Cage-shaped nanogel immobilized enzyme as well as preparation method and application thereof

Abstract

The invention discloses a cage-shaped nanogel immobilized enzyme as well as a preparation method and application thereof. The preparation method comprises the following steps: carrying out Schiff base reaction on transglutaminase and oxidized sodium alginate to prepare an OSA-TG conjugate; carrying out electrostatic self-assembly on the methacrylate / quaternized double modified dextrin and the OSA-TG conjugate, so as to prepare MQD / OSA-TG nanoparticles; the MQD / OSA-TG nano particles are subjected to a free radical polymerization reaction and a calcium ion cross-linking reaction, and MQD / OSA-TG double-cross-linked nano gel is prepared; and degrading the MQD / OSA-TG double-crosslinking nanogel by adopting a chelating agent and a pH (Potential of Hydrogen) regulator, so as to prepare the cage-shaped nanogel immobilized enzyme. The cage-shaped nanogel provided by the invention can protect the protein structure of the enzyme, maintain the enzyme activity and improve the enzyme stability, and meanwhile, the provided method has the advantages of simplicity in operation, safety, reliability and the like, and has huge industrial application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of immobilized enzymes, and particularly relates to a cage-like nanogel immobilized enzyme, a preparation method thereof and an application thereof. Background Art

[0002] Transglutaminase (TG, EC 2.3.2.13), as an efficient biocatalyst, can effectively catalyze the formation of isopeptide bonds between proteins, endow the product with proteolytic stability, provide an innovative approach for the green synthesis of amide bonds, and has irreplaceable application value in the food industry, pharmaceuticals and green chemical industries. However, the inherent limitations of natural enzymes (including low thermal stability, narrow optimal pH range and poor organic solvent tolerance) severely limit their application in high-temperature processing and extreme pH scenarios.

[0003] Enzyme immobilization technology confines enzyme molecules within a specific region through covalent bonds, physical adsorption or other methods, thereby significantly improving their stability while maintaining catalytic activity. Compared with planar materials (such as membrane materials, electrode surfaces), nanocarriers exhibit more prominent advantages due to their unique physicochemical properties. Nanocarrier-immobilized enzymes utilize their high surface curvature and surface area-to-volume ratio to reduce the contact area with enzymes, thereby effectively maintaining the native conformation and activity of enzymes. Currently, inorganic / organic nanocarriers, such as silica nanotubes, metal-organic frameworks (MOFs), gold nanoparticles, polymer nanoparticles (polymethyl methacrylate, polystyrene, polyacrylamide, etc.) and polysaccharide nanogels have been successfully used for enzyme immobilization, and they all show higher stability. However, the dense network formed by crosslinked nanocarriers may cause substrate mass transfer barriers, resulting in reduced accessibility of enzyme active sites, loss of enzyme kinetic properties and alteration of conformational integrity, thereby affecting catalytic efficiency. Summary of the Invention

[0004] The main objective of the present invention is to provide a cage-like nanogel immobilized enzyme, a preparation method thereof and an application thereof to overcome the deficiencies of the prior art.

[0005] To achieve the foregoing invention objective, the technical solutions adopted by the present invention include:

[0006] An embodiment of the present invention provides a preparation method of a cage-like nanogel immobilized enzyme, which includes:

[0007] Carrying out a Schiff base reaction between transglutaminase (TG) and oxidized sodium alginate (OSA) to obtain an OSA-TG conjugate;

[0008] Carrying out electrostatic self-assembly between methacrylate / quaternized double-modified dextrin (MQD) and the OSA-TG conjugate to obtain MQD / OSA-TG nanoparticles;

[0009] Subject the MQD / OSA-TG nanoparticles to free radical polymerization reaction and calcium ion crosslinking reaction to obtain MQD / OSA-TG double crosslinked nanogels;

[0010] In addition, degrade the MQD / OSA-TG double crosslinked nanogels using a chelating agent and a pH regulator to obtain cage-shaped nanogel immobilized enzymes.

[0011] An embodiment of the present invention also provides a cage-shaped nanogel immobilized enzyme prepared by the foregoing preparation method.

