Immobilized enzyme catalyst based on copper-based metal organic framework as well as preparation method and application of immobilized enzyme catalyst

Abstract

The invention belongs to the technical field of enzyme composite materials and biological catalysis, and particularly relates to an immobilized enzyme catalyst based on a copper-based metal organic framework as well as a preparation method and application of the immobilized enzyme catalyst. Cytochrome c (Cyt c), squaric acid and copper ions are uniformly mixed through an in-situ co-precipitation method, in-situ embedding of Cyt c is achieved through hydrogen bonds and coordinate bonds generated in the Cu-MOF forming process, the Cyt c-coated Cu-MOF compound enzyme catalyst is prepared, a constructed Cu-MOF carrier is of a mesoporous structure, a stable protective microenvironment can be provided for Cyt c, enzyme leaching is effectively inhibited, and the catalyst has the advantages of being capable of being used for preparing the complex enzyme catalyst. The operation stability and the environmental tolerance are remarkably improved, and the catalyst shows efficient catalytic degradability in organic pollutants including phenolic compounds and dyes, and has wide application prospects in the fields of biological catalysis and the like. Moreover, the method is mild in condition, simple and convenient to operate, low in cost and easy for large-scale production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme composite materials and biocatalysis, and particularly relates to an immobilized enzyme catalyst based on a copper-based metal organic framework and a preparation method and application thereof. BACKGROUND

[0002] Cytochrome c (peroxidase, Cyt c) as an important heme protein, with its excellent redox activity, has shown broad application prospects in the fields of biocatalysis, biosensing and environmental governance. However, the free state of Cyt c is easily disturbed by environmental factors such as temperature and organic solvents in practical application, resulting in unstable structure and easy loss of activity, and is difficult to recycle and reuse, which seriously restricts its industrialization promotion.

[0003] In order to improve the stability and reusability of Cyt c, the commonly used immobilization strategies include physical adsorption, chemical covalent combination and embedding method. The physical adsorption method is simple in operation and mild in conditions, but the interaction between the protein and the carrier is weak, and leakage is easy to occur, and the stability is poor; the chemical covalent combination method can enhance the fixing strength, but the reaction process often involves severe modification, which may destroy the spatial conformation of Cyt c, resulting in a significant reduction in its redox activity; the traditional embedding method can provide certain protection to the protein, but it generally has problems such as large mass transfer resistance, low catalytic efficiency and protein leakage.

[0004] In recent years, porous materials such as metal organic frameworks (MOFs) and covalent organic frameworks (COFs) have been widely used as protein immobilization carriers due to their high specific surface area and confinement effect. Existing research attempts to introduce Cyt c into MOF or COF materials through post-synthesis loading or simple mixing, which has improved the stability of Cyt c to a certain extent, but such methods often rely on external adsorption or non-in situ embedding, which can easily lead to uneven protein distribution, low immobilization efficiency and insufficient microenvironment regulation, and is difficult to effectively improve the catalytic performance of Cyt c. For example, a method for immobilizing laccase on a functionalized MOF is disclosed in a Chinese patent application, which includes hydrothermal synthesis, amino functionalization, alkali etching, silanization modification, and uses glutaraldehyde as a crosslinking agent to immobilize laccase. Although this method improves the environmental stability and recycling of the enzyme, it involves multiple complex processes and uses toxic crosslinking agents, which limits its practical application potential.

[0005] In the field of catalytic degradation of phenolic compounds and organic dyes, due to the wide existence of such pollutants in industrial wastewater, even at low concentrations, they still have strong toxicity, carcinogenicity and biological accumulation, which pose a serious threat to the ecological environment and human health, and it is urgent to develop efficient catalytic removal technology. However, the existing immobilized Cyt c system still generally faces problems such as low catalytic efficiency, poor reusability, weak environmental adaptability and the like when treating such pollutants, which is difficult to meet the dual demands of high efficiency and stability for actual wastewater treatment. In addition, the currently widely used batch degradation mode has the limitations of long production cycle, inability to run continuously, low mass transfer efficiency and the like, which leads to insufficient overall catalytic degradation capacity and limits its industrial application.

[0006] Therefore, developing a new immobilization strategy to realize efficient and stable encapsulation of Cyt c while significantly improving its catalytic activity, especially in the degradation of phenolic pollutants and organic dyes, has become an important direction of current research. The ideal immobilization method should take into account multiple requirements such as simple operation, environmental friendliness, and adaptation to continuous treatment, in order to promote the practical application of Cyt c in the field of environmental catalysis. SUMMARY

[0007] The technical problem to be solved by the present application is to overcome the defects and shortcomings of the existing enzyme immobilization method, which relies on complex chemical modification and toxic cross-linking agents, easily damages the protein activity during the immobilization process, and is complicated to operate, and it is difficult to balance environmental friendliness, stability and high efficiency catalysis. The primary object is to provide a preparation method of a copper-based metal organic framework immobilized enzyme catalyst.

[0008] The second object of the present application is to provide a copper-based metal organic framework immobilized enzyme catalyst prepared by the preparation method.

[0009] The third object of the present application is to provide the application of the copper-based metal organic framework immobilized enzyme catalyst.

[0010] The fourth object of the present application is to provide a method for catalytic degradation of organic pollutants.

[0011] The above objects of the present application are achieved by the following technical solutions: The present application protects a preparation method of a copper-based metal organic framework immobilized enzyme catalyst, comprising the following steps: Mixing cytochrome c and square acid or salt solution, adding copper salt solution, fully reacting at room temperature, solid-liquid separation, washing the precipitate, collecting the solid product for drying, and the obtained copper-based metal organic framework immobilized enzyme catalyst is obtained; The pH condition for mixing cytochrome c and square acid or salt solution is 5.5-7.5.

