Co-immobilized laccase-mediator system as well as preparation method and application thereof
By using the metal-organic framework material MnMOF to prepare a co-immobilized laccase-mediator system, the problems of poor stability and reusability of laccase were solved, and a highly efficient dye degradation effect was achieved, filling the gap in the research on laccase-mediator system immobilization.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ANHUI NORMAL UNIV
- Filing Date
- 2025-10-31
- Publication Date
- 2026-05-12
AI Technical Summary
In existing technologies, laccase has poor stability and reusability, and traditional carrier materials do not have good tunability and crystallinity, resulting in low immobilized enzyme activity. Furthermore, insufficient research on the immobilization of laccase-mediator systems limits its application potential in dye wastewater treatment.
Using metal-organic framework material MnMOF as an immobilization carrier, a co-immobilized laccase-mediator system MnMOF@Lac5@M@PEG was prepared by biomineralization. Combined with PEG encapsulation, the stability and catalytic activity of the enzyme were improved.
The co-immobilized laccase-mediator system prepared under ambient temperature and pressure exhibits good chemical and thermal stability, a catalytic activity increase of 190.78%, good reusability, and high degradation efficiency, making it suitable for the field of dye degradation.
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Figure CN122012420A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of immobilized enzyme preparation technology, specifically relating to a method for preparing a co-immobilized laccase-mediator system and its application. Background Technology
[0002] Alizarin Red S is an anthraquinone dye widely used for staining biological tissues, as a calcium ion indicator, and for dyeing textiles such as wool and silk. It is well-known for its vibrant color and specific chemical binding properties. However, as a synthetic dye with potential environmental risks, Alizarin Red is difficult to degrade naturally in the environment, exhibits bioaccumulation, and may cause mutagenicity and ecotoxicity in aquatic organisms. Therefore, its residues have become a concern in water pollution. However, compared to traditional physical adsorption and chemical oxidation methods, which have limited efficiency, high costs, and potential for producing toxic byproducts, biodegradation is considered an effective way to treat Alizarin Red pollution, offering advantages such as environmental friendliness, relatively simple operation, and more thorough degradation.
[0003] Lacase is a copper-containing polyphenol oxidase capable of catalyzing the oxidation of various aromatic compounds and is widely used in the paper industry, textile decolorization, bioremediation, and food processing. Lacase can catalyze the oxidative degradation of dye molecules, especially those containing phenols or aromatic amines. Of particular interest is the laccase-mediator system formed in the presence of mediator molecules (such as small molecule compounds like ABTS and HBT), which significantly expands the substrate range of laccase, enabling it to efficiently degrade dyes with more complex structures and higher oxidation potentials (such as anthraquinone and triphenylmethane dyes), destroying their chromophores and forming low-color or even colorless degradation products. However, the catalytic activity of free laccase is often significantly affected by various environmental factors such as pH, temperature, and inhibitors (such as halides), resulting in poor stability in practical applications. Furthermore, as a water-soluble protein, laccase is difficult to effectively separate and recover from complex dye wastewater systems after the reaction, leading to non-reusability, increased treatment costs, and limiting its potential for large-scale dye wastewater treatment. Crucially, current research on the overall immobilization of laccase-mediator systems is relatively limited. Exploring how to co-immobilize laccase and mediator molecules on a carrier is vital for fully realizing the degradation potential of the laccase-mediator system and overcoming its application bottlenecks, making it a direction worthy of in-depth research. Therefore, immobilizing laccase and its mediator system on a water-insoluble carrier using immobilization technology can effectively overcome the limitations of free enzymes / systems, significantly improving their stability, reusability, and ease of operation, thereby reducing processing costs while increasing the efficiency of dye degradation processes.
[0004] However, traditional carrier materials lack good tunability and crystallinity, which may lead to low protein loading efficiency, instability, and easy enzyme leaching, thus affecting the activity of immobilized enzymes. Metal-organic frameworks (MOFs) are novel porous crystalline hybrid materials composed of metal ions and organic ligands through strong coordination bonds. They possess many advantages such as tunable pore size, high specific surface area, and good chemical and thermal stability, making them promising new and excellent carriers for immobilized enzyme preparations. However, the surface charge and chemical properties of proteins or enzymes determine their ability to be encapsulated in MOFs. Therefore, the enzyme activity of most immobilized enzymes is not high. Doping MOFs with different metal ions, through competitive coordination between metal ions and organic ligands, is an effective way to improve the activity and stability of immobilized enzymes. Summary of the Invention
[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a novel laccase-mediator system, its preparation method and application, so as to solve the problems mentioned in the background technology.
