A copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity and a preparation method and application thereof

Abstract

The application provides a copper-penicillamine / imidazole nanometer enzyme with laccase-like activity and a preparation method and application thereof, and the preparation method comprises the following steps: S1, solution preparation: S1a, dissolving copper chloride in deionized water to obtain a copper chloride solution; S1b, dissolving penicillamine in deionized water to obtain a penicillamine solution; S1c, dissolving dimethyl imidazole in deionized water to obtain a dimethyl imidazole solution; S2, mixing the copper chloride solution, the penicillamine solution and the dimethyl imidazole solution with deionized water in a volume ratio of (15-2):(8-1):1:(18-2), and stirring at a temperature of 20-50 DEG C to obtain the copper-penicillamine / imidazole nanometer enzyme with laccase-like activity. The copper-penicillamine / imidazole nanometer enzyme with laccase-like activity prepared by the application can catalyze the degradation of various phenolic compounds, reduce the concentration of the phenolic compounds, and thus achieve the purpose of reducing the pollution of the phenolic compounds in water.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nano-enzymes, in particular to a copper-penicillamine / imidazole nano-enzyme with laccase-like activity and a preparation method and application thereof. BACKGROUND

[0002] Phenolic compounds are widely used in chemical, pharmaceutical and papermaking industries, and the wastewater discharged by these related industries contains a high content of phenolic compounds. Phenolic compounds have strong biological toxicity, and improper discharge of wastewater containing phenolic compounds can seriously pollute water bodies and thus endanger human health. For example, chlorophenolic compounds have a hormone-like effect, which can affect the level of sex hormones in the body and the expression of sex hormone synthesis-related genes, and affect the number of reproductive cells, and have been included in the list of priority pollutants for monitoring by countries around the world. Therefore, the degradation and treatment of phenolic compounds in related industrial wastewater is a problem worthy of attention.

[0003] Natural laccase is a polyphenol oxidase containing four copper ions, which can be used for the catalytic oxidation of phenolic compounds. Therefore, laccase is widely used to solve the problem of water pollution caused by phenolic compounds. However, laccase is essentially a protein, which has the problems of poor stability, high production cost and non-reusability. At present, there are mainly two strategies to solve the above problems, one is to immobilize laccase by using polymers, magnetic beads and the like, and the other is to synthesize nano-enzymes with laccase activity. Nano-enzymes are nano-materials with enzyme-like activity, and the nano-enzymes with laccase activity reported so far include cerium dioxide (Chem. Eng. Sci. 2015, 262, 747-755), cuprous oxide (ACS Sustainable Chem. Eng. 2022, 10, 4, 1398-1407), copper and nucleotide coordination nanoparticles (ACS Appl. Mater. Interfaces 2017, 9, 1352-1360), copper and amino acid coordination nanoparticles (patent CN202211264345.9) and copper and peptide coordination nanoparticles (Applied Catalysis B: Environmental, 2019, 254, 452; patent CN202011262686.3) and the like. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a preparation method of a copper-penicillamine / imidazole nano-enzyme with laccase-like activity, so as to solve the problems of poor stability and high cost of natural laccase for phenolic water pollutants, and to meet the market demand for nano-enzymes with laccase-like activity.

[0005] To solve the above problems, the present application provides a preparation method of a copper-penicillamine / imidazole nano-enzyme with laccase-like activity, comprising the following steps:

[0006] S1: solution preparation:

[0007] S1a: dissolving copper chloride in deionized water to obtain a copper chloride solution, the concentration of the copper chloride solution being 0.1-7 mg / mL;

[0008] S1b: dissolving penicillamine in deionized water to obtain a penicillamine solution, the concentration of the penicillamine solution being 0.1-5 mg / mL;

[0009] S1c: dissolving dimethylimidazole in deionized water to obtain a dimethylimidazole solution, the concentration of the dimethylimidazole being 0.1-5 mg / mL;

[0010] S2: mixing the copper chloride solution, the penicillamine solution and the dimethylimidazole solution prepared in step S1 with deionized water in a volume ratio of (15-2):(8-1):1:(18-2), and stirring at a temperature of 20-50℃ to obtain the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity.

