Enzyme immobilization carrier, immobilized enzyme as well as preparation method and application of immobilized enzyme

Abstract

The invention belongs to the field of biological catalysis, and particularly relates to an enzyme immobilization carrier, an immobilized enzyme and a preparation method and application of the enzyme immobilization carrier, the enzyme immobilization carrier and the immobilized enzyme, the enzyme immobilization carrier comprises aminated magnetic Fe-Ni nanoparticles, the enzyme immobilization carrier has good stability and is easy to separate and recycle, and the enzyme activity can still be kept at 90% or above after the enzyme immobilization carrier is recycled 12 times.

Description

Technical Field

[0001] The invention belongs to the field of biocatalysis, and more specifically, relates to an enzyme immobilization carrier, an immobilized enzyme, and a preparation method and application thereof. Background Art

[0002] Enzyme immobilization technology refers to the process of embedding free enzymes or binding and restricting them on the surface and micropores of a carrier, making them immobile, but still maintaining the integrity of the enzyme's space and activity center. It is a mainstream enzyme catalysis technology in enzyme engineering applications. Compared with free enzymes, immobilized enzymes are not only more stable, but also easy to separate from the reaction solution. They can be recycled multiple times for batch reactions or column loading for continuous reactions, providing the possibility for widespread application in industrial production. The performance of immobilized enzymes mainly depends on the immobilization method and the carrier material used, among which the performance of the carrier material can directly affect the catalytic activity of the immobilized enzyme.

[0003] The ideal carrier material should have good mechanical strength, thermal stability, chemical stability, biodegradability, high affinity for enzymes, and be able to maintain high enzyme activity. Common immobilization carriers in industry are divided into organic carrier materials, inorganic carrier materials and composite carrier materials. Organic carrier materials and inorganic carrier materials are the most common traditional immobilization materials at present. Organic carrier materials are generally natural polysaccharides, synthetic organic polymer compounds, etc., which have a wide range of applications and easy access to raw materials, but low mechanical strength and easy to cause reaction product pollution; inorganic carrier materials are generally mesoporous materials, non-metallic minerals, metal oxides and their salts, which have stable chemical properties, sustainable utilization, and low cost, but enzymes are easy to fall off from the system. Composite carrier materials are new carrier materials, such as magnetic polymer microspheres, magnetic mesoporous materials, magnetic nanomaterials, etc., which can have the excellent properties of both organic carrier materials and inorganic carrier materials, and can break through the limitation of immobilized enzymes on a single carrier, but their complicated preparation process is not conducive to industrial production.

[0004] CN201810038098.8 discloses an enzyme immobilization method using diatomaceous earth as a carrier and glutaraldehyde as a cross-linking agent. Although this method has low cost, it has the problem of difficult enzyme recovery. CN201710958924.6 discloses an embedding and solidification method through membrane filtration and modified starch solidification. It uses diatomaceous earth as a carrier, and also has the problem of difficult separation and recovery. CN202211356017.1 uses Fe3O4 / polymethyl methacrylate composite microspheres modified with grafted polyvinyl alcohol and thiol as immobilized enzyme carriers, and adsorbs the enzyme between the polyvinyl alcohol chains through the interaction between the carrier and the DL-pantoic acid splitting enzyme, and then uses the cross-linking agent's diepoxy group to react with the thiol modified on the polyvinyl alcohol chain, and encapsulates the DL-pantoic acid splitting enzyme on the surface of the magnetic microspheres, and the enzyme can be recovered by using an external magnetic field. Although this method solves the problem of difficult enzyme recovery, the preparation method of the immobilization method is complicated, which is not conducive to industrialization.

[0005] How to develop a carrier material that is stable, easy to recycle, and maintains high enzyme activity after recycling is one of the focuses of immobilized enzyme research. Summary of the invention

[0006] In order to solve the deficiencies in the prior art, the present invention provides an enzyme immobilization carrier, an immobilized enzyme, and a preparation method and application thereof. The enzyme immobilization carrier not only has good stability, but is also easy to separate and recycle. After being recycled 12 times, the enzyme activity can still be maintained at more than 90%.

[0007] In order to achieve the above object, the present invention provides an enzyme immobilization carrier in a first aspect, wherein the immobilized enzyme carrier comprises aminated magnetic Fe-Ni nanoparticles.

