A hydrogen-bonded organic framework enzyme biocomposite material and its preparation method and application

Abstract

The present invention discloses a hydrogen-bonded organic framework enzyme biocomposite material, and its preparation method and application. The hydrogen-bonded organic framework enzyme biocomposite material uses an organic building unit as the framework body, and the enzyme is wrapped inside the framework body; the organic building unit is a carboxylic acid hydrogen-bonded organic framework material; and the enzyme is laccase. The raw materials used in this synthesis method are easily available, the synthesis method is mild and simple, and the enzyme activity can be retained to the maximum extent. The Lac@HOF is applied to the catalytic degradation of organic pollutants in water bodies, not only exhibiting excellent catalytic performance, but also significantly improving resistance to environmental factors, and has extremely high value in practical industrial applications.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogen-bonded organic framework materials, and more specifically, relates to a hydrogen-bonded organic framework enzyme biocomposite material, a preparation method thereof, and an application thereof. Background Art

[0002] Wastewater generated by the textile industry is one of the main sources of pollution of surface and groundwater. Synthetic dyes used in the dyeing process are the main component of wastewater pollutants. The reason is that in actual production, very little dye adheres to the fabric, and most of the dye remains in the wastewater. 8 Kilograms of dyes are discharged into wastewater. Furthermore, due to their high solubility in water, wastewater containing these dyes can cause serious environmental problems when discharged directly into rivers and natural streams. The complex aromatic structure and chemical composition of dye molecules make them difficult to decompose by oxygen, heat, light, and certain oxidizing agents. Therefore, removing or degrading these dyes is a major challenge in wastewater treatment. Malachite green is a commonly used triphenylmethane dye widely used in the printing and dyeing industry. Numerous studies have reported that it can cause mutagenic, carcinogenic, and teratogenic effects in humans.

[0003] Currently, wastewater treatment methods mainly include physical and chemical methods and biological methods. Physical and chemical methods such as chemical oxidation, membrane separation technology, and adsorption have been used for wastewater treatment. Although these methods have certain treatment effects, they are very expensive and may cause secondary pollution. Compared with other methods, biological methods use plants, microorganisms, and enzymes to treat wastewater, which are easy to operate, lower in cost, environmentally friendly, and safe. Unfortunately, plants and microorganisms are very sensitive to some toxic pollutants in wastewater, which greatly limits their scope of application.

[0004] In contrast, enzymes are effective biocatalysts that can act quickly and selectively in harsh environments. They have specific active sites that can bind to specific substrates and reduce the activation energy of the reaction during the enzymatic process to promote the degradation of toxic environmental pollutants. Therefore, they have high reaction kinetics and specificity. In addition, enzymatic biodegradation has the advantages of more flexible operating conditions, no need for nutrient supply, less by-product formation, and fast mass transfer rate. However, enzymes are easily soluble in water, difficult to separate after use, have high environmental requirements, are easily inactivated, and purification technology is expensive, all of which hinder their application in actual production. Immobilized enzymes are a method developed to overcome a series of problems encountered in the use of enzymes. They are mainly divided into the following categories: adsorption method, embedding method, covalent binding method, etc.

[0005] Therefore, finding a framework carrier with excellent performance and combining it with biological enzymes using a suitable fixation method to obtain a biocomposite material with excellent catalytic activity is an important issue that needs to be solved urgently. Summary of the Invention

[0006] In response to the above-mentioned existing technical problems, the first purpose of the present invention is to provide a hydrogen-bonded organic framework enzyme biocomposite material, which not only effectively retains the activity of the biological enzyme and has excellent removal ability for organic pollutants in sewage, but also the hydrogen-bonded organic framework enzyme biocomposite material has significantly improved resistance to environmental factors, excellent thermal stability, storage stability and reusability, and is resistant to strong acids, strong alkalis and metal ions, and has extremely high value in actual industrial applications.

[0007] The second object of the present invention is to provide a method for preparing a hydrogen-bond organic framework enzyme biocomposite material.

