Nanobubble-regulated hydrogen-bonded organic framework immobilized enzyme and preparation method thereof

Abstract

The application discloses a kind of nano bubble regulated hydrogen bond organic framework immobilized enzyme, and hydrogen bond organic framework immobilized enzyme is prepared by in-situ self-assembly and embedding method in water environment containing nano bubble from four (4-amidinophenyl) methane and four (4-carboxyphenyl) methane.The hydrogen bond organic framework is used to realize the immobilization of enzyme by in-situ embedding method, and a multi-level pore immobilized enzyme system based on nano bubble is constructed.Nano bubble can expand the pore size and pore volume of hydrogen bond organic framework as pore-forming template, while maintaining the stability of enzyme structure, significantly improving the mass transfer rate of substrate molecules in the framework, thereby enhancing the catalytic activity and substrate affinity of immobilized enzyme.The method effectively improves the enzyme catalytic reaction efficiency while maintaining the system immobilization efficiency, pH stability, temperature stability and reusability.The process is simple and suitable for the construction of multi-level pore immobilized system for various enzymes.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme technology, and particularly relates to a method for in-situ embedding of immobilized enzymes using a hydrogen-bonded organic framework regulated by nanobubbles. Background Technology

[0002] Enzymes are a major discovery in the field of biotechnology. Due to their ease of production, mild reaction conditions, substrate specificity, and high catalytic efficiency, they are widely used in industries such as chemicals, pharmaceuticals, food, cosmetics, detergents, and papermaking. However, enzymes are relatively expensive and difficult to recycle. Furthermore, the molecular structure of enzymes is unstable and sensitive to high temperatures and pH. The most effective way to overcome these problems is to immobilize enzymes on carrier materials of different sizes and shapes. Immobilization increases the stability of enzymes to environmental changes and can minimize or prevent product contamination. Simultaneously, immobilized enzymes can be separated from their substrates and reused, effectively reducing the cost of enzyme use.

[0003] In the synthesis of immobilized enzymes, the synthesis method and materials are the two main factors affecting the system performance. Common synthesis methods include adsorption, covalent bonding, cross-linking, and encapsulation, and the form and type of carrier materials used in different methods also vary. Developing novel conversion systems based on immobilized enzyme technology to achieve enzyme reuse is an urgent problem to be solved. Hydrogen-bonded organic frameworks (HOFs) are porous crystalline materials assembled through hydrogen bond interactions. They are prepared under mild conditions, are metal-free, and have good biocompatibility. Therefore, they can be used as bio-encapsulation materials to protect natural enzymes from harsh in vitro environments and improve enzyme stability. Summary of the Invention

[0004] To address the limitations of existing technologies in enzyme immobilization, particularly the limited application of hydrogen-bonded organic frameworks (HOFs) and their lack of performance enhancement for immobilized enzyme systems, this invention proposes a method for immobilizing enzymes using HOFs regulated by nanobubbles. CAT@HOF-1 / NBs immobilized enzyme particles were constructed, achieving in-situ embedding and immobilization of enzyme molecules within the HOF framework, significantly enhancing the enzymatic conversion process. The nanobubbles, acting as pore-forming templates, effectively expand the pore size and volume of the HOF framework, significantly increasing the mass transfer rate of substrate molecules within the framework while maintaining enzyme structural stability, thereby improving the catalytic activity and substrate affinity of the immobilized enzyme. This method effectively improves enzyme catalytic reaction efficiency and reduces the cost of using biocatalysts without affecting system immobilization efficiency, pH stability, temperature stability, or reusability. Furthermore, this invention employs a physical pressurization method to prepare nanobubbles, avoiding the use of chemical additives, resulting in low energy consumption and environmental friendliness, aligning with green chemistry principles.