[0012] The application realizes uniform embedding and efficient fixing of protein molecules in the MOF matrix by innovatively using in-situ embedding method to induce self-assembly of SA and copper acetate into Cyt c@Cu-MOF in the presence of Cyt c, and has good loading capacity and embedding rate. The strategy not only significantly enhances the structural stability of Cyt c, but more importantly, effectively optimizes the secondary structure of the enzyme, significantly improves the affinity between the catalyst and the substrate, and thus greatly improves the catalytic activity of Cyt c, and the catalytic efficiency is significantly higher than that of free enzyme. The composite catalyst exhibits excellent performance in the efficient catalytic degradation of difficult-to-degrade pollutants such as phenolic compounds and organic dyes, can realize rapid and complete conversion of pollutants, and has good recycling stability.

[0013] The application has the advantages of combining high stability immobilization with high catalytic activity improvement, solving the contradiction between environmental friendliness, catalytic activity and stability in the prior art, providing an efficient, green and recyclable new catalyst system for industrial biological catalysis and environmental pollutant treatment, especially in the catalytic degradation of phenolic compounds and organic dyes, and proposing a continuous degradation method to improve the mass transfer efficiency and catalytic degradation capacity of the degradation method, providing a new idea for industrial application, and having important application value and broad industrialization prospect.

[0014] Preferably, the molar ratio of the squaric acid or its salt and the copper salt is (1-2): 1, more preferably (1-1.5): 1, and further more preferably 1:(1.2-1.4).

[0015] Preferably, the mass molar ratio of the cytochrome c and the copper salt is (10-30) g: 1 mol, more preferably (15-25) g: 1 mol, and further more preferably (18-22) g: 1 mol.

[0016] Further, the salt form of the squaric acid is preferably sodium squarate. The salt form of the squaric acid has better water solubility.

[0017] As an optional embodiment, commercially available squaric acid salt can be directly used, or squaric acid can be converted into squaric acid salt. Taking sodium squarate as an example, the preparation of the sodium squarate specifically includes the following steps: uniformly mixing a squaric acid aqueous solution and a sodium hydroxide solution to obtain the sodium squarate.

[0018] The cytochrome c and the squaric acid or salt thereof solution are mixed under the pH 5.5-7.5 environment. On the one hand, the activity of the enzyme is better maintained during the embedding process, and a relatively mild environment is provided for the enzyme to avoid denaturation and inactivation of the enzyme during the embedding process due to too high or too low pH. On the other hand, too high or too low pH can lead to the destruction of the Cu-MOF structure. The near neutral or neutral environment with pH 5.5-7.5 is relatively mild, which helps to maintain the structural stability of the Cu-MOF and promotes the coordination reaction, which is conducive to the synthesis of Cu-MOF.

[0019] Preferably, the pH condition for mixing the cytochrome c and the squaric acid or salt thereof solution is 6-7.

[0020] Further, the pH condition is adjusted by using a basic reagent (preferably sodium hydroxide solution) or an acidic reagent (preferably squaric acid solution).

[0021] Preferably, the copper salt includes copper acetate, copper chloride, copper sulfate, copper nitrate, or a hydrate of any of the above copper salts.

[0022] Further, the concentration of the squaric acid or salt thereof solution is 0.07-2M, preferably 0.08-1M, and more preferably 0.1-0.2M.

[0023] Further, the concentration of the copper salt solution is 0.05-1M, preferably 0.08-0.5M, and more preferably 0.1-0.15M.

[0024] Further, the mass-volume ratio of the cytochrome c, squaric acid or salt thereof solution, and copper salt solution is (1-3) mg:(0.8-1.2) mL:1 mL, preferably (1.5-2.5) mg:1 mL:1 mL.

[0025] Further, the sufficient reaction time is 10-30 min, preferably 15-25 min.

[0026] Further, the sufficient reaction condition is stirring, and the stirring speed is 400-1200 rpm, preferably 600-1000 rpm.

[0027] As an optional embodiment, the mixing is first mixing the cytochrome c and the squaric acid or salt thereof solution, and then adding the copper salt solution to the system for mixing.

[0028] Further, the solid-liquid separation is performed by centrifugation.

[0029] Preferably, the centrifugation condition is 8000-15000 rpm for 5-15 min. The appropriate centrifugation condition can be selected according to the actual processing condition.

[0030] Further, the drying is freeze-drying.

[0031] Preferably, the temperature of the freeze-drying is -35 ~ -60℃, more preferably -40 ~ -50℃.

[0032] Preferably, the time of the freeze-drying is 20 ~ 40h, more preferably 24 ~ 30h.

[0033] Further, the solvent of the washing is water.

[0034] Further, the solid product is collected after the washing through solid-liquid separation, and the solid-liquid separation is defined as the same as the aforementioned.

[0035] The present application protects the immobilized enzyme catalyst of copper-based metal organic framework prepared by the preparation method.

[0036] Further, the loading of the immobilized enzyme catalyst of copper-based metal organic framework is ≥ 85mg / g.

[0037] Further, the loading of the immobilized enzyme catalyst of copper-based metal organic framework is ≥ 90%.

[0038] The present application also protects the application of the immobilized enzyme catalyst of copper-based metal organic framework in degrading organic pollutants; preferably, the organic pollutants include phenolic compounds and / or organic dyes.

[0039] Further, the phenolic compound is a phenolic compound containing aromatic structure.

