Amino-modified diatomite-based method for immobilizing d-allulose-3-epimerase

Abstract

An amino-modified diatomite-based method for immobilizing a D-allulose-3-epimerase, relating to the technical field of enzyme engineering. The method for immobilizing a D-allulose-3-epimerase specifically comprises using amino-modified diatomite to immobilize the D-allulose-3-epimerase. In addition, an immobilized D-allulose-3-epimerase prepared by using the method has enzyme activity which can reach 1168.98 U / g, high recovery of 64.94%, and a protein immobilization rate of 52.96%.

Description

A method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth Technical Field

[0001] The invention relates to a method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth, belonging to the technical field of enzyme engineering. Background Art

[0002] D-psicose, a key member of the rare sugar family, is a novel low-energy sweetener. Due to its high sweetness and low energy content, D-psicose is considered an ideal sweetener and an effective alternative to sucrose, useful in the development of low-calorie foods and beverages. Furthermore, D-psicose can undergo the Maillard reaction, which helps improve food quality. Furthermore, D-psicose exhibits multiple physiological functions and holds great potential for application. For example, D-psicose can inhibit intestinal α-glucosidase activity, preventing elevated blood sugar; it can reduce lipase activity, lowering blood lipids; and it also has neuroprotective effects. However, D-psicose is extremely scarce and difficult to extract naturally. Therefore, it is necessary to utilize biotechnology to produce D-psicose to meet growing market demand.

[0003] D-psicose-3-epimerase catalyzes the C3 epimerization of various ketose sugars and is an excellent catalyst for producing rare sugars. It can produce high-value-added D-psicose using D-fructose as a substrate. However, in industrial production, free D-psicose-3-epimerase exhibits significantly reduced activity, poor stability, non-reusability, and difficulty separating it from the product. These drawbacks significantly limit its practical application.

[0004] Therefore, the development of immobilization technology for D-psicose-3-epimerase can increase enzyme stability while allowing the enzyme to be used repeatedly, thereby enabling continuous production of D-psicose while reducing costs, which is of great significance to the industrial development of allulose.

[0005] Currently, D-psicose-3-epimerase is mostly immobilized using anion exchange resin as a carrier, but the resulting immobilized enzyme typically exhibits low enzymatic activity and low production efficiency. Metal-organic frameworks and titanium dioxide have also been used to immobilize D-psicose-3-epimerase. These biocatalysts offer good operational stability, but these materials are relatively expensive. In recent years, existing enzyme immobilization technologies have mostly used sodium alginate as an immobilization carrier. However, sodium alginate is unstable in highly concentrated electrolyte solutions and easily softens, making it unsuitable for the industrial application of D-psicose-3-epimerase.

[0006] The chemical composition of diatomaceous earth is mainly silicon dioxide, which can be used as a catalyst carrier. It has the porous and hollow structure of diatom cone fossils, and has high porosity, low cost, high reusability and chemical stability. The surface of diatomaceous earth has hydroxyl functional groups, which mainly play an adsorption role in the adsorption process. In order to further increase the adsorption performance and stability of diatomaceous earth, diatomaceous earth is usually modified to improve its adsorption performance. For example, Chinese patent CN111440785A discloses a method for immobilizing cells containing glucose isomerase on modified diatomaceous earth, which is obtained by modifying natural diatomaceous earth to immobilize glucose isomerase wet bacteria; however, the modified diatomaceous earth is used to fix the wet bacteria, and there are still problems such as low immobilization efficiency and low enzyme activity of the immobilized enzyme.

[0007] Therefore, there is an urgent need to find a simple, efficient, low-cost, and mechanically stable immobilization method suitable for the industrial production of D-psicose-3-epimerase.

[0008] Summary of the Invention

[0009] In response to the defects and shortcomings of the existing technology, the present invention provides a method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth. This method enables the immobilized D-psicose-3-epimerase to have high enzymatic activity and stability, and can be reused multiple times, thereby reducing the cost of enzymatic production of D-psicose.

