Biocatalyst for the degradation of acridines and method for its preparation

Abstract

This invention discloses a highly efficient biocatalyst for acridine degradation, comprising the following components by mass fraction: 10 parts 1-ethyl-3-methylimidazolium acetate; 2-4 parts cellulose; 2-4 parts chitosan; 10-20 parts dopamine; 10 parts Tris-HCl buffer solution; 3-5 parts glucose oxidase; 5-7 parts horseradish peroxidase; and 10 parts PBS buffer solution. The invention also discloses a method for preparing this highly efficient acridine-degrading biocatalyst. This invention is applicable to the field of wastewater treatment technology. The preparation method is simple and does not require complex equipment. It not only retains the properties of cellulose and chitosan but also solves the problems of chitosan's acid solubility and poor mechanical strength, and cellulose's smooth surface and poor adsorption performance. A novel cellulose-chitosan composite microsphere is successfully prepared, reducing the cost of enzyme use, accelerating the degradation rate, and improving reusability.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically a biocatalyst for degrading acridine and its preparation method. Background Technology

[0002] Acridine is a nitrogen-containing heterocyclic compound widely found in industrial wastewater such as coking wastewater, dye wastewater, pharmaceutical wastewater, and pesticide wastewater. It possesses carcinogenic, teratogenic, and mutagenic properties, and its high concentration, strong toxicity, and recalcitrant nature pose potential hazards to the environment and human health. Due to its recalcitrant nature, research on acridine degradation is currently scarce.

[0003] In recent years, the main methods applied to treat nitrogen-containing heterocyclic compounds include physical treatment, chemical treatment, and biological treatment. Physical treatment techniques, such as adsorption, extraction, and coagulation-sedimentation, are suitable for wastewater pretreatment because they can only transfer pollutants and cannot completely eliminate them. Chemical treatment involves using chemical oxidants under the action of catalysts to treat organic wastewater, thereby improving its biodegradability, or directly oxidizing and degrading organic matter in wastewater to stabilize it or convert it into less harmful compounds. This method is widely used in actual wastewater treatment, but it suffers from drawbacks such as complex catalyst preparation, harsh reaction conditions, high operating costs, poor pollutant removal efficiency, and a tendency to cause secondary pollution. Biological treatment utilizes the metabolism of microorganisms to convert organic pollutants in wastewater into the microorganisms' own biomass or energy substances, thereby purifying the wastewater. This technology offers mild reaction conditions, low cost, wide applicability to different wastewater qualities, and is less likely to cause secondary pollution. However, in actual wastewater treatment environments, the wastewater composition is complex, making it impossible to guarantee high activity of microorganisms and efficient degradation of pollutants. Enzyme biocatalysis technology, with its outstanding advantages such as high catalytic capacity for specific reactions, short reaction time, mild conditions, and environmental friendliness, has received much attention in recent years as an efficient and feasible method for degrading recalcitrant organic pollutants.

[0004] Enzymes are expensive, highly water-soluble, and difficult to separate from substrates and products. Directly using free enzymes in wastewater treatment results in significant waste due to the inability to recycle and reuse them. Furthermore, enzymes are highly sensitive to their environment, easily denatured and inactivated, severely limiting their practical applications. To address this problem, recent research both domestically and internationally has focused on enzyme immobilization. Enzyme immobilization technology refers to the use of carrier materials to bind or confine enzymes within a specific space, preserving their catalytic activity, improving stability, and achieving reusability. The most common carrier forms are microspheres and membranes, which facilitate filtration and separation. Immobilizing enzymes not only preserves their unique catalytic activity and improves their stability but also enables recycling and reuse. Therefore, the preparation of highly efficient immobilized enzymes is of great significance to the rapid development of the field of biocatalysis.

[0005] Among the many oxidoreductases used in wastewater treatment, HRP is relatively inexpensive, has high specific activity, and is easy to prepare. It maintains catalytic activity over a wide range of pollutant concentrations, temperatures, and pH levels, making it considered a green, environmentally friendly, and economically safe enzyme catalyst with great potential in water treatment. However, due to its low redox potential, HRP can only catalyze phenols, anilines, and their substituted derivatives in the presence of H₂O₂, and cannot or has difficulty catalyzing aromatic and heterocyclic compounds. Researchers have discovered that some redox mediators, such as 2,2-azobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT), and 2,2,6,6-tetramethylpiperidine-1-oxy (TEMPO), can synergize with the enzyme, broadening the substrate range and improving the catalytic performance of the oxidoreductase. This allows HRP to be widely used in the degradation of aromatic and heterocyclic compounds. However, HRP can only function in the presence of H2O2, which is a major challenge for its application in actual wastewater treatment. This is because H2O2 is a corrosive reagent that is difficult to handle and store, and excessive H2O2 can deactivate the HRP biocatalyst, resulting in a significant reduction in process efficiency. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a biocatalyst for degrading acridine and its preparation method.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Firstly, the biocatalyst for degrading acridine comprises, by mass fraction, the following components:

[0009] 10 parts of 1-ethyl-3-methylimidazolium acetate;

[0010] 2-4 parts cellulose;

[0011] 2-4 parts chitosan;

[0012] 10-20 parts dopamine;

[0013] 10 portions of Tris-HCl buffer solution;

[0014] 3-5 parts glucose oxidase;

[0015] 5-7 parts horseradish peroxidase;

[0016] 10 portions of PBS buffer solution.