[0040] Preferably, the phenolic compound containing aromatic structure includes one or more of 2,4-dichlorophenol, 2,6-dimethylphenol, phenol, 1-naphthol, 4-methoxyphenol, hydroquinone, o-nitrophenol.

[0041] Preferably, the organic dye includes one or more of methyl orange, indigo carmine, rhodamine B, methylene blue, reactive blue.

[0042] Preferably, the reactive blue includes reactive blue 4.

[0043] The present application also protects a method for catalytically degrading organic pollutants, which uses the immobilized enzyme catalyst of copper-based metal organic framework to catalytically degrade organic pollutants.

[0044] Further, the organic pollutants include phenolic compounds and / or organic dyes.

[0045] Further, the phenolic compound is a phenolic compound containing aromatic structure.

[0046] Preferably, the phenolic compound containing aromatic structure includes one or more of 2,4-dichlorophenol, 2,6-dimethylphenol, phenol, 1-naphthol, 4-methoxyphenol, hydroquinone, o-nitrophenol.

[0047] Preferably, the organic dye includes one or more of methyl orange, indigo carmine, rhodamine B, methylene blue, reactive blue.

[0048] Preferably, the reactive blue includes reactive blue 4.

[0049] Further, the catalytic degradation mode includes batch reaction or continuous flow mode.

[0050] Further, the continuous flow mode is performed using a continuous flow device.

[0051] Further, the continuous flow device is a continuous flow reactor, which can be a glass chromatographic column.

[0052] Preferably, the flow rate of the continuous flow mode is 100-500 μL / min, preferably 100-300 μL / min, and further more preferably 150-250 μL / min.

[0053] Further, when the organic pollutant is an organic dye, the concentration of the organic pollutant in the catalytic degradation system is ≥0.1 mM, preferably 0.1-1 mM, and more preferably 0.1-0.2 mM.

[0054] Further, when the organic pollutant is a phenolic compound, the concentration of the organic pollutant in the catalytic degradation system is ≥0.1 mg / mL, preferably ≥0.18 mg / mL.

[0055] Further, the catalytic degradation of organic matter is carried out at a respective suitable pH.

[0056] Compared with the prior art, the present application has the following beneficial effects: The present application realizes efficient immobilization of Cyt c by constructing a Cyt c@Cu-MOF composite system, has high embedding rate and loading capacity, maintains high catalytic activity, and greatly improves stability and practicality, provides a new efficient and sustainable catalyst solution for the application of biological catalysis technology in the field of environmental remediation and green chemistry, and specific advantages are as follows: (1) The present application significantly improves the stability and repeated use performance of Cyt c. By in-situ embedding method, Cyt c is uniformly embedded in Cu-MOF matrix to form ordered mesoporous structure, which effectively limits enzyme molecule leaching. Cu 2+The metal organic framework constructed with squaric acid provides a rigid skeleton for Cyt c, effectively shields the enzyme molecules from the adverse effects of the external environment, limits the thermal motion and solvent-induced denaturation, and thus significantly enhances the stability and reusability of the enzyme.

[0057] (2) The catalytic activity of the immobilized enzyme is significantly improved. Based on the structural characteristics, functional mechanism and application selection of cytochrome c, the MOF formed by copper salt and squaric acid organic ligand is used to embed cytochrome c to achieve better catalytic effect. The reason for the improvement of the catalytic activity may be that the Cu node in Cyt c@Cu-MOF may optimize the electron transfer path by synergistic effect with the Fe center of Cyt c, promote the decomposition of H2O2, and improve the catalytic activity. Or in the embedding process, the microenvironment of the active center of hematin may be slightly disturbed, the secondary structure of the enzyme is changed, the affinity of hematin with the substrate is improved when the hematin is exposed, and then the catalytic activity is enhanced.

[0058] (3) The preparation process of the application is simple and low in cost. The raw materials (such as copper acetate and squaric acid) are widely available and low in price, the synthesis process is carried out at normal temperature and pressure, without the need for complex equipment, and without the need for complex operations such as hydrothermal reaction, amino functionalization, etching, silanization modification and addition of toxic curing agent (such as glutaraldehyde), simple operation, fast reaction speed, good industrial application prospect.

[0059] (4) The application has a wide application prospect in efficiently degrading difficult-to-treat pollutants. The prepared Cyt c@Cu-MOF catalyst shows excellent catalytic degradation ability for phenolic compounds and organic dyes, and has important application value in the field of environmental governance. Especially in the continuous flow reaction system, the catalyst can still maintain high catalytic activity, realizing continuous and efficient removal of pollutants, reducing the amount of enzyme used, and improving the economy and practicability of the catalytic process. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 The figure is the nitrogen adsorption-desorption isotherm and pore size distribution curve of the Cu-MOF of Example 1.

[0061] Figure 2 The figure is the nitrogen adsorption-desorption isotherm and pore size distribution curve of the enzyme catalyst Cyt c@Cu-MOF prepared in Example 2.

[0062] Figure 3 The figure is the standard curve of Cyt c.

[0063] Figure 4Graphs of the inclusion rate and loading amount of Cyt c in the enzyme catalysts prepared for Example 2, Comparative Example 1, Comparative Example 2.

[0064] Figure 5 Bar graph of the relative enzyme activity of Cyt c & Cu-MOF of Example 1, enzyme catalysts prepared for Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, free enzyme, Cu-MOF catalyst of Example 1, copper salt, squarate.

[0065] Figure 6 Graphs of the enzyme reaction kinetics of Cyt c & Cu-MOF of Example 1, enzyme catalyst Cyt c@Cu-MOF prepared for Example 2, Cyt c & Cu-MOF of Comparative Example 3, and free enzyme.