[0010] The object of the present invention is to provide a method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth, the method comprising the following steps:

[0011] (1) Diatomite modification

[0012] The modified diatomaceous earth is obtained by adding natural diatomaceous earth to a H2SO4 solution with a concentration of 1 to 5 mol / L, stirring for a certain period of time, and then ultrasonically treating the solution. The modified diatomaceous earth is then washed with deionized water until neutral, filtered, dried, and sieved.

[0013] (2) Preparation of amino-modified diatomaceous earth carrier

[0014] The modified diatomaceous earth obtained in step (1) is placed in a solvent, and an amination reagent is added, shaken for reaction, and washed after the reaction is completed to obtain an amination-modified diatomaceous earth carrier;

[0015] (3) Immobilization of D-psicose-3-epimerase

[0016] D-psicose-3-epimerase is dissolved in a Tris-HCl buffer solution to form an enzyme solution; a cross-linking agent is first added to the enzyme solution to carry out a cross-linking reaction to form an enzyme solution adduct; and then the amino-modified diatomaceous earth carrier prepared in step (2) is added to carry out an immobilization reaction. After the reaction is completed, the solution is washed and dried to obtain a finished immobilized enzyme.

[0017] In one embodiment, the material-liquid ratio of the diatomaceous earth to the H2SO4 solution in step (1) is 1:10-30 g / mL.

[0018] In one embodiment, the stirring time in step (1) is 4 to 6 hours.

[0019] In one embodiment, the solvent in step (2) comprises any one of an aqueous solution, anhydrous ethanol, an acid solution and an alkaline solution.

[0020] In one embodiment, the material-liquid ratio of the modified diatomaceous earth to the solvent in step (2) is 1:10-100 g / mL.

[0021] In one embodiment, the amination reagent in step (2) includes any one of 3-aminopropyltriethoxysilane, 3-aminopropyl(diethoxy)methylsilane, chitosan, polyethylenediamine, polydiethylenetriamine, polyethanolamine, and polyethyleneimine.

[0022] In one embodiment, in step (2), when the amination reagent is 3-aminopropyltriethoxysilane, the selected solvent is anhydrous ethanol; when the amination reagent is chitosan, the selected solvent is a weak acid solution; when the amination reagent is polyethyleneimine, the selected solvent is an alkaline solution.

[0023] In one embodiment, the mass concentration of the amination reagent added in step (2) is 1 to 15%.

[0024] In one embodiment, the oscillation reaction conditions in step (2) are: 15-50° C., 100-400 r constant temperature oscillation for 1-72 h.

[0025] In one embodiment, the D-psicose-3-epimerase in step (3) is derived from: recombinant bacteria producing D-psicose-3-epimerase are inoculated into LB liquid culture medium and cultured at 37°C and 200 rpm for 12 hours to obtain a primary seed solution; the seed solution is inoculated into a secondary seed culture medium at a volume concentration of 3% inoculation amount, and cultured at 37°C and 200 rpm for 12 hours to obtain a secondary seed solution; the secondary seed solution is inoculated into a fermentation medium at an inoculum concentration of 3% inoculation amount, and cultured at 37°C and 200 rpm for 12 hours. The fermentation broth is centrifuged to collect wet cells, resuspend the cells in 50 mM Tris-HCl buffer solution (pH = 7.5), and then treated with 20 mg / mL lysozyme solution to break the enzyme. The supernatant of the centrifugation is filtered through a membrane to obtain a crude enzyme solution, and the crude enzyme solution is freeze-dried to obtain enzyme powder.

[0026] In one embodiment, the cross-linking agent in step (3) is any one of dialdehyde starch, polyethylene glycol diglycidyl ether, genipin, disuccinimidyl suberate, and glutaraldehyde.

[0027] In one embodiment, the volume concentration of the cross-linking agent in the enzyme solution system in step (3) is 0.01-1% (V / V), and the cross-linking reaction time is 0.1-24 hours.

[0028] In one embodiment, the D-psicose-3-epimerase in step (3) is added to the reaction system in the form of an enzyme solution, and the concentration of the enzyme in the enzyme solution is 1 to 40 mg / ml; the ratio of the amino-modified diatomaceous earth carrier to the enzyme in the reaction system is: 1 to 3 g of the amino-modified diatomaceous earth carrier is added for every 10 to 100 mg of enzyme.