[0008] The third object of the present invention is to provide the use of the above hydrogen-bond organic framework enzyme biocomposite material in removing organic pollutants.

[0009] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0010] A hydrogen-bond organic framework enzyme biocomposite material, wherein the composite material uses an organic building unit as a framework body, and an enzyme is wrapped inside the framework body; the organic building unit is a carboxylic acid hydrogen-bond organic framework material; and the enzyme is laccase.

[0011] Hydrogen-bonded organic frameworks (HOFs) are a class of porous materials with periodic structures formed by self-assembly of organic building blocks through hydrogen bonding, π-π stacking, and van der Waals interactions. Compared to MOFs, which are composed of inorganic metal and organic units, HOFs contain no metal nodes, are synthesized under mild conditions, and have lower density and larger theoretical pore volume, which are highly advantageous for certain biological applications. Laccase is a polyphenol oxidase containing multiple copper molecules with strong catalytic ability, which enables it to act on a wide range of substrates; it can catalyze over 250 substrates. However, laccase has poor stability and its catalytic ability is easily affected by external environmental factors such as extreme pH and temperature. In addition, free laccase is highly water-soluble, making it difficult to separate from the system after the reaction, making it impossible to reuse, which increases costs and significantly limits its application in industrial fields.

[0012] The inventors found through a large number of screening and experiments that when a hydrogen-bonded organic framework is used as an immobilization carrier and laccase is preferably used as an immobilization object, the hydrogen-bonded organic framework enzyme biocomposite material obtained by the preparation has excellent performance. The inventors found that by adopting a biomimetic mineralization method and controlling the pH within a specific range during the preparation process, laccase can be better dispersed in an aqueous solution of an organic ligand of the hydrogen-bonded organic framework, and a one-pot encapsulation is performed, thereby better encapsulating the laccase in a hydrogen-bonded organic framework with an organic building unit as the framework body, and effectively retaining the activity of the laccase in the hydrogen-bonded organic framework. The hydrogen-bonded organic framework enzyme biocomposite material obtained by the preparation has excellent removal ability for organic pollutants, and does not need to add additional reaction medium during the reaction to quickly remove pollutants. In addition, the hydrogen-bonded organic framework enzyme biocomposite material not only has excellent removal ability for organic pollutants in sewage, but also has significantly improved resistance to environmental factors, has excellent thermal stability, storage stability and reusability, and can withstand strong acids, strong bases and metal ions. The hydrogen-bond organic framework enzyme biocomposite material has a material dosage of only 10 mg / L for a malachite green solution with a concentration of 50 mg / L. Under the same conditions, the material dosage is much lower than that of other reported materials, and has extremely high value in actual industrial applications.

[0013] Preferably, the organic building block is 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene.

[0014] Preferably, in the hydrogen-bonding organic framework enzyme biocomposite material, the mass fraction of the laccase is 8-10%.

[0015] Further preferably, in the hydrogen-bonding organic framework enzyme biocomposite material, the mass fraction of the laccase is 10%.

[0016] In addition, the present invention also provides a method for preparing a hydrogen-bonded organic framework enzyme biocomposite material, comprising the following preparation steps:

[0017] (1) dissolving the organic building unit in a solvent to obtain solution A;

[0018] (2) dissolving laccase in a buffer solution with a pH of 3.5 to 4.5 to obtain solution B;

[0019] (3) Solution A and solution B are mixed and stirred, and then allowed to stand for a period of time, and then centrifuged to obtain a hydrogen-bonded organic framework enzyme biocomposite material.

[0020] Preferably, in the step (1), the organic building unit (1,3,6,8-tetrakis(4-carboxyphenyl)pyrene) is dissolved in a solvent by ultrasonic treatment.

[0021] Preferably, in step (1), the solvent is N,N-dimethylformamide.

[0022] Preferably, the mass ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene to laccase is 1.5-3:1.

[0023] Preferably, in step (2), the buffer solution is a phosphoric acid / phosphate buffer solution.

[0024] In the step (2), the pH of the buffer solution is 4.