[0006] To achieve the above objectives, the present invention provides a method for preparing a co-immobilized laccase-mediator system (MnMOF@Lac5@M@PEG), using laccase as the enzyme protein and metal-organic framework material MnMOF as the immobilization carrier, comprising the following steps: (1) Mix the laccase solution and 2-methylimidazole solution evenly and shake on a vortex mixer for 30-40s to obtain mixed solution a; (2) Add manganese chloride solution to the mixed solution a obtained in step (1), mix evenly and shake on a vortex shaker for 30-40s, then place in a horizontal shaker at 30℃ for 30 min to obtain mixed solution b; (3) Add equimolar amounts of p-coumaric acid and ABTS to the mixed solution b obtained in step (2), mix and stir evenly, react at 30°C, then add polyethylene glycol PEG, mix and stir evenly to obtain mixed solution c; (4) Centrifuge the mixed solution c obtained in step (3) at 10,000 rpm for 5-8 min, discard the supernatant, and obtain a brown precipitate. Rinse the brown precipitate with deionized water several times until it turns brownish-red, and dry it in a constant temperature oven at 37°C for 10-12 h to obtain the immobilized laccase-mediator system MnMOF@Lac5@PEG@M.
[0007] Further, in step (2), the mass concentration of laccase in the mixed solution b is 0.2-1.4 µg / mL, the molar concentration of 2-methylimidazole is 25-175 mM, and the molar concentration of manganese chloride is 50-350 mM.
[0008] Further, in step (3), the molar concentrations of ABTS and p-coumaric acid in the mixed solution c are 0.5-12 mM, and the mass concentration of PEG is 20 mg / mL.
[0009] Furthermore, in step (3), the reaction time at the 30°C temperature is 3-5 hours.
[0010] This invention discloses a method for preparing the co-immobilized laccase-mediator system obtained by the above preparation method.
[0011] Furthermore, the immobilized laccase-mediator system has a hexagonal morphology with a diameter of 0.05-0.1 μm.
[0012] The present invention also discloses the application of the above-mentioned co-immobilized laccase-mediator system in the field of dye degradation.
[0013] Further, specifically including: uniformly dispersing alizarin red in immobilized lipase solution, shaking the reaction mixture to react, reacting at 30 ℃ for 15 min, and centrifuging to determine the alizarin red content in the supernatant after the reaction is completed.
[0014] The beneficial effects of this invention are: 1. Co-immobilized laccase MnMOF@Lac5 was successfully prepared under ambient temperature and pressure via biomineralization. It exhibits excellent chemical and thermal stability (retaining 54.17% of its initial activity after incubation at 30 °C for 100 h), non-toxicity, and a simple synthesis process. Compared to the free enzyme, it further improves catalytic activity (190.78% of the free enzyme's activity) and stability, demonstrating good reusability; after being reused 8 times, it still retains 50% of its initial activity.
[0015] 2. PEG encapsulation of MnMOF@M@Lac5 solves the problem of mediator loss, has better industrial applicability, and fills the gap in the research on immobilization of laccase-mediator systems.