[0012] An embodiment of the present invention also provides an application of the foregoing cage-shaped nanogel immobilized enzyme in high-temperature processing or an extreme pH environment.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] (1) The present invention utilizes the dynamic reversible characteristics of Schiff base bonds to achieve the directional coupling of TG and OSA, and maintains the catalytic activity by regulating the conformational rigidity of enzyme molecules; a weak acidic environment response mechanism is introduced, so that the immobilized enzyme undergoes controllable bond cleavage under specific pH conditions, and the intelligent adaptation of enzyme-substrate is achieved through the dynamic exposure of active sites;

[0015] (2) Based on the sacrificial template strategy, the present invention selectively dissociates the OSA ionic crosslinked network through the chelation of EDTA-mediated Ca 2+ to construct a cage-shaped nanoconfinement system with a cavity structure, effectively balancing the contradiction between substrate mass transfer and enzyme immobilization;

[0016] (3) The cage-shaped nanogel immobilized enzyme prepared by the present invention significantly improves the stability of the enzyme at a wider pH and high temperature through the spatial confinement effect, enhanced conformational rigidity, and microenvironment buffering effect;

[0017] (4) The protein hydrogel formed by crosslinking the cage-shaped nanogel immobilized enzyme prepared by the present invention exhibits stronger mechanical strength and functional properties;

[0018] (5) The preparation method provided by the present invention has mild reaction conditions, a simple preparation method, and is easy to realize large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1are transmission electron microscope images of immobilized TG (free TG, OSA-TG, MQD / OSA-TG, single-crosslinked MQD / OSA-TG, double-crosslinked MQD / OSA-TG, and cage-like nanogel-immobilized TG) at each stage in Example 1 of the present invention;

[0021] Figures 2A - 2E are particle size and Zeta potential diagrams of immobilized TG at each stage in Example 1 of the present invention;

[0022] Figures 3A - 3C are enzymatic property diagrams of immobilized TG at each stage in Example 1 of the present invention;

[0023] Figures 4A - 4C are gel strength, fracture stress, and scanning electron microscope microstructural diagrams of crosslinked whey protein isolate hydrogels with immobilized TG at each stage in Example 1 of the present invention. Detailed implementation manners

[0024] In view of the deficiencies of the prior art, the inventors of this case have, through long-term research and a large amount of practice, been able to propose the technical solution of the present invention. The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0025] Specifically, as an aspect of the technical solution of the present invention, a preparation method of a cage-like nanogel-immobilized enzyme involved therein includes:

[0026] Performing a Schiff base reaction between transglutaminase (TG) and oxidized sodium alginate (OSA) to obtain an OSA-TG conjugate;

[0027] Performing electrostatic self-assembly between methacrylate / quaternized double-modified dextrin (MQD) and the OSA-TG conjugate to obtain MQD / OSA-TG nanoparticles;

[0028] Performing a radical polymerization reaction and a calcium ion crosslinking reaction on the MQD / OSA-TG nanoparticles to obtain double-crosslinked MQD / OSA-TG nanogels;

[0029] And degrading the double-crosslinked MQD / OSA-TG nanogels using a chelating agent and a pH regulator to obtain cage-like nanogel-immobilized enzymes.

[0030] In some preferred embodiments, the preparation method specifically includes: dissolving transglutaminase and oxidized sodium alginate in water and mixing them, and then adjusting the pH of the obtained mixed solution to weakly alkaline with an alkaline solution and carrying out a Schiff base reaction at 25-55 °C for 12-24 h to obtain an OSA-TG conjugate.

[0031] Further, the mass ratio of transglutaminase to oxidized sodium alginate is 1:0.2-1.

[0032] Further, the oxidation degree of the oxidized sodium alginate is 10-60%.

[0033] Further, the pH value of the mixed solution is 7-8.

[0034] In some preferred embodiments, the preparation method specifically includes: under the condition of magnetic stirring, dropping methacrylate / quaternized double-modified dextrin into the solution of OSA-TG conjugate for self-assembly to obtain MQD / OSA-TG nanoparticles.

[0035] Further, the mass ratio of transglutaminase to methacrylate / quaternized double-modified dextrin is 1:0.2-1.2.

[0036] Further, the methacrylate substitution degree of the methacrylate / quaternized double-modified dextrin is 0-50%.

[0037] Further, the quaternization degree of the methacrylate / quaternized double-modified dextrin is 0-50%.

[0038] Further, the rotation speed of the magnetic stirring is 200-800 rpm, and the stirring time is 1-4 h.

[0039] In some preferred embodiments, the preparation method specifically includes: adding N,N'-methylenebisacrylamide and potassium persulfate solution to the solution of OSA-TG conjugate to carry out a free radical polymerization reaction, and then adding CaCl2 solution for calcium ion cross-linking reaction to obtain MQD / OSA-TG double-crosslinked nanogels.

[0040] Further, the concentration of N,N'-methylenebisacrylamide is 0.5-5 mg / mL.

[0041] Further, the concentration of the potassium persulfate solution is 0.5-5 mg / mL.

[0042] Further, the temperature of the free radical polymerization reaction is 25-50 °C, and the time is 12-24 h.