[0029] In one embodiment, the fixed reaction conditions of step (3) are: 4-30° C., 100-300 r constant temperature oscillation for 1-48 h.

[0030] In one embodiment, step (2) and step (3) may further include:

[0031] Step (2) Preparation of Aminated Modified Diatomaceous Earth Carrier

[0032] The modified diatomaceous earth prepared in step (1) is dissolved in an anhydrous ethanol solution, and an amination reagent is added and shaken at a constant temperature; then a cross-linking agent solution is added and the reaction is shaken at a constant temperature; after taking out the sample, it is washed with deionized water 3 to 5 times to obtain an amino-modified diatomaceous earth carrier;

[0033] Step (3) Immobilization of D-psicose-3-epimerase

[0034] D-psicose-3-epimerase was dissolved in a Tris-HCl buffer solution to form an enzyme solution, and then the amino-treated diatomaceous earth carrier obtained in step (2) was added and the immobilization reaction was carried out under constant temperature oscillation. After the reaction was completed, the solution was washed with deionized water several times and dried naturally to obtain the immobilized enzyme product.

[0035] In one embodiment, the amination agent is polyethyleneimine.

[0036] The second object of the present invention is to provide an immobilized D-psicose-3-epimerase prepared by the above method.

[0037] The third object of the present invention is to provide an application of the above-mentioned method in preparing immobilized D-psicose-3-epimerase.

[0038] A fourth object of the present invention is to provide a method for improving the enzymatic activity of immobilized D-psicose-3-epimerase, comprising the following steps:

[0039] (1) Diatomite modification

[0040] The modified diatomaceous earth is obtained by adding natural diatomaceous earth to a H2SO4 solution with a concentration of 1 to 5 mol / L, stirring for a certain period of time, and then ultrasonically treating the solution. The modified diatomaceous earth is then washed with deionized water until neutral, filtered, dried, and sieved.

[0041] (2) Preparation of amino-modified diatomaceous earth carrier

[0042] The modified diatomaceous earth obtained in step (1) is placed in a solvent, and an amination reagent is added, shaken for reaction, and washed after the reaction is completed to obtain an amination-modified diatomaceous earth carrier;

[0043] (3) Immobilization of D-psicose-3-epimerase

[0044] D-psicose-3-epimerase is dissolved in a Tris-HCl buffer solution to form an enzyme solution; a cross-linking agent is first added to the enzyme solution to carry out a cross-linking reaction to form an enzyme solution adduct; and then the amino-modified diatomaceous earth carrier prepared in step (2) is added to carry out an immobilization reaction. After the reaction is completed, the solution is washed and dried to obtain a finished immobilized enzyme.

[0045] Beneficial effects of the present invention:

[0046] The method for immobilizing D-psicose-3-epimerase provided by the present invention is simple, efficient, economical, and practical. The immobilized D-psicose-3-epimerase prepared using this method has an enzymatic activity of up to 1168.98 U / g, a high recovery rate of 64.94%, and a protein immobilization rate of 52.96%, demonstrating great potential for industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG1 is a physical diagram of Comparative Example 2. DETAILED DESCRIPTION

[0048] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0049] The culture medium formula of the present invention is:

[0050] LB liquid medium (g / L): peptone 15 g / L, glucose 10 g / L, yeast extract 20 g / L, sodium chloride 10 g / L, K2HPO4·12H2O 1 g / L, MgSO4·7H2O 1 g / L, solvent: deionized water, pH = 7.

[0051] Fermentation medium (g / L): peptone 15 g / L, glucose 15 g / L, yeast extract 20 g / L, sodium chloride 10 g / L, K2HPO4·12H2O 1 g / L, MgSO4·7H2O 1 g / L, solvent is deionized water, pH = 7.

[0052] The recombinant strain Bacillus subtilis 1A751(dal-) / pUB-P43-dpe-dal producing D-psicose-3-epimerase was prepared according to the method described in HE W, MU W, JIANG B, et al. Construction of a food grade recombinant Bacillus subtilis based on replicative plasmids with an auxotrophic marker for biotransformation of D-Fructose to D-Allulose[J]. J Agric Food Chem, 2016, 64(16):3243-3250.) and obtained from the fermentation broth through activation culture, seed culture, and fermentation culture.

