain Fe3O4 magnetic nanoparticles.

[0007] S2. Add sodium alginate to water, let it swell, then heat and stir until clear and transparent to obtain sodium alginate solution;

[0008] S3. Add alkali and chitosan to the above sodium alginate solution, stir evenly to obtain a gel, freeze, take out, thaw, add Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0009] S4. The above mixture is dripped into a metal salt solution and solidified to obtain magnetic chitosan gel beads. The magnetic chitosan gel beads are recovered, washed, and stored in water.

[0010] S5. Weigh clean magnetic chitosan gel beads and add them to the EGDE solution. React to obtain chitosan composite beads. Remove the chitosan composite beads and remove the residual EGDE.

[0011] S6. Dilute the amylase solution with phosphate buffer solution, add the chitosan complex beads washed in step S5 to the diluted amylase solution, remove the chitosan complex beads after the reaction, and wash them thoroughly with phosphate buffer solution until no enzyme protein is washed out, thus obtaining heat-resistant magnetic chitosan immobilized enzyme.

[0012] Furthermore, Fe in step S1 3+ and Fe 2+ The molar ratio is 2:1.

[0013] Furthermore, the heating and stirring described in step S1 involves a heating temperature of 70°C and a stirring time of 30 minutes.

[0014] Furthermore, in step S1, the alkaline solution used to adjust the pH of the reaction system is an ammonia solution; the final pH is adjusted to 9.

[0015] Furthermore, in step S2, the mass ratio of sodium alginate to water is (0.03–0.06):8.4.

[0016] Furthermore, the base mentioned in step S3 is KOH and urea.

[0017] Furthermore, in step S3, the mass ratio of KOH to urea is 1:(0.4-0.8).

[0018] Furthermore, in step S3, the mass ratio of KOH to chitosan powder is 1:(0.4-0.6).

[0019] Furthermore, in step S3, the mass ratio of KOH to Fe3O4 magnetic nanoparticles is 1:(0.2-0.4).

[0020] Furthermore, the curing reaction temperature in step S4 is 60°C, and the curing reaction time is 1–4 hours.

[0021] Furthermore, the metal salt solution mentioned in step S4 is one or more of copper sulfate aqueous solution, magnesium sulfate aqueous solution, and zinc sulfate aqueous solution; the concentration of the metal salt solution is 10-40 g / L.

[0022] Furthermore, in step S4, the magnetic chitosan gel beads are recycled using an external magnetic field.

[0023] Furthermore, the mass-to-volume ratio of the clean magnetic chitosan gel beads and the EGDE solution in step S5 is (0.8–1.2 g):(2–4 mL).

[0024] Furthermore, the mass fraction of the EGDE solution in step S5 is 0.5% to 3%.

[0025] Furthermore, the reaction conditions described in step S5 are as follows: using a shaker, reacting for 1 hour at a temperature of 25°C and a rotation speed of 200 rpm.

[0026] Furthermore, the amylase solution mentioned in step S6 is a heat-resistant α-amylase solution.

[0027] Furthermore, the concentration of the phosphate buffer solution in step S6 is 0.2 mol / L and the pH is 6; the concentration of the diluted amylase solution is 1 mg / mL.

[0028] Furthermore, the reaction conditions described in step S6 are as follows: using a shaker, reacting for 12 hours at a temperature of 25°C and a rotation speed of 200 rpm.

[0029] This invention also provides a chitosan-immobilized enzyme prepared by the above method. This chitosan-immobilized enzyme exhibits good biocompatibility, as well as certain mechanical strength and heat resistance, allowing for continuous use at high temperatures. Furthermore, the addition of magnetic Fe3O4 particles facilitates rapid separation of the enzyme from the substrate and also enables repeated or continuous use of the enzyme.

[0030] Furthermore, the chitosan immobilized enzyme prepared by this invention has applications in the food, pharmaceutical, and chemical industries.

[0031] The beneficial effects achieved by this invention are as follows:

[0032] (1) The present invention utilizes the method of dissolution and hydrogen bond rearrangement under alkaline solution freezing conditions to prepare chitosan composite hydrogel with excellent mechanical properties and high temperature resistance.