[0016] 3. This co-immobilized laccase-mediator system provides a green and efficient method for dye degradation. Attached Figure Description
[0017] Figure 1 For the determination of the 2-methylimidazole molar concentration during enzyme immobilization in Example 3 (■, relative enzyme activity; ●, immobilization efficiency); Figure 2 For example 4, the determination of the molar concentration of manganese chloride during enzyme immobilization (■, relative enzyme activity; ●, immobilization efficiency); Figure 3For example 5, the determination of laccase protein mass concentration during enzyme immobilization (■, relative enzyme activity; ●, immobilization efficiency); Figure 4 Fourier transform infrared image of MnMOF@Lac5@M@PEG prepared in Example 1; Figure 5 The effect of reaction pH on the activity of laccase Lac5 and MnMOF@Lac5@M@PEG prepared in Example 1 is shown in the figure. Figure 6 The graph shows the effect of reaction temperature on the activity of laccase Lac5 and the MnMOF@Lac5@M@PEG enzyme prepared in Example 1. Figure 7 The stability of laccase Lac5 and MnMOF@Lac5@M@PEG prepared in Example 1 in buffer solution at 4°C and pH 6.5; Figure 8 Thermal stability of Lacase Lac5 and MnMOF@Lac5@M@PEG prepared in Example 1 at 30°C; Figure 9 The effect of mediator molar concentration on the activity of immobilized laccase-mediator system; Figure 10 A graph showing the number of times MnMOF@Lac5@M@PEG prepared for laccase Lac5 and Example 1 can be reused. Figure 11 Time gradient diagram of alizarin red degradation by laccase Lac5 and MnMOF@Lac5@M@PEG prepared in Example 1. Detailed Implementation
[0018] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention. Example 1
[0019] A method for preparing a co-immobilized laccase-mediator system includes the following steps: (1) Take 500 uL of laccase solution with a mass concentration of 2 µg / mL and 75 uL of 1 M 2-methylimidazole solution, mix them evenly and shake them on a vortex shaker for 30s to obtain mixed solution a; (2) Add 100 μL of 2 M manganese chloride solution to the mixed solution a obtained in step (1), mix well and shake on a vortex shaker for 30 s, then place in a horizontal shaker at 30 ℃ for 30 min to obtain mixed solution b; (3) Add equimolar amounts of p-coumaric acid and ABTS to the mixed solution b obtained in step (2), mix and stir until homogeneous, react at room temperature for 3 hours, then add polyethylene glycol PEG with a mass concentration of 20 mg / mL, mix and stir until homogeneous to obtain mixed solution c; wherein, the molar concentration of ABTS and p-coumaric acid in mixed solution c is 2.0 mM. (4) Place the mixed solution c obtained in step (3) into a centrifuge at 10,000 rpm for 5 min, discard the supernatant, and obtain a brown precipitate. Rinse the brown precipitate three times with deionized water until it turns brownish-red. Place it in a constant temperature oven at 37℃ and dry for 12 h to obtain the immobilized laccase-mediator system MnMOF@Lac5@M@PEG.
[0020] Take 75 μL of 1 M 2-methylimidazole solution and 100 μL of 2 M manganese chloride solution, mix them thoroughly and shake them on a vortex mixer for 30 s, then place them in a horizontal shaker at 30 ℃ for 30 min to obtain the MnMOF vector without laccase immobilization, and set it aside for later use. Example 2
[0021] The experiment for determining catalytic activity includes the following steps: The catalytic activity of free or immobilized laccase was determined using the guaiacol method. One enzyme activity unit (U) was defined as the amount of enzyme required to produce 1 μmol of guaiacol dimer per unit time under optimal pH (6.5 for both free and immobilized enzymes) and 25 °C. The standard reaction system consisted of 1 mL of 1 mg / mL laccase or an equivalent amount of immobilized laccase, 10 μL of 50 mM guaiacol, and 980 μL of 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (6.5 for both free and immobilized enzymes). The mixture was thoroughly mixed and incubated in a 25 °C water bath for 5 min. The OD470 nm of the free enzyme was immediately measured, while the OD470 nm of the immobilized enzyme was measured immediately after centrifugation at 10,000 rpm for 30 s. A mixture without enzyme was used as a control. All experiments were independently repeated three times, and the measured values were the average of the three independent experiments. The conclusion is that the catalytic activity of immobilized laccase is higher than that of free laccase. Example 3
[0022] The comparative experiment on optimizing the molar concentration of immobilized laccase 2-methylimidazole included the following steps: (1) Take 500 μL of laccase solution with a protein concentration of 2 µg / mL and mix it with a series of volumes of 1 M 2-methylimidazole solution in an Ep tube. Mix the mixture thoroughly and shake it on a vortex mixer for 30 s to obtain mixed solution a1. (2) Add 100 μL of 2 M manganese chloride solution to the mixed solution a1 obtained in step (1), add ultrapure water to make the final volume 1 mL, mix well and shake on a vortex shaker for 30 s, then place in a horizontal shaker at 30 ℃ for 30 min to obtain mixed solution d. The reaction solution changes from clear to turbid. Centrifuge at 10,000 rpm for 5 min to obtain a brown precipitate. Wash three times with ultrapure water and dry at 37°C to obtain immobilized laccase MnMOF@Lac5; In the mixed solution d, the mass concentration of laccase was 1 µg / mL, the molar concentration of 2-methylimidazole was 25-175 mM, and the molar concentration of manganese chloride was 200 mM.