[0066] Figure 7 Bar graphs of the relative enzyme activity and stability test data; wherein a~b graphs are bar graphs of the relative enzyme activity of enzyme catalysts prepared for Example 2, Comparative Example 1, Comparative Example 2, and free enzyme under different temperatures (a) and different solvents (b) environments; c graph is a data graph of the cycle stability of enzyme catalysts prepared for Example 2, Comparative Example 2; d graph is a data graph of the storage stability of enzyme catalyst prepared for Example 2 and free enzyme.

[0067] Figure 8 Data graph of the catalytic degradation of phenolic compounds by enzyme catalysts prepared for Example 2, Comparative Example 1, Comparative Example 2, and free enzyme.

[0068] Figure 9 Data graph of the catalytic degradation of different types of phenolic compounds by enzyme catalyst prepared for Example 2 and free enzyme.

[0069] Figure 10 Data graph of the catalytic degradation of different types of organic dyes by enzyme catalyst prepared for Example 2 and free enzyme.

[0070] Figure 11 Graph of the efficiency comparison results of the enzyme catalyst prepared for Example 2 in the continuous flow (Flow reactor) and batch catalysis (batch reactor) modes for degrading organic dyes.

[0071] Figure 12Figure 2. The graph of the fitting curve of the amide I band (a, b) and the data statistics of the secondary structure proportion (c) of free Cyt c and the FTIR spectra of the enzyme catalyst prepared in Example 2; the fluorescence spectra (d) and UV-Vis absorption spectra (e) of free Cyt c, Cu-MOF of Example 1 and Cyt c@Cu-MOF of Example 2. DETAILED DESCRIPTION

[0072] The present application is further illustrated by the following description with reference to the accompanying drawings and specific examples. The reagents, methods and apparatuses used in the present application are conventional in the art unless otherwise specified. The examples do not limit the present application in any form.

[0073] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0074] Figure 7 a represents Figure 7 the graph of a, Figure 7 b represents Figure 7 the graph of b, and the naming of other graphs is similar.

[0075] Example 1. A copper-based metal organic framework material Cu-MOF The preparation method of the Cu-MOF catalyst comprises the following steps: A 15 mg / mL aqueous solution of squaric acid was adjusted to pH 6 with a 30 mg / mL aqueous solution of sodium hydroxide to obtain a sodium squarate aqueous solution (the change in the volume of the solution after the addition of sodium hydroxide can be ignored). 1 mL of the above-mentioned sodium squarate (0.13 M) aqueous solution and 1 mL of a 0.1 M aqueous solution of copper acetate monohydrate were mixed uniformly, and after stirring (at a speed of 800 rpm) the mixture at room temperature for 20 min, the supernatant was removed by centrifugation (at 12000 rpm for 5 min), and the solid product was collected and freeze-dried (pre-freezing at -40°C to -50°C for 24 h) to obtain the Cu-MOF catalyst.

[0076] Example 2. An immobilized enzyme catalyst Cyt c@Cu-MOF based on a copper-based metal organic framework The preparation method of the Cyt c@Cu-MOF catalyst comprises the following steps: Cyt c 2 mg was added to 1 mL of sodium formate (0.13 M, prepared according to the method of Example 1) aqueous solution, mixed well, then 1 mL of copper acetate monohydrate aqueous solution (0.1 M) was added, and the mixture was stirred (at a speed of 800 rpm) at room temperature for 20 min, then centrifuged (at 12000 rpm for 5 min) to remove the supernatant, washed once with 4 mL of deionized water and centrifuged again, and the solid product was collected and freeze-dried (pre-freezing at -40 to -50 °C for 24 h) to obtain the Cyt c@Cu-MOF catalyst.

[0077] Comparative Example 1 Cyt c@ZIF-8 The preparation of Cyt c@ZIF-8 includes the following steps: Cyt c 2 mg was added to 1 mL of deionized water, mixed well, then 8 mL of dimethylimidazole aqueous solution was added, and finally 1 mL of zinc nitrate hexahydrate aqueous solution was added, wherein the molar concentration of the dimethylimidazole aqueous solution was 1 M, and the molar concentration of the zinc nitrate hexahydrate aqueous solution was 0.15 M, the mixture was stirred (at a speed of 1000 rpm) at room temperature for 30 min, then centrifuged (at 10000 rpm for 15 min) to remove the supernatant, washed once with 10 mL of deionized water and centrifuged again, and the solid product was collected and freeze-dried (pre-freezing at -40 to -50 °C for 24 h) to obtain the Cyt c@ZIF-8 catalyst.

[0078] Comparative Example 2 Cyt c@ZIF-L The preparation of Cyt c@ZIF-L includes the following steps: Cyt c 2 mg was added to 1 mL of deionized water, mixed well, then 8 mL of dimethylimidazole aqueous solution was added, and finally 1 mL of zinc nitrate hexahydrate aqueous solution was added, wherein the molar concentration of the dimethylimidazole aqueous solution was 0.5 M, and the molar concentration of the zinc nitrate hexahydrate aqueous solution was 0.15 M, the mixture was stirred (at a speed of 1000 rpm) at room temperature for 30 min, then centrifuged (at 10000 rpm for 15 min) to remove the supernatant, washed once with 10 mL of deionized water and centrifuged again, and the solid product was collected and freeze-dried (pre-freezing at -40 to -50 °C for 24 h) to obtain the Cyt c@ZIF-L catalyst.