[0033] (2) The addition of magnetic Fe3O4 particles in this invention facilitates the rapid separation of enzyme and substrate and also makes it easier for enzyme to be used repeatedly or continuously.

[0034] (3) By introducing polyanionic sodium alginate, the two are mixed and attracted to each other by charge to form a gel. Strong charge adsorption can not only improve the mechanical properties of the blended gel membrane, but also make the blended gel membrane retain the ion exchange properties of sodium alginate and improve the overall thermal stability.

[0035] (4) The present invention soaks chitosan hydrogel in a metal ion solution, and utilizes the metal ions to form metal coordination bonds with the amino groups in chitosan, so that the chitosan molecular chain segments are more tightly bound, thereby improving the mechanical properties of the hydrogel. Attached Figure Description

[0036] Figure 1 This is a scanning electron microscope image (magnification 3000x) of the chitosan-immobilized enzyme obtained in Example 2 of the present invention.

[0037] Figure 2 Thermogravimetric analysis (TGA) diagrams of chitosan immobilized enzyme and sodium alginate obtained in Examples 2 and 8 of this invention are shown.

[0038] Figure 3 Repeated reaction experiments were conducted on the chitosan immobilized enzyme obtained in Example 2 of this invention.

[0039] Figure 4 This is a comparison of the chitosan immobilized enzyme and the free enzyme obtained in Example 2 of the present invention at different pH values.

[0040] Figure 5 This is a comparison of the chitosan immobilized enzyme and the free enzyme obtained in Example 2 of the present invention at different temperatures. Detailed Implementation

[0041] Materials and Instruments

[0042] α-Amylase (3500 U / mL) was purchased from Henan Zanyun Food Co., Ltd.; chitosan powder (BR, degree of deacetylation 90%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; sodium alginate (AR, 90%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; ethylene glycol diglycidyl ether (EGDE) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; other biochemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0043] The following instruments were purchased: a reciprocating water bath constant temperature incubator shaker (from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.); a DF-101S thermal collector-type constant temperature magnetic stirrer (from Gongyi Yuhua Instrument Co., Ltd.); a UV-1200 ultraviolet-visible spectrophotometer (from Shanghai Meipuda Instrument Co., Ltd.); a Fourier transform infrared spectrometer (from Nicolet Instruments, Inc., USA); a thermogravimetric analyzer (from Mettler Toledo, Switzerland); and a differential scanning calorimeter (from TA Instruments, Inc., USA).

[0044] I. Preparation process of chitosan immobilized enzyme in a specific embodiment

[0045] Example 1

[0046] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0047] S2. Add 0.03g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0048] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0049] S4. Use a 5mL syringe to draw the above mixture and drop it into a 20g / L ZnSO4 solution at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0050] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0051] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0052] Example 2

[0053] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0054] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0055] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0056] S4. Use a 5mL syringe to draw the above mixture and drop it into a 20g / L ZnSO4 solution at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0057] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0058] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0059] Example 3

[0060] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0061] S2. Add 0.06g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0062] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0063] S4. Use a 5mL syringe to draw the above mixture and drop it into a 20g / L ZnSO4 solution at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0064] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0065] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0066] Example 4

[0067] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0068] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0069] S3. Add 1g KOH, 0.6g urea and 0.4g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0070] S4. Use a 5mL syringe to draw the above mixture and drop it into a 20g / L ZnSO4 solution at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0071] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0072] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0073] Example 5

[0074] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0075] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0076] S3. Add 1g KOH, 0.6g urea and 0.6g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0077] S4. Use a 5mL syringe to draw the above mixture and drop it into a 20g / L ZnSO4 solution at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0078] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0079] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0080] Example 6

[0081] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0082] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0083] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0084] S4. Use a 5mL syringe to draw the above mixture and drop it into a 20g / L CuSO4 solution at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0085] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0086] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0087] Example 7

[0088] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0089] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0090] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0091] S4. Use a 5mL syringe to draw the above mixture and drop it into a 20g / L MgSO4 solution at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0092] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0093] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0094] Example 8