[0005] To address the aforementioned technical problems, this invention proposes a hydrogen-bonded organic framework immobilized enzyme controlled by nanobubbles. The immobilized enzyme is prepared using hydrogen-bonded organic framework monomers through in-situ self-assembly and embedding methods. The hydrogen-bonded organic framework monomers are tetra(4-amidinylphenyl)methane and tetra(4-carboxyphenyl)methane. The enzyme is immobilized in an aqueous environment containing nanobubbles using tetra(4-amidinylphenyl)methane and tetra(4-carboxyphenyl)methane.

[0006] Furthermore, this invention also proposes a method for preparing the enzyme immobilized by the aforementioned nanobubble-regulated hydrogen-bonded organic framework, comprising the following steps:

[0007] Step 1) Dissolve tetra(4-amidinylphenyl)methane in water containing nanobubbles at a mass-to-volume ratio of 4 mg / mL, add lyophilized enzyme powder, wherein the mass ratio of lyophilized enzyme powder to tetra(4-amidinylphenyl)methane is 1:2; stir at room temperature at a stirring speed of 300 rpm for 10 minutes to obtain mixture A;

[0008] Step 2) Dissolve tetra(4-carboxyphenyl)methane in water containing nanobubbles at a mass-to-volume ratio of 60 mg / 19 mL, and add 1% ammonia solution to obtain mixture B, in which the mass concentration of tetra(4-carboxyphenyl)methane is 3 mg / mL.

[0009] Step 3) While stirring at room temperature, add mixture B dropwise to mixture A at a volume ratio of 1:1, and then continue stirring at room temperature for 1 hour to obtain mixture C, wherein the stirring speed is 300 rpm;

[0010] Step 4) After centrifuging the mixture C, retain the precipitate, wash the precipitate three times with water, freeze-dry it, and obtain a particulate nanobubble-regulated hydrogen bonded organic framework immobilized enzyme.

[0011] Furthermore, in the preparation method described in this invention, wherein:

[0012] In step 1), the lyophilized enzyme powder is one of catalase (CAT), glucose oxidase (GOD), carbonic anhydrase (CA), and formate dehydrogenase (FDH).

[0013] In step 4), the centrifugation speed is 10,000 rpm and the centrifugation time is 5 minutes; the freeze-drying time is 12 hours.

[0014] In this invention, the solvent water containing nanobubbles is prepared by a physical pressure cycling method.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The preparation method described in this invention is mild and simple to implement. By introducing nanobubbles as dynamic templates during the immobilized enzyme synthesis process, the pore structure of the hydrogen-bonded organic framework is regulated, significantly improving the pore size and pore volume of the material, thereby enhancing the substrate mass transfer rate and enzyme catalytic efficiency. Compared with existing HOF-immobilized enzymes, the CAT@HOF-1 / NBs immobilized enzyme designed in this invention effectively combines the biocompatibility and structural tunability of the hydrogen-bonded organic framework with the physical pore-forming advantages of nanobubbles. While maintaining the original stability and reusability of the enzyme, it significantly enhances the catalytic performance of the enzymatic reaction, providing a new strategy for developing efficient, stable, and environmentally friendly industrial enzyme preparations. Attached Figure Description

[0017] Figure 1 The image shows a scanning electron microscope (SEM) image of HOF-1 prepared in Comparative Example 1.

[0018] Figure 2 The image shows a scanning electron microscope (SEM) image of CAT@HOF-1 prepared in Comparative Example 2.

[0019] Figure 3 The image shows a scanning electron microscope (SEM) image of CAT@HOF-1 / NBs prepared in Example 1.

[0020] Figure 4 The XRD spectra of CAT@HOF-1 and CAT@HOF-1 / NBs are those of HOF-1 prepared in Comparative Examples 1-2 and Example 1.

[0021] Figure 5 The nitrogen adsorption-desorption curves and average pore size and pore volume obtained by BET test are shown for HOF-1 prepared in Comparative Example 1.

[0022] Figure 6 The nitrogen adsorption-desorption curve of CAT@HOF-1 prepared in Comparative Example 2 and the average pore size and pore volume obtained by BET test are shown.