[0025] Preferably, the standing treatment time is 10 to 30 minutes.

[0026] More preferably, the standing treatment time is 20 minutes.

[0027] In addition, the present invention also protects the use of the hydrogen-bond organic framework enzyme biocomposite material in removing organic pollutants.

[0028] Preferably, the organic pollutants are one or more of malachite green, methylene blue, Congo red, and bisphenol A.

[0029] Further preferably, the organic pollutant is malachite green.

[0030] Preferably, the pH of the organic pollutants is 3-11.

[0031] More preferably, the pH of the organic pollutants is 3-9.

[0032] Further preferably, the organic pollutants are removed under light conditions.

[0033] Preferably, the reaction time of the hydrogen-bonding organic framework enzyme biocomposite material for removing organic pollutants is ≥0.5 h.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention provides a hydrogen-bonded organic framework enzyme biocomposite material, which not only effectively retains the activity of laccase and has excellent removal ability for organic pollutants in sewage, but also significantly improves resistance to environmental factors, has excellent thermal stability, storage stability and reusability, is resistant to strong acids, strong bases and metal ions, and has extremely high value in practical industrial applications.

[0036] (2) The hydrogen-bonded organic framework enzyme biocomposite provided by the present invention requires only 10 mg / L of material to be added to a 50 mg / L malachite green solution. Under the same conditions, this material dosage is far lower than that of other reported materials, making it highly valuable in practical industrial applications.

[0037] (3) The present invention promotes the synthesis of the hydrogen-bonding organic framework enzyme biocomposite material by optimizing the pH value during the preparation of the hydrogen-bonding organic framework enzyme biocomposite material and increasing the static conditions to age the hydrogen-bonding organic framework enzyme biocomposite material. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the infrared spectrum of the hydrogen-bonded organic framework enzyme biocomposite material in Example 1.

[0039] Figure 2 This is the CLSM image of the hydrogen-bonded organic framework enzyme biocomposite material in Example 1.

[0040] Figure 3 The removal rates of malachite green by the hydrogen-bonded organic framework enzyme biocomposite material in Example 1, the Lac+HOF prepared in Comparative Example 1, and the HOF in the control group at different times.

[0041] Figure 4 The removal rates of malachite green by the hydrogen-bonded organic framework enzyme biocomposite material, laccase, and HOF in Example 1 at different reaction times are shown.

[0042] Figure 5 The effect of sodium chloride and its mass fraction on the removal of malachite green by the hydrogen-bonded organic framework enzyme biocomposite material in Example 1.

[0043] Figure 6 This is the effect of natural organic matter and its concentration on the removal of malachite green by the hydrogen-bonded organic framework enzyme biocomposite material in Example 1.

[0044] Figure 7 The effect of different temperatures on the removal of malachite green by the hydrogen-bonded organic framework enzyme biocomposite material in Example 1 is shown.

[0045] Figure 8 This is the effect of different reaction pH on the removal of malachite green by the hydrogen-bonded organic framework enzyme biocomposite material in Example 1.

[0046] Figure 9 This is an investigation into the stability of the hydrogen-bonded organic framework enzyme biocomposite material in Example 1 at different pH values.

[0047] Figure 10 The effect of different metal ions on the removal of malachite green by the hydrogen-bonded organic framework enzyme biocomposite material in Example 1.

[0048] Figure 11 The removal of malachite green by the hydrogen-bonding organic framework enzyme biocomposite material in Example 1 at different storage times.

[0049] Figure 12 Removal of malachite green by hydrogen-bonded organic framework enzyme biocomposites with different reuse times. DETAILED DESCRIPTION

[0050] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0051] Example 1

[0052] (1) 10 mg of the organic linker 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene was dissolved in 1 mL of N,N-dimethylformamide (DMF) by ultrasonic treatment to obtain solution A;

[0053] (2) Dissolve 5 mg of laccase (Lac) in a phosphate / phosphate buffer solution at pH 4 to obtain solution B;

[0054] (3) The solution A in step (1) and the solution B in step (2) were mixed and stirred for 5 minutes, and then allowed to stand for 20 minutes, and then collected by centrifugation to obtain a hydrogen-bonded organic framework enzyme biocomposite material (Lac@HOF).