[0011] As a preferred scheme, in step S1a, the concentration of the copper chloride solution is 5-7 mg / mL.

[0012] As a preferred scheme, in step S1b, the concentration of the penicillamine solution is 3-4 mg / mL.

[0013] As a preferred scheme, in step S1c, the concentration of the dimethylimidazole is 1-3 mg / mL.

[0014] As a preferred scheme, in step S2, the volume ratio of the copper chloride solution, the penicillamine solution and the dimethylimidazole solution to deionized water is 10:5:1:18.

[0015] As a preferred scheme, in step S2, the stirring temperature is 30-40℃, and the stirring time is 0.25-1.5 h.

[0016] The second technical problem to be solved by the present application is to provide a copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity to solve the problem of high cost of conventional enzymes in solving the water pollution problem of phenol and other phenolic compounds, and to provide a copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity with low preparation cost.

[0017] To solve the above problems, the present application provides a copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, which is prepared by the above preparation method.

[0018] The copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity prepared in the application is a special nanomaterial with enzyme-like activity, and its special structure endows it with the activity of natural laccase, which can catalyze the reaction of laccase substrates.

[0019] The third technical problem to be solved by the application is to provide an application of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, so as to solve the problems of difficulty in degrading phenol by conventional enzymes and high degradation cost.

[0020] In order to solve the above problems, the application provides the application of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, and the application includes catalyzing the degradation of phenol.

[0021] As a preferred scheme, the application includes catalyzing the degradation of 2,4-dichlorophenol, including the following steps: taking 4-aminoantipyrine as a chromogenic agent, taking 2,4-dichlorophenol as a phenolic compound representative substrate, testing the degradation performance of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity on 2,4-dichlorophenol (2,4-DP), including:

[0022] A1: dissolving 4-aminoantipyrine in deionized water to obtain a 4-aminoantipyrine solution, and dissolving 2,4-dichlorophenol in deionized water to obtain a 2,4-dichlorophenol solution;

[0023] A2: mixing the 4-aminoantipyrine solution, the 2,4-dichlorophenol solution, the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, a Tris-HCl buffer with a pH of 7.4, and deionized water to obtain a total reaction system, and after stirring, observing the color change of the solution to determine the degradation capacity of the copper-penicillamine / imidazole nanoscale enzyme on dichlorophenol;

[0024] The volume of the total reaction system is 0.8-1.2 mL, the copper-penicillamine / imidazole nanoscale enzyme is added in an amount of 30-100 muL, the concentration of 2,4-dichlorophenol is 0.05-0.15 mg / mL, and the concentration of 4-aminoantipyrine is 0.05-0.15 mg / mL.

[0025] The reaction temperature of the total reaction system is 20-50 DEG C, and the stirring time is 0.25-1.5 h.

[0026] As a preferred scheme, the volume of the total reaction system is 1.0 mL, the copper-penicillamine / imidazole nanoscale enzyme is added in an amount of 60 muL, the concentration of 2,4-dichlorophenol is 0.1 mg / mL, and the concentration of 4-aminoantipyrine is 0.1 mg / mL.

[0027] The reaction temperature of the total reaction system is 30-40 DEG C, and the stirring time is 1 h.

[0028] By configuring the copper-penicillamine / imidazole nanoscale enzyme and degrading 2,4-dichlorophenol, the effect is significantly better than that of laccase. The application has the advantages of simple preparation, simple operation, low cost and the like.