[0017] Secondly, a method for preparing a biocatalyst for degrading acridine includes the following steps:

[0018] (I) Preparation of cellulose-chitosan composite microspheres:

[0019] The prescribed amount of ionic liquid 1-ethyl-3-methylimidazolium acetate was placed in a reaction vessel, and the prescribed amounts of cellulose and chitosan were added and stirred. The mixture was reacted at 80-100℃ for 2-3 hours until the cellulose and chitosan were completely dissolved. Heating was then stopped to obtain a clear gel-like solution. After the solution was cooled to room temperature, it was added dropwise to distilled water using a peristaltic pump to obtain cellulose-chitosan composite microspheres. The microspheres were allowed to stand for 0.5-2 hours to harden, and then washed to obtain cellulose-chitosan composite microspheres.

[0020] (II) Preparation of dopamine-modified cellulose-chitosan composite microspheres:

[0021] The cellulose-chitosan composite microspheres obtained in step (I) and the prescribed amount of dopamine were added sequentially to the reaction vessel, followed by the prescribed amount of Tris-HCl buffer solution. The reaction was carried out in a constant temperature air shaker, and the microspheres were separated by filtration and washed to obtain dopamine-modified cellulose-chitosan composite microspheres.

[0022] (III) Preparation of biocatalysts for the degradation of acridine:

[0023] The dopamine-modified cellulose-chitosan composite microspheres obtained in step (II) were added sequentially to the reaction vessel, along with a formulation amount of glucose oxidase and a formulation amount of horseradish peroxidase, and then a formulation amount of PBS buffer solution was added. The reaction was carried out in a constant temperature air shaker. The microspheres were separated by filtration and washed with PBS buffer solution at pH 6.0-7.0 to obtain the biocatalyst for degrading acridine.

[0024] Preferably, in step (i), the reaction vessel is a 25-50 mL three-necked flask, the stirring rate is 800-1000 r / min, and the mass concentration of the clear colloidal solution is 4-8 wt%.

[0025] Preferably, in step (ii), the rotation speed of the constant temperature air oscillator is 150-200 r / min, the reaction time in the constant temperature air oscillator is 2-3 h, and the reaction temperature is 20-30℃.

[0026] Preferably, in step (ii), the concentration of the Tris-HCl buffer solution is 0.01-0.02 mol / L, the pH value is 8.0-9.0, the temperature is 20-30℃, and the reaction time is 2-3h.

[0027] Preferably, in step (iii), the concentration of the PBS buffer solution is 0.1-0.2 mol / L, the pH value is 6.0-7.0, the temperature is 20-30℃, and the reaction time is 2-3 h.

[0028] Preferably, in step (iii), the rotation speed of the constant temperature air oscillator is 150-200 r / min, the reaction time is 12-24 h, and the reaction temperature is 20-30℃.

[0029] Preferably, the washing is performed 3 times.

[0030] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0031] (1) The preparation method of this biocatalyst is simple and does not require complex instruments and equipment;

[0032] (2) This biocatalyst combines two different carrier materials, cellulose and chitosan, which not only retains the properties of cellulose and chitosan, but also solves the problems of chitosan dissolving in acid and having poor mechanical strength, and cellulose having a smooth surface and poor adsorption performance, thus successfully preparing a novel cellulose-chitosan composite microsphere.

[0033] (3) This biocatalyst is the first to use horseradish peroxidase (HRP) and glucose oxidase (GOD) together to prepare a co-immobilized dual-enzyme biocatalyst based on cascade reaction. This not only reduces the cost of enzyme use and accelerates the degradation rate, but also improves enzyme stability and reusability.

[0034] (4) This biocatalyst combines horseradish peroxidase with the redox mediator HBT, thus broadening the substrate range of horseradish peroxidase.

[0035] (5) This biocatalyst achieves efficient degradation of the recalcitrant compound acridine, providing new technical support and theoretical basis for the application of horseradish peroxidase-mediator in heterocyclic aromatic wastewater treatment;

[0036] (6) The biocatalyst can maintain catalytic activity over a wide range of pollutant concentrations, temperatures and pH levels, and has good storage stability and reusability. Attached Figure Description

[0037] Figure 1 This is a scanning electron microscope (SEM) image of the outer surface of the present invention;

[0038] Figure 2 This is a scanning electron microscope (SEM) image of the cross-section of the present invention;

[0039] Figure 3 This is a scanning electron microscope (SEM) image of the sheet structure of the present invention;

[0040] Figure 4 This is the X-ray diffraction (XRD) pattern of the present invention;