[0043] Further, the concentration of the CaCl2 solution is 0.1-1 mmol / L.

[0044] Furthermore, the temperature of the calcium ion cross-linking reaction is room temperature, and the time is 1 to 4 h.

[0045] In some preferred embodiments, the preparation method specifically includes: adding a chelating agent solution to the MQD / OSA-TG double-crosslinked nanogel, and adding a pH regulator to adjust the pH value to 5 to 7 and reacting for 1 to 4 h to obtain a cage-shaped nanogel immobilized enzyme.

[0046] Furthermore, the chelating agent solution includes an EDTA solution, and the concentration of the EDTA solution is 0.2 to 2.0 mmol / L.

[0047] In some more specific embodiments, the preparation method of the cage-shaped nanogel immobilized enzyme includes the following steps:

[0048] (1) Dissolve transglutaminase (TG) and oxidized sodium alginate (OSA) in deionized water respectively, mix them, adjust the pH to weakly alkaline with 1 mol / L NaOH, and carry out covalent binding at 25 °C to form an OSA-TG conjugate.

[0049] (2) Under magnetic stirring, drop methacrylate / quaternized double-modified dextrin (MQD) into the OSA-TG conjugate solution, and form MQD / OSA-TG nanoparticles through electrostatic self-assembly.

[0050] (3) Add N,N'-methylenebisacrylamide and potassium persulfate solution to the solution in step (2), covalently crosslink MQD, and then add CaCl2 solution to crosslink OSA at room temperature to form MQD / OSA-TG double-crosslinked nanogel.

[0051] (4) At room temperature, add an EDTA solution and adjust the pH to the solution in step (3) to degrade the OSA crosslinked network, and obtain a cage-shaped nanogel immobilized enzyme through purification and freeze-drying.

[0052] Preferably, in step (1), the mass ratio of transglutaminase to oxidized sodium alginate is 1:0.2 to 1; the oxidation degree of the oxidized sodium alginate is 10 to 60%; the reaction conditions include a pH range of 7 to 8 and a time of 12 to 24 h.

[0053] Preferably, in step (2), the mass ratio of transglutaminase to methacrylate / quaternized double-modified dextrin is 1:0.2 to 1.2; the methacrylate substitution degree of the double-modified dextrin is 0 to 50%; the quaternization degree of the double-modified dextrin is 0 to 50%; the magnetic stirring speed is 200 - 800 rpm; the stirring time is 1 - 4 h.

[0054] Preferably, in step (3), the concentration of N,N'-methylenebisacrylamide is 0.5-5 me / mL, and the concentration of the potassium persulfate solution is 0.5-5 mg / mL; the reaction conditions include a temperature of 25-50 °C and a time of 12-24 h.

[0055] Preferably, in step (3), the concentration of the CaCl2 solution is 0.1-1 mM; the reaction time is 1-4 h.

[0056] Preferably, in step (4), the concentration of EDTA is 0.2-2 mM; the reaction conditions include a pH range of 5-7 and a time of 1-4 h.

[0057] Another aspect of the embodiments of the present invention also provides a cage-shaped nanogel-immobilized enzyme prepared by the aforementioned preparation method.

[0058] The cage-shaped nanogel-immobilized enzyme in the present invention can protect the protein structure of the enzyme, maintain the enzyme activity, and improve the enzyme stability.

[0059] Another aspect of the embodiments of the present invention also provides the application of the aforementioned cage-shaped nanogel-immobilized enzyme in high-temperature processing or an environment with extreme pH values.

[0060] The technical solutions of the present invention will be further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. These embodiments are implemented on the premise of the technical solutions of the present invention, and detailed implementation manners and specific operation procedures are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0061] The experimental materials used in the following embodiments can be obtained from conventional biochemical reagent companies without special instructions.

[0062] Example 1

[0063] Prepare TG solution, 2 mg / mL OSA solution (oxidation degree 60%) and 4 mg / mL MQD solution (methacrylate substitution degree 30%, quaternization degree 30%) with a concentration of 4 mg / mL each. Mix TG and OSA in equal volumes and stir evenly. Adjust the pH of the solution to 7.5 with 1 mol / L NaOH, and let it stand at room temperature for 24 h to obtain OSA-TG solution. Under magnetic stirring at 600 rpm, add MQD dropwise to the OSA-TG solution and continue stirring for 3 h to obtain MQD / OSA-TG solution. Subsequently, add N,N′-methylenebisacrylamide (MBA, 20 mg / mL) and potassium persulfate solution (KPS, 50 mg / mL) in sequence, and supplement an appropriate amount of deionized water, where OSA-TG∶MQD∶MBA∶KPS∶H2O = 1∶0.25∶0.2∶0.03∶0.52 (v / v). React at 25 °C for 12 h to form a single-crosslinked nanogel, and then construct a double-crosslinked structure through 0.1 mM Ca 2+ ion crosslinking for 2 h; finally, add 0.2 mM EDTA and adjust the pH to 6 to degrade the OSA crosslinked network, and obtain cage-like nanogel-immobilized TG enzyme after purification and freeze-drying.