[0095] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0096] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0097] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0098] S4. Use a 5mL syringe to draw the above mixture and drop it into pure water at 60℃. Continue to solidify for 1 hour to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0099] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0100] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0101] Example 9

[0102] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0103] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0104] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0105] S4. Use a 5mL syringe to draw the above mixture and drop it into 10g / L ZnSO4 at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0106] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0107] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0108] Example 10

[0109] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0110] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0111] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0112] S4. Use a 5mL syringe to draw the above mixture and drop it into 40g / L ZnSO4 at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0113] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.8% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0114] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0115] Example 11

[0116] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0117] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0118] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0119] S4. Use a 5mL syringe to draw the above mixture and drop it into 40g / L ZnSO4 at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0120] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 0.6% EGDE solution. Place the solution in a shaker and react for 1h at a temperature of 25℃ and a rotation speed of 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0121] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0122] Example 12

[0123] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0124] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0125] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0126] S4. Use a 5mL syringe to draw the above mixture and drop it into 40g / L ZnSO4 at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0127] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 1% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0128] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0129] Example 13

[0130] S1. Dissolve anhydrous FeCl3 and FeSO4·7H2O in deionized water (Fe 3+ and Fe 2+ The reaction mixture was prepared by adding ammonia solution to adjust the pH of the reaction system to 9 (molar ratio 2:1), heating and stirring at 70°C for 30 min, filtering, washing the filter residue with deionized water, and then drying the filter residue in an oven to obtain magnetic nanoparticles Fe3O4.

[0131] S2. Add 0.05g of sodium alginate powder to 8.4g of deionized water, let it swell for 1 hour, then heat and stir until clear and transparent to obtain sodium alginate solution.

[0132] S3. Add 1g KOH, 0.6g urea and 0.5g chitosan powder to the above sodium alginate solution, stir evenly to obtain a gel, freeze in a -20℃ freezer overnight, take it out, thaw it, add 0.3g Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture.

[0133] S4. Use a 5mL syringe to draw the above mixture and drop it into 40g / L ZnSO4 at 60℃. Continue to solidify for 1h to obtain magnetic chitosan gel beads. Then, recover the magnetic chitosan gel beads by applying an external magnetic field, wash with deionized water, and store them in water.

[0134] S5. Weigh 0.1g of clean magnetic chitosan gel beads and add them to 3mL of 3% EGDE solution. Place the solution in a shaker and react for 1h at 25℃ and 200rpm to obtain chitosan composite beads. After removing the beads, rinse them repeatedly with deionized water to remove any remaining EGDE.

[0135] S6. Dilute the heat-resistant α-amylase (3500 U / mL) solution to 1 mg / mL amylase solution with 0.2 mol / L phosphate buffer solution (pH=6). Add the chitosan complex beads washed in step S5 to 3 mL of the prepared amylase solution, place in a shaker, and react for 12 h at 25 °C and 200 rpm. Then remove the chitosan complex beads and wash thoroughly with 0.2 mol / L phosphate buffer solution until no enzyme protein is washed out, thus obtaining the heat-resistant magnetic chitosan immobilized enzyme.

[0136] II. Determination of the enzyme activity of the chitosan-immobilized enzymes in Examples 1-12 above.

[0137] The absorbance was determined spectrophotometrically as follows: α-amylase hydrolyzed starch solution into glucose under specific temperature and pH conditions. Immediately after the reaction, 200 μL of the reaction solution was taken out, and 600 μL of DNS reagent was added to terminate the reaction. The solution was boiled for 5 min, cooled with cold water, and then diluted with water to 15 mL. The absorbance was measured at 540 nm. The specific steps are as follows.

[0138] 1. Phosphate buffer solution pH 6.5

[0139] Take 16.435g of sodium dihydrogen phosphate (NaH2PO4) and 8.943g of disodium hydrogen phosphate (Na2HPO4), dissolve them in water, and make up to 1000ml.