[0008] In order to obtain a better recycling and reuse effect, in the present invention, the particle size of the amino-containing magnetic Fe-Ni nanoparticles is preferably 10 to 60 nm, and further preferably, the particle size of the amino-containing magnetic Fe-Ni nanoparticles is 10 to 50 nm.

[0009] The second aspect of the present invention provides a method for preparing the above enzyme immobilization carrier, which comprises: chelating and adsorbing -NH2 groups on magnetic Fe-Ni nanoparticles through coordination to obtain amino magnetic Fe-Ni nanoparticles.

[0010] The third aspect of the present invention provides an immobilized enzyme, which comprises the above enzyme immobilization carrier and an enzyme covalently linked to the enzyme immobilization carrier.

[0011] The immobilized enzyme of the present invention is not strictly limited to the applicable enzyme.

[0012] According to the present invention, preferably, the mass ratio of the enzyme immobilization carrier to the enzyme is 1:0.05-0.3.

[0013] The fourth aspect of the present invention provides a method for preparing the above-mentioned immobilized enzyme, which comprises: reacting the above-mentioned enzyme immobilization carrier with an aqueous solution of a cross-linking agent, washing, vacuum drying, and then reacting with an enzyme solution to obtain the immobilized enzyme.

[0014] Preferably, the preparation method comprises: mixing the above enzyme immobilization carrier with an aqueous solution of a crosslinking agent, shaking at 25-35°C for 2-5h, washing, vacuum drying, mixing with an enzyme solution, and shaking at 25-35°C for 2-5h to obtain the immobilized enzyme.

[0015] In order to achieve a full reaction between the aminated magnetic Fe-Ni nanoparticles and the aqueous solution of the crosslinking agent, in order to obtain better stability and maintain the activity of the enzyme, preferably, the concentration of the aqueous solution of the crosslinking agent is 1% to 15%. In the present invention, any crosslinking agent that can be compatible with the aminated magnetic Fe-Ni nanoparticles can be applied to the present invention. Preferably, the crosslinking agent is at least one of glutaraldehyde, acetic anhydride, diglycidyl ethyl ether and methyl succinimidyl. Preferably, the mass ratio of the aminated magnetic Fe-Ni nanoparticles to the crosslinking agent is 1:5 to 1:10.

[0016] According to the present invention, preferably, the vacuum drying temperature is 50-70° C. and the time is 1-5 h.

[0017] The fifth aspect of the present invention provides the use of the above-mentioned immobilized enzyme in the preparation of D-pantolactone and NMN.

[0018] The immobilized enzyme technology provided by the present invention does not limit the type of enzyme, and it shows better performance in the preparation of D-pantolactone and NMN. The specific applications are as follows:

[0019] In the preparation of D-pantolactone, after DL-pantolactone hydrolase is immobilized on the aminated magnetic Fe-Ni nanoparticles, the resolution reaction of DL-pantolactone is preferably carried out at a reaction temperature of 20-45°C and a pH of 5-8.

[0020] In the preparation of NMN, nicotinamide riboside kinase NRK and polyphosphate kinase PPK are respectively fixed on amino-modified magnetic Fe-Ni nanoparticles, and then the NMN synthesis reaction is preferably carried out at a reaction temperature of 30 to 40°C and a pH of 4 to 7.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) The present invention uses amino-modified magnetic Fe-Ni nanoparticles as enzyme immobilization carriers, which have higher mechanical strength, larger specific surface area, higher enzyme loading capacity, and less magnetism and dispersibility than magnetic Fe3O4 nanoparticle carriers.

[0023] 2) The preparation method of the enzyme immobilization carrier of the present invention is simple, the raw materials for preparation are simple and easy to obtain, and it is easy to expand industrial production.

[0024] 3) The immobilized enzyme of the present invention has strong stability. After being recycled and reused for 12 times, the enzyme activity of the immobilized enzyme can still be maintained at more than 90%.

[0025] 4) Generally, biological enzymes fixed on immobilized carriers cannot be efficiently recovered by conventional centrifugation and filtration methods. Special equipment such as ultrafiltration machines and nanofiltration machines are required for recovery, and the operation steps are complicated and the recovery rate is low. However, the immobilized enzyme of the present invention can be separated and recovered from the reaction solution under the action of an external magnetic field, which not only avoids mechanical damage to the enzyme structure during the enzyme recovery process, but also has a simple recovery method and a high recovery rate.