[0041] Figure 5 This is the Fourier transform infrared (FT-IR) spectrum of the present invention;

[0042] Figure 6 This is a graph showing the effect of different catalysts of the present invention on the degradation rate of acridine;

[0043] Figure 7 This is a graph showing the effect of temperature on the catalytic activity of free and immobilized dual enzymes according to the present invention;

[0044] Figure 8 This is a graph showing the effect of pH on the catalytic activity of free and immobilized dual enzymes according to the present invention;

[0045] Figure 9 This is a graph showing the effect of different amounts of mediator added in this invention on the catalytic activity of the catalyst;

[0046] Figure 10 This is a graph showing the effect of different substrate concentrations on the catalytic activity of the catalyst in this invention;

[0047] Figure 11 This is a diagram illustrating the reusability and storage stability of the biocatalyst of this invention. Detailed Implementation

[0048] The following is in conjunction with the appendix Figure 1-11 The following describes specific embodiments of the biocatalyst for acridine degradation and its preparation method. The biocatalyst for acridine degradation and its preparation method are not limited to the descriptions in the following examples.

[0049] Example 1:

[0050] This embodiment provides a specific implementation of a biocatalyst for degrading acridine, which includes the following components by mass fraction:

[0051] 10 parts of 1-ethyl-3-methylimidazolium acetate;

[0052] 2 parts cellulose;

[0053] 2 parts chitosan;

[0054] 10 parts dopamine;

[0055] 10 portions of Tris-HCl buffer solution;

[0056] 3 portions of glucose oxidase;

[0057] 5 portions of horseradish peroxidase;

[0058] 10 portions of PBS buffer solution.

[0059] Example 2:

[0060] This embodiment provides a specific implementation of a biocatalyst for degrading acridine, which includes the following components by mass fraction:

[0061] 10 parts of 1-ethyl-3-methylimidazolium acetate;

[0062] 3 parts cellulose;

[0063] 3 parts chitosan;

[0064] 15 parts dopamine;

[0065] 10 portions of Tris-HCl buffer solution;

[0066] 4 portions of glucose oxidase;

[0067] 6 portions of horseradish peroxidase;

[0068] 10 portions of PBS buffer solution.

[0069] Example 3:

[0070] This embodiment provides a specific implementation of a biocatalyst for degrading acridine, which includes the following components by mass fraction:

[0071] 10 parts of 1-ethyl-3-methylimidazolium acetate;

[0072] 4 parts cellulose;

[0073] 4 parts chitosan;

[0074] 20 parts dopamine;

[0075] 10 portions of Tris-HCl buffer solution;

[0076] 5 portions of glucose oxidase;

[0077] 7 portions of horseradish peroxidase;

[0078] 10 portions of PBS buffer solution.

[0079] Example 4:

[0080] This embodiment provides a specific implementation method for preparing a biocatalyst for degrading acridine, including the following steps:

[0081] (I) Preparation of cellulose-chitosan composite microspheres:

[0082] The prescribed amount of ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]) was placed in a reaction vessel, and the prescribed amounts of cellulose and chitosan were added and stirred. The mixture was reacted at 80°C for 2 hours until the cellulose and chitosan were completely dissolved. Heating was then stopped, and a clear gel-like solution was obtained. After the solution was cooled to room temperature, it was added dropwise to distilled water using a peristaltic pump to obtain cellulose-chitosan composite microspheres. The microspheres were allowed to stand for 0.5 hours to harden, and after washing, the cellulose-chitosan composite microspheres were obtained, denoted as Ce-Cs beads.

[0083] (II) Preparation of dopamine-modified cellulose-chitosan composite microspheres:

[0084] The cellulose-chitosan composite microspheres obtained in step (I) and the prescribed amount of dopamine were added sequentially to the reaction vessel, followed by the prescribed amount of Tris-HCl buffer solution. The reaction was carried out in a constant temperature air shaker, and the microspheres were separated by filtration and washed to obtain dopamine-modified cellulose-chitosan composite microspheres, denoted as Ce-Cs@DAbeads.

[0085] (III) Preparation of biocatalysts for the degradation of acridine:

[0086] The dopamine-modified cellulose-chitosan composite microspheres obtained in step (II) were added sequentially to a reaction vessel, along with a specified amount of glucose oxidase (GOD) and horseradish peroxidase (HRP), and then a specified amount of PBS buffer solution was added. The reaction was carried out in a constant-temperature air shaker. The microspheres were separated by filtration and washed with PBS buffer solution at pH 6.0 to obtain acridine-degrading biocatalyst, denoted as Ce-Cs@DA / HRP-GODbeads, with the structure shown below. Figure 1-3 As shown;

[0087] Furthermore, in step (i), the reaction vessel is a 25 mL three-necked flask, the stirring rate is 800 r / min, and the mass concentration of the clear gel solution is 4 wt%.

[0088] Furthermore, in step (ii), the rotation speed of the constant temperature air oscillator is 150 r / min, the reaction time in the constant temperature air oscillator is 2 h, and the reaction temperature is 20 °C.