[0023] Figure 7 The nitrogen adsorption-desorption curves of CAT@HOF-1 / NBs prepared in Example 1 and the average pore size and pore volume obtained by BET test are shown.

[0024] Figure 8 Pore ​​size distribution diagrams of HOF-1, CAT@HOF-1, and CAT@HOF-1 / NBs prepared in Comparative Examples 1-2 and Example 1;

[0025] Figure 9 The diagram shows the enzyme-catalyzed reaction kinetics of free CA in Test Example 1;

[0026] Figure 10The enzyme reaction kinetics test diagram of CAT@HOF-1 prepared in Comparative Example 2 is shown in Test Example 2.

[0027] Figure 11 This is a kinetic diagram of the enzymatic reaction of CAT@HOF-1 / NBs prepared in Example 1 in Test Example 2;

[0028] Figure 12 This is a comparison chart of the temperature stability of CAT@HOF-1 and CAT@HOF-1 / NBs prepared in Test Example 4, Comparative Example 2, and Example 1.

[0029] Figure 13 This is a comparison chart of the pH stability of free CAT, CAT@HOF-1 prepared in Example 5, Comparative Example 2, and CAT@HOF-1 / NBs prepared in Example 1. Detailed Implementation

[0030] This invention proposes a method for preparing hydrogen-bonded organic framework (HBO) immobilized enzymes controlled by nanobubbles. The method utilizes HBO monomers via in-situ embedding to prepare immobilized enzymes. The HBO monomers are tetra(4-amidinylphenyl)methane and tetra(4-carboxyphenyl)methane. The enzymes are immobilized in an aqueous environment containing nanobubbles using tetra(4-amidinylphenyl)methane and tetra(4-carboxyphenyl)methane. The main steps involve dissolving tetra(4-amidinylphenyl)methane and the enzyme in a solvent containing nanobubbles, then mixing the tetra(4-carboxyphenyl)methane nanobubble solution with the enzyme under weakly alkaline conditions. Immobilization of the enzyme is achieved through in-situ self-assembly and embedding, constructing a hierarchical porous enzyme immobilization system based on nanobubbles.

[0031] In this invention, nanobubbles are used as dynamic pore-forming templates during framework formation, effectively controlling and expanding the pore size and volume of the hydrogen-bonded organic framework material. While maintaining enzyme structural stability, this significantly enhances the mass transfer rate of substrate molecules within the framework, thereby strengthening the catalytic activity and substrate affinity of the immobilized enzyme. This method effectively improves enzyme catalytic reaction efficiency while maintaining system immobilization efficiency, pH stability, temperature stability, and reusability. The process is simple and applicable to the construction of multi-level pore immobilization systems for various enzymes. Furthermore, the process is simple, operates under mild conditions, and is environmentally friendly, making it suitable for the immobilization and performance enhancement of various oxidoreductases and hydrolases.

[0032] In this invention, one of catalase (CAT), glucose oxidase (GOD), carbonic anhydrase (CA), and formate dehydrogenase (FDH) can be used as a model enzyme.

[0033] In this invention, solvent water containing nanobubbles is prepared by physical pressure cycling.

[0034] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The specific embodiments described are only for explanation and illustration of the present invention and are not intended to limit the present invention.

[0035] Comparative Example 1

[0036] The steps for preparing enzyme-free hydrogen-bonded organic frameworks (HOF-1) are as follows:

[0037] 4 mg of tetrakis(4-amidinylphenyl)methane was dissolved in 1 mL of deionized water to obtain solution I. 3 mg of tetrakis(4-carboxyphenyl)methane was dissolved in 0.95 mL of deionized water, and 0.05 mL of 1% (w / w) ammonia solution was added to obtain solution II. Solutions I and II were mixed at room temperature and a stirring speed of 300 rpm, and stirring was continued for 1 hour. After centrifugation, the precipitate was retained, washed three times with deionized water, and lyophilized to obtain the hydrogen-bonded organic framework (HOF-1).