[0026] Other features and advantages of the present invention will be described in detail in the following detailed description. DETAILED DESCRIPTION

[0027] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0028] The methods for determining various enzyme activities in the embodiments of the present invention are as follows:

[0029] 1) Method for determining the activity of immobilized D-pantolactone hydrolase:

[0030] To 1g of immobilized enzyme, add 50g of racemic D,L-pantolactone (prepared to 20g / L concentration with Tris-HCl buffer), and react at 28℃ and 200r / min for 60min. The immobilized enzyme is removed by an external magnetic field, and the amount of D-pantolactone produced is determined by HPLC analysis, and the enzyme activity units (U / g) per gram of immobilized enzyme are calculated.

[0031] 2) Method for determining the activity of immobilized nicotinamide riboside kinase NRK:

[0032] 1g immobilized enzyme, add 20g nicotinamide riboside chloride, 4.8g ATP, 8g magnesium chloride hexahydrate, 12g sodium hexametaphosphate, dilute to 200mL, shake at 38℃, 200r / min for 60min, remove the immobilized enzyme by external magnetic field, and determine the β-nicotinamide monohydrate by HPLC.Nucleotides The amount of enzyme produced was calculated and the enzyme activity units per gram of immobilized enzyme (U / g).

[0033] 3) The activity determination method of immobilized polyphosphate kinase PPK is as follows:

[0034] To 1g of immobilized enzyme, add 21.3g of adenosine diphosphate (ADP) and 30.5g of sodium hexametaphosphate, make the volume to 200ML, and react at 38℃ and 200r / min for 60min. Remove the immobilized enzyme by external magnetic field, determine the amount of adenosine triphosphate (ATP) generated by HPLC, and calculate the enzyme activity units (U / g) per gram of immobilized enzyme.

[0035] Example 1-Preparation of enzyme immobilization carrier-1

[0036] Weigh 1 mol of EDTA-2K salt and dissolve it in a beaker containing 5000 mL of deionized water, and stir for 10 min. Then, add 1 mol of magnetic Fe-Ni nanoparticles with a particle size of 10 nm to the beaker, stir for 15 min, filter, wash with water, and transfer to an oven, and bake at 80 ° C for about 24 h until dry to obtain enzyme immobilization carrier-1.

[0037] Example 2-Preparation of enzyme immobilization carrier-2

[0038] Weigh 1 mol of 3-aminopropyltriethoxysilane (APTES) and dissolve it in a beaker containing 5000 mL of deionized water / ethanol (volume ratio of 1 / 1), and stir for 10 minutes. Then, add 1 mol of magnetic Fe-Ni nanoparticles with a particle size of 30 nm to the beaker, stir and react for 7 hours, filter, wash with ethanol, wash with water, and transfer to an oven, bake at 80°C for about 24 hours, until dry, to obtain enzyme immobilization carrier-2.

[0039] Example 3 - Preparation of immobilized enzyme-1 (D-pantolactone hydrolase)

[0040] 1) 10 g of the enzyme immobilized carrier-1 prepared in Example 1 was mixed with 80 g of a 2% aqueous solution prepared with glutaraldehyde, and then shaken at room temperature of 25° C. for 2 h, and then subjected to magnetic separation and washing, and then vacuum dried at 60° C. for 2 h to obtain a cross-linked carrier.

[0041] 2) 900 mL of a 20 g / L solution of D-pantolactone hydrolase from Gibberella (initial enzyme activity of 54.1 U / g) was mixed with 60 g of the cross-linked carrier prepared in step 2), and the mixture was shaken at room temperature of 25° C. for 2 h. After separation and washing, immobilized enzyme-1 was obtained.

[0042] The immobilized enzyme-1 was detected to have a loading of 290 mg / g on the amino-magnetic Fe-Ni nanoparticle carrier, an enzyme activity of 53 U / g, and a single recovery rate of 98.4%. The enzyme activity of the immobilized enzyme-1 was measured to be 50.4 U / g and 53 U / g after being placed at 25°C for 24 hours and 4°C for 1 month, respectively.