[0089] Furthermore, in step (ii), the concentration of the Tris-HCl buffer solution is 0.01 mol / L, the pH value is 8.0, the temperature is 20℃, and the reaction time is 2h.

[0090] Furthermore, in step (iii), the concentration of the PBS buffer solution is 0.1 mol / L, the pH value is 6.0, the temperature is 20℃, and the reaction time is 2h.

[0091] Furthermore, in step (iii), the rotation speed of the constant temperature air oscillator is 150 r / min, the reaction time is 12 h, and the reaction temperature is 20 °C.

[0092] Furthermore, the washing cycle is twice.

[0093] Example 5:

[0094] This embodiment provides a specific implementation method for preparing a biocatalyst for degrading acridine, including the following steps:

[0095] (I) Preparation of cellulose-chitosan composite microspheres:

[0096] The prescribed amount of ionic liquid 1-ethyl-3-methylimidazolium acetate was placed in a reaction vessel, and the prescribed amounts of cellulose and chitosan were added and stirred. The mixture was reacted at 90℃ for 2.5 h until the cellulose and chitosan were completely dissolved. Heating was stopped to obtain a clear gel-like solution. After the solution was cooled to room temperature, it was added dropwise to distilled water using a peristaltic pump to obtain cellulose-chitosan composite microspheres. The microspheres were allowed to stand for 1 h to harden, and then washed to obtain cellulose-chitosan composite microspheres.

[0097] (II) Preparation of dopamine-modified cellulose-chitosan composite microspheres:

[0098] The cellulose-chitosan composite microspheres obtained in step (I) and the prescribed amount of dopamine were added sequentially to the reaction vessel, followed by the prescribed amount of Tris-HCl buffer solution. The reaction was carried out in a constant temperature air shaker, and the microspheres were separated by filtration and washed to obtain dopamine-modified cellulose-chitosan composite microspheres.

[0099] (III) Preparation of biocatalysts for the degradation of acridine:

[0100] The dopamine-modified cellulose-chitosan composite microspheres obtained in step (II) were added sequentially to a reaction vessel, along with a specified amount of glucose oxidase and horseradish peroxidase, and then a specified amount of PBS buffer solution was added. The reaction was carried out in a constant-temperature air shaker. The microspheres were separated by filtration and washed with PBS buffer solution at pH 6.5 to obtain a biocatalyst for degrading acridine, the structure of which is shown below. Figure 1-3 As shown;

[0101] Furthermore, in step (i), the reaction vessel is a 30mL three-necked flask, the stirring rate is 900r / min, and the mass concentration of the clear gel solution is 6wt%.

[0102] Furthermore, in step (ii), the rotation speed of the constant temperature air oscillator is 175 r / min, the reaction time in the constant temperature air oscillator is 2.5 h, and the reaction temperature is 25 °C.

[0103] Furthermore, in step (ii), the concentration of the Tris-HCl buffer solution is 0.0015 mol / L, the pH value is 8.5, the temperature is 25℃, and the reaction time is 2.5 h.

[0104] Furthermore, in step (iii), the concentration of the PBS buffer solution is 0.15 mol / L, the pH value is 6.5, the temperature is 25℃, and the reaction time is 2.5 h.

[0105] Furthermore, in step (iii), the rotation speed of the constant temperature air oscillator is 175 r / min, the reaction time is 18 h, and the reaction temperature is 25 °C.

[0106] Furthermore, the washing cycle is twice.

[0107] Example 6:

[0108] This embodiment provides a specific implementation method for preparing a biocatalyst for degrading acridine, including the following steps:

[0109] (I) Preparation of cellulose-chitosan composite microspheres:

[0110] The prescribed amount of ionic liquid 1-ethyl-3-methylimidazolium acetate was placed in a reaction vessel, and the prescribed amounts of cellulose and chitosan were added and stirred. The mixture was reacted at 100℃ for 3 hours until the cellulose and chitosan were completely dissolved. Heating was stopped to obtain a clear gel-like solution. After the solution was cooled to room temperature, it was added dropwise to distilled water using a peristaltic pump to obtain cellulose-chitosan composite microspheres. The microspheres were allowed to stand for 2 hours to harden and then washed to obtain cellulose-chitosan composite microspheres.

[0111] (II) Preparation of dopamine-modified cellulose-chitosan composite microspheres:

[0112] The cellulose-chitosan composite microspheres obtained in step (I) and the prescribed amount of dopamine were added sequentially to the reaction vessel, followed by the prescribed amount of Tris-HCl buffer solution. The reaction was carried out in a constant temperature air shaker, and the microspheres were separated by filtration and washed to obtain dopamine-modified cellulose-chitosan composite microspheres.