[0029] In summary, the copper-penicillamine / imidazole nanoscale enzyme and the application thereof provided by the application are very convenient in preparation and operation, can be produced in a lower cost and a more simple and rapid manner, are faster and more thorough in degrading phenol, and are obviously higher than existing laccase in the degradation capacity of phenol. The above advantages all indicate that the copper-penicillamine / imidazole nanoscale enzyme has high potential value in the current environmental remediation market. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 : Transmission electron microscope image of copper-penicillamine / imidazole nanoscale enzyme in experimental example 4;

[0031] Figure 2 : Dynamic light scattering diagram of copper-penicillamine / imidazole nanoscale enzyme obtained in experimental example 4;

[0032] Figure 3 : Ultraviolet absorption spectrum diagram and sample diagram of samples a, b, c, d and e in experimental example 4;

[0033] Figure 4 : Ultraviolet absorption spectrum diagram and sample diagram of copper-penicillamine / imidazole nanoscale enzyme and laccase after degrading dichlorophenol in experimental example 4;

[0034] Figure 5 : Schematic diagram of degradation of 3-nitrophenol, 4-nitrophenol and acetaminophen by copper-penicillamine / imidazole nanoscale enzyme with 4-aminoantipyrine as a chromogenic agent in experimental example 4;

[0035] Figure 6 : Stability comparison diagram of copper-penicillamine / imidazole nanoscale enzyme and natural laccase stored for 30 minutes at different storage temperatures. DETAILED DESCRIPTION

[0036] The technical solutions of the application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0037] The application provides a preparation method of copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, comprising the following steps:

[0038] S1: solution preparation:

[0039] S1a: dissolving copper chloride in deionized water to obtain a copper chloride solution, the concentration of the copper chloride solution being 0.1-7 mg / mL;

[0040] S1b: dissolving penicillamine in deionized water to obtain a penicillamine solution, the concentration of the penicillamine solution being 0.1-5 mg / mL;

[0041] S1c: dissolving dimethylimidazole in deionized water to obtain a dimethylimidazole solution, the concentration of the dimethylimidazole being 0.1-5 mg / mL;

[0042] S2: mixing the copper chloride solution, the penicillamine solution and the dimethylimidazole solution prepared in the step S1 with deionized water in a volume ratio of (15-2):(8-1):1:(18-2), and stirring at a temperature of 20-50℃ to obtain the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity.

[0043] Preferably, in the step S1a, the concentration of the copper chloride solution is 5-7 mg / mL.

[0044] Preferably, in the step S1b, the concentration of the penicillamine solution is 3-4 mg / mL.

[0045] Preferably, in the step S1c, the concentration of the dimethylimidazole is 1-3 mg / mL.

[0046] Preferably, in the step S2, the volume ratio of the copper chloride solution, the penicillamine solution, the dimethylimidazole solution and deionized water is 10:5:1:18.

[0047] Preferably, in the step S2, the stirring temperature is 30-40℃, and the stirring time is 0.25-1.5 h.

[0048] The application provides a copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, which is prepared by the above preparation method.

[0049] The application provides an application of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, which includes catalyzing the degradation of phenol.

[0050] Preferably, the application includes catalyzing the degradation of 2,4-dichlorophenol, including the following steps: taking 4-aminoantipyrine as a chromogenic agent, taking 2,4-dichlorophenol as a phenolic compound representative substrate, testing the degradation performance of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity on 2,4-dichlorophenol, including:

[0051] A1: 4-aminoantipyrine is dissolved in deionized water to obtain a 4-aminoantipyrine solution, and 2,4-dichlorophenol is dissolved in deionized water to obtain a 2,4-dichlorophenol solution;

[0052] A2: the 4-aminoantipyrine solution, the 2,4-dichlorophenol solution, the copper-penicillamine / imidazole nanozyme with laccase-like activity, the Tris-HCl buffer with a pH of 7.4, and deionized water are mixed to obtain a total reaction system, after stirring, the color change of the solution is observed to determine the degradation capacity of the copper-penicillamine / imidazole nanozyme on dichlorophenol;

[0053] The volume of the total reaction system is 0.8-1.2 mL, the copper-penicillamine / imidazole nanozyme is added in an amount of 30-100 μL, the 2,4-dichlorophenol concentration is 0.05-0.15 mg / mL, and the 4-aminoantipyrine concentration is 0.05-0.15 mg / mL.