[0023] The results of determining the molar concentration of 2-methylimidazolium during the synthesis of the immobilized laccase prepared in this step are as follows: Figure 1 As shown, when the final molar concentration of 2-methylimidazole is 75 mM, the relative activity and protein immobilization efficiency of the immobilized laccase are the highest, and the immobilization effect is the best. Example 4
[0024] The comparative experiment on optimizing the molar concentration of immobilized laccase manganese chloride included the following steps: (1) Take 500 μL of laccase solution with a protein concentration of 2 µg / mL and 75 μL of 1 M 2-methylimidazole solution in an Ep tube, mix them evenly, and shake them on a vortex mixer for 30 s to obtain mixed solution a; (2) Add a series of volumes of 2M manganese chloride solution to the mixed solution a obtained in step (1), add ultrapure water to make the final volume 1 mL, mix well and shake on a vortex shaker for 30 s, then place in a horizontal shaker at 30 °C for 30 min to obtain mixed solution d1. The reaction solution changes from clear to turbid. Centrifuge at 10,000 rpm for 5 min to obtain a brown precipitate. Wash three times with ultrapure water and dry at 37 °C to obtain immobilized laccase MnMOF@Lac5. In mixed solution d1, the mass concentration of laccase was 1 µg / mL, the molar concentration of 2-methylimidazole was 75 mM, and the molar concentration of manganese chloride was 50-350 mM.
[0025] The results of determining the molar concentration of manganese chloride during the synthesis of the immobilized laccase prepared in this step are as follows: Figure 2 As shown, when the amount of manganese chloride added was 0.2 mL (final concentration of 200 mM), the relative activity of immobilized laccase and the protein immobilization efficiency were the highest, and the immobilization effect was the best. Example 5
[0026] The comparative experiment on optimizing the mass concentration of immobilized laccase included the following steps: (1) Take a series of volumes of laccase solution with a protein concentration of 2 µg / mL and 75 µL of 1 M 2-methylimidazole solution in an Ep tube, mix them evenly, and shake them on a vortex mixer for 30 s to obtain mixed solution a2; (2) Add 100 µL of 2 M manganese chloride solution to the mixed solution a2 obtained in step (1), add ultrapure water to make the final volume 1 mL, mix well and shake on a vortex shaker for 30 s, then place in a horizontal shaker at 30 ℃ for 30 min to obtain mixed solution d2. The reaction solution changes from clear to turbid. Centrifuge at 10,000 rpm for 5 min to obtain a brown precipitate. Wash three times with ultrapure water and dry at 37 ℃ to obtain immobilized laccase MnMOF@Lac5; In mixed solution d2, the mass concentration of laccase was 0.2-1.4 µg / mL, the molar concentration of 2-methylimidazole was 75 mM, and the molar concentration of manganese chloride was 200 mM. The results of determining the protein concentration of the immobilized laccase prepared in this step during synthesis are as follows: Figure 3 As shown, when the final protein concentration of laccase is 1 µg / mL, the relative activity and protein immobilization efficiency of the immobilized laccase are the highest, and the immobilization effect is the best. Example 6
[0027] Take 1-2 mg of dried comparative MnMOF, MnMOF@Lac5 prepared in Example 2, and MnMOF@Lac5@M@PEG prepared in Example 1, mix and grind with dried KBr powder, press into transparent thin films, and perform Fourier transform infrared analysis; turn on the instrument and preheat, perform background scanning, and set the parameters (number of scans: resolution: 4 cm⁻¹). -1 Wavenumber range: 4000-400 cm⁻¹ -1 ), the result is as follows Figure 4 As shown, compared to MnMOF, MnMOF@Lac5 and MnMOF@Lac5@M@PEG exhibit better performance in the amide I band (1600-1700 cm⁻¹). -1 There is a clear difference, representing the successful fixation of Lac5; secondly, at 1100 cm -1 The presence of (-COC-) indicates a difference between MnMOF@Lac5 and MnMOF@Lac5@M@PEG, demonstrating the successful modification of PEG. Example 7