[0079] Since ZIF-8 and ZIF-L are the most thoroughly studied MOF materials, their synthesis and application are mature, which helps ensure the reproducibility of experiments and can serve as reliable performance benchmarks. Although ZIF-L has a more open structure, theoretically beneficial for mass transfer, experimental results show that its protection of cytochrome C activity is lower than that of ZIF-8, possibly due to stronger confinement effects, unfavorable interfacial interactions, or higher mass transfer tortuosity. This indicates that structural openness does not directly equate to higher enzyme activity, highlighting the importance of regulating MOF crystal structure, surface chemistry, and growth behavior. If Cyt c@Cu-MOF outperforms ZIF-8 and ZIF-L in both stability and catalytic activity, it strongly demonstrates the superiority and innovation of its structural design. Therefore, using these two as control groups is an effective strategy for evaluating the catalytic and application performance of Cyt c@Cu-MOF prepared in this scheme.

[0080] Comparative Example 3: Cyt c & Cu-MOF Cyt c&Cu-MOF is obtained by physically mixing a cytochrome solution with the Cu-MOF catalyst prepared in Example 1. The specific method includes the following steps: 25 μL of a 0.125 mg / mL Cyt c solution was physically mixed with an appropriate amount of the Cu-MOF catalyst prepared in Example 1 to obtain Cyt c&Cu-MOF. This system contained 3.125 μg Cyt c, and the Cu-MOF content was consistent with that in 25 μL of the Cyt c@Cu-MOF catalyst prepared in Example 2.

[0081] Experimental Example 1: Aperture Measurement 1. Experimental Methods Using Cu-MOF prepared in Example 1 and Cyt c@Cu-MOF prepared in Example 2 as test objects, the adsorption-desorption isotherms and pore size distribution curves (BJH) of nitrogen at 77 K were measured on a Micromeritics ASAP2460 analyzer.

[0082] 2. Experimental Results The results are as follows Figures 1-2 As shown, the nitrogen adsorption isotherm of the Cu-MOF prepared in Example 1 is type IV, accompanied by H3 / H4 type hysteresis loops, indicating that the material has a mesoporous structure; the BJH pore size distribution shows that the main pore size is concentrated in 10-15 nm, which is typical of the mesoporous range (2-50 nm). Figure 1 ).

[0083] The nitrogen adsorption isotherm of Cytc@Cu-MOF prepared in Example 2 is type IV with a H3 hysteresis loop, indicating the presence of meso / macro-pores. BJH pore size distribution shows that the pore size ranges from 5 to 100 nm, in which mesopores of 20-50 nm coexist with macropores of >50 nm, and the main pore size is about 70 nm. This multi-level pore structure may be due to the stacking gap between MOF particles or the macroscopic channels induced by Cyt c Figure 2 ).

[0084] Experimental Example 2: Encapsulation rate and loading capacity test 1. Experimental method The calculation method of the loading capacity and encapsulation rate of Cyt c is as follows: After the first centrifugation of the MOF self-assembly of the enzyme, the supernatant remaining was measured by ultraviolet-visible spectrophotometer for its absorbance at 409 nm. By measuring the absorbance at 409 nm of a series of gradient concentrations of Cyt c solution, a Cyt c standard curve was drawn Figure 3 According to the Cyt c standard curve, the enzyme concentration contained in the supernatant was calculated, and the encapsulation rate and loading capacity were calculated.

[0085] The calculation method of the encapsulation rate and loading capacity is shown in Formula 1 and Formula 2: Encapsulation rate (%) = (m-cV) / m*100% (Formula 1) Loading rate (%) = (m-cV) / w*100% (Formula 2) In the formula, m (mg) represents the mass of Cyt c initially added to the solution; c (mg / mL) and V (mL) represent the concentration and volume of Cyt c in the supernatant after the immobilization process, respectively; w (g) represents the mass of the powder obtained after freeze-drying of the immobilized enzyme (Cyt c@Cu-MOF, Cyt c@ZIF-L, Cyt c@ZIF-8).

[0086] 2. Experimental results The results are shown in Table 1 Figure 4 As shown in Table 1, Cyt c@Cu-MOF showed the highest encapsulation rate (91.8%) and loading capacity (85.4 mg / g), indicating that it has excellent encapsulation performance. Cyt c@ZIF-8 has the lowest encapsulation rate (49.8%) and loading capacity (37.3 mg / g), indicating that it has poor encapsulation efficiency. The encapsulation rate of Cyt c@ZIF-L is 65.8%, and the loading capacity is 69.2 mg / g, which is between the two. Overall, Cu-MOF is significantly superior to ZIF-8 and ZIF-L in terms of encapsulation efficiency and loading capacity.

[0087] Experimental Example 3: Enzyme activity test 1. Experimental method (1) Enzyme activity test The enzyme activity was determined by using ABTS and H2O2 as substrates, and by catalyzing the conversion of colorless ABTS to greenish ABTS+ oxidation state, which shows a characteristic ultraviolet absorption peak at 420 nm.

[0088] In a 1 mL reaction system, 100 μL of 10 mM ABTS aqueous solution, 100 μL of 50 mM H2O2 aqueous solution and 775 μL of 0.1 M PBS buffer (pH 7.0) were sequentially added to make the final concentrations of ABTS and H2O2 1 mM and 5 mM, respectively; then 25 μL of the sample to be tested was added, and the sample to be tested was 0.125 mg / mL free Cytc solution, Cytc@Cu-MOF, Cytc@ZIF-8, Cytc@ZIF-L catalyst containing the same amount of Cytc (3.125 μg) according to the catalyst loading rate, Cyt c&Cu-MOF prepared in Comparative Example 3, Cu-MOF catalyst, copper ions or squarate of Example 1. After mixing uniformly, the change in absorbance at 420 nm of the product was recorded at room temperature for 2 min to calculate the activity of the enzyme (Formula 3).