[0038] Figure 1 Here is a scanning electron microscope (SEM) image of HOF-1. Figure 4 The XRD pattern of HOF-1 is shown in the figure.

[0039] Comparative Example 2

[0040] Using catalase (CAT) as a model enzyme, a CAT@hydrogen-bonded organic framework immobilized enzyme (CAT@HOF-1) was prepared, and the steps are as follows:

[0041] 4 mg of tetrakis(4-amidinylphenyl)methane was dissolved in 1 mL of deionized water, and 2 mg of lyophilized CAT enzyme powder was added. The mixture was stirred at 300 rpm for 10 minutes at room temperature to obtain solution III. 3 mg of tetrakis(4-carboxyphenyl)methane was dissolved in 0.95 mL of deionized water, and 0.05 mL of 1% (w / w) ammonia solution was added to obtain solution IV. Solution IV was added dropwise to solution III, and the mixture was stirred at 300 rpm for 1 hour at room temperature. After centrifugation, the precipitate was retained, washed three times with deionized water, and lyophilized to obtain CAT@HOF-1.

[0042] Figure 2 Here is a scanning electron microscope (SEM) image of CAT@HOF-1. Figure 4 The XRD pattern of CAT@HOF-1 is shown in the figure.

[0043] Example 1

[0044] Using catalase (CAT) as a model enzyme, a CAT@HOF-1 / NBs enzyme immobilized by a nanobubble was prepared. The steps are as follows:

[0045] Preparation of solvent water containing nanobubbles: Fill a 10 mL syringe with pure water and expel the air inside. Seal the syringe tip with a Luer cap. Quickly pull out the syringe plunger to depressurize the water, then immediately release the plunger. Under vacuum pressure, the plunger moves at a relatively high speed, generating nanobubbles. Repeat this pressure change process in the syringe five times to finally obtain solvent water containing nanobubbles.

[0046] Preparation of CAT@HOF-1 / NBs: 4 mg of tetrakis(4-amidinephenyl)methane was dissolved in 1 mL of the above-prepared solvent containing nanobubbles, and 2 mg of lyophilized CAT enzyme powder was added. The mixture was stirred for 10 minutes at room temperature and a stirring speed of 300 rpm to obtain solution V. 3 mg of tetrakis(4-carboxyphenyl)methane was dissolved in 0.95 mL of the above-prepared solvent containing nanobubbles, and 0.05 mL of a 1% (w / w) ammonia solution was added to obtain solution VI. Solution VI was added dropwise to solution V, and the mixture was stirred for 1 hour at room temperature and a stirring speed of 300 rpm. After centrifugation, the precipitate was retained, washed three times with deionized water, and lyophilized to obtain CAT@HOF-1 / NBs.

[0047] Figure 3 Here are scanning electron microscope (SEM) images of CAT@HOF-1 / NBs. Figure 4 The XRD patterns of CAT@HOF-1 / NBs are shown.

[0048] Depend on Figures 1 to 3 It can be seen that the three solid particles HOF-1, CAT@HOF-1, and CAT@HOF-1 / NBs prepared in Comparative Examples 1-2 and Example 1 all have the same rod-shaped morphology, with a length of 1-3 μm and a width of about 100 nm. Figure 1 and Figure 2 A comparison shows that the addition of CAT does not affect the morphology of HOF-1. And... Figure 3 This clearly shows that the rod-like morphology of CAT@HOF-1 / NBs exhibits a clustered structure similar to tree branches, with overlapping layers on the surface. This may be due to the interference of nanobubbles with the crystallization of the material.