[0064] Characterize the structure and enzymatic properties of the obtained immobilized TG, Figure 1 which are the transmission electron microscope images of immobilized TG (free TG, OSA-TG, MQD / OSA-TG, single-crosslinked MQD / OSA-TG, double-crosslinked MQD / OSA-TG, and cage-like nanogel-immobilized TG) at each stage in Example 1 of this application; Figures 2A - 2E which are the particle sizes and Zeta potentials of immobilized TG at each stage in Example 1 of this application: ( Figure 2A ) The particle sizes and Zeta potentials of OSA-TG at different ratios of TG and OSA; ( Figure 2B ) The particle sizes and Zeta potentials of MQD / OSA-TG at different ratios of MQD and OSA-TG; ( Figure 2C ) The particle sizes of single-crosslinked MQD / OSA-TG at different MBA concentrations; ( Figure 2D ) The particle sizes of double-crosslinked MQD / OSA-TG at different CaCl2 concentrations; ( Figure 2E ) The particle sizes of cage-like nanogel-immobilized TG after degrading the OSA network with different concentrations of EDTA. Figures 3A - 3C which are the enzymatic properties of immobilized TG at each stage in Example 1 of this invention: ( Figure 3A ) The enzyme activities of immobilized TG at each stage; ( Figure 3B ) The relative enzyme activities of immobilized TG incubated at 65 °C for 6 h at each stage; ( Figure 3C ) The relative enzyme activities of immobilized TG incubated at pH 3 for 6 h at each stage. Figures 4A - 4CThese are the characteristics of the immobilized TG crosslinked whey protein isolate hydrogel at each stage in Example 1 of the present invention:( Figure 4A ) Gel strength;( Figure 4B ) Breaking stress;( Figure 4C ) Scanning electron microscope microstructure. The results show that the particle size of the immobilized enzyme is 201.2±2.4 nm; the Zeta potential is -29.3±1.2 mV; the enzyme immobilization rate is 91.5±2.6%; the enzyme activity is 48.7±2.7 U / g, which is 97.4% of the initial enzyme activity; after heat incubation at 65 °C for 6 h, 91.2% of the initial enzyme activity can still be retained, and free TG retains 12.1% of the initial enzyme activity under the same conditions; after incubation at pH 3 for 6 h, 89.5% of the initial enzyme activity can still be retained, and free TG retains 8.1% of the initial enzyme activity under the same conditions; after the immobilized enzyme is stored sealed at 4 °C for 60 days, the enzyme activity is maintained at 94.8% of the initial enzyme activity, while free TG can only maintain 83.1% of the initial enzyme activity. Compared with free TG (31.4±3.2 g, 3.1±0.4 kPa), the whey protein isolate hydrogel crosslinked with immobilized TG has stronger gel strength (582.1±4.6 g) and breaking stress (35.1±2.6 kPa).

[0065] Example 2

[0066] Prepare TG solution, 4 mg / mL OSA solution (oxidation degree 60%) and 4 mg / mL MQD solution (methacrylate substitution degree 30%, quaternization degree 30%) with a concentration of 4 mg / mL each. Mix TG and OSA in equal volumes and adjust the pH of the solution to 7.5 with 1 mol / L NaOH, and let it stand at room temperature for 24 h to obtain OSA-TG solution. Under magnetic stirring at 600 rpm, add MQD dropwise to the OSA-TG solution and continue stirring for 3 h to obtain MQD / OSA-TG solution. Subsequently, add N,N′-methylenebisacrylamide (MBA, 20 mg / mL) and potassium persulfate solution (KPS, 50 mg / mL) in sequence, and supplement an appropriate amount of deionized water, where OSA-TG∶MQD∶MBA∶KPS∶H2O = 1∶0.25∶0.2∶0.03∶0.52 (v / v). React at 25 °C for 12 h to form a single-crosslinked nanogel, and then construct a double-crosslinked structure through 0.5 mM Ca 2+ ion crosslinking for 2 h; finally, add 1 mM EDTA and adjust the pH to 6 to degrade the OSA crosslinked network, and obtain cage-like nanogel-immobilized TG enzyme after purification and freeze-drying.