[0043] Example 4-Preparation of immobilized enzyme-2 (D-pantolactone hydrolase)

[0044] 1) 10 g of the enzyme immobilized carrier-1 prepared in Example 1 was mixed with 50 g of glutaraldehyde in a 5% aqueous solution and shaken at room temperature of 25° C. for 2 h. After magnetic separation and washing, the mixture was vacuum dried at 60° C. for 1 h to obtain a cross-linked carrier.

[0045] 2) 100 mL of a 20 g / L solution of D-pantolactone hydrolase from Fusarium verticillioides (initial enzyme activity of 47.8 U / g) was added thereto and 20 g of the cross-linked carrier prepared in step 2) was added thereto, and the mixture was shaken at room temperature of 25° C. for 2 h. After separation and washing, immobilized enzyme-2 was obtained.

[0046] The immobilized enzyme-2 was detected to have a loading of 195 mg / g on the amino-magnetic Fe-Ni nanoparticle carrier, an enzyme activity of 46.9 U / g, and a single recovery rate of 98%. The enzyme activity of the immobilized enzyme-2 was measured to be 44.6 U / g and 46.9 U / g after being placed at 25°C for 24 hours and at 4°C for 1 month, respectively.

[0047] Example 5-Preparation of immobilized enzyme-3 (D-pantolactone hydrolase)

[0048] 1) 15 g of the enzyme immobilization carrier-1 prepared in Example 1 was mixed with 150 g of a 10% solution of acetic anhydride and shaken at room temperature of 25° C. for 2 h. After magnetic separation and washing, the mixture was vacuum dried at 60° C. for 5 h to obtain a cross-linked carrier.

[0049] 2) Take 250 mL of a 20 g / L solution of D-pantolactone hydrolase from Aspergillus (initial enzyme activity is 48.4 U / g) and add 100 g of the cross-linked carrier prepared in step 2) thereto, shake at room temperature of 25°C for 2 h, separate and wash to obtain immobilized enzyme-3.

[0050] The immobilized enzyme-1 was detected to have a loading capacity of 72 mg / g on the amino-magnetic Fe-Ni nanoparticle carrier, an enzymatic activity of 47.4 U / g, and a single recovery rate of enzymatic activity of 97.5%. The enzymatic activity of immobilized enzyme-3 was measured to be 46.2 U / g and 47.4 U / g respectively after being placed at 25°C for 24 hours and at 4°C for 1 month.

[0051] Example 6-Preparation of immobilized enzyme-4 (D-pantolactone hydrolase)

[0052] 1) 10 g of the enzyme immobilized carrier-1 prepared in Example 2 was mixed with 80 g of a 2% aqueous solution prepared with glutaraldehyde, and then shaken at room temperature of 25° C. for 2 h. After magnetic separation and washing, the mixture was vacuum dried at 60° C. for 2 h to obtain a cross-linked carrier.

[0053] 2) 900 mL of a 20 g / L solution of D-pantolactone hydrolase from Gibberella (initial enzyme activity of 54.1 U / g) was mixed with 60 g of the cross-linked carrier prepared in step 2), and the mixture was shaken at room temperature of 25° C. for 2 h. After separation and washing, immobilized enzyme-1 was obtained.

[0054] The immobilized enzyme-1 was detected to have a loading of 260 mg / g on the amino-magnetic Fe-Ni nanoparticle carrier, an enzyme activity of 52.5 U / g, and a single recovery rate of 98.4%. The enzyme activity of the immobilized enzyme-1 was measured to be 49.4 U / g and 52.5 U / g after being placed at 25°C for 24 hours and at 4°C for 1 month, respectively.

[0055] Example 7-Preparation of Immobilized Enzyme-5 (Nicotinamide Riboside Kinase NRK)

[0056] 1) 10 g of the enzyme immobilized carrier-2 prepared in Example 2 was mixed with 80 g of a 2% solution prepared with glutaraldehyde, and then shaken at room temperature of 25° C. for 2 h. After magnetic separation and washing, the mixture was vacuum dried at 60° C. for 5 h to obtain a cross-linked carrier.

[0057] 2) 900 mL of a 20 g / L solution of nicotinamide riboside kinase NRK from Escherichia coli (initial enzyme activity of 100.6 U / g) was mixed with 60 g of the cross-linked carrier prepared in step 2), and the mixture was shaken at room temperature of 25° C. for 2 h. After separation and washing, immobilized enzyme-5 was obtained.