[0049] Figure 4 XRD patterns of HOF-1, CAT@HOF-1, and CAT@HOF-1 / NBs prepared in Comparative Examples 1-2 and Example 1. The crystal structures of the three materials were compared by XRD. Figure 4The results show that HOF-1 without enzyme molecules has higher peak heights, sharper peak shapes, and better crystallinity. The characteristic peaks of CAT@HOF-1 shift slightly to the left, indicating a larger lattice constant. This is typically due to the inclusion of heteroatoms larger than the host atoms in the unit cell. The presence of elements with high atomic numbers, such as Fe and S, in the enzyme molecule causes the spectral shift. HOF-1 exhibits diffraction peaks at 9.4° and 19.0°, while CAT@HOF-1 / NBs shows peaks at 8.5° and 17.1°. The presence of diffraction peaks at all four positions for CAT@HOF-1 suggests the possible presence of some pure HOF-1 without encapsulated enzyme molecules, while the synthesis of CAT@HOF-1 / NBs crystals is more uniform. Overall, the characteristic peaks of the three crystal materials are largely consistent, and the presence of CAT and nanobubbles does not alter the crystal structure.

[0050] Figure 5 , Figure 6 , Figure 7 The figures show nitrogen adsorption-desorption curves and average pore size and pore volume obtained by BET test for HOF-1, CAT@HOF-1, and CAT@HOF-1 / NBs prepared in Comparative Examples 1-2 and Example 1, respectively. Figure 8 Pore ​​size distribution diagrams for HOF-1, CAT@HOF-1, and CAT@HOF-1 / NBs. (Source: [Insert source here]) Figure 5 , Figure 6 , Figure 7 It can be seen that the average pore size and pore volume of HOF-1, CAT@HOF-1, and CAT@HOF-1 / NBs are 14.538 nm, 14.079 nm, 23.323 nm, and 0.0478 nm, respectively. 3 g -1 0.0513 m 3 g -1 0.0740 m 3 g -1 Comparing the pore sizes of the three, it can be seen that the average pore size of CAT@HOF-1 is slightly lower than that of HOF-1. This is mainly because CAT occupies a portion of the pore space. Even with CAT occupying a portion of the pore space, the pore size of CAT@HOF-1 / NBs still exceeds that of HOF-1 by about 60%. At the same time, the pore volume of CAT@HOF-1 / NBs is 1.5 times that of CAT@HOF-1. Figure 8It can be seen that within the 0-10 nm range, the pore size of HOF-1 is concentrated between 1.5-2 nm, while the pore size of CAT@HOF-1 and CAT@HOF-1 / NBs is dispersed between 2-4 nm, with the highest number of pores at 2.995 nm and 3.225 nm. CAT@HOF-1 / NBs has a greater number of pores in the 4-10 nm range than CAT@HOF-1. The differences in pore volume and pore size simultaneously confirm that the manipulation of nanobubbles significantly improves the pore parameters of the framework material, proving that nanobubbles successfully play their template role in improving the structure of crystalline materials.

[0051] Test Example 1

[0052] Enzymatic reaction kinetics of free CAT

[0053] CAT is an enzyme that catalyzes the decomposition of H2O2 into H2O and O2. H2O2, as a substrate of horseradish peroxidase (HRP), oxidizes 3,3',5,5'-tetramethylbenzidine (TMB) to a blue product (oxTMB) with strong absorbance at 652 nm. Within a certain concentration range, the absorbance of the blue product shows a good linear relationship with the concentration of hydrogen peroxide. This experiment determined the change in H2O2 concentration by measuring the absorbance of the product at 652 nm and calculated the enzyme activity.

[0054] Add 10 μL of CAT (concentration 0.2 mg / mL) -1 ) and different volumes (10 μL, 20 μL, 30 μL, 40 μL, 50 μL) of H2O2 aqueous solution (concentration 50 mmol L) -1 After mixing, use Tris-HCl buffer (100 mmol / L) -1 The solution was diluted to 1 mL with a pH of 7.8, and the reaction was allowed to proceed for 1 min. 200 μL of the reaction solution was then taken and 700 μL of Tris-HCl (100 mmol / L) was added. -1 pH 7.8), 50 μL HRP (concentration 2 mg / mL) -1 ), 50 μL TMB (concentration 1.2 mg / mL) -1 The absorbance was measured at 652 nm using a UV spectrophotometer. The Michaelis constant (K) was calculated by plotting the double reciprocal Lineweaver-Burk equation. m mmol L -1 ) and maximum reaction rate (V max mmol L -1 min -1 The kinetic parameters of enzyme catalysis processes, such as those for enzymes like α, are calculated using the following equations:

[0055]

[0056] Where: V represents the catalytic reaction rate (mmol / L) corresponding to different substrate concentrations. -1 min -1 [S] represents different substrate concentrations (mmol / L). -1 ), K m Michaelis constant (mmol / L) -1 ), V max Maximum reaction rate (mmol / L) -1 min -1 ).