[0089] activity = ⊿A / ⊿t * εABTS (Formula 3) In the formula, ⊿A represents the change in absorbance at 420 nm; ⊿t represents the reaction time; and εABTS represents the molar extinction coefficient of ABTS, wherein the molar extinction coefficient of ABTS at A420 is 36800 M -1 cm -1 .

[0090] (2) Stability test ① Temperature and organic solvent stability: the sample to be tested (free Cytc solution, Cytc@Cu-MOF, Cytc@ZIF-8, Cytc@ZIF-L catalyst (Cyt c concentration is 0.125 mg / mL)) was incubated at a temperature (60-90℃) and in different organic solvents (DMSO, DMF, THF, acetone, urea) for a known period of time, and then its activity was determined according to the above enzyme activity test method. The activity before treatment was taken as 100%, and the relative activity was calculated.

[0091] ②Cycling stability: after each use, Cyt c@Cu-MOF and Cyt c@ZIF-L were separated by centrifugation, washed with deionized water, and reused in the next cycle. Then its activity was determined according to the above enzyme activity test method, and this process was repeated for 7 times. The activity of the catalyst that was not recycled was taken as 100%, and the relative activity was calculated.

[0092] ③Storage stability: Cyt c and Cyt c@Cu-MOF were dispersed in deionized water and stored at 4°C environment, and the sample was recovered every 24 h, and its activity was determined according to the above enzyme activity test method. The relative activity was calculated based on the enzyme activity of the freshly prepared Cyt c and Cyt c@Cu-MOF as 100%.

[0093] 2、Experimental results The relative enzyme activity column chart is shown in Figure 5 The relative activity of the Cu-MOF catalyst of Example 1, Cu salt and squarate as catalysts was only 25%, 15% and close to 0% respectively, which showed that the Cu-MOF or the raw material constituting it itself almost did not have the ability to catalyze the oxidation of ABTS. The relative activity of the Cyt c@Cu-MOF prepared in Example 2 was the highest, reaching about 230%, which was significantly higher than that of other groups. In particular, compared with the free enzyme, the Cyt c@Cu-MOF prepared in Example 2 was 2.3 times the activity of the free enzyme, which showed good catalytic activity. This result showed that for improving the catalytic performance of Cyt c, the overall structure of the Cyt c@Cu-MOF composite material played a key role in improving the catalytic performance of Cyt c, rather than each component constituting the Cyt c@Cu-MOF.

[0094] In addition, the present application also tested the enzymatic reaction kinetics of the Cyt c@Cu-MOF of Example 2, the Cyt c@ZIF-8 of Comparative Example 1, the Cyt c@ZIF-L of Comparative Example 2, the free Cyt c and the Cyt c&Cu-MOF of Comparative Example 3, and the results are shown in Figure 6 The Km of the Cyt c in the free state was 28.5 mM, the Km of the Cyt c&Cu-MOF obtained by physical mixing was 9.3 mM, and the Km of the Cyt c@Cu-MOF was 7.6 mM. The significant reduction of the Km value proved that the Cyt c@Cu-MOF had a higher affinity for the substrate than the free Cyt c or the Cyt c&Cu-MOF obtained by physical mixing, and it was speculated that the secondary structure of the immobilized enzyme changed favorably, which increased the contact between the enzyme active center and the substrate, which showed that the Cyt c@Cu-MOF prepared according to the method described in Example 2 had high catalytic activity.

[0095] It can be seen from Figure 7 that taking the enzyme catalytic activity before each treatment as 100%, the Cyt c@Cu-MOF prepared in Example 2 had a relative activity of 100% at different temperatures (60~90°C, Figure 7 a) and different solvent (DMSO, DMF, THF, acetone, urea) environments Figure 7The enzyme activities of b) are significantly higher than that of free Cyt c, and also higher than those of Cyt c@ZIF-8 of Comparative Example 1 and Cyt c@ZIF-L of Comparative Example 2, indicating that the Cyt c@Cu-MOF obtained in the application has the characteristic of high stability.

[0096] Taking the catalytic activity of the enzyme used for the first time as 100%, with the increase of the number of cycles, the activity of Cyt c@ZIF-L of Comparative Example 2 decreases more rapidly, and after the 7th cycle, it decreases to about 45%, while the Cyt c@Cu-MOF of Example 2 still maintains about 72% (Example 2: 72% vs. Comparative Example 2: 45%). Figure 7 c) indicating that the Cyt c@Cu-MOF obtained in the application has better recycling stability, which is conducive to the efficient reuse of the enzyme, reduces the cost, and provides economic feasibility support for industrial application.

[0097] Taking the catalytic activity of the enzyme used for the first time as 100%, with the increase of the number of cycles, the activity of Cyt c@ZIF-L of Comparative Example 2 decreases more rapidly, and after the 7th cycle, it decreases to about 45%, while the Cyt c@Cu-MOF of Example 2 still maintains about 72% (Example 2: 72% vs. Comparative Example 2: 45%). Figure 7 d), which provides economic feasibility support for industrial application.