[0055] (4) Post-treatment: The hydrogen-bonded organic framework enzyme biocomposite was washed twice with deionized water and once with ethanol to remove excess solvent and impurities. The hydrogen-bonded organic framework enzyme biocomposite was directly dispersed in deionized water and stored at 4°C.

[0056] The copper ion content in Lac@HOF was detected by inductively coupled plasma emission spectrometry (ICP), which confirmed that the mass fraction of laccase in the hydrogen-bonded organic framework enzyme biocomposite material was 10%.

[0057] Figure 1 This is the infrared spectrum of the hydrogen bond organic framework enzyme biocomposite material prepared in Example 1. Figure 1 It can be seen that the absorption peaks of hydrogen-bonded organic framework (HOF) and hydrogen-bonded organic framework enzyme biocomposite (Lac@HOF) are basically the same, indicating that the addition of laccase (Lac) does not change the structure of HOF. In addition, the absorption peak of Lac also appears in Lac@HOF, indicating that laccase is successfully encapsulated.

[0058] To further prove the presence of laccase in Lac@HOF, we used confocal laser scanning microscopy (CLSM) to observe the Lac@HOF samples. Figure 2 The CLSM image of the hydrogen bond organic framework enzyme biocomposite prepared in Example 1 is shown in FIG. Figure 2 It can be seen that the blue fluorescence emission produced by laccase can be clearly observed in the fluorescence field, which confirms the successful doping of laccase.

[0059] Example 2

[0060] The difference between this embodiment and embodiment 1 is that in step (2), the amount of laccase added is 5.5 mg; the pH is 3.5; and in step (3), the standing time is 30 minutes. The mass fraction of laccase in the hydrogen-bonded organic framework enzyme biocomposite material is 9%.

[0061] Example 3

[0062] The difference between this embodiment and embodiment 1 is that: in step (2), the amount of laccase added is 4.5 mg; the pH is 4.5; in step (3), the static treatment time is 10 minutes. The mass fraction of laccase in the hydrogen-bonded organic framework enzyme biocomposite material is 8%.

[0063] Comparative Example 1

[0064] The difference between this comparative example and Example 1 is:

[0065] (1) Obtaining HOF material: 10 mg of the organic linker 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) was dissolved in 1 mL of N,N-dimethylformamide (DMF) by ultrasonication and then allowed to stand for 20 minutes. The resulting HOF-101 material was collected by centrifugation and washed twice with deionized water and once with ethanol. HOF-101 was directly dispersed in deionized water and stored at 4°C.

[0066] (2) Weigh the same amounts of HOF-101 material and laccase synthesized in step (1) of this example as in Example 1, then add the HOF-101 material to 5 mL of pure water dissolved with laccase, and shake for 30 minutes to obtain Lac+HOF, which is then used to remove malachite green.

[0067] Figure 3 The removal rates of organic pollutants in sewage were obtained by adding Lac@HOF prepared in Example 1, Lac+HOF prepared in Comparative Example 1, and HOF in the control group at different times, where C0 is the absorbance after the addition of the materials and C is the absorbance after only malachite green was added. Figure 3 As shown in the figure, the removal rate of Lac+HOF prepared by physical mixing in Comparative Example 1 is basically the same as that of HOF, indicating that laccase and HOF do not produce a good synergistic effect in the process. This proves that in the preparation process of hydrogen-bonded organic framework enzyme biocomposite materials, a specific preparation method (bionic mineralization method) and control of specific process conditions (such as pH, static treatment, etc.) are required to synthesize Lac@HOF, and the synthesized Lac@HOF has unique advantages and can significantly improve the catalytic activity of the material.