[0057] Figure 9 This is a kinetic diagram of the enzymatic reaction of free CA.

[0058] Test Example 2

[0059] Enzyme kinetics of immobilized enzymes

[0060] 20 mg of the immobilized enzymes CAT@HOF-1 and CAT@HOF-1 / NBs prepared in Comparative Example 2 and Example 1, respectively, were added to different volumes (10 μL, 20 μL, 30 μL, 40 μL, 50 μL) of H2O2 aqueous solution (concentration 50 mmol / L). -1 The solution was brought to a final volume of 1 mL with Tris-HCl buffer (100 mmol L⁻¹, pH 7.8), and centrifuged (10000 rpm, 2 min) after reacting for 1 min. 200 μL of the supernatant was then added to 700 μL of Tris-HCl (100 mmol L⁻¹, pH 7.8). -1 pH 7.8), 50 μL HRP (concentration 2 mg / mL) -1 ), 50 μL TMB (concentration 1.2 mg / mL) -1 The absorbance was measured at 652 nm using a UV spectrophotometer.

[0061] The procedure for determining catalytic reaction kinetics is the same as in Test Example 1.

[0062] Figure 10 , Figure 11 The figures show the enzyme reaction kinetics of CAT@HOF-1 and CAT@HOF-1 / NBs, respectively.

[0063] Enzyme kinetic parameters are the main parameters for evaluating catalytic performance, such as... Figure 9 , 10As shown in Figure 11, the Km values ​​of free CAT, CAT@HOF-1, and CAT@HOF-1 / NBs were 0.095 mmol / L. -1 0.957 mmol / L -1 2.895 mmol / L -1 Michaelis constant K m The Michaelis constant reflects the affinity between the enzyme and its substrate; a smaller Michaelis constant indicates a higher affinity. Compared to free enzymes, immobilized enzyme structures significantly reduce the enzyme-substrate affinity, while the regulation by nanobubbles restores this affinity. Enzyme catalytic constant k cat The k of CAT@HOF-1 represents the number of substrate molecules that a single enzyme molecule can catalyze per unit time. cat The value is only 1.3% of free CAT, while the k of CAT@HOF-1 / NBs cat The value reached 15.1% of free CAT, approximately ten times that of CAT@HOF-1, further demonstrating the significant improvement in mass transfer efficiency of immobilized enzymes regulated by nanobubbles. In summary, CAT@HOF-1 / NBs increased the affinity and mass transfer efficiency between the enzyme and substrate. Studies on enzyme encapsulation using porous crystalline materials have shown that pore structure restricts enzyme conformational changes and substrate contact, thus reducing affinity and catalytic efficiency. The ability of channels in the carrier material structure to allow rapid passage of substrate molecules also affects catalytic efficiency. Therefore, the improved catalytic performance of CAT@HOF-1 / NBs is due to the regulation of carrier pore size, pore volume, or microstructure by nanobubbles.

[0064] Test Example 3

[0065] Temperature and pH stability studies of free CAT

[0066] The temperature stability test procedure is as follows: CAT was incubated in water baths at temperatures of 30℃, 40℃, 50℃, 60℃, and 70℃ for 120 min, respectively, and the enzyme activity was tested according to the activity determination procedure in Test Example 1. The highest enzyme activity under these operating conditions was used as the standard for each group, and the temperature stability was calculated using the relative enzyme activity formula.