[0054] The reaction temperature of the total reaction system is 20-50 °C, and the stirring time is 0.25-1.5 h.

[0055] Preferably, the volume of the total reaction system is 1.0 mL, the copper-penicillamine / imidazole nanozyme is added in an amount of 60 μL, the 2,4-dichlorophenol concentration is 0.1 mg / mL, and the 4-aminoantipyrine concentration is 0.1 mg / mL.

[0056] The reaction temperature of the total reaction system is 30-40 °C, and the stirring time is 1 h.

[0057] and the ultraviolet absorption spectrum of the copper-penicillamine / imidazole nanozyme and laccase after degrading dichlorophenol is respectively determined:

[0058] The ultraviolet absorption spectrum of the copper-penicillamine / imidazole nanozyme after degrading dichlorophenol is measured by an ultraviolet-visible absorption spectrometer, the copper-penicillamine / imidazole nanozyme, the 4-aminoantipyrine solution, the 2,4-dichlorophenol solution, and the Tris-HCl buffer with a pH of 7.4 are taken, deionized water is added, and 2 mL of a to-be-tested solution is prepared; the laccase, the 4-aminoantipyrine solution, the 2,4-dichlorophenol solution, and the Tris-HCl buffer with a pH of 7.4 are taken, deionized water is added, and 2 mL of a to-be-tested solution is prepared. 2 mL of deionized water is used as a reference solution.

[0059] The copper-penicillamine / imidazole solution is 50-70 μL, the 2,4-dichlorophenol solution is 100-200 μL, the 4-aminoantipyrine solution is 100-200 μL, the deionized water is 100-150 μL, and the Tris-HCl buffer with a pH of 7.4 is 1000-1500 μL.

[0060] The following embodiments are provided to further develop the above-mentioned scope to develop the present application in more detail:

[0061] Embodiment 1:

[0062] The present embodiment provides a copper-penicillamine / imidazole nanozyme with laccase-like activity and a preparation method thereof, the preparation method comprising the following steps:

[0063] S1: solution preparation:

[0064] S1a: dissolving copper chloride in deionized water to obtain a copper chloride solution, the concentration of the copper chloride solution being 6 mg / mL;

[0065] S1b: dissolving penicillamine in deionized water to obtain a penicillamine solution, the concentration of the penicillamine solution being 3.5 mg / mL;

[0066] S1c: dissolving dimethyl imidazole in deionized water to obtain a dimethyl imidazole solution, the concentration of the dimethyl imidazole being 2 mg / mL;

[0067] S2: mixing the copper chloride solution, the penicillamine solution and the dimethyl imidazole solution prepared in step S1 with deionized water in a volume ratio of 10:5:1:18, and stirring at a temperature of 35°C to obtain the copper-penicillamine / imidazole nanozyme with laccase-like activity.

[0068] The present application provides an application of the copper-penicillamine / imidazole nanozyme with laccase-like activity, which comprises catalyzing the degradation of phenol.

[0069] Preferably, the application comprises catalyzing the degradation of 2,4-dichlorophenol, which comprises the following steps: testing the degradation performance of the copper-penicillamine / imidazole nanozyme with laccase-like activity on 2,4-dichlorophenol with 4-aminoantipyrine as a chromogenic agent, which comprises:

[0070] A1: dissolving 4-aminoantipyrine in deionized water to obtain a 4-aminoantipyrine solution, and dissolving 2,4-dichlorophenol in deionized water to obtain a 2,4-dichlorophenol solution;

[0071] A2: mixing the 4-aminoantipyrine solution, the 2,4-dichlorophenol solution, the copper-penicillamine / imidazole nanozyme with laccase-like activity, a Tris-HCl buffer with a pH of 7.4 and deionized water to obtain a total reaction system, and observing the color change of the solution after stirring to determine the degradation capacity of the copper-penicillamine / imidazole nanozyme on dichlorophenol;

[0072] The reaction temperature of the total reaction system is 35°C, and the stirring time is 1 h.