[0067] The structure and enzymatic properties of the obtained immobilized TG were characterized. The results showed that the particle size of the immobilized enzyme was 253.4 ± 4.9 nm; the Zeta potential was -36.7 ± 2.1 mV; the enzyme immobilization rate was 92.5 ± 1.7%; the enzyme activity was 44.1 ± 1.2 U / g, which was 88.2% of the initial enzyme activity; after heat incubation at 65 °C for 6 h, 86.9% of the initial enzyme activity was still retained, while the free TG retained 12.1% of the initial enzyme activity under the same conditions; after incubation at pH 3 for 6 h, 86.1% of the initial enzyme activity was still retained, while the free TG retained 8.1% of the initial enzyme activity under the same conditions; after the immobilized enzyme was stored sealed at 4 °C for 60 days, the enzyme activity was maintained at 92.2% of the initial enzyme activity, while the free TG could only maintain 83.1% of the initial enzyme activity. Compared with the free TG (31.4 ± 3.2 g, 3.1 ± 0.4 kPa), the whey protein isolate hydrogel crosslinked with the immobilized TG had stronger gel strength (562.1 ± 12.3 g) and breaking stress (31.6 ± 1.9 kPa).

[0068] Example 3

[0069] Prepare TG solutions, 2 mg / mL OSA solutions (oxidation degree 30%), and 4 mg / mL MQD solutions (methacrylate substitution degree 30%, quaternization degree 30%) with concentrations all being 4 mg / mL. Mix TG and OSA in equal volumes and stir evenly, adjust the pH of the solution to 7.8 with 1 mol / L NaOH, and let it stand at room temperature for reaction for 24 h to obtain the OSA-TG solution. Under magnetic stirring at 800 rpm, add MQD dropwise to the OSA-TG solution and continuously stir for 4 h to obtain the MQD / OSA-TG solution. Subsequently, add N,N'-methylenebisacrylamide (MBA, 20 mg / mL) and potassium persulfate solution (KPS, 50 mg / mL) in sequence, and supplement an appropriate amount of deionized water, where OSA-TG∶MQD∶MBA∶KPS∶H2O = 1∶0.25∶0.2∶0.03∶0.52 (v / v). React at 25 °C for 12 h to form a single-crosslinked nanogel, and then construct a double-crosslinked structure through 0.25 mM Ca 2+ ion crosslinking for 2 h; finally, add 0.5 mM EDTA and adjust the pH to 5.5 to degrade the OSA crosslinked network, and obtain the cage-shaped nanogel-immobilized TG enzyme after purification and lyophilization.

[0070] The structure and enzymatic properties of the obtained immobilized TG were characterized. The results showed that the particle size of the immobilized enzyme was 172.6 ± 5.5 nm; the Zeta potential was -23.2 ± 1.9 mV; the enzyme immobilization rate was 85.1 ± 3.1%; the enzyme activity was 46.2 ± 1.5 U / g, which was 92.4% of the initial enzyme activity; after heat incubation at 65 °C for 6 h, 86.0% of the initial enzyme activity was still retained, while the free TG retained 12.1% of the initial enzyme activity under the same conditions; after incubation at pH 3 for 6 h, 87.2% of the initial enzyme activity was still retained, while the free TG retained 8.1% of the initial enzyme activity under the same conditions; after the immobilized enzyme was stored sealed at 4 °C for 60 days, the enzyme activity was maintained at 91.5% of the initial enzyme activity, while the free TG could only maintain 83.1% of the initial enzyme activity. Compared with the free TG (31.4 ± 3.2 g, 3.1 ± 0.4 kPa), the whey protein isolate hydrogel crosslinked with the immobilized TG had stronger gel strength (549.1 ± 7.7 g) and breaking stress (29.2 ± 1.3 kPa).

[0071] Example 4

[0072] Prepare TG solutions, 2 mg / mL OSA solutions (oxidation degree 60%) and 4 mg / mL MQD solutions (methacrylate substitution degree 30%, quaternization degree 30%) with concentrations of 4 mg / mL each. Mix TG and OSA in equal volumes and stir evenly, adjust the pH of the solution to 7.5 with 1 mol / L NaOH, and let it stand and react at room temperature for 24 h to obtain the OSA-TG solution. Under magnetic stirring at 600 rpm, add MQD dropwise to the OSA-TG solution and continue stirring for 3 h to obtain the MQD / OSA-TG solution. Subsequently, add N,N'-methylenebisacrylamide (MBA, 20 mg / mL) and potassium persulfate solution (KPS, 50 mg / mL) in sequence, and supplement an appropriate amount of deionized water, where OSA-TG∶MQD∶MBA∶KPS∶H2O = 1∶0.25∶0.4∶0.05∶0.3 (v / v). React at 25 °C for 12 h to form a single cross-linked nanogel, and then construct a double cross-linked structure by ionic cross-linking with 0.1 mM Ca 2+ ions for 2 h; finally, add 0.2 mM EDTA and adjust the pH to 6 to degrade the OSA cross-linked network, and obtain the cage-shaped nanogel-immobilized TG enzyme after purification and freeze-drying.