[0068] Test example

[0069] (1) Effects of different reaction times on the removal of malachite green by hydrogen-bonded organic framework enzyme biocomposites

[0070] 0.05 mg of the hydrogen-bonded organic framework enzyme biocomposite material prepared in Example 1, 0.01 mg of laccase, and 0.05 mg of the hydrogen-bonded organic framework (HOF) synthesized in step (1) of Comparative Example 1 were added to 5 mL of malachite green solution having a concentration of 50 mg / L, and the reaction solution was shaken in the dark for 30 minutes to reach adsorption equilibrium. Next, the laccase reaction system was placed in dark conditions, and the hydrogen-bonded organic framework enzyme biocomposite material reaction system was placed under illumination conditions (40 W UV lamp with a wavelength of 365 nm). The sample was taken out at different reaction times, and the absorbance of the malachite green solution was then measured at a maximum absorption wavelength of 617 nm using an ultraviolet-visible spectrophotometer to calculate the malachite green removal rate.

[0071] like Figure 4 As shown, Figure 4 Figure 2 shows the removal efficiency of organic pollutants by the hydrogen-bonded organic framework enzyme biocomposite, laccase, and HOF at different reaction times. As shown in the figure, laccase has a very low removal efficiency for malachite green, reaching only 3.4% after 2 hours of reaction. HOF also has a removal efficiency of only 40.8%. However, the hydrogen-bonded organic framework enzyme biocomposite has a very good removal efficiency for malachite green, removing 85% of malachite green after 30 minutes of reaction, and reaching a removal efficiency of 96% after 2 hours. This demonstrates that the combination of laccase and HOF significantly improves the removal performance of malachite green.

[0072] (2) Effects of sodium chloride and its mass fraction on the removal of malachite green by hydrogen-bonded organic framework enzyme biocomposites

[0073] Respectively, appropriate amount of sodium chloride is dissolved in 5mL concentration and is 50mg / L malachite green solution, obtain the reaction solution of mass fraction of sodium chloride and be respectively 0%, 3.5%, 10%, 20%, then add the hydrogen bond organic framework enzyme biocomposite material and 0.04mg laccase of 0.05mg embodiment 1 preparation respectively, and the reaction solution is shaken in the dark for 30 minutes to reach adsorption equilibrium. Then, the laccase reaction system reacts 24 hours under dark conditions, and the hydrogen bond organic framework enzyme biocomposite material reaction system reacts 2 hours under illumination conditions (40w wavelength is 365nm ultraviolet lamp). After the reaction is completed, adopt ultraviolet visible spectrophotometer to measure the absorbance of malachite green solution under maximum absorption wavelength 617nm, and then calculate malachite green removal rate.

[0074] like Figure 5As shown, laccase is highly sensitive to sodium chloride, with its removal rate decreasing rapidly with increasing sodium chloride concentration. Even at a sodium chloride concentration of 3.5%, the enzyme's removal rate plummets to 2.2%. The hydrogen-bonded organic framework enzyme biocomposite exhibits significantly improved tolerance to high sodium chloride concentrations, consistently maintaining a removal rate exceeding 96%. This demonstrates that the hydrogen-bonded organic framework provides a relatively mild microenvironment for laccase, making it less susceptible to drastic conformational changes and thus significantly enhancing its stability. This holds great value for the treatment of dye wastewater.

[0075] (3) Effects of natural organic matter and its concentration on the removal of malachite green by hydrogen-bonded organic framework enzyme biocomposites

[0076] Respectively, appropriate natural organic matter is dissolved in 5mL concentration and is the malachite green solution of 50mg / L, so that the concentration of natural organic matter in solution is respectively 0,10,50,100mg / L, then add the hydrogen bond organic framework enzyme biocomposite material prepared by 0.05mg embodiment 1 and 0.04mg laccase, and the reaction solution is shaken in the dark for 30 minutes to reach adsorption equilibrium. Then, the laccase reaction system reacts 24 hours under dark conditions, and the hydrogen bond organic framework enzyme biocomposite material reaction system reacts 2 hours under illumination conditions (40w wavelength is 365nm ultraviolet lamp). After the reaction is completed, the absorbance of malachite green solution is measured at a maximum absorption wavelength of 617nm using an ultraviolet-visible spectrophotometer, and then the malachite green removal rate is calculated.