[0098] Experimental Example 4: Test of degradation of phenolic compounds 1. Experimental method Take 180 μL of 4-AAP (4-aminoantipyrine, as a chromogenic agent) aqueous solution and 180 μL of phenolic compound aqueous solution, add to 600 μL of PBS buffer (pH = 7), then add 30 μL of H2O2 aqueous solution, finally add 10 μL of Cyt c@Cu-MOF (or Cyt c@ZIF-8 prepared in Comparative Example 1 or Cyt c@ZIF-L prepared in Comparative Example 2) aqueous dispersion prepared according to the method of Example 2 (the Cyt c concentration in the three enzyme catalyst aqueous dispersions is 0.125 mg / mL), mix well, the final reaction system is 1 mL, after standing at room temperature for 2 h, centrifugal separation (8000 r / min, 3 min) is carried out, and the absorbance of the supernatant at 510 nm is measured. The principle of the determination is that the quinone intermediate of the phenolic compound is produced by catalytic oxidation of the phenolic compound, the quinone intermediate couples with 4-aminoantipyrine (4-AAP) to generate red quinonimine dye which has a maximum absorption peak at 510 nm. Therefore, in the application of phenol degradation, the larger the absorbance value, the better the degradation effect.

[0099] Before mixing, the concentrations of the 4-AAP aqueous solution and the aqueous solutions of phenolic compounds (including 2,4-dichlorophenol, 2,6-dimethylphenol, hydroquinone, phenol, 1-naphthol, o-nitrophenol, and 2,6-dichlorophenol) were both 1 mg / mL; the molar concentration of the PBS buffer was 0.1 M; the molar concentration of the H2O2 aqueous solution was 50 mM; and the concentration of the immobilized enzyme in the Cyt c@Cu-MOF (or Cyt c@ZIF-8 or Cyt c@ZIF-L) aqueous dispersion was 0.125 mg / mL.

[0100] 2. Experimental Results The enzyme catalysts prepared according to the methods of Example 2, Comparative Example 1, and Comparative Example 2, and the liquid products of free Cyt c catalyzing the degradation of phenolic compounds were used as experimental subjects. The absorbance of the obtained products at 510 nm was measured using a UV-Vis spectrophotometer (n=3 for each experiment, average value was taken). This was to evaluate the degradation effect of the prepared catalysts on phenolic compounds; a higher absorbance value indicated a better catalytic degradation effect. Experimental results are as follows: Figure 8 and Figure 9 .

[0101] Depend on Figure 8 It can be seen that Cyt c@Cu-MOF exhibits the highest reaction rate throughout the entire reaction process, especially in the initial stage, where its reaction rate is significantly higher than that of the other two enzyme catalysts and free Cyt c. This phenomenon indicates that Cyt c@Cu-MOF has a significant catalytic degradation effect on phenolic compounds.

[0102] Figure 9 The results clearly show that the absorbance values ​​of different phenolic compounds treated with Cyt c@Cu-MOF in Example 2 were significantly higher than those of free Cyt c, ranging from 1.6 to 14 times. This indicates that Cyt c@Cu-MOF significantly enhances the catalytic degradation ability of various phenolic compounds. This result demonstrates that Cyt c@Cu-MOF exhibits good catalytic effects on a variety of phenolic compounds, showcasing its broad application potential in the degradation of phenolic compounds.

[0103] Experimental Example 5: Degradation Test of Organic Dyes (1) Catalytic degradation of different types of organic dyes Take 100 μL of organic dye aqueous solution, add 150 μL of hydrogen peroxide aqueous solution, then add 725 μL of PBS buffer (use the buffer with the optimal pH of each dye), and finally add 25 μL of Cyt c@Cu-MOF (or free Cyt c aqueous solution) aqueous dispersion prepared according to the method in Example 2. Mix well, let stand at room temperature for 2 h, and centrifuge (8000 r / min, 5 min) to collect the degraded liquid product.

[0104] Wherein, the molar concentration of the organic dye aqueous solution (including methyl orange, indigo carmine, rhodamine B, methylene blue, reactive blue 4) is 1 mM; the molar concentration of the PBS buffer (taking the optimal pH value of each dye, the optimal PBS buffer pH value of methyl orange, indigo carmine, rhodamine B, methylene blue, and reactive blue 4 dyes is 8, 7, 8, 8, and 8, respectively) is 0.1 M; the molar concentration of the H2O2 aqueous solution is 30 mM; the concentration of the enzyme in the Cyt c@Cu-MOF dispersion (or free Cyt c solution) is 0.125 mg / mL.

[0105] (2) Catalytic degradation of organic dyes based on continuous flow device The method for constructing a continuous flow device to catalytically degrade dyes is as follows: the organic dye solution (including methyl orange (MO), indigo carmine (IC), rhodamine B (RhB), methylene blue (MB), and reactive blue 4 (RB4)) and the H2O2 solution are mixed uniformly to obtain a reaction liquid, which is placed in a raw material bottle. The continuous flow reactor is filled with 1.2 mg of Cyt c@Cu-MOF solid prepared according to the method of Example 2. The peristaltic pump is started, and the reaction liquid in the raw material bottle is pumped into the continuous flow reactor at a flow rate of 200 μL / min. After running the reaction at room temperature for 2 h, the liquid product after degradation in the product bottle is collected.

[0106] Wherein, the molar concentration of the organic dye solution (including methyl orange, indigo carmine, rhodamine B, methylene blue, and reactive blue 4) is 0.4 mM; the molar concentration of the H2O2 solution is 20 mM; both are prepared using a PBS buffer with a molar concentration of 0.1 M (taking the optimal pH value of each dye, the optimal PBS buffer pH value of methyl orange, indigo carmine, rhodamine B, methylene blue, and reactive blue 4 dyes is 8, 7, 8, 8, and 8, respectively), and the volume ratio of the two is 1:1.