[0113] (III) Preparation of biocatalysts for the degradation of acridine:

[0114] The dopamine-modified cellulose-chitosan composite microspheres obtained in step (II) were added sequentially to a reaction vessel, along with a specified amount of glucose oxidase and horseradish peroxidase, and then a specified amount of PBS buffer solution was added. The reaction was carried out in a constant-temperature air shaker. The microspheres were separated by filtration and washed with PBS buffer solution at pH 7.0 to obtain a biocatalyst for degrading acridine, the structure of which is shown below. Figure 1-3 As shown;

[0115] Furthermore, in step (i), the reaction vessel is a 25-50 mL three-necked flask, the stirring rate is 1000 r / min, and the mass concentration of the clear gel solution is 8 wt%.

[0116] Furthermore, in step (ii), the rotation speed of the constant temperature air oscillator is 200 r / min, the reaction time in the constant temperature air oscillator is 3 h, and the reaction temperature is 30 °C.

[0117] Furthermore, in step (ii), the concentration of the Tris-HCl buffer solution is 0.02 mol / L, the pH value is 9.0, the temperature is 30℃, and the reaction time is 3h.

[0118] Furthermore, in step (iii), the concentration of the PBS buffer solution is 0.2 mol / L, the pH value is 7.0, the temperature is 30℃, and the reaction time is 3h.

[0119] Furthermore, in step (iii), the rotation speed of the constant temperature air oscillator is 200 r / min, the reaction time is 24 h, and the reaction temperature is 30 °C.

[0120] Furthermore, the washing cycle is 3 times.

[0121] Figure 4 The image shows the X-ray diffraction (XRD) pattern of this biocatalyst. The curves corresponding to Ce, Cs, and Ce-Cs are the spectra of cellulose, chitosan, and cellulose-chitosan composite microspheres, respectively. Figure 5 The image shows the Fourier transform infrared (FT-IR) spectrum of the biocatalyst. The curves Ce-Cs@DA / HRP-GOD, Ce-Cs@DA, Ce, and Cs correspond to the immobilized dual enzymes of dopamine-modified cellulose-chitosan composite microspheres, the dopamine-modified cellulose-chitosan composite microspheres, and the spectra of cellulose and chitosan, respectively.

[0122] It can be seen that: Figure 4In the diffraction pattern, Ce exhibits typical characteristic peaks of type I cellulose diffraction, namely a sharp peak at 2θ = 22.5°, a small peak at 2θ = 34.5°, and a broad peak between 2θ = 14.9° and 2θ = 16.2°. Cs exhibits typical characteristic peaks of chitosan diffraction, with a strong diffraction peak at 2θ = 20.5° and a weak diffraction peak at 2θ = 10.5°. The diffraction peaks of the Ce-Cs composite microspheres are similar to those of type II cellulose, exhibiting a characteristic diffraction peak at 2θ = 20.5°, which is similar to the results found in previously reviewed articles. XRD analysis indicates that the functional modification of cellulose and chitosan with the ionic liquid ([Emim][OAc]) only involves physical changes, without altering the crystal structure.

[0123] exist Figure 5 Comparing the Ce, Cs, and Ce-Cs@DA spectra, it can be seen that in the characteristic absorption peak of Ce-Cs@DA, the 1086 cm⁻¹ peak is... -1 2910cm -1 3420cm -1 1420cm -1 1594cm -1 The characteristic absorption peaks of Ce and Cs are basically the same, indicating that the dissolution of cellulose and chitosan by the ionic liquid is only a physical process, and no new functional groups are formed in the regenerated cellulose and chitosan, meaning that no derivatization reaction of cellulose and chitosan occurs during the dissolution process. Furthermore, the Ce-Cs@DA spectrum is located at 1334 cm⁻¹. -1 1240cm -1 The absorption peak at 3420 cm⁻¹ is essentially the same as the typical absorption peak in the dopamine structure, indicating that dopamine is coated on the surface of the microspheres. In the spectrum of Ce-Cs@DA / HRP-GOD, in addition to the aforementioned signal, there is also an absorption peak at 3420 cm⁻¹. -1 A significant reduction in peak values ​​was observed, which is attributed to the co-immobilization of the two enzymes HRP-GOD on Ce-Cs@DA beads. Therefore, FT-IR spectroscopy confirmed the successful preparation of the co-immobilized two-enzyme biocatalyst.

[0124] Experimental Investigation 1: Investigation into the high efficiency of this biocatalyst in degrading acridine:

[0125] Prepare a simulated acridine wastewater solution of 5-15 mg / L, pH 7.0. Add 10 mL of the simulated wastewater to a 25 mL Erlenmeyer flask. Add a certain amount of glucose (Glu) and 1-hydroxybenzotriazole (HBT) to the flask. Under reaction conditions of 20-30℃ and 150-200 r / min, degrade acridine with 1 g of the biocatalyst Ce-Cs@DA / HRP-GOD beads (wet weight) until the remaining acridine content is almost zero as detected by high performance liquid chromatography. Filter to separate the biocatalyst Ce-Cs@DA / HRP-GOD beads. (Detection conditions: mobile phase methanol:water = 8:2; detection wavelength: 250 nm; flow rate: 1-2 mL / min; injection volume: 10-20 μL. Filter the sample with a 0.45 μm syringe filter before injecting into the column.) The acridine degradation rate is calculated using the following formula:

[0126] D(%)=[(C0-C t ) / C0]×100%

[0127] Where C0 is the initial concentration of acridine, C t This represents the concentration of acridine after degradation.