[0077] like Figure 6 As shown, the addition of natural organic matter significantly impacts the removal of malachite green by laccase. With increasing natural organic matter concentration, the laccase removal rate decreases rapidly. When the concentration of natural organic matter exceeds 50 mg / L, the laccase is essentially inactive. This is because natural organic matter drastically alters the spatial structure of the laccase, leading to laccase inactivity. In contrast, the hydrogen-bonded organic framework enzyme biocomposite maintains a high malachite green removal rate of 96% even at a natural organic matter concentration of 100 mg / L. This is because the encapsulation of the hydrogen-bonded organic framework prevents the laccase from direct contact with the natural organic matter, thus preventing denaturation due to excessive water loss. Furthermore, because the spatial structure of the laccase is constrained by the carrier, the laccase structure is less susceptible to structural changes, thus preserving the activity of the material.

[0078] (4) Effects of different temperatures on the removal of malachite green by hydrogen-bonded organic framework enzyme biocomposites

[0079] The hydrogen bond organic framework enzyme biocomposite material prepared by 0.05mg embodiment 1 and the laccase of 0.04mg are dissolved in 5mL ultrapure water respectively, and are hatched (20,30,40,50,60,80 ℃) 24 hours with different temperatures in a water-bath, after hatching, malachite green dye is added, and dye concentration is 50mg / L.Then the reaction solution is shaken 30 minutes to reach adsorption equilibrium under darkness.Then, the laccase reaction system reacts 24 hours under dark conditions, and the hydrogen bond organic framework enzyme biocomposite material reaction system reacts 2 hours under illumination conditions (40w wavelength is 365nm ultraviolet lamp).After reaction is completed, adopt ultraviolet visible spectrophotometer to measure the absorbance of malachite green solution under maximum absorption wavelength 617nm, and then calculate malachite green removal rate.

[0080] like Figure 7 As shown, when the incubation temperature is below 40°C, the malachite green removal rate of laccase increases with increasing temperature, and exhibits optimal stability at 40°C, with a removal rate of 45.3%. However, as the temperature rises further, its activity decreases rapidly, and at 80°C, the laccase is essentially inactive. In contrast, due to the protection of the carrier, the hydrogen-bonded organic framework enzyme biocomposite exhibits extremely strong thermal stability, with a malachite green removal rate greater than 93% within the range of 20-80°C. This is likely because the hydrogen-bonded organic framework restricts the enzyme's spatial structure, preventing the enzyme from folding at high temperatures.

[0081] (5) Effect of reaction pH on the removal of malachite green by hydrogen-bonded organic framework enzyme biocomposites

[0082] The buffer solution of pH value is 3, 5, 6, 7, 8, 9, 11 configured with phosphoric acid and phosphate buffer respectively for standby use. The malachite green solution of 5mL concentration is 50mg / L using the buffer configuration of different pH values. The hydrogen bond organic framework enzyme biocomposite material and laccase prepared in Example 1 are then added respectively, and the reaction solution is shaken in the dark for 30 minutes to reach adsorption equilibrium. Then, the laccase reaction system is reacted for 24 hours under dark conditions, and the hydrogen bond organic framework enzyme biocomposite material reaction system is reacted for 2 hours under illumination conditions. After the reaction is completed, the absorbance of malachite green solution is measured at a maximum absorption wavelength of 617nm using an ultraviolet-visible spectrophotometer, and then the malachite green removal rate is calculated.

[0083] like Figure 8 As shown in the figure, changes in pH have a significant impact on laccase. When the pH is less than 4 or greater than 9, the removal efficiency of laccase for organic pollutants is significantly reduced. However, the removal of organic pollutants by the hydrogen-bonded organic framework enzyme biocomposite material has no significant change at pH 3-9, but the removal rate of organic pollutants is significantly reduced when the pH is greater than 9.