[0107] (3) Batch catalytic degradation of organic dyes The difference between the above (2) and the operation of catalytically degrading organic dyes based on a continuous flow device is that the reaction is not carried out using a continuous flow device, but a certain amount of organic dye solution (indigo carmine (IC)), H2O2 solution, and Cyt c@Cu-MOF solid prepared according to the method of Example 2 are mixed uniformly. The volume of the organic dye solution and the hydrogen peroxide solution is 12 mL, and the other conditions are the same.

[0108] Wherein, the calculation method of the catalytic efficiency (CE) is shown in formula 4: CE= (C0-C t ) *V / m*t (formula 4) Wherein, C0 is the initial concentration of the dye, C twherein V is the volume of the collected product, m is the mass of Cyt c@Cu-MOF catalyst in the system, and t is the reaction time. The dye concentration is calculated by the IC dye standard curve (which is obtained by following the conventional method).

[0109] 2. Experimental results The liquid product collected by the above operation was used as the experimental object to test the degradation degree (n = 3 for each experiment, and the average value was taken). The absorbance was measured at each maximum absorption wavelength by using a UV spectrophotometer, and the degradation degree was evaluated by calculating the dye degradation rate. The wavelengths of the maximum absorption peaks of methyl orange (MO), indigo carmine (IC), rhodamine B (RhB), methylene blue (MB), and reactive blue 4 (RB4) dyes were 489 nm, 608 nm, 554 nm, 664 nm, and 659 nm, respectively, and the experimental results are shown in Figures 10-11

[0110] wherein, Figure 10 The degradation rates of different types of organic dyes catalyzed by Cyt c@Cu-MOF and free Cyt c were compared according to the method of Example 2, and the results showed that the Cyt c@Cu-MOF catalyst exhibited significant advantages in catalytic degradation of various dyes.

[0111] Figure 11 The results of the comparison of the efficiency of the enzyme catalyst Cyt c@Cu-MOF of Example 2 in degrading organic dyes in continuous flow and batch catalysis modes are shown in the figure, which shows that the catalytic efficiency (CE) of the continuous flow is significantly higher than that of the batch catalysis, indicating that the above-mentioned continuous flow reactor exhibits higher efficiency in catalytic degradation of dyes, providing a new idea for its industrial application, and has important application value and broad industrialization prospect.

[0112] Example 6: Determination of the secondary structure of Cyt c The secondary structures of Cyt c and Cyt c@Cu-MOF were analyzed by performing spectral scanning of Cyt c and Cyt c@Cu-MOF by using a fluorescence spectrophotometer and a UV-UV spectrophotometer, and performing peak fitting of the amide I band of the FTIR spectrum.

[0113] The results are shown in Figure 12 ​As shown in a-e, after embedding, the secondary structure of Cytc has slightly changed, the fluorescence and UV-Vis spectra show that the heme microenvironment is slightly disturbed, and the iron changes from low spin to high spin; FTIR analysis shows that the a-helix decreases (50% to 20%) and the beta-turn increases (18% to 50%). This indicates that the embedding of Cu-MOF regulates the secondary structure of cytochrome c. This structural remodeling enhances the flexibility of the active site and the substrate affinity, optimizes the active conformation, and thus significantly improves the catalytic activity of the enzyme.

[0114] In summary, by constructing the Cyt c@Cu-MOF composite system, the present application realizes efficient immobilization of the enzyme, maintains high catalytic activity, greatly improves the stability and practicality, and provides a new type of efficient and sustainable catalyst solution for the application of biological catalysis technology in the fields of environmental remediation and green chemistry.

[0115] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods and shall be included in the protection scope of the present application.

Claims

1. A method for preparing an immobilized enzyme catalyst based on a copper-based metal-organic framework, characterized in that, Includes the following steps: Cytochrome c and squaric acid or its salt solution are mixed and then added to a copper salt solution. The mixture is allowed to react fully at room temperature. Solid-liquid separation is performed, the precipitate is washed, and the solid product is collected and dried. The result is an immobilized enzyme catalyst based on a copper-based metal-organic framework. The pH condition for mixing the cytochrome c and squaric acid or its salt solution is 5.5 to 7.

5.

2. The preparation method according to claim 1, characterized in that, The molar ratio of the squaric acid or its salt to the copper salt is (1~2):

1.

3. The preparation method according to claim 1, characterized in that, The molar ratio of cytochrome c to copper salt is (10~30) mg: 1 mol.

4. The preparation method according to claim 1, characterized in that, The copper salt includes copper acetate, copper chloride, copper sulfate, copper nitrate, or a hydrate of any of the above copper salts.

5. The copper-based metal-organic framework immobilized enzyme catalyst prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the copper-based metal-organic framework immobilized enzyme catalyst of claim 5 in the degradation of organic pollutants; preferably, the organic pollutants include phenolic compounds and / or organic dyes.

7. The application according to claim 6, characterized in that, The phenolic compounds are phenolic compounds containing aromatic structures.

8. The application according to claim 7, characterized in that, The aromatic phenolic compounds include one or more of 2,4-dichlorophenol, 2,6-dimethylphenol, phenol, 1-naphthol, 4-methoxyphenol, hydroquinone, and o-nitrophenol.

9. The application according to claim 6, characterized in that, The organic dyes include one or more of methyl orange, indigo carmine, rhodamine B, methylene blue, and reactive blue.

10. A method for catalytically degrading organic pollutants, characterized in that, The catalytic degradation of organic pollutants is carried out using an immobilized enzyme catalyst comprising the copper-based metal-organic framework of claim 5.

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