[0073] Preferably, the total reaction system has a volume of 1.0 mL, the copper-penicillamine imidazole nanoscale enzyme is added in an amount of 60 μL, the 2,4-dichlorophenol has a concentration of 0.1 mg / mL, and the 4-aminoantipyrine has a concentration of 0.1 mg / mL.

[0074] Embodiment 2

[0075] The embodiment provides a copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity and a preparation method thereof, and the preparation method comprises the following steps:

[0076] S1: solution preparation

[0077] S1a: dissolving copper chloride in deionized water to obtain a copper chloride solution, wherein the concentration of the copper chloride solution is 0.1 mg / mL;

[0078] S1b: dissolving penicillamine in deionized water to obtain a penicillamine solution, wherein the concentration of the penicillamine solution is 0.1 mg / mL;

[0079] S1c: dissolving dimethyl imidazole in deionized water to obtain a dimethyl imidazole solution, wherein the concentration of the dimethyl imidazole is 0.1-5 mg / mL;

[0080] S2: mixing the copper chloride solution, the penicillamine solution and the dimethyl imidazole solution prepared in the step S1 with deionized water in a volume ratio of 15:8:1:18, and stirring at a temperature of 20°C to obtain the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity.

[0081] The embodiment provides an application of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, and the application comprises catalyzing the degradation of phenol.

[0082] Preferably, the application comprises catalyzing the degradation of 2,4-dichlorophenol, and comprises the following steps: taking 4-aminoantipyrine as a chromogenic agent to test the degradation performance of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity on 2,4-dichlorophenol, and the steps comprise:

[0083] A1: dissolving 4-aminoantipyrine in deionized water to obtain a 4-aminoantipyrine solution, and dissolving 2,4-dichlorophenol in deionized water to obtain a 2,4-dichlorophenol solution;

[0084] A2: mixing the 4-aminoantipyrine solution, the 2,4-dichlorophenol solution, the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, a Tris-HCl buffer solution with a pH of 7.4 and deionized water to obtain a total reaction system, stirring, and observing the color change of the solution to determine the degradation capacity of the copper-penicillamine / imidazole nanoscale enzyme on dichlorophenol.

[0085] The volume of the total reaction system is 0.8 mL, the copper-penicillamine / imidazole nanoscale enzyme is added in an amount of 30 μL, the concentration of 2,4-dichlorophenol is 0.05 mg / mL, and the concentration of 4-aminoantipyrine is 0.05 mg / mL.

[0086] The reaction temperature of the total reaction system is 20℃, and the stirring time is 0.25 h.

[0087] Embodiment 3:

[0088] The embodiment provides a copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity and a preparation method thereof, and the preparation method comprises the following steps:

[0089] S1: solution preparation:

[0090] S1a: dissolving copper chloride in deionized water to obtain a copper chloride solution, wherein the concentration of the copper chloride solution is 0.1-7 mg / mL;

[0091] S1b: dissolving penicillamine in deionized water to obtain a penicillamine solution, wherein the concentration of the penicillamine solution is 0.1-5 mg / mL;

[0092] S1c: dissolving dimethyl imidazole in deionized water to obtain a dimethyl imidazole solution, wherein the concentration of the dimethyl imidazole is 0.1-5 mg / mL;

[0093] S2: mixing the copper chloride solution, the penicillamine solution and the dimethyl imidazole solution prepared in the step S1 with deionized water in a volume ratio of 2:1:1:2, and stirring at a temperature of 50℃ to obtain the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity.

[0094] The application provides an application of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, and the application comprises catalyzing the degradation of phenol.