[0128] Actual wastewater has a complex composition, and catalysts are easily affected by other pollutants in the wastewater. In order to facilitate the study of the catalyst's catalytic performance, simulated wastewater was used in the experiment instead of actual wastewater.

[0129] Experimental Investigation 2: Effect of Substrate Concentration on the Degradation Performance of Acridine by Biocatalysts

[0130] Excessive substrate concentration may cause acridine molecules to cover the active site of the enzyme, affecting the effectiveness of the biocatalyst. At a reaction temperature of 30℃, a reaction pH of 7.0, and with glucose and HBT additions of 20 mg each, and Ce-Cs@DA / HRP-GODbeads addition of 1 g, the effect of Ce-Cs@DA / HRP-GODbeads on the acridine degradation performance of reaction systems with different substrate concentrations (5-55 mg / L) was investigated to determine the optimal substrate concentration for acridine degradation by the biocatalyst.

[0131] Experimental Investigation 3: Effect of Temperature on the Degradation Performance of Acridine by Biocatalysts

[0132] like Figure 7As shown, the curves corresponding to Ce-Cs@DA / HRP-GOD and free HRP-GOD represent the degradation rates of acridine by immobilized and free enzymes at different temperatures. Too low a temperature weakens enzyme activity, while too high a temperature causes protein denaturation, leading to enzyme inactivation and affecting the effectiveness of the biocatalyst. With an acridine concentration of 15 mg / L, a reaction pH of 7.0, 20 mg of glucose and 20 mg of HBT added, and 1 g of Ce-Cs@DA / HRP-GOD beads added, the effect of Ce-Cs@DA / HRP-GOD beads on the acridine degradation performance in reaction systems at different temperatures (10-60℃) was investigated to determine the optimal temperature for acridine degradation by the biocatalyst.

[0133] Experiment 4: Effect of pH on the performance of biocatalysts in degrading acridine

[0134] like Figure 8 As shown, the curves corresponding to Ce-Cs@DA / HRP-GOD and free HRP-GOD represent the degradation rates of acridine by immobilized and free enzymes at different pH values. Acidity and alkalinity can alter the electrostatic properties of protein surfaces, thereby changing enzyme activity or stability, leading to a decrease in the effectiveness of the biocatalyst. This study investigated the effect of Ce-Cs@DA / HRP-GOD beads on the degradation performance of acridine in reaction systems at different pH values ​​(4.0-9.0) with an acridine concentration of 15 mg / L, a reaction temperature of 30℃, glucose and HBT additions of 20 mg each, and Ce-Cs@DA / HRP-GOD beads addition of 1 g, to determine the optimal pH for acridine degradation by the biocatalyst.

[0135] Experimental Investigation 5: Effects of Reusability and Storage Stability on the Performance of Biocatalysts in Degrading Acridine

[0136] like Figure 11 As shown, the black and white columns represent the degradation rates of acridine under different number of repetitions and different storage times, respectively. The reusability and storage stability of biocatalysts have significant practical application value in wastewater treatment. At a reaction temperature of 30℃, a reaction pH of 7.0, and with glucose and HBT additions of 20 mg and Ce-Cs@DA / HRP-GOD beads of 1 g, a 15 mg / L acridine solution was catalytically degraded. After 8 h of reaction, the Ce-Cs@DA / HRP-GOD beads were separated and washed three times with pH 7.0 PBS buffer solution. The reaction was repeated nine times. After each reaction, the concentration of acridine in the solution was determined by HPLC.

[0137] A certain amount of Ce-Cs@DA / HRP-GOD beads (wet weight) were immersed in 0.1M pH 7.0 PBS buffer solution and stored in a 4℃ refrigerator. 1g of microspheres were taken out periodically to determine the degradation rate of 15mg / L acridine solution and to compare the changes in degradation rate before and after storage.

[0138] Experimental Investigation 6: Effect of HBT Addition Amount on the Degradation Performance of Acridine by Biocatalyst

[0139] Because of its low redox potential, HRP can only catalyze phenols, anilines, and their substituted derivatives in the presence of H₂O₂, and cannot or has difficulty catalyzing aromatic and nitrogen-containing heterocyclic compounds. Researchers have discovered that some redox mediators, such as 2,2-azobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT), and 2,2,6,6-tetramethylpiperidine-1-oxy (TEMPO), can broaden the substrate range and improve the catalytic performance of redox enzymes, enabling HRP to be widely used in the degradation of aromatic and nitrogen-containing heterocyclic compounds. However, most mediators are toxic, unstable, or expensive, and large-scale use will inevitably cause secondary environmental pollution. Therefore, measures such as reducing the concentration of the mediator and lowering the reaction temperature are usually taken to prevent the mediator from affecting the enzyme catalysis process. This study investigated the effect of Ce-Cs@DA / HRP-GOD beads on the degradation performance of acridine in reaction systems with different mediator amounts (0-4 mg / mL) at a concentration of 15 mg / L, a reaction temperature of 30℃, a reaction pH of 7.0, a glucose addition of 20 mg, and a Ce-Cs@DA / HRP-GOD beads addition of 1 g, to determine the optimal amount of mediator for the degradation of acridine by the biocatalyst.