[0084] (6) Effect of the stability of hydrogen-bonded organic framework enzyme biocomposites at different pH

[0085] The hydrogen-bonded organic framework enzyme biocomposite material and laccase prepared in Example 1 were respectively dissolved in 5 mL of buffer solutions of different pH values. After incubation for 24 hours, malachite green dye was added to make a dye concentration of 50 mg / L, and the reaction solution was shaken in the dark for 30 minutes to reach adsorption equilibrium. Then, the laccase reaction system was reacted in the dark for 24 hours, and the laccase@HOF reaction system was reacted under light conditions for 2 hours. After the reaction was completed, the absorbance of the malachite green solution was measured at a maximum absorption wavelength of 617 nm using an ultraviolet-visible spectrophotometer, and the malachite green removal rate was calculated.

[0086] like Figure 9 As shown, incubation at different pH levels has a significant impact on the effectiveness of laccase. When the incubation pH is less than 4 or greater than 10, the stability of the laccase is significantly affected, thereby affecting its removal rate of organic pollutants. The removal rate of the hydrogen-bonded organic framework enzyme biocomposite for organic pollutants gradually decreases with increasing incubation pH, but it still maintains a high removal rate (>52%).

[0087] (7) Effects of metal ions on the removal of malachite green by hydrogen-bonded organic framework enzyme biocomposites

[0088] Respectively by a certain amount of FeSO 4, CuSO 4, MgSO 4, CaCl 2, KCl is dissolved in 5mL ultrapure water, and the concentration of every kind of metal ion is 2mmol / L, is then added malachite green dye, makes dye concentration be 50mg / L.Then in reaction soln, add the hydrogen bond organic framework enzyme biocomposite material prepared by 0.05mg embodiment 1 and the laccase of 0.04mg respectively, and it is shaken 30 minutes to reach adsorption equilibrium under dark conditions.Finally, laccase reaction system is reacted 24 hours under dark conditions, hydrogen bond organic framework enzyme biocomposite material reaction system reacts 50 minutes under illumination condition (40w wavelength is 365nm ultraviolet lamp).After reaction is completed, adopt ultraviolet visible spectrophotometer to measure the absorbance of malachite green solution under maximum absorption wavelength 617nm, and then calculate malachite green removal rate.

[0089] like Figure 10As shown, nearly all metal ions inhibit laccase's removal of malachite green. Laccase is strongly inhibited in copper and calcium ion solutions, with removal rates reduced to 22% and 10% respectively. In particular, in ferrous ion solutions, laccase has almost no removal effect on malachite green. However, the hydrogen-bonded organic framework enzyme biocomposite maintains a high removal rate in various metal ion solutions, with only a slight decrease (66%) in copper ion solution. This suggests that the hydrogen-bonded organic framework significantly enhances the rigidity of laccase, making it less likely for its functional groups to bind to external metal ions, thereby maintaining its stability.

[0090] (8) Study on the storage stability of hydrogen-bonded organic framework enzyme biocomposites

[0091] The hydrogen bond organic framework enzyme biocomposite material and laccase prepared in Example 1 are placed in an external environment where room temperature is about 20 DEG C and is not subjected to lucifuge treatment and stored. When the storage time is 3, 7, 14, 21, and 35 days, 0.05mg of the hydrogen bond organic framework enzyme biocomposite material and 0.04mg of the laccase are respectively taken and dissolved in 5mL malachite green and are shaken in the dark for 30 minutes to reach adsorption equilibrium. Then the laccase reaction system is reacted 24 hours under dark conditions, and the hydrogen bond organic framework enzyme biocomposite material reaction system is reacted 2 hours under illumination conditions (40w wavelength is 365nm ultraviolet lamp). After the reaction is completed, the absorbance of malachite green solution is measured using an ultraviolet-visible spectrophotometer at a maximum absorption wavelength of 617nm, and then the malachite green removal rate is calculated.