[0095] Preferably, the application comprises catalyzing the degradation of 2,4-dichlorophenol, and comprises the following steps: taking 4-aminoantipyrine as a chromogenic agent, testing the degradation performance of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity on 2,4-dichlorophenol (2,4-DP), and comprising the following steps:

[0096] A1: dissolving 4-aminoantipyrine in deionized water to obtain a 4-aminoantipyrine solution, and dissolving 2,4-dichlorophenol in deionized water to obtain a 2,4-dichlorophenol solution;

[0097] A2: mixed 4-aminoantipyrine solution, 2,4-dichlorophenol solution, copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, Tris-HCl buffer solution with pH 7.4 and deionized water to obtain a total reaction system, after stirring, observed the color change of the solution to determine the degradation capacity of copper-penicillamine / imidazole nanoscale enzyme on dichlorophenol;

[0098] The volume of the total reaction system was 1.2 mL, the copper-penicillamine / imidazole nanoscale enzyme was added in an amount of 100 μL, the 2,4-dichlorophenol concentration was 0.15 mg / mL, and the 4-aminoantipyrine concentration was 0.15 mg / mL.

[0099] The reaction temperature of the total reaction system was 50°C, and the stirring time was 1.5 h.

[0100] The following application uses actual data to further expand the above examples and the technical solutions of the present application:

[0101] Example 4:

[0102] The present embodiment provides a copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity and a preparation method and application thereof, and the preparation method comprises:

[0103] S1: solution preparation: S1a: 67.2 mg of copper chloride was dissolved in 10 mL of deionized water to obtain a copper chloride solution with a concentration of 6.72 / mL;

[0104] S1b: 37.3 mg of penicillamine was dissolved in 10 mL of deionized water to obtain a penicillamine solution with a concentration of 3.73 mg;

[0105] S1c: 2.05 mg of 2-methylimidazole was dissolved in 10 mL of deionized water to obtain a 2-methylimidazole solution;

[0106] S2: mixed the above solutions with deionized water in a ratio of 10:5:1:18, stirred at room temperature for 1 h to obtain the desired copper-penicillamine / imidazole nanoscale enzyme.

[0107] The copper-penicillamine / imidazole nanoscale enzyme obtained was imaged using a transmission electron microscope, and the results are shown in Figure 1 .

[0108] The copper-penicillamine / imidazole nanoscale enzyme obtained was tested by dynamic light scattering, and the results are shown in Figure 2 .

[0109] The degradation performance of copper-penicillamine / imidazole nanoscale enzyme on 2,4-dichlorophenol was determined:

[0110] The degradation performance of copper-penicillamine / imidazolium nanozyme (Cu-PA / IMD) on 2,4-dichlorophenol was tested using 4-aminoantipyrine as a chromogenic agent. 10 mg of 4-aminoantipyrine was dissolved in 10 mL of deionized water to obtain a 1 mg / mL 4-aminoantipyrine solution (4-AP), and 10 mg of 2,4-dichlorophenol was dissolved in 10 mL of deionized water to obtain a 1 mg / mL 2,4-dichlorophenol solution (2,4-DP). Five solutions (a, e, and d) were prepared according to Table 1 below. After reacting for 1 h, photographs were taken and UV-Vis absorption spectra were measured.

[0111] Table 1:

[0112]

[0113] like Figure 3 As shown, sample e in the experiment, after being mixed with 2,4-dichlorophenol solution, copper-penicillamine / imidazolium nanozyme, 4-aminoantipyrine solution, deionized water, and Tris-HCl, turned deep red after reacting at room temperature for 1 hour. Other samples remained transparent with no obvious color change. UV absorption spectroscopy analysis revealed that sample e had a significantly higher absorbance than the other samples. This indicates that the copper-penicillamine / imidazolium nanozyme has a good degradation effect on 2,4-dichlorophenol.