[0053] The present invention relates to a determination method:

[0054] 1. Determination of enzyme activity of immobilized enzyme

[0055] (1) A 100 g / L fructose solution was prepared using a phosphate buffer solution having a pH of 7.5 and a 50 mM Na2HPO4-NaH2PO4 solution;

[0056] (2) Add 50 mg of immobilized enzyme to 5 mL of 100 g / L fructose solution. After incubation in a 55°C water bath for 10 min, the immobilized enzyme was separated from the fructose solution to terminate the reaction. The sample was centrifuged (12,000 rpm, 5 min), and the supernatant was filtered through a 0.22 μm aqueous microporous filter membrane and analyzed by high performance liquid chromatography (HPLC) using a differential detector for D-psicose.

[0057] Enzyme activity definition: Under the above conditions, the amount of enzyme required to produce 1 μmol of D-psicose in 1 min is one enzyme activity unit.

[0058] 2. Determination of enzyme activity recovery rate

[0059] The enzyme activity recovery rate refers to the percentage of the total activity of the immobilized enzyme to the total activity of the enzyme solution used for immobilization.

[0060] Enzyme activity recovery rate (%) = (total activity of immobilized enzyme / total enzyme activity used for immobilization) * 100%

[0061] 3. Determination of protein immobilization efficiency

[0062] The protein immobilization rate refers to the percentage of enzyme protein content immobilized on the immobilized enzyme to the initial enzyme protein content used for immobilization. The enzyme protein content is determined and calculated using the BCA protein assay kit.

[0063] Protein immobilization rate (%) = ((total enzyme protein content for immobilization - enzyme protein content in supernatant - enzyme protein content in washing liquid) / total enzyme protein content for immobilization) * 100%

[0064] Example 1

[0065] The preparation of crude D-psicose-3-epimerase enzyme solution specifically comprises the following steps:

[0066] The recombinant bacteria producing D-psicose-3-epimerase were inoculated into LB liquid medium and cultured at 37°C and 200 rpm for 12 hours to obtain a primary seed solution; the seed solution was inoculated into a secondary seed medium at a volume concentration of 3% and cultured at 37°C and 200 rpm for 12 hours to obtain a secondary seed solution; the secondary seed solution was inoculated into a fermentation medium at a volume concentration of 3% and cultured at 37°C and 200 rpm for 12 hours. The fermentation broth was centrifuged (8000 rpm, 15 minutes, 4°C), the wet cells were collected, the cells were resuspended in 50 mM Tris-HCl buffer solution (pH = 7.5), and the enzyme was broken by treatment with 20 mg / mL lysozyme solution. The supernatant was filtered through a 0.45 μm aqueous microporous filter membrane to obtain a crude enzyme solution, which was freeze-dried to obtain freeze-dried enzyme powder of D-psicose-3-epimerase.

[0067] After testing, the enzyme activity of the freeze-dried enzyme powder of D-psicose-3-epimerase was 36U / mg.

[0068] Example 2

[0069] A method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth, comprising the following steps:

[0070] (1) Modification of diatomite

[0071] 20 g of natural diatomaceous earth was added to 5 times the volume of 5 mol / L H2SO4 solution, stirred thoroughly for 4 h, ultrasonically treated at 50 ° C for 30 min, cooled to room temperature, and then washed with deionized water until neutral, then filtered, dried, and sieved to obtain modified diatomaceous earth;

[0072] (2) Preparation of amino-modified diatomaceous earth carrier

[0073] The modified diatomaceous earth obtained in step (1) was weighed and dissolved in anhydrous ethanol to a mass concentration of 4%, and 3-aminopropyltriethoxysilane was added to a final concentration of 4% (V / V). The mixture was shaken at 25°C and 200r for 24 hours. After the reaction, the sample was removed and washed three times with deionized water to obtain an amino-modified diatomaceous earth carrier.