[0058] The immobilized enzyme-5 was detected to have a loading capacity of 243 mg / g on the amino-magnetic Fe-Ni nanoparticle carrier, an activity of 100.4 U / g, and a single recovery rate of 97.5%. The enzyme activity of immobilized enzyme-1 was measured to be 97.5 U / g and 99.6 U / g after being placed at 25°C for 24 hours and 4°C for 1 month, respectively.

[0059] Example 8-Preparation of immobilized enzyme-6 (polyphosphate kinase PPK)

[0060] 1) 10 g of the enzyme immobilized carrier-2 prepared in Example 2 was mixed with 80 g of a 2% solution prepared with glutaraldehyde, and then shaken at room temperature of 25° C. for 2 h. After magnetic separation and washing, the mixture was vacuum dried at 60° C. for 5 h to obtain a cross-linked carrier.

[0061] 2) 900 mL of a 20 g / L solution of polyphosphate kinase PPK from Escherichia coli (initial enzyme activity of 80.4 U / g) was mixed with 60 g of the cross-linked carrier prepared in step 2), and the mixture was shaken at room temperature of 25° C. for 2 h. After separation and washing, immobilized enzyme-6 was obtained.

[0062] The immobilized enzyme-6 was detected to have a loading capacity of 262 mg / g on the amino-magnetic Fe-Ni nanoparticle carrier, an enzymatic activity of 79.2 U / g, and a single recovery rate of enzymatic activity of 97.1%. After the immobilized enzyme-6 was placed at 25°C for 24 hours and at 4°C for 1 month, its enzymatic activity was measured to be 77.2 U / g and 79.2 U / g, respectively.

[0063] Comparative Example 1 - Using aminated magnetic Fe3O4 nanoparticles as carrier

[0064] 1) Preparation of aminated magnetic Fe3O4 nanoparticles: Weigh 1 mol of EDTA-2K salt and dissolve it in a beaker containing 5000 mL of deionized water, and stir for 10 min. Then, add 1 mol of magnetic Fe3O4 nanoparticles with a particle size of 10 nm to the beaker, stir for 15 min, filter, wash with water, transfer to an oven, and dry at 80°C for 24 h to obtain aminated magnetic Fe3O4 nanoparticles.

[0065] 2) 10 g of the amino-modified magnetic Fe3O4 nanoparticles prepared in step 1) were mixed with 80 g of a 2% aqueous solution prepared with glutaraldehyde and shaken at room temperature of 25°C for 2 h. After magnetic separation and washing, the mixture was vacuum dried at 60°C for 2 h to obtain a cross-linked carrier.

[0066] 3) Compared with Example 3, 900 mL of a 20 g / L solution of D-pantolactone hydrolase from Gibberella (initial enzyme activity of 54.1 U / g) was mixed with 60 g of the cross-linked carrier prepared in step 2), and the mixture was shaken at room temperature of 25°C for 2 h. After separation and washing, the immobilized enzyme-7 was obtained.

[0067] The loading amount of immobilized enzyme-7 on the amino-modified magnetic Fe3O4 nanoparticle carrier was detected to be 250 mg / g, the enzyme activity of the immobilized enzyme was 52.6 U / g, and the single recovery rate of the immobilized enzyme was 97.3%.

[0068] After the immobilized enzyme-7 was placed at 25°C for 24 hours and at 4°C for 1 month, its enzyme activity was measured to be 46.5 U / g and 47.8 U / g, respectively.

[0069] Comparative Example 2 - Direct use of glutaraldehyde to fix the enzyme

[0070] Compared with Example 3, 900 mL of a 20 g / L solution of D-pantolactone hydrolase from Gibberella (initial enzyme activity of 54.1 U / g) was mixed with 80 g of a 2% aqueous solution prepared with glutaraldehyde, and stirred evenly. The mixture was stirred for 5 minutes every hour, kept at 4°C for 4 hours, and finally washed with water, pressed, and dried to obtain immobilized enzyme-8.

[0071] The enzyme activity of immobilized enzyme-8 was detected to be 48.71U / g, and the single recovery rate of immobilized enzyme was 82%. After the immobilized enzyme-8 was placed at 25°C for 24 hours and at 4°C for 1 month, its enzyme activity was measured to be 39.45U / g and 43.37U / g respectively.