[0140] Experimental Investigation 7: Effect of Different Catalysts on Acridine Degradation Rate

[0141] like Figure 6 As shown, the curves corresponding to Ce-Cs@DA / HRP-GOD+HBT+Glu, Ce-Cs@DA / HRP+HBT+H2O2, and Ce-Cs@DA / GOD+Glu represent the degradation rates of acridine by immobilized dual enzymes and immobilized single enzymes under the synergistic effect of the mediator, respectively; the curve corresponding to Ce-Cs@DA / HRP-GOD+Glu represents the degradation rate of acridine by immobilized dual enzymes without the mediator.

[0142] Experimental Investigation 8: Effect of Different Mediator Amounts on the Catalytic Activity of the Catalyst (Figure)

[0143] like Figure 9The figures show the degradation rates of acridine with different amounts of mediators. It can be seen that without mediators, the degradation rates of acridine by both the free and immobilized dual enzymes are very low, with degradation rates of only 7.5% and 20.0% respectively after 8 hours of reaction. The slight increase in the degradation rate of acridine by the immobilized dual enzymes may be due to the physical adsorption of acridine by the microbeads. However, in the presence of HBT, the removal rate gradually increased to 99.9% as the HBT concentration increased from 0.5 mg / mL to 2 mg / mL. When the HBT concentration increased to 3 mg / mL or more, the removal rate did not significantly improve, and acridine was completely degraded after 8 hours of reaction. This indicates that the redox medium HBT can significantly improve the degradation efficiency of acridine by horseradish peroxidase (HRP). However, excessive HBT did not significantly promote the improvement of degradation efficiency.

[0144] Experimental Investigation 9: Effect of Different Substrate Concentrations on the Catalytic Activity of the Catalyst (Figure)

[0145] like Figure 10 The figures show the degradation rates of the catalyst for substrates at different concentrations. It can be seen that as the acridine solution concentration increases from 5 mg / L to 15 mg / L, acridine can be completely degraded by the catalyst. However, the removal rate gradually decreases with further increases in acridine concentration. When the acridine concentration is 35 mg / L, only 91.1% of the acridine is degraded after 8 hours of reaction. When the acridine concentration reaches 55 mg / L, the acridine removal rate decreases to 83.6%. These results indicate that high concentrations of acridine may adversely affect enzyme activity. Therefore, the performance of the catalyst is affected by relatively high concentrations of acridine.

[0146] Relevant experiments have verified that this biocatalyst exhibits catalytic activity under different substrate concentrations, temperatures, pH levels, number of cycles, and storage times.

[0147] Principle Analysis:

[0148] To address the problems of existing technologies, we attempted to explore a cascade reaction based on dual-enzyme catalysis. In this system, the first enzyme generates H₂O₂ during the reaction, and the second enzyme (HRP) utilizes the newly generated H₂O₂ for oxidation. Using both enzymes together not only weakens the product inhibition during the first enzyme's reaction, ensuring the smooth progress of the enzymatic reaction, but also reduces the substrate inhibition during the second enzyme's reaction, shifting the equilibrium towards product formation. This improves enzyme stability and accelerates the degradation rate, making it a promising dual-enzyme catalytic system. Glucose oxidase (GOD) is an aerobic dehydrogenase that specifically oxidizes β-D-glucose to gluconic acid and H₂O₂ under aerobic conditions, and is considered a green, environmentally friendly, economical, and safe enzyme catalyst. HRP-GOD co-immobilization technology fully leverages the synergistic catalytic effect of the dual-enzyme system. It not only rapidly eliminates product inhibition by GOD, ensuring the smooth progress of the enzymatic reaction, but also utilizes GOD oxidation to activate the H2O2 required for HRP, preventing HRP inactivation in the presence of excess H2O2, thus improving enzyme stability and enabling continuous operation of the enzyme-catalyzed oxidation process. Most importantly, when using the same total amount of enzyme, the degradation efficiency of the dual-enzyme catalytic system is higher than that of the single-enzyme system, which reduces enzyme usage costs to some extent, resulting in a higher cost-effectiveness ratio. Therefore, combining horseradish peroxidase (HRP) with oxidoreductase (GOD) is a promising enzyme catalytic system for wastewater treatment.