[0074] (3) Immobilization of D-psicose-3-epimerase

[0075] 100 mg of the freeze-dried enzyme powder of D-psicose-3-epimerase prepared in Example 1 was weighed and dissolved in 50 mmol / L Tris-HCl buffer solution (pH = 7.5) to form an enzyme solution (the amount of D-psicose-3-epimerase added was 3600 U); a glutaraldehyde solution with a final concentration of 0.05% (V / V) was first added to the enzyme solution, and the solution was allowed to stand for cross-linking for 20 minutes to form an enzyme solution adduct; then the solution was added to the amino-modified diatomaceous earth support obtained in step (2), and the immobilization reaction was carried out at 25°C and 200r constant temperature for 4 hours. After the reaction, the solution was washed with deionized water several times and then dried naturally to obtain the immobilized enzyme product.

[0076] Comparative Example 1

[0077] The only difference from Example 2 is that the modified diatomaceous earth in step (2) is replaced by natural diatomaceous earth, and the other parameters and conditions are the same as those in Example 2.

[0078] The properties of the immobilized enzyme products prepared in Example 2 and Comparative Example 1 are shown in Table 1:

[0079] Table 1. Properties of immobilized enzyme products

[0080] Example 3

[0081] A method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth, comprising the following steps:

[0082] (1) Modification of diatomite

[0083] Same as step (1) in Example 2;

[0084] (2) Preparation of amino-modified diatomaceous earth carrier

[0085] Chitosan was dissolved in 1% (v / v) acetic acid to a mass concentration of 1% (m / v), and magnetically stirred at 60°C for 1 hour to form a chitosan colloidal solution; then the modified diatomaceous earth prepared in step (1) was weighed and immersed in the prepared chitosan colloidal solution to a mass concentration of 4%, and then stirred at a constant temperature of 25°C for 24 hours. After taking out the sample, it was washed with deionized water for 3 to 5 times to obtain an amino-modified diatomaceous earth carrier;

[0086] (3) Immobilization of D-psicose-3-epimerase

[0087] 100 mg of the freeze-dried enzyme powder of D-psicose-3-epimerase prepared in Example 1 was weighed and dissolved in 50 mmol / L Tris-HCl buffer solution (pH = 7.5) to form an enzyme solution (the amount of D-psicose-3-epimerase added was 3600 U); a glutaraldehyde solution with a final concentration of 0.05% (V / V) was first added to the enzyme solution, and the solution was allowed to stand for cross-linking for 20 minutes to form an enzyme solution adduct; then the solution was added to the amino-modified diatomaceous earth carrier obtained in step (2), and the immobilization reaction was carried out at 25°C and 200r constant temperature for 4 hours. After the reaction, the solution was washed with deionized water several times and then dried naturally to obtain the immobilized enzyme product.

[0088] The performance of the immobilized enzyme in this example is as follows: the enzyme activity of the immobilized enzyme is 187.22 U / g, the enzyme activity recovery rate is 10.40%, and the protein immobilization rate is 21.76%.

[0089] Example 4

[0090] A method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth, comprising the following steps:

[0091] (1) Modification of diatomite

[0092] Same as step (1) in Example 2;

[0093] (2) Preparation of amino-modified diatomaceous earth carrier

[0094] The modified diatomaceous earth prepared in step (1) was weighed and dissolved in a 100 mmol / L NaHCO3 solution to a mass concentration of 4%. A polyethyleneimine solution with a final concentration of 3.6% (v / v) was added and the mixture was shaken at 200 rpm at 25°C for 24 h. After the sample was removed, it was washed with deionized water for 3 to 5 times to obtain an amino-modified diatomaceous earth carrier.

[0095] (3) Immobilization of D-psicose-3-epimerase

[0096] 100 mg of the freeze-dried enzyme powder of D-psicose-3-epimerase prepared in Example 1 was weighed and dissolved in 50 mmol / L Tris-HCl buffer solution (pH = 7.5) to form an enzyme solution (the amount of D-psicose-3-epimerase added was 3600 U); a glutaraldehyde solution with a final concentration of 0.05% (V / V) was first added to the enzyme solution, and the solution was allowed to stand for cross-linking for 20 minutes to form an enzyme solution adduct; then the solution was added to the amino-modified diatomaceous earth carrier obtained in step (2), and the immobilization reaction was carried out at 25°C and 200r constant temperature for 4 hours. After the reaction, the solution was washed with deionized water several times and then dried naturally to obtain the immobilized enzyme product.