[0140] 2. DNS reagent

[0141] Solution A: Dissolve 6.9g of crystalline phenol in 15.2mL of 10% sodium hydroxide solution, and dilute with water to 69mL. Add 6.9g of sodium bisulfite to this solution.

[0142] Solution B: Weigh 255g of potassium sodium tartrate and add it to 300mL of 10% sodium hydroxide solution, then add 880mL of 1% 3,5-dinitrosalicylic acid solution.

[0143] Mixing solutions A and B yields a yellow reagent, which should be stored in a brown bottle for later use. Let it stand at room temperature for 7–10 days before use.

[0144] 3. Starch solution

[0145] Take 20g of soluble starch, add it to the above phosphate buffer solution to dissolve it, and make up to 1000ml.

[0146] 4. Measurement Procedure

[0147] Using 10 mL of 2% starch solution as a substrate, add 0.1 g of immobilized enzyme or an enzyme solution with equivalent activity, and incubate in a water bath at 80℃ and 300 rpm for 10 min. Immediately after the reaction, remove 200 μL of the reaction solution, add 600 μL of DNS reagent to terminate the reaction, boil for 5 min, cool with cold water, and then dilute with water to 15 mL. Measure the absorbance at 540 nm.

[0148] Blank experiment: Place 10 mL of 2% starch solution in a water bath at 80℃ and 300 rpm for 10 min. After the reaction, immediately remove 200 μL of the solution, add 600 μL of DNS reagent, boil for 5 min, cool with cold water, and then dilute with water to 15 mL. Measure the absorbance at 540 nm.

[0149] Enzyme activity is defined as the amount of glucose produced by 1g of immobilized enzyme in 1min.

[0150] Relative enzyme activity: Under certain conditions, the highest activity of an enzyme is defined as 100%, and the remaining percentage content corresponding to this is the relative enzyme activity.

[0151] The test results are shown in Table 1 below:

[0152] Table 1 Immobilized enzyme activity

[0153]

[0154]

[0155] As can be seen from Examples 1-13 above, the optimized combination obtained from the single-factor experiment is 5% chitosan by mass, 0.5% sodium alginate by mass, 20 g / L ZnSO4 by mass concentration, and 0.8% ethylene glycol diglycidyl ether by volume.

[0156] As can be seen from Examples 1-3 above, the activity of the immobilized enzyme first increases and then decreases with the addition of sodium alginate. This is because when we connect the carrier and the enzyme using the cross-linking agent EGDE, the cross-linking sites are provided by the amino groups of chitosan. Therefore, the addition of sodium alginate hinders the connection between the enzyme molecule and the carrier, or the active center of the enzyme molecule may be partially covered, preventing sufficient contact with the substrate, thus affecting the activity of the immobilized enzyme.

[0157] As can be seen from Examples 2, 4, and 5 above, the immobilized enzyme activity is highest when the chitosan mass fraction is 5%, while the immobilized enzyme activity decreases as the chitosan mass fraction continues to increase. When the mass fraction exceeds 5%, its viscosity increases, making it difficult to extrude into spheres, and the resulting gel spheres are too large, affecting the full binding of the enzyme to the substrate, leading to a decrease in enzyme activity.

[0158] As can be seen from Examples 2, 6, and 7 above, different metal ions have different effects on enzyme activity. When the metal ion is Zn... 2+ and Mg 2+ At that time, the enzyme activity increased relative to pure water, of which Mg 2+ The enzyme activity is highest when the metal ion is Cu. 2+ At this time, the enzyme activity is at its lowest. Because the alkaline system for dissolving chitosan contains KOH, when the mixture is added dropwise to a metal salt solution, the Zn in the solution... 2+ Mg 2+ and Cu 2+ It will adsorb onto the carrier in the form of Zn(OH)2, Mg(OH)2, and Cu(OH)2. While increasing the strength of the carrier, it will also change the microstructure of the carrier, thereby affecting the connection between the carrier and the enzyme, and thus affecting the activity of the immobilized enzyme.