[0114] The UV absorption spectra of copper-penicillamine / imidazolium nanozyme and laccase after degradation of dichlorophenol were measured respectively:

[0115] The UV absorption spectrum of copper-penicillamine / imidazolium nanozyme after degradation of dichlorophenol was measured using a UV-Vis absorption spectrometer. The copper-penicillamine / imidazolium nanozyme was prepared using the same method as in Example 1. 120 μL of copper-penicillamine / imidazolium nanozyme, 200 μL of 4-aminoantipyrine solution, 200 μL of 2,4-dichlorophenol solution, and 40 μL of deionized water were added, followed by the addition of 1400 μL of Tris-HCl (pH=7) to prepare 2 mL of test solution a. 120 μL of laccase solution, 200 μL of 4-aminoantipyrine solution, and 200 μL of 2,4-dichlorophenol solution were added, followed by the addition of Tris-HCl (pH=7.4) buffer and 80 μL of deionized water to prepare 2 mL of test solution b. 2 mL of deionized water was used as a reference solution. The UV-Vis absorption spectrometer was preheated for 10 minutes, and the scanning wavelength range was 300-800 nm. The results are as follows: Figure 4 As shown, the reaction system catalyzed by copper-penicillamine / imidazolium nanozyme has higher absorbance compared to the laccase catalysis system.

[0116] The degradation efficiency of copper-penicillamine / imidazolium nanozyme on 3-nitrophenol (3-NP), 4-nitrophenol (4-NP) and p-acetylphenol (APAP) using 4-aminoantipyrine as substrate.

[0117] 10 mg of 4-aminoantipyrine was dissolved in 10 mL of deionized water to obtain a 1 mg / mL 4-aminoantipyrine solution. 10 mg of 3-nitrophenol was dissolved in 10 mL of deionized water to obtain a 1 mg / mL 3-nitrophenol solution. 10 mg of 4-nitrophenol was dissolved in 10 mL of deionized water to obtain a 1 mg / mL 4-nitrophenol solution. 10 mg of acetaminophen was dissolved in 10 mL of deionized water to obtain a 1 mg / mL acetaminophen solution. 100 μL of 3-nitrophenol, 4-nitrophenol, and acetaminophen solutions were mixed with 40 μL of deionized water, 60 μL of copper-penicillamine / imidazolium nanozyme, 100 μL of aminopyridine, and 700 μL of Tris-HCl pH 7.4 buffer, respectively. The mixtures were stirred at 40 °C for 1 h. The degradation activity of the copper-penicillamine / imidazolium nanozyme against 2,4-dichlorophenol was taken as 100%. The degradation capabilities of various other substrates were then calculated. Figure 5 The overall degradation efficiency shown has exceeded 80%, proving that copper-penicillamine / imidazolium nanozymes have high degradation performance for a variety of phenolic compounds.

[0118] This invention also compared the stability of the prepared copper-penicillamine / imidazolium nanozyme and natural laccase after storage at different storage temperatures for 30 minutes, such as... Figure 6 As shown, Figure 6 A comparison of the stability of copper-penicillamine / imidazolium nanozyme and natural laccase stored at different storage temperatures for 30 minutes.

[0119] The specific experimental procedure involved storing natural laccase and the copper-penicillamine / imidazolium nanozyme prepared in Example 4 at different temperatures for 30 minutes, and then storing them at 30°C. O Based on the temperature after storage (i.e., 30°C) O The activity of C was 100%, and its activity was measured after storage at different temperatures. Figure 6 The prepared copper-penicillamine / imidazolium nanozyme can be observed to be within the range of 30-90. O C exhibits strong stability and is significantly superior to conventional natural laccase.

[0120] The above embodiments further illustrate that the present invention provides a copper-penicillamine / imidazolium nanozyme with laccase-like activity that is low in preparation cost and simple to operate, as well as a method for its preparation. Furthermore, the nanozyme prepared by the present invention can react with a variety of phenolic compounds, reducing the harm of phenolic compounds and thus achieving the purpose of reducing pollution.

[0121] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited to this. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present disclosure.