[0097] The performance of the immobilized enzyme in this example is as follows: the enzyme activity of the immobilized enzyme is 1168.98 U / g, the enzyme activity recovery rate is 64.94%, and the protein immobilization rate is 52.96%.

[0098] Example 5

[0099] A method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth, comprising the following steps:

[0100] (1) Modification of diatomite

[0101] Same as step (1) in Example 2;

[0102] (2) Preparation of amino-modified diatomaceous earth carrier

[0103] The modified diatomaceous earth prepared in step (1) was weighed and dissolved in an anhydrous ethanol solution to a mass concentration of 4%, and a polyethyleneimine solution with a final concentration of 3.6% (v / v) was added, and the mixture was shaken at a constant temperature of 25°C and 200r for 12h; then a glutaraldehyde solution with a final concentration of 1.2% (v / v) was added, and the mixture was shaken at a constant temperature of 25°C and 200r for 12h; after removing the sample, the sample was washed with deionized water for 3 to 5 times to obtain an amino-modified diatomaceous earth carrier;

[0104] (3) Immobilization of D-psicose-3-epimerase

[0105] 100 mg of the freeze-dried enzyme powder of D-psicose-3-epimerase prepared in Example 1 was weighed and dissolved in 50 mmol / L Tris-HCl buffer solution (pH = 7.5) to form an enzyme solution (the added enzyme amount of D-psicose-3-epimerase was 3600 U), and then added to the amino-treated diatomaceous earth carrier obtained in step (2). The immobilization reaction was carried out at 25°C and 200r constant temperature shaking for 4 h. After the reaction was completed, the mixture was washed with deionized water several times and naturally dried to obtain the immobilized enzyme product.

[0106] The performance of the immobilized enzyme in this example is as follows: the enzyme activity of the immobilized enzyme is 945.08 U / g, the enzyme activity recovery rate is 52.50%, and the protein immobilization rate is 28.17%.

[0107] Comparative Example 2

[0108] The only difference from Example 5 is that the anhydrous ethanol in step (2) is replaced by a 100 mmol / L NaHCO3 solution or aqueous solution; other parameters and conditions are the same as those in Example 5.

[0109] The results showed that polyethyleneimine and glutaraldehyde cross-linked into clusters in aqueous solution, resulting in the inability of polyethyleneimine to be effectively grafted onto the surface of diatomite, and the preparation of amino-modified diatomite failed (as shown in Figure 1).

[0110] Comparative Example 3

[0111] A method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth, comprising the following steps:

[0112] (1) Modification of diatomite

[0113] Same as step (1) in Example 2;

[0114] (2) Immobilization of D-psicose-3-epimerase

[0115] 100 mg of the freeze-dried enzyme powder of D-psicose-3-epimerase prepared in Example 1 was weighed and dissolved in 50 mmol / L Tris-HCl buffer solution (pH = 7.5) to form an enzyme solution (the amount of D-psicose-3-epimerase added was 3600 U); the modified diatomaceous earth prepared in step (1) was added to the enzyme solution to a mass concentration of 4%, adsorbed and shaken for 1 hour, and then a polyethyleneimine solution with a final concentration of 3.6% (v / v) was added and shaken at a constant temperature of 25°C and 200r for 2 hours; then a glutaraldehyde solution with a final concentration of 0.05% (v / v) was added, and the mixture was shaken at a constant temperature of 25°C and 200r for 1 hour. After the reaction was completed, the mixture was washed with deionized water several times and naturally dried to obtain the immobilized enzyme product.

[0116] The properties of the immobilized enzyme prepared in this comparative example are as follows: the enzyme activity of the immobilized enzyme is 63.34 U / g, and the enzyme activity recovery rate is 3.52%.