[0159] As can be seen from Examples 2 and 8-10 above, the immobilized enzyme activity in Zn 2+ Within the concentration range, the activity first increases and then gradually decreases. The immobilized enzyme exhibits the highest relative activity and best immobilization effect when the ZnSO4 concentration is 20 g / L. When the ZnSO4 solution concentration is below 20 g / L, as the concentration increases, more ions are adsorbed onto the support, resulting in higher enzyme activity. However, the ZnSO4 solution also affects the degree of cross-linking; the higher the concentration, the denser the immobilized enzyme structure, leading to insufficient contact between the immobilized enzyme and the substrate, thus affecting the immobilized enzyme activity.

[0160] As can be seen from Examples 2 and 11-13 above, the immobilized enzyme activity first increases and then decreases with the concentration of the crosslinking agent. When the volume concentration of ethylene glycol diglycidyl ether is 0.8%, the immobilized enzyme exhibits the highest relative activity and the best immobilization effect. By replacing glutaraldehyde with EGDE, while retaining the excellent properties of the hydrogel material, this crosslinking agent has lower enzyme toxicity and a higher crosslinking effect than glutaraldehyde, which can further improve enzyme activity. This epoxy crosslinking agent can undergo crosslinking reactions with various groups in the chitosan molecule. Compared with the groups of traditional crosslinking agents (such as aldehyde groups), the epoxy groups have lower reactivity, relatively milder reaction conditions, and easily transform the chain structure of chitosan into a network structure.

[0161] III. Experiments were conducted on the repeated use of the chitosan immobilized enzyme obtained in Example 2 above.

[0162] An experiment was conducted on the reusability of the chitosan-immobilized enzyme obtained in Example 2 above, and the results are as follows: Figure 3 As shown, the immobilized enzyme obtained in Example 2 showed a decrease in enzyme activity after several repetitions, but its relative enzyme activity remained above 75% after 8 repetitions, indicating that the immobilized enzyme had a good reusability.

[0163] IV. Comparison of chitosan immobilized enzyme and free enzyme obtained in Example 2 of the present invention at different pH values.

[0164] The chitosan-immobilized enzyme and free enzyme obtained in Example 2 were subjected to different pH values ​​to determine their enzyme activities. The experimental results are shown in [Figure number missing]. Figure 4 .Depend on Figure 4 It can be seen that the optimal reaction pH for both free enzymes and immobilized enzymes is 6.5. Compared with the relative activity of free enzymes, the relative enzyme activity of immobilized enzymes does not change much. That is, immobilized enzymes can maintain relatively high enzyme activity within a pH range and have a wider adaptability than free enzymes.

[0165] V. Comparison of chitosan immobilized enzyme and free enzyme obtained in Example 2 of the present invention at different temperatures.

[0166] The activities of the chitosan-immobilized enzyme and the free enzyme obtained in Example 2 were measured at different temperatures. The results are shown in [Figure number missing]. Figure 5 Enzyme activity increases with increasing reaction temperature. The optimal temperature for immobilized enzymes is 95℃, which is 5℃ higher than that for free enzymes (90℃). Immobilized enzymes also exhibit good activity at reaction temperatures below the optimal temperature. This may be because the immobilization carrier improves the thermal stability of the enzyme's spatial structure.

[0167] VI. The chitosan immobilized enzymes obtained in Examples 2 and 8 were subjected to thermogravimetric analysis.

[0168] The TGA curve of the immobilized enzyme is as follows: Figure 2 As shown. All three materials exhibit a large weight loss region between 250 and 350°C; the lost substance should be water adsorbed by the copolymer hydrogel network. Sodium alginate has poor heat resistance, but due to its excellent gelling properties, especially in Ca... 2+ Under ionic conditions, the resulting cross-linking significantly improves gel strength. Compared to sodium alginate gel, the immobilized enzymes prepared in Examples 8 and 2 show improved thermal stability because chitosan is the only natural polycationic polysaccharide, while sodium alginate is a polyanion. When mixed, they attract each other through charge interactions, forming a gel. This strong charge adsorption not only improves the mechanical properties of the blended gel membrane but also allows the composite system to maintain the ion exchange capacity of sodium alginate and enhance overall thermal stability. Compared to Example 8, Example 2 shows further improved thermal stability and a smaller weight loss of only 39.27%. This is because metal ions can form metal-ligand coordination bonds with appropriate ligands under certain conditions. This is a non-covalent interaction with strong bonding. Chitosan's molecular chain is rich in free bases, which readily coordinate with transition metals and rare earth metal ions. The resulting chitosan-metal complex material provides excellent mechanical and heat resistance properties.