[0028] The experiment to test the effect of reaction pH on the enzyme activity of MnMOF@Lac5@M@PEG prepared in Example 1 was conducted as follows: The activities of Lac5 and MnMOF@Lac5@M@PEG were measured under different pH conditions. The buffer formulations were as follows: 50 mM Sodium phosphate dibasic-citric acid buffer (pH 2.5-8.0); 100 mM Tris-HCl buffer (pH 8.0-9.0). The relative enzyme activity was calculated with the enzyme activity at the optimal pH conditions as 100%. The results are as follows. Figure 5 As shown, the optimal pH for Lac5, MnMOF@Lac5, and MnMOF@Lac5@M@PEG is 6.5 (Sodium phosphate dibasic-citric acid buffer). Example 8
[0029] The experiment to test the effect of reaction temperature on the enzyme activity of MnMOF@Lac5@M@PEG prepared in Example 1 was conducted as follows: Under pH 6.5 conditions, the activities of Lac5 and MnMOF@Lac5 in the temperature range of 10-80℃ were determined using a standard reaction system (the same as in Example 1). The enzyme activity at the optimal temperature was taken as 100%, and the relative enzyme activity was calculated. The results are as follows: Figure 6 As shown, the optimal reaction temperature for Lac5 and MnMOF@Lac5@M@PEG remains 65℃, and MnMOF@Lac5@M@PEG exhibits excellent protective effects on the enzyme at all temperatures. Example 9
[0030] The experiment to test the effect of ambient temperature (30℃) on the stability of MnMOF@Lac5@M@PEG prepared in Example 1 was conducted as follows: Lac5 and MnMOF@Lac5@M@PEG prepared in Example 1 were separately prepared into solutions using a pH 6.5 buffer. These solutions were placed in a 30°C water bath, and samples were taken periodically. The activities of Lac5 and MnMOF@Lac5@M@PEG were determined using a standard reaction system (the same as in Example 1). Relative enzyme activities were calculated with the untreated enzyme activity as 100%. Results are as follows: Figure 7 As shown, the immobilized laccase MnMOF@Lac5@M@PEG exhibits good thermostability, retaining 69% of its initial activity after incubation at 30℃ for 100 h, while free laccase loses its activity more rapidly under the same conditions. Example 10
[0031] The experiment to test the effect of pH 6.5 buffer on the stability of MnMOF@Lac5@M@PEG prepared in Example 1 was conducted as follows: Lacase 5 and MnMOF@Lac5@M@PEG prepared in Example 1 were prepared into solutions using a pH 6.5 buffer. These enzyme solutions were stored at 4°C. Samples were taken periodically, and the activities of laccase 5 and MnMOF@Lac5@M@PEG prepared in Example 1 were determined using a standard reaction system (the same as in Example 1). The relative enzyme activity was calculated with the untreated enzyme activity as 100%. Results are as follows: Figure 8 As shown, the immobilized laccase MnMOF@Lac5@M@PEG exhibits good thermostability, retaining 85% of its initial activity after incubation in pH 6.5 buffer for 100 h. Example 11
[0032] The steps for optimizing the molar concentration of the mediator in the immobilized laccase-mediator system are as follows: The degradation performance of the co-immobilized laccase-mediator system was evaluated by decolorizing alizarin red, a typical structural pollutant. The loading capacity of MnMOF@Lac5@M@PEG was investigated by sequentially adding p-coumaric acid and ABTS (in equal proportions). Figure 9 As shown, when both ABTS and p-coumaric acid were used at 2.0 mM, the immobilization amounts of ABTS and p-coumaric acid on the MOF were approximately 0.59 mM and 0.62 mM, respectively, with the highest removal rate of alizarin red reaching 366.29% compared to the free enzyme. With increasing concentrations of ABTS and p-coumaric acid, the removal capacity of MnMOF@Lac5@M@PEG for alizarin red decreased. This phenomenon is because higher concentrations of the synthesis medium may lead to laccase inactivation; therefore, a co-immobilized laccase-mediator system was prepared using ABTS and p-coumaric acid at 2.0 mM. Example 12