Claims

1. A method for immobilizing D-psicose-3-epimerase based on amino-modified diatomaceous earth, characterized in that: The method comprises the following steps: (1) Diatomite modification The natural diatomaceous earth is added to a H2SO4 solution with a concentration of 1 to 5 mol / L, and then ultrasonically treated after stirring for a certain period of time, and then washed with deionized water until neutral, filtered, dried, and sieved to obtain modified diatomaceous earth; (2) Preparation of amino-modified diatomaceous earth carrier The modified diatomaceous earth obtained in step (1) is placed in a solvent, and an amination reagent is added, and the mixture is shaken for reaction, and washed after the reaction is completed, thereby obtaining an amination-modified diatomaceous earth carrier; (3) Immobilization of D-psicose-3-epimerase The D-psicose-3-epimerase is dissolved in a Tris-HCl buffer solution to form an enzyme solution; a cross-linking agent is first added to the enzyme solution to perform a cross-linking reaction to form an enzyme solution adduct; and then the amino-modified diatomaceous earth carrier prepared in step (2) is added to perform an immobilization reaction. After the reaction is completed, the carrier is washed and dried to obtain an immobilized enzyme product.

2. The method according to claim 1, characterized in that The solvent in step (2) includes any one of an aqueous solution, anhydrous ethanol, an acid solution and an alkaline solution.

3. The method according to claim 1, characterized in that The amination reagent in step (2) includes any one of 3-aminopropyltriethoxysilane, 3-aminopropyl(diethoxy)methylsilane, chitosan, polyethylenediamine, polydiethylenetriamine, polyethanolamine, and polyethyleneimine.

4. The method according to claim 1, characterized in that: The oscillation reaction conditions of step (2) are: 15-50° C., 100-400 r constant temperature oscillation for 1-72 h.

5. The method according to claim 1, characterized in that The cross-linking agent in step (3) is any one of dialdehyde starch, polyethylene glycol diglycidyl ether, genipin, disuccinimidyl suberate, and glutaraldehyde.

6. The method according to claim 1, characterized in that In step (3), the D-psicose-3-epimerase is added to the reaction system in the form of an enzyme solution, wherein the concentration of the enzyme in the enzyme solution is 1 to 40 mg / ml; and the ratio of the amount of the amino-modified diatomaceous earth carrier to the enzyme in the reaction system is: 1 to 3 g of the amino-modified diatomaceous earth carrier is added for every 10 to 100 mg of the enzyme.

7. The method according to claim 1, characterized in that Step (2) and step (3) may also be: Step (2) Preparation of Aminated Modified Diatomaceous Earth Carrier The modified diatomite prepared in step (1) is placed in an anhydrous ethanol solution, and an amination reagent is added to the solution, followed by constant temperature shaking; then a cross-linking agent solution is added to the solution, followed by constant temperature shaking reaction; after taking out the sample, the sample is washed with deionized water to obtain an amination-modified diatomite carrier; Step (3) Immobilization of D-psicose-3-epimerase Dissolve D-psicose-3-epimerase in Tris-HCl buffer solution to form an enzyme solution, and then add the enzyme solution in step (2) The obtained amino diatomaceous earth carrier is subjected to constant temperature oscillation for immobilization reaction. After the reaction is completed, it is washed with deionized water for multiple times and dried naturally to obtain the immobilized enzyme product.

8. Immobilized D-psicose-3-epimerase prepared by the method according to any one of claims 1 to 7.

9. Use of the method according to any one of claims 1 to 7 in the preparation of immobilized D-psicose-3-epimerase.

10. A method for improving the activity of immobilized D-psicose-3-epimerase, characterized in that: The method comprises the following steps: (1) Diatomite modification The natural diatomaceous earth is added to a H2SO4 solution with a concentration of 1 to 5 mol / L, and then ultrasonically treated after stirring for a certain period of time, and then washed with deionized water until neutral, filtered, dried, and sieved to obtain modified diatomaceous earth; (2) Preparation of amino-modified diatomaceous earth carrier The modified diatomaceous earth obtained in step (1) is placed in a solvent, and an amination reagent is added, and the mixture is shaken for reaction, and washed after the reaction is completed, thereby obtaining an amination-modified diatomaceous earth carrier; (3) Immobilization of D-psicose-3-epimerase The D-psicose-3-epimerase is dissolved in a Tris-HCl buffer solution to form an enzyme solution; a cross-linking agent is first added to the enzyme solution to carry out a cross-linking reaction to form an enzyme solution adduct; and then the amino-modified diatomaceous earth carrier prepared in step (2) is added to carry out an immobilization reaction. After the reaction is completed, the carrier is washed and dried to obtain an immobilized enzyme product.

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