[0067] The pH stability determination procedure is as follows: CA was incubated in buffer solutions with pH values ​​of 4, 5, 6, 7, 8, 9, and 10 for 120 min, and its enzyme activity was tested according to the activity determination procedure in Test Example 1. The highest enzyme activity under the specified operating conditions was used as the standard for each group, and pH stability was calculated using the relative enzyme activity formula.

[0068] Stability is generally expressed as relative catalytic activity. Relative enzyme activity refers to the ratio of its activity after incubation under certain operating conditions to its peak activity, usually expressed as a percentage. The calculation formula is: Relative catalytic activity (%) = A t / A max ×100%, where A t and A max The values ​​represent the absorbance of the reaction solution after incubation under different conditions and the highest absorbance value under those conditions, respectively.

[0069] Test Example 4

[0070] Temperature stability study of immobilized enzymes

[0071] The CAT@HOF-1 and CAT@HOF-1 / NBs prepared in Comparative Example 2 and Example 1 were dispersed in Tris-HCl buffer (100 mmol / L). -1 The enzymes were incubated in water baths at temperatures of 30°C, 40°C, 50°C, 60°C, and 70°C for 120 min, respectively, at pH 7.8. Then, the activity of the immobilized enzymes was determined according to the method in Test Example 2, and the stability was calculated.

[0072] Figure 12 A comparison of the temperature stability of free CAT, CAT@HOF-1, and CAT@HOF-1 / NBs.

[0073] Figure 12 The relative activities of free and immobilized enzymes after incubation in the range of 30-70 °C are shown. As can be seen from the figure, the optimal reaction temperature for both free and immobilized CAT is 30 °C. When the temperature is increased to 60 °C and 70 °C, the relative activity of free CAT decreases to 44.0% and 35.7%, respectively, while the relative activities of CAT@HOF-1 are 53% and 44%, respectively. This indicates that HOF-1 provides some protection to the enzyme under high temperature conditions. The activities of CAT@HOF-1 / NBs at 60 °C and 70 °C are 48.17% and 40.96%, respectively, slightly lower than the relative activities of CAT@HOF-1.

[0074] Test Example 5

[0075] pH stability study of immobilized enzymes

[0076] The CAT@HOF-1 and CAT@HOF-1 / NBs prepared in Comparative Examples 1-2 and Example 1 were incubated for 120 min in buffer solutions at pH=4, 5, 6, 7, 8, 9, and 10, respectively; then, the activity of the immobilized enzymes was determined according to the method in Test Example 2, and the stability was calculated.

[0077] Figure 13 A comparison of the pH stability of free CAT, CAT@HOF-1, and CAT@HOF-1 / NBs.

[0078] Figure 13The relative activities of free and immobilized enzymes were demonstrated within the pH range of 4-10. The optimal pH for both free and immobilized CAT was 8, at which point their relative activity was 100%. CAT showed significantly lower tolerance to acidic environments compared to alkaline environments. At pH 4-7, the relative activities of free CAT were 58.2%, 80.4%, 80.7%, and 91.2%, respectively. CAT@HOF-1 and CAT@HOF-1 / NBs showed activities of 90.9% and 93.8% at pH 5, respectively, and maintained relative enzyme activity above 98% within the pH range of 6-8. This indicates that HOF-1 can maximize enzyme catalytic activity under weakly acidic conditions, making this material most valuable for application within the pH range of 5-8. Comparing the relative activities of CAT@HOF-1 and CAT@HOF-1 / NBs, they were essentially identical within the pH range of 6-9. However, under strongly acidic and alkaline conditions at pH 4 and pH 10, the relative activity of CAT@HOF-1 / NBs was slightly higher than that of CAT@HOF-1.