[0169] This invention is not limited to the specific embodiments described above. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any simple changes or modifications made to the design structure and concept of this invention fall within the protection scope of this invention.

Claims

1. A method for preparing a heat-resistant magnetic chitosan-immobilized enzyme, characterized in that, The steps include: S1. Dissolve FeCl3 and FeSO4·7H2O in water, adjust the pH of the reaction system with an alkaline solution, heat and stir, filter, wash the filter residue, and dry the filter residue to obtain Fe3O4 magnetic nanoparticles. S2. Add sodium alginate to water, let it swell, then heat and stir until clear and transparent to obtain a sodium alginate solution; the mass ratio of sodium alginate to water is 0.05:8.

4. S3. Add alkali and chitosan to the above sodium alginate solution, stir evenly to obtain a gel, freeze, remove, thaw, add Fe3O4 magnetic nanoparticles to the thawed gel, stir until uniform to obtain a mixture; the alkali is KOH and urea; S4. The above mixture is dripped into a metal salt solution and solidified to obtain magnetic chitosan gel beads. The magnetic chitosan gel beads are recovered, washed, and stored in water. The metal salt solution is one or more of magnesium sulfate aqueous solution and zinc sulfate aqueous solution. S5. Weigh the gel beads stored in water and add them to the EGDE solution. React to obtain chitosan composite beads. Remove the chitosan composite beads and remove the residual EGDE. S6. Dilute the amylase solution with phosphate buffer solution, add the chitosan complex beads washed in step S5 to the diluted amylase solution, react, then remove the chitosan complex beads and wash with phosphate buffer solution to obtain heat-resistant magnetic chitosan immobilized enzyme.

2. The preparation method according to claim 1, characterized in that, Fe in step S1 3+ and Fe 2+ The molar ratio is 2:1; the heating and stirring conditions in step S1 are a heating temperature of 70°C and a stirring time of 30 min; the alkaline solution used to adjust the pH of the reaction system in step S1 is an ammonia solution; the final pH is adjusted to 9.

3. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of KOH to urea is 1:0.4–0.8; the mass ratio of KOH to chitosan in step S3 is 1:0.4–0.6; and the mass ratio of KOH to Fe3O4 magnetic nanoparticles in step S3 is 1:0.2–0.

4.

4. The preparation method according to claim 1, characterized in that, The curing reaction temperature in step S4 is 60℃, and the curing reaction time is 1 to 4 hours; the concentration of the metal salt solution in step S4 is 10 to 40 g / L.

5. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the gel beads and EGDE solution preserved in water in step S5 is 0.8–1.2 g: 2–4 mL; the mass fraction of the EGDE solution in step S5 is 0.5–3%; the reaction conditions in step S5 are as follows: using a shaker, reacting at a temperature of 25°C and a rotation speed of 200 rpm for 1 h.

6. The preparation method according to claim 1, characterized in that, The amylase solution mentioned in step S6 is a heat-resistant α-amylase solution with an enzyme activity of 3500 U / mL; the phosphate buffer solution mentioned in step S6 has a concentration of 0.2 mol / L and a pH of 6; the concentration of the diluted amylase solution is 1 mg / mL.

7. The preparation method according to claim 1, characterized in that, The reaction conditions described in step S6 are as follows: using a shaker, reacting at a temperature of 25°C and a rotation speed of 200 rpm for 12 hours.

8. A heat-resistant magnetic chitosan immobilized enzyme prepared by the method according to any one of claims 1 to 7.

9. The application of the heat-resistant magnetic chitosan immobilized enzyme according to claim 8 in the food, pharmaceutical, and chemical industries.

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