[0072] Test Example 1-DL-pantolactone resolution efficiency test

[0073] The immobilized enzymes prepared in Examples 3-5 and Comparative Examples 1-2 were applied to the resolution of DL-pantolactone, and the specific steps were as follows:

[0074] 1) Take 6 three-necked flasks, add DL-pantolactone respectively, control the temperature at 28-30° C., and then add the immobilized enzymes prepared in Examples 3-5 and Comparative Examples 1-2 respectively. In each reaction, the mass ratio of DL-pantolactone to immobilized enzyme is 4:1, and water is added until the content of DL-pantolactone reaches 25-28%. During this process, the pH value is maintained between 6.8 and 7.2.

[0075] 2) After the mixture was reacted at 30°C for 30 hours, it was sampled and centrifuged, and the supernatant was taken and the amount of D-pantolactone produced was determined by HPLC.

[0076] 3) After the reaction is completed, the immobilized enzyme is adsorbed in a three-necked flask by an external magnetic field for recovery. The specific steps are: pour out the reaction solution, wash the immobilized enzyme remaining in the beaker with a small amount of deionized water, and pour out the washing water.

[0077] 4) Using the recovered immobilized enzyme, repeat steps 1), 2), and 3). Since a small amount of immobilized enzyme is lost during the recovery process, when it is reused, fresh immobilized enzyme can be added to the reaction system according to the loss amount.

[0078] The immobilized enzyme was reused 12 times, and the yields of D-pantolactone in the first and twelfth times are shown in Table 1 below.

[0079] Table 1

[0080]

[0081]

[0082] Test Example 2-Synthetic NMN Efficiency Test

[0083] The immobilized enzyme prepared in Example 7 was applied to the synthesis of NMN, and the specific steps were as follows:

[0084] 1) Take a three-necked flask, add nicotinamide riboside chloride, magnesium chloride hexahydrate, and ATP, control the temperature at 37-38°C, and then add the immobilized enzyme. Each reaction is added according to the mass ratio of nicotinamide riboside chloride to immobilized enzyme of 5:1, and add water until the content of nicotinamide riboside chloride reaches 60g / L. During this process, keep the pH value between 4.8 and 5.2.

[0085] 2) After the mixture was reacted at 37°C for 8 hours, it was sampled and centrifuged, and the supernatant was used to determine the β-nicotinamide monohydrate by HPLC. Nucleosides acid Amount generated.

[0086] 3) After the reaction is completed, the immobilized enzyme is adsorbed in a three-necked flask by an external magnetic field for recovery. The specific steps are: pour out the reaction solution, wash the immobilized enzyme remaining in the beaker with a small amount of deionized water, and pour out the washing water.

[0087] 4) Using the recovered immobilized enzyme, repeat steps 1), 2), and 3). Since a small amount of immobilized enzyme is lost during the recovery process, fresh immobilized enzyme can be added to the reaction system according to the loss when it is reused.

[0088] The immobilized enzyme was reused 12 times, and the NMN production rates for the 1st and 12th times are shown in Table 2.

[0089] Table 2

[0090] category NMN production rate per reaction NMN production rate after 12 reactions Example 7 95.2％ 93.8％

[0091] Test Example 3- ATP synthesis efficiency test

[0092] The immobilized enzyme prepared in Example 8 was applied to the synthesis of ATP, and the specific steps were as follows:

[0093] 1) Take a three-necked flask, add adenosine diphosphate (ADP) and sodium hexametaphosphate, control the temperature at 35-38°C, and then add the immobilized enzyme. Each reaction is carried out at a mass ratio of adenosine diphosphate (ADP) to immobilized enzyme of 5:1, and add water until the content of adenosine diphosphate (ADP) reaches 30g / L. During this process, keep the pH value between 4.0 and 5.0.

[0094] 2) After the mixture was reacted at 36°C for 4 hours, it was sampled and centrifuged, and the supernatant was taken to determine the amount of adenosine triphosphate (ATP) generated by HPLC.