[0079] In summary, the method for immobilizing enzymes using a hydrogen-bonded organic framework regulated by nanobubbles proposed in this invention is mild, simple, and environmentally friendly. By introducing physically prepared nanobubbles as dynamic templates during the synthesis of the hydrogen-bonded organic framework, the pore structure of the framework material is controlled, effectively improving the mass transfer efficiency and catalytic activity of the immobilized enzyme system. The immobilized enzyme constructed by this method successfully combines the excellent biocompatibility and structural tunability of the hydrogen-bonded organic framework with the physical pore-forming advantages of nanobubbles. While significantly enhancing the enzyme's catalytic performance, it perfectly maintains the enzyme's immobilization efficiency, operational stability, and reusability, providing a new strategy for developing efficient, stable, and green chemistry-compliant industrial enzyme preparations.

[0080] Although the method for immobilizing enzymes with hydrogen-bonded organic frameworks regulated by nanobubbles and its application in enhancing enzyme catalytic performance have been described above with reference to specific embodiments, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. The preparation method proposed in this invention is also applicable to constructing hierarchical porous immobilized enzyme systems based on different hydrogen-bonded organic framework monomers and multiple enzymes. Those skilled in the art, under the guidance of this invention, can make various adjustments and modifications to the nanobubble preparation parameters, the ratio of framework monomers, and the enzyme loading process without departing from the spirit and scope of the claims; all of these modifications are within the protection scope of this invention.

Claims

1. A hydrogen-bonded organic framework immobilized enzyme regulated by nanobubbles, characterized in that, Immobilized enzymes were prepared by in-situ self-assembly and embedding of hydrogen-bonded organic framework monomers, wherein the hydrogen-bonded organic framework monomers were tetra(4-amidinylphenyl)methane and tetra(4-carboxyphenyl)methane. The enzymes were immobilized in an aqueous environment containing nanobubbles using tetra(4-amidinylphenyl)methane and tetra(4-carboxyphenyl)methane.

2. A method for preparing a hydrogen-bonded organic framework immobilized enzyme regulated by nanobubbles as described in claim 1, characterized in that, Includes the following steps: Step 1) Dissolve tetra(4-amidinylphenyl)methane in water containing nanobubbles at a mass-to-volume ratio of 4 mg / mL, add lyophilized enzyme powder, wherein the mass ratio of lyophilized enzyme powder to tetra(4-amidinylphenyl)methane is 1:2; stir at room temperature at a stirring speed of 300 rpm for 10 minutes to obtain mixture A; Step 2) Dissolve tetra(4-carboxyphenyl)methane in water containing nanobubbles at a mass-to-volume ratio of 60 mg / 19 mL, and add 1% ammonia solution to obtain mixture B, in which the mass concentration of tetra(4-carboxyphenyl)methane is 3 mg / mL. Step 3) While stirring at room temperature, add mixture B dropwise to mixture A at a volume ratio of 1:1, and then continue stirring at room temperature for 1 hour to obtain mixture C, wherein the stirring speed is 300 rpm; Step 4) After centrifuging the mixture C, retain the precipitate, wash the precipitate three times with water, freeze-dry it, and obtain a particulate nanobubble-regulated hydrogen bonded organic framework immobilized enzyme.

3. The preparation method according to claim 2, characterized in that, In step 1), the lyophilized enzyme powder is one of catalase, glucose oxidase, carbonic anhydrase, and formate dehydrogenase.

4. The preparation method according to claim 2, characterized in that, In step 4), the centrifugation speed is 10,000 rpm and the centrifugation time is 5 minutes; the freeze-drying time is 12 hours.

5. The preparation method according to claim 2, characterized in that, The solvent water containing nanobubbles is prepared by a physical pressure cycling method, and the preparation steps are as follows: Step a) Fill the syringe with pure water, expel the internal air, and then seal the syringe tip with a Luer lock cap; Step b) Quickly pull out the syringe plunger. Immediately after the water pressure is reduced, the plunger is released. Under vacuum pressure, the plunger moves at a relatively high speed, generating nanobubbles inside the syringe. Step c) Repeat step b) multiple times to obtain solvent water containing nanobubbles.

6. The preparation method according to claim 5, characterized in that, Repeat step b) 5 times.

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