[0092] like Figure 11 As shown, the activity of free laccase decreases rapidly over time, with its malachite green removal rate dropping from 31% to 6.9% in just 7 days, and after 21 days, the laccase has essentially lost its activity. In contrast, the hydrogen-bonded organic framework enzyme biocomposite exhibits excellent storage stability, maintaining a high malachite green removal rate (96%) even after five weeks at 20°C. The improved stability of the immobilized enzyme may be related to the reduced fluidity of the protein structure. Because the laccase is embedded in the carrier, its rigidity is increased, and the immobilized laccase's ability to maintain a stable protein conformation is enhanced, thus preventing enzyme denaturation.

[0093] (9) Study on the reusability of hydrogen-bonded organic framework enzyme biocomposites

[0094] The hydrogen bond organic framework enzyme biocomposite material prepared by 0.1mg embodiment 1 is added to the malachite green solution with a concentration of 50mg / L, and the reaction solution is then shaken in the dark for 30 minutes to reach adsorption equilibrium. Then, the hydrogen bond organic framework enzyme biocomposite material reaction system is reacted for 2 hours under illumination conditions (50w blue light with a wavelength of 420nm). After the reaction completes, 0.25mL of the reacted solution is pipetted and diluted with 2.75mL ultrapure water for subsequent detection. Then, malachite green is added again in the reaction system and illumination is carried out again. The operating steps are the same as before, and are reused 10 times. After each reaction completes, the addition of malachite green is reduced in proportion to keep unchanged the pollutant concentration in the control reaction system. All diluted samples to be tested adopt ultraviolet-visible spectrophotometer to measure the absorbance of malachite green solution under the maximum absorption wavelength of 617nm, and then calculate the malachite green removal rate.

[0095] like Figure 12 As shown, the hydrogen-bonded organic framework enzyme biocomposite maintained a high removal rate of malachite green (>92%) even after 10 reuses. This is likely due to the material's large number of active sites, which allows for rapid removal of pollutants and prevents their accumulation on the material's surface. Furthermore, the protective effect of the hydrogen-bonded organic framework on the enzyme prevents the functional groups on the enzyme's surface from binding to small organic molecules in the solution, and reduces the damage to the active substances produced by the reaction on the laccase activity. This excellent reusability can reduce the cost of practical applications.

[0096] The foregoing examples are merely illustrative, serving to illustrate some of the features of the method of the present invention. The appended claims are intended to claim the widest possible scope that can be envisioned, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the present invention. Some numerical ranges used in the claims also include subranges therein, and variations in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A hydrogen-bonded organic framework enzyme biocomposite material, characterized in that: The composite material uses an organic structural unit as a framework body, and an enzyme is wrapped inside the framework body; the enzyme is laccase; The organic building block is 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene; The preparation method of the hydrogen-bond organic framework enzyme biocomposite material comprises the following preparation steps: (1) dissolving the organic building unit in a solvent to obtain solution A; (2) Dissolve laccase in a buffer solution with a pH of 3.5 to 4.5 to obtain solution B; (3) Solution A and solution B are mixed and stirred, and then allowed to stand for a period of time, and then centrifuged to obtain a hydrogen-bonded organic framework enzyme biocomposite material; In the step (1), the solvent is N,N-dimethylformamide; The mass ratio of the organic building unit to the laccase is 1.5-3:

1.

2. The hydrogen-bonded organic framework enzyme biocomposite material according to claim 1, characterized in that: In the hydrogen-bond organic framework enzyme biocomposite material, the mass fraction of the laccase is 8-10%.

3. The hydrogen-bonded organic framework enzyme biocomposite material according to claim 1, characterized in that: In the step (2), the buffer solution is a phosphoric acid / phosphate buffer solution.

4. The hydrogen-bonded organic framework enzyme biocomposite material according to claim 1, characterized in that: In the step (2), the pH of the buffer solution is 4.

5. Use of the hydrogen-bonding organic framework enzyme biocomposite material according to any one of claims 1 to 4 in removing organic pollutants.

6. The application according to claim 5, characterized in that The organic pollutants are one or more of malachite green, methylene blue, Congo red, and bisphenol A.

Images