[0073] The structure and enzymatic properties of the obtained immobilized TG were characterized. The results showed that the particle size of the immobilized enzyme was 299.5 ± 11.2 nm; the Zeta potential was -28.1 ± 2.7 mV; the enzyme immobilization rate was 90.1 ± 3.2%; the enzyme activity was 45.8 ± 1.9 U / g, which was 91.6% of the initial enzyme activity; after heat incubation at 65 °C for 6 h, 88.7% of the initial enzyme activity was still retained, while the free TG retained 12.1% of the initial enzyme activity under the same conditions; after incubation at pH 3 for 6 h, 86.9% of the initial enzyme activity was still retained, while the free TG retained 8.1% of the initial enzyme activity under the same conditions; after the immobilized enzyme was sealed and stored at 4 °C for 60 days, the enzyme activity was maintained at 90.3% of the initial enzyme activity, while the free TG could only maintain 83.1% of the initial enzyme activity. Compared with free TG (31.4 ± 3.2 g, 3.1 ± 0.4 kPa), the whey protein isolate hydrogel crosslinked with immobilized TG had stronger gel strength (551.6 ± 12.3 g) and breaking stress (30.0 ± 2.4 kPa).

[0074] Example 5

[0075] Prepare TG solutions, 2 mg / mL OSA solutions (oxidation degree 60%), and 4 mg / mL MQD solutions (methacrylate substitution degree 15%, quaternization degree 15%) with concentrations of 4 mg / mL each. Mix TG and OSA in equal volumes and adjust the solution pH to 7.5 with 1 mol / L NaOH. Let it stand and react at room temperature for 24 h to obtain the OSA-TG solution. Under magnetic stirring at 600 rpm, add MQD dropwise to the OSA-TG solution and continue stirring for 3 h to obtain the MQD / OSA-TG solution. Subsequently, add N,N'-methylenebisacrylamide (MBA, 20 mg / mL) and potassium persulfate solution (KPS, 50 mg / mL) in sequence, and supplement an appropriate amount of deionized water, where OSA-TG∶MQD∶MBA∶KPS∶H2O = 1∶0.25∶0.2∶0.06∶0.49 (v / v). React at 40 °C for 6 h to form a single-crosslinked nanogel, and then construct a double-crosslinked structure through 0.4 mM Ca 2+ ion crosslinking for 4 h; finally, add 0.8 mM EDTA and adjust the pH to 6 to degrade the OSA crosslinked network, and obtain cage-shaped nanogel-immobilized TG enzyme after purification and lyophilization.

[0076] The structure and enzymatic properties of the obtained immobilized TG were characterized. The results showed that the particle size of the immobilized enzyme was 532.4 ± 23.5 nm; the Zeta potential was -34.7 ± 2.2 mV; the enzyme immobilization rate was 90.1 ± 3.8%; the enzyme activity was 45.9 ± 2.4 U / g, which was 91.8% of the initial enzyme activity; after heat incubation at 65 °C for 6 h, 88.3% of the initial enzyme activity was still retained, while the free TG retained 12.1% of the initial enzyme activity under the same conditions; after incubation at pH 3 for 6 h, 86.7% of the initial enzyme activity was still retained, while the free TG retained 8.1% of the initial enzyme activity under the same conditions; after the immobilized enzyme was stored sealed at 4 °C for 60 days, the enzyme activity was maintained at 89.7% of the initial enzyme activity, while the free TG could only maintain 83.1% of the initial enzyme activity. Compared with the free TG (31.4 ± 3.2 g, 3.1 ± 0.4 kPa), the whey protein isolate hydrogel crosslinked with the immobilized TG had stronger gel strength (533.7 ± 12.4 g) and breaking stress (27.6 ± 3.2 kPa).

[0077] Comparative Example 1

[0078] TG solution with a concentration of 4 mg / mL and OSA solution (oxidation degree of 60%) with a concentration of 2 mg / mL were prepared. TG and OSA were mixed evenly in equal volumes, and the pH of the solution was adjusted to 7.5 with 1 mol / L NaOH, and the reaction was allowed to stand at room temperature for 24 h. After purification and lyophilization, OSA-TG was obtained.