[0033] The experiment on the number of times MnMOF@Lac5 prepared in the comparative example and MnMOF@Lac5@M@PEG prepared in Example 1 were repeated was conducted, and the steps were as follows: At 25°C and pH 8.5, the hydrolytic activity of laccase Lac5, comparatively prepared MnMOF@Lac5, and MnMOF@Lac5@M@PEG prepared in Example 1 was detected in a standard reaction system (the same as in Example 1). After the reaction, the immobilized enzyme was separated from the reaction solution by centrifugation at 10,000 rpm for 3 min. After washing once with ultrapure water, the above reaction process was repeated. The OD470 value of the supernatant after each reaction was recorded. Taking the enzyme activity of the first reaction as 100%, the relative enzyme activity of the subsequent reaction processes was calculated to determine the reusability of MnMOF@Lac5 and MnMOF@Lac5@M@PEG. The results are as follows: Figure 10As shown, the PEG-modified immobilized laccase MMnMOF@Lac5@M@PEG has better reusability than MnMOF@Lac5, retaining 50% of its initial activity after 8 reuses. Example 13
[0034] The time gradient detection experiment for testing laccase Lac5 and the MnMOF@Lac5@M@PEG degradation dye prepared in Example 1 was conducted as follows: Take a 100 mg / L anthraquinone dye (Alizarin Red) solution, and add isoenzymatically active free laccase, MnMOF@Lac5 prepared in Example 2, and MnMOF@Lac5@M@PEG prepared in Example 1, respectively. React at 30℃. Samples are taken periodically and centrifuged, and the OD520 of the supernatant is measured to calculate the degradation rate. The results are as follows: Figure 11 The immobilized system MnMOF@Lac5@M@PEG degraded 100% of the dye within 15 min, which is twice as efficient as free Lac5.
[0035] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.
Claims
1. A method for preparing a co-immobilized laccase-mediator system, characterized in that, Includes the following steps: (1) Mix the laccase solution and 2-methylimidazole solution evenly and shake on a vortex mixer for 30-40s to obtain mixed solution a; (2) Add manganese chloride solution to the mixed solution a obtained in step (1), mix evenly and shake on a vortex shaker for 30-40s, then place in a horizontal shaker at 30℃ for 30 min to obtain mixed solution b; (3) Add equimolar amounts of p-coumaric acid and ABTS to the mixed solution b obtained in step (2), mix and stir evenly, react at 30°C, then add polyethylene glycol (PEG), mix and stir evenly to obtain mixed solution c; (4) Centrifuge the mixed solution c obtained in step (3) at 10,000 rpm for 5-8 min, discard the supernatant, and obtain a brown precipitate. Rinse the brown precipitate with deionized water several times until it turns brownish-red. Dry it in a constant temperature oven at 37℃ for 10-12 h to obtain the immobilized laccase-mediator system MnMOF@Lac5@PEG@M.
2. The method for preparing a co-immobilized laccase-mediator system according to claim 1, characterized in that, In step (2), the mixed solution b contains laccase at a mass concentration of 0.2-1.4 µg / mL, 2-methylimidazole at a molar concentration of 25-175 mM, and manganese chloride at a molar concentration of 50-350 mM.
3. The method for preparing a co-immobilized laccase-mediator system according to claim 1, characterized in that, In step (3), the molar concentrations of ABTS and p-coumaric acid in the mixed solution c are 0.5-12 mM, and the mass concentration of PEG is 20 mg / mL.
4. The method for preparing a co-immobilized laccase-mediator system according to claim 1, characterized in that, In step (3), the reaction time at the 30°C temperature is 3-5 hours (to expand the protection range).
5. A method for preparing a co-immobilized laccase-mediator system according to any one of claims 1-4.
6. The application of the co-immobilized laccase-mediator system according to any one of claims 1-5 in the field of dye degradation.
7. The application of the co-immobilized laccase-mediator system according to claim 6 in the field of dye degradation, characterized in that... Alizarin red was uniformly dispersed in the immobilized lipase solution, the reaction mixture was shaken to react, and the content of alizarin red in the supernatant was determined by centrifugation after the reaction was completed.