[0095] 3) After the reaction is completed, the immobilized enzyme is adsorbed in a three-necked flask by an external magnetic field for recovery. The specific steps are: pour out the reaction solution, wash the immobilized enzyme remaining in the beaker with a small amount of deionized water, and pour out the washing water.

[0096] 4) Using the recovered immobilized enzyme, repeat steps 1), 2), and 3). Since a small amount of immobilized enzyme is lost during the recovery process, fresh immobilized enzyme can be added to the reaction system according to the loss when it is reused.

[0097] The immobilized enzyme was reused 12 times, and the ATP generation rates for the first and 12th times are shown in Table 3 below.

[0098] Table 3

[0099] category ATP generation rate per reaction ATP generation rate after 12 reactions Example 8 99.1％ 98.2％

[0100] Test Example 4-Stability Test

[0101] The enzyme activities of the immobilized enzymes in the above Examples 3-8 and Comparative Examples 1-2 after the first and 12 consecutive reactions were detected, as shown in Table 4 below:

[0102] Table 4

[0103] category Residual enzyme activity after 1 reaction Residual enzyme activity after 12 reactions Example 3 99.5％ 97.6％ Example 4 98.7％ 96.5％ Example 5 98.2％ 95.2％ Example 6 98.1％ 94.8％ Example 7 98.6％ 94.7％ Example 8 99.1％ 95.6％ Comparative Example 1 98.4％ 88.4％ Comparative Example 2 98.3％ 80.7％

[0104] It can be seen from the above Examples 3-8 and Comparative Examples 1-2 that the activity of the immobilized enzyme prepared using the immobilized carrier of the present invention does not decrease significantly after being maintained in multiple continuous reactions. It can be seen that the immobilized carrier of the present invention can effectively immobilize the enzyme and stably maintain the activity of the enzyme in multiple continuous enzyme-catalyzed reactions.

[0105] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An enzyme immobilization carrier, characterized in that: The enzyme immobilization carrier comprises amino magnetic Fe-Ni nanoparticles.

2. The enzyme immobilization carrier according to claim 1, characterized in that: The particle size of the aminated magnetic Fe-Ni nanoparticles is 10 to 60 nm. Preferably, the particle size of the aminated magnetic Fe-Ni nanoparticles is 10 to 50 nm.

3. A method for preparing the enzyme immobilization carrier according to claim 1 or 2, characterized in that: The preparation method comprises: chelating and adsorbing -NH2 groups on magnetic Fe-Ni nanoparticles through coordination to obtain aminated magnetic Fe-Ni nanoparticles.

4. An immobilized enzyme, characterized in that The immobilized enzyme comprises the enzyme immobilization carrier according to claim 1 or 2 and an enzyme covalently linked to the enzyme immobilization carrier.

5. The immobilized enzyme according to claim 4, characterized in that The mass ratio of the enzyme immobilization carrier to the enzyme is 1:0.05-0.

3.

6. A method for preparing the immobilized enzyme according to claim 4 or 5, characterized in that: The preparation method comprises: The enzyme immobilization carrier is reacted with an aqueous solution of a cross-linking agent, washed, vacuum dried, and then reacted with an enzyme solution to obtain the immobilized enzyme.

7. The preparation method according to claim 6, characterized in that: The preparation method comprises: mixing the enzyme immobilization carrier with an aqueous solution of a crosslinking agent, shaking at 25-35°C for 2-5h, washing, vacuum drying, mixing with an enzyme solution, shaking at 25-35°C for 2-5h, and obtaining the immobilized enzyme.

8. The preparation method according to claim 6, characterized in that: The mass ratio of the enzyme immobilization carrier to the cross-linking agent is 1:5 to 1:10; Preferably, the vacuum drying temperature is 50-70°C and the time is 1-5h; Preferably, the cross-linking agent is at least one of glutaraldehyde, acetic anhydride, diglycidyl ethyl ether and methyl succinimidyl; Preferably, the concentration of the aqueous solution of the cross-linking agent is 1% to 15%.

9. Use of the immobilized enzyme according to claim 4 or 5 in the preparation of D-pantolactone and NMN.

10. The use according to claim 9, characterized in that: When the immobilized enzyme is used to prepare D-pantolactone, the reaction temperature is 20-40° C. and the pH is 5-8; When the immobilized enzyme is used to prepare NMN, the reaction temperature is 30-45° C. and the pH is 4-7.

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