[0079] The structure and enzymatic properties of the obtained OSA-TG were characterized. The results showed that the particle size of the immobilized enzyme was 156.7 ± 5.2 nm; the Zeta potential was -40.3 ± 3.4 mV; the enzyme immobilization rate was 88.7 ± 1.6%; the enzyme activity was 47.9 ± 1.1 U / g, which was 95.8% of the initial enzyme activity; after heat incubation at 65 °C for 6 h, 75.6% of the initial enzyme activity was still retained, while the free TG retained 12.1% of the initial enzyme activity under the same conditions; after incubation at pH 3 for 6 h, 72.1% of the initial enzyme activity was retained, while the free TG retained 8.1% of the initial enzyme activity under the same conditions; after the immobilized enzyme was stored sealed at 4 °C for 60 days, the enzyme activity was maintained at 86.3% of the initial enzyme activity, while the free TG could only maintain 83.1% of the initial enzyme activity. Compared with the free TG (31.4 ± 3.2 g, 3.1 ± 0.4 kPa), the whey protein isolate hydrogel crosslinked with OSA-TG had stronger gel strength (359 ± 10.4 g) and breaking stress (16.7 ± 0.9 kPa).

[0080] Comparative Example 2

[0081] Prepare TG solution, 2 mg / mL OSA solution (oxidation degree 60%) and 4 mg / mL MQD solution (methacrylate substitution degree 30%, quaternization degree 30%) with the same concentration of 4 mg / mL. Mix TG and OSA evenly in equal volumes, adjust the pH of the solution to 7.5 with 1 mol / L NaOH, and let it stand at room temperature for reaction for 24 h to obtain OSA-TG solution. Under magnetic stirring at 600 rpm, drop MQD into the OSA-TG solution and continuously stir for 3 h, and obtain MQD / OSA-TG after purification and freeze-drying.

[0082] Characterize the structure and enzymatic properties of the obtained MQD / OSA-TG. The results show that the particle size of the immobilized enzyme is 95.6 ± 2.2 nm; the Zeta potential is -29.7 ± 0.5 mV; the enzyme immobilization rate is 94.9 ± 0.3%; the enzyme activity is 47.2 ± 0.9 U / g, which is 94.4% of the initial enzyme activity; after heat incubation at 65 °C for 6 h, 80.6% of the initial enzyme activity can still be retained, and free TG retains 12.1% of the initial enzyme activity under the same conditions; after incubation at pH 3 for 6 h, 82.7% of the initial enzyme activity is retained, and free TG retains 8.1% of the initial enzyme activity under the same conditions; after the immobilized enzyme is stored sealed at 4 °C for 60 days, the enzyme activity is maintained at 88.3% of the initial enzyme activity, while free TG can only maintain 83.1% of the initial enzyme activity. Compared with free TG (31.4 ± 3.2 g, 3.1 ± 0.4 kPa), the whey protein isolate hydrogel crosslinked by immobilized TG has stronger gel strength (487.2 ± 10.2 g) and breaking stress (25.7 ± 0.5 kPa).

[0083] Comparative Example 3

[0084] Prepare TG solution, 2 mg / mL OSA solution (oxidation degree 60%) and 4 mg / mL MQD solution (methacrylate substitution degree 30%, quaternization degree 30%) with the same concentration of 4 mg / mL. Mix TG and OSA evenly in equal volumes, adjust the pH of the solution to 7.5 with 1 mol / L NaOH, and let it stand at room temperature for reaction for 24 h to obtain OSA-TG solution. Under magnetic stirring at 600 rpm, drop MQD into the OSA-TG solution and continuously stir for 3 h to obtain MQD / OSA-TG solution. Subsequently, add N,N'-methylenebisacrylamide (MBA, 20 mg / mL) and potassium persulfate solution (KPS, 50 mg / mL) in sequence, and supplement an appropriate amount of deionized water, where OSA-TG∶MQD∶MBA∶KPS∶H2O = 1∶0.25∶0.2∶0.03∶0.52 (v / v). React at 25 °C for 12 h to form a single-crosslinked nanogel, and then through 0.1 mM Ca 2+A double-crosslinked structure was constructed by ionic crosslinking for 2 h, and the double-crosslinked nanogel-immobilized TGase was obtained through purification and lyophilization.