[0149] Currently, many co-immobilization supports are used for HRP-GOX, such as silica, polypyrrole membranes, epoxy resins, chitosan, and magnetic particles. However, very few immobilized enzyme supports have been successfully applied to industrial production. Cellulose (Ce) and chitosan (Cs) are abundant in nature and possess important properties such as biodegradability, biocompatibility, and bioactivity, making them important carriers for immobilized enzymes. Cellulose has good chemical stability, is non-toxic, biodegradable, inexpensive, and easy to modify. However, cellulose-based supports have drawbacks such as smooth surfaces and poor adsorption performance. Chitosan is a natural cationic flocculant that can rapidly adsorb enzymes to the carrier for subsequent covalent bonding. However, chitosan has disadvantages such as low mechanical strength, poor chemical resistance, and high crystallinity. Since cellulose and chitosan have similar molecular structures and are compatible, we considered mixing cellulose and chitosan to prepare cellulose-chitosan composite microspheres. This novel composite microsphere not only retains the properties of cellulose and chitosan, but also solves the problems of chitosan dissolving in acid and having poor mechanical strength, and cellulose having a smooth surface and poor adsorption performance.

[0150] Dopamine can undergo oxidative self-polymerization in an alkaline aerobic environment to form highly adhesive polydopamine, which adheres to various matrix surfaces. The dopamine-modified matrix surface possesses functional groups such as amino, imino, catechol, and diquinone. These groups can undergo Michael addition and Schiff base reactions in appropriate solution environments, increasing the complexing and reducing abilities of the matrix material and providing more adsorption sites. Dopamine-modified functional materials have very broad application prospects; therefore, we chose to modify cellulose-chitosan composite microspheres with dopamine. Ionic liquids are green solvents with great potential application value, possessing advantages such as non-volatility, stability, and strong polarity. They can effectively dissolve cellulose and chitosan, which have strong intermolecular and intramolecular hydrogen bonds, making them excellent solvents for dissolving cellulose-chitosan.

[0151] Compared with conventional immobilized horseradish peroxidase biocatalysts, the dopamine-modified cellulose-chitosan composite microsphere co-immobilized horseradish peroxidase and glucose oxidase biocatalysts not only produced a better carrier type, perfectly preserving the advantages of cellulose and chitosan, but also modified the carrier with dopamine, achieving stable enzyme linkage by introducing functional groups that can covalently bind to the enzyme. Most importantly, this technology attempts a dual-enzyme immobilization technique, saving raw materials and improving the degradation rate.

[0152] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A biocatalyst for degrading acridines, characterized in that, By mass fraction, comprising the following components: 10 parts of 1-ethyl-3-methyl imidazole acetate; 2-4 parts of cellulose; 2-4 parts of chitosan; 10-20 parts of dopamine; 10 parts of Tris-Hcl buffer solution; 3-5 parts of glucose oxidase; 5-7 parts of horseradish peroxidase; 10 parts of PBS buffer solution; The preparation method of the biocatalyst for degrading acridine comprises the following steps: (1) Preparation of cellulose-chitosan composite microspheres: Put the formula amount of ionic liquid 1-ethyl-3-methyl imidazole acetate into the reaction container, add the formula amount of cellulose and the formula amount of chitosan and stir, react at 80-100℃ for 2-3h until the cellulose and chitosan are completely dissolved, stop heating, obtain a clear gelatinous solution, after the solution is cooled to room temperature, add distilled water drop by drop with a peristaltic pump to obtain cellulose-chitosan composite microspheres, let it harden for 0.5-2h, and then wash to obtain cellulose-chitosan composite microspheres; (2) Preparation of dopamine-modified cellulose-chitosan composite microspheres: Add the cellulose-chitosan composite microspheres obtained in step (1) and the formula amount of dopamine into the reaction container in sequence, then add the formula amount of Tris-Hcl buffer solution, react in a constant-temperature air shaker, separate and wash the microspheres to obtain dopamine-modified cellulose-chitosan composite microspheres; (3) Preparation of biocatalyst for degrading acridine: Add the dopamine-modified cellulose-chitosan composite microspheres obtained in step (2), the formula amount of glucose oxidase and the formula amount of horseradish peroxidase into the reaction container in sequence, then add the formula amount of PBS buffer solution, react in a constant-temperature air shaker, separate and wash the microspheres with PBS buffer solution with pH value of 6.0-7.0 to obtain the biocatalyst for degrading acridine.

2. The biocatalyst degrading acridine according to claim 1, characterized in that, In step (1), the reaction container is a 25-50mL three-necked flask, the stirring rate is 800-1000r / min, and the mass concentration of the clear gelatinous solution is 4-8wt%.

3. The biocatalyst degrading acridine according to claim 1, characterized in that, In step (2), the rotation speed of the constant-temperature air shaker is 150-200r / min, the reaction time in the constant-temperature air shaker is 2-3h, and the reaction temperature is 20-30℃.

4. The biocatalyst degrading acridine according to claim 1, characterized in that, In step (2), the concentration of the Tris-HCl buffer solution is 0.01-0.02mol / L, the pH value is 8.0-9.0, the temperature is 20-30℃, and the reaction time is 2-3h.

5. The biocatalyst degrading acridine according to claim 1, characterized in that, The number of washing is 2-3 times.

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