Claims

1. A method for preparing a copper-penicillamine / imidazole nanoscale enzyme having laccase-like activity, characterized by: The method comprises the following steps: S1: solution preparation S1a: dissolving copper chloride in deionized water to obtain a copper chloride solution, the concentration of the copper chloride solution being 0.1-7 mg / mL; S1b: dissolving penicillamine in deionized water to obtain a penicillamine solution, the concentration of the penicillamine solution being 0.1-5 mg / mL; S1c: dissolving dimethylimidazole in deionized water to obtain a dimethylimidazole solution, the concentration of the dimethylimidazole being 0.1-5 mg / mL; S2: mixing the copper chloride solution, the penicillamine solution and the dimethylimidazole solution prepared in step S1 with deionized water in a volume ratio of (15-2):(8-1):1:(18-2), and stirring at a temperature of 20-50°C to obtain the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity.

2. The method for preparing copper-penicillamine / imidazole nanoszyme with laccase-like activity according to claim 1, characterized by: In step S1a, the concentration of the copper chloride solution is 5-7 mg / mL.

3. The method for preparing copper-penicillamine / imidazole nanoszyme with laccase-like activity according to claim 1, characterized by: In step S1b, the concentration of the penicillamine solution is 3-4 mg / mL.

4. The method for preparing copper-penicillamine / imidazole nanoszyme with laccase-like activity according to claim 1, characterized by: In step S1c, the concentration of the dimethylimidazole is 1-3 mg / mL.

5. The method for preparing copper-penicillamine / imidazole nanoszyme with laccase-like activity according to claim 1, characterized by: In step S2, the volume ratio of the copper chloride solution, the penicillamine solution, the dimethylimidazole solution and deionized water is 10:5:1:

18.

6. The method for preparing copper-penicillamine / imidazole nanoszyme with laccase-like activity according to claim 1, characterized by: In step S2, the stirring temperature is 30-40°C, and the stirring time is 0.25-1.5 h.

7. A copper-penicillamine / imidazole nanoszyme having laccase-like activity, characterized by: The copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity is prepared by any one of the preparation methods of claims 1-6.

8. Use of the copper-penicillamine / imidazole nanoszyme having laccase-like activity according to claim 7, characterized by: The application includes catalyzing the degradation of phenolic compounds.

9. Use of a copper-penicillamine / imidazole nanoszyme having laccase-like activity according to claim 8, characterized in that: The application includes catalyzing the degradation of 2,4-dichlorophenol, which comprises the following steps: using 4-aminoantipyrine as a chromogenic agent to test the degradation performance of the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity on 2,4-dichlorophenol, which comprises: A1: dissolving 4-aminoantipyrine in deionized water to obtain a 4-aminoantipyrine solution, and dissolving 2,4-dichlorophenol in deionized water to obtain a 2,4-dichlorophenol solution; A2: mixing the 4-aminoantipyrine solution, the 2,4-dichlorophenol solution, the copper-penicillamine / imidazole nanoscale enzyme with laccase-like activity, a Tris-HCl buffer with a pH value of 7.4 and deionized water to obtain a total reaction system, and observing the color change of the solution after stirring to determine the degradation capacity of the copper-penicillamine / imidazole nanoscale enzyme on dichlorophenol; The volume of the total reaction system is 0.8-1.2 mL, the copper-penicillamine / imidazole nanoscale enzyme is added in an amount of 30-100 µL, the concentration of 2,4-dichlorophenol is 0.05-0.15 mg / mL, and the concentration of 4-aminoantipyrine is 0.05-0.15 mg / mL. The reaction temperature of the total reaction system is 20-50°C, and the stirring time is 0.25-1.5 h.

10. Use of a copper-penicillamine / imidazole nanoszyme having laccase-like activity according to claim 9, characterized in that: The volume of the total reaction system is 1.0 mL, the copper-penicillamine / imidazole nanoscale enzyme is added in an amount of 60 µL, the 2,4-dichlorophenol concentration is 0.1 mg / mL, and the 4-aminoantipyrine concentration is 0.1 mg / mL; The reaction temperature of the total reaction system is 30-40 ℃, and the stirring time is 1 h.

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