[0085] The structure and enzymatic properties of the obtained double-crosslinked nanogel-immobilized TGase were characterized. The results showed that the particle size of the immobilized enzyme was 180.7 ± 5.1 nm; the Zeta potential was -28.2 ± 2.0 mV; the enzyme immobilization rate was 90.3 ± 1.4%; the enzyme activity was 42.6 ± 3.2 U / g, which was 85.2% of the initial enzyme activity; after heat incubation at 65 °C for 6 h, 82.3% of the initial enzyme activity was retained, while free TG retained 12.1% of the initial enzyme activity under the same conditions; after incubation at pH 3 for 6 h, 82.6% of the initial enzyme activity was still retained, while free TG retained 8.1% of the initial enzyme activity under the same conditions; after the immobilized enzyme was stored sealed at 4 °C for 60 days, the enzyme activity was maintained at 83.7% of the initial enzyme activity, while free TG could only maintain 83.1% of the initial enzyme activity. Compared with free TG (31.4 ± 3.2 g, 3.1 ± 0.4 kPa), the whey protein isolate hydrogel crosslinked by immobilized TG had stronger gel strength (377.6 ± 20.1 g) and breaking stress (23.9 ± 1.7 kPa).

[0086] In addition, the inventors of this case also referred to the foregoing embodiments and conducted tests with other raw materials, process operations, and process conditions described in this specification, and all obtained relatively ideal results.

[0087] It should be understood that the technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention without departing from the purpose of the present invention and the scope protected by the claims falls within the protection scope of the present invention.

Claims

1. A preparation method of cage-shaped nanogel-immobilized enzyme, characterized in that, Including: Making transglutaminase react with oxidized sodium alginate to obtain an OSA-TG conjugate; Making methacrylate / quaternized double-modified dextrin and the OSA-TG conjugate undergo electrostatic self-assembly to obtain MQD / OSA-TG nanoparticles; Making the MQD / OSA-TG nanoparticles undergo free radical polymerization reaction and calcium ion cross-linking reaction to obtain MQD / OSA-TG double-crosslinked nanogels; And, using a chelating agent and a pH regulator to degrade the MQD / OSA-TG double-crosslinked nanogels to obtain cage-like nanogel immobilized enzymes.

2. The preparation method according to claim 1, characterized in that Specifically including: Dissolving transglutaminase and oxidized sodium alginate in water respectively and mixing them, then adjusting the pH of the obtained mixed solution to weakly alkaline with an alkaline solution and carrying out a Schiff base reaction at 25-55 °C for 12-24 h to obtain an OSA-TG conjugate.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the transglutaminase to the oxidized sodium alginate is 1:0.2-1; And / or, the oxidation degree of the oxidized sodium alginate is 10-60%; And / or, the pH value of the mixed solution is 7-8.

4. The preparation method according to claim 1, characterized in that, Specifically including: Under the condition of magnetic stirring, dropping methacrylate / quaternized double-modified dextrin into the solution of the OSA-TG conjugate for self-assembly to obtain MQD / OSA-TG nanoparticles.

5. The preparation method according to claim 4, characterized in that: The mass ratio of the transglutaminase to the methacrylate / quaternized double-modified dextrin is 1:0.2-1.2; And / or, the methacrylate substitution degree of the methacrylate / quaternized double-modified dextrin is 0-50%; and / or, the quaternization degree of the methacrylate / quaternized double-modified dextrin is 0-50%; And / or, the rotation speed of the magnetic stirring is 200-800 rpm, and the stirring time is 1-4 h.

6. The preparation method according to claim 1, wherein Specifically including: Adding N,N'-methylenebisacrylamide and a potassium persulfate solution to the solution of the OSA-TG conjugate to carry out a free radical polymerization reaction, and then adding a CaCl2 solution to carry out a calcium ion cross-linking reaction to obtain MQD / OSA-TG double-crosslinked nanogels.

7. The preparation method according to claim 6, characterized in that: The concentration of the N,N′-methylenebisacrylamide is 0.5-5 mg / mL; And / or, the concentration of the potassium persulfate solution is 0.5-5 mg / mL; And / or, the temperature of the free radical polymerization reaction is 25-50 °C, and the time is 12-24 h; And / or, the concentration of the CaCl2 solution is 0.1-1 mmol / L; And / or, the temperature of the calcium ion cross-linking reaction is room temperature, and the time is 1-4 h.

8. The preparation method according to claim 1, wherein Specifically including: Adding a chelating agent solution to the MQD / OSA-TG double-crosslinked nanogels, and adding a pH regulator to adjust the pH value to 5-7 and reacting for 1-4 h to obtain cage-like nanogel immobilized enzymes; preferably, the chelating agent solution includes an EDTA solution, and the concentration of the EDTA solution is 0.2-2.0 mmol / L.

9. Cage-like nanogel immobilized enzymes prepared by the preparation method according to any one of claims 1-8.

10. Application of the cage-like nanogel immobilized enzyme according to claim 9 in high-temperature processing or an extreme pH value environment.

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