A zinc-doped graphite carbon nitride material for loading laccase, immobilized laccase and preparation method and application thereof

Abstract

The application belongs to the field of environmental catalytic materials and wastewater treatment technology, and particularly relates to a zinc-doped graphite carbon nitride material for loading laccase, immobilized laccase and a preparation method and application thereof. The zinc-doped graphite carbon nitride material for loading laccase is prepared by mixing zinc salt, urea and deionized water in a ratio of (0.04g-0.08g):8g:20mL, drying, and calcining under an inert atmosphere to obtain a solid material, and then silanizing the solid material to obtain the zinc-doped graphite carbon nitride material. The immobilized laccase is used to degrade pollutants with a concentration of 10mg / L-30mg / L and a pH of 2-11.

Description

Technical Field

[0001] This invention belongs to the field of environmental catalytic materials and wastewater treatment technology, specifically relating to a zinc-doped graphitic carbon nitride material for loading laccase, immobilized laccase, its preparation method and application. Background Technology

[0002] Methylene blue, or MB for short, is a widely used cationic dye. Due to its high water solubility and stability, it is prevalent in wastewater from the textile and paper industries. It is difficult to degrade naturally, posing a serious threat to aquatic ecosystems and human health.

[0003] Traditional water treatment methods have low efficiency in removing dye wastewater (MB). Enzymatic degradation is an effective method for degrading MB, including laccase degradation. However, laccase alone is easily deactivated and struggles to adapt to complex dye wastewater environments, resulting in low MB degradation efficiency.

[0004] Existing technologies utilize composite materials formed by laccase and other loading materials to load laccase, aiming to address the issue of laccase's easy inactivation when used alone. Primitive g-C3N4 is a common loading material, but its slow charge generation and migration rates result in low efficiency when loading laccase in practice. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention provides a zinc-doped graphitic carbon nitride material for loading laccase, immobilized laccase, its preparation method, and its application, which can solve the technical problem of low efficiency of pristine g-C3N4 when loading laccase due to its slow charge generation and migration rate.

[0006] This invention is achieved by adopting the following technical solution: A zinc-doped graphitic carbon nitride material for loading laccase is prepared by mixing zinc salt, urea and deionized water in a ratio of (0.04g~0.08g):8g:20mL, drying the mixture, calcining it under an inert atmosphere to obtain a solid material, and then silanizing the solid material to obtain the zinc-doped graphitic carbon nitride material.

[0007] Furthermore, the zinc salt is ZnCl2.

[0008] A method for preparing zinc-doped graphitic carbon nitride materials, characterized by comprising the following steps: Urea and ZnCl2 were dissolved in deionized water and dried to obtain a solid precipitate. The ratio of zinc salt, urea and deionized water was (0.04g~0.08g): 8g: 20mL. The solid precipitate was ground to obtain a solid powder, which was then calcined at 550°C for 4 hours under an inert atmosphere and cooled to 30°C to obtain a solid material. Solid materials, alcohol, and 3-aminopropyltriethoxysilane were silanized in a ratio of 50 mg: 10 mL: 1 mL to obtain zinc-doped graphitic carbon nitride materials.

[0009] Furthermore, the calcination conditions are as follows: heating to 550°C at a rate of 2.5°C / min under a nitrogen atmosphere.

[0010] Furthermore, the specific steps of the silanization are as follows: Zinc-doped graphite carbon nitride material was added to alcohol and ultrasonically dispersed. 3-aminopropyltriethoxysilane was added, stirred, and heated at 70°C for 12 hours to carry out silanization reaction. After centrifugation and drying, zinc-doped graphite carbon nitride material was obtained.

[0011] An immobilized laccase comprising the zinc-doped graphitic carbon nitride material is prepared by mixing zinc-doped graphitic carbon nitride material, disodium hydrogen phosphate-citrate buffer, glutaraldehyde solution and laccase in a ratio of 20 mg:(0.72 mL~5.52 mL):(0.04 mL~0.64 mL):(2.4 mL~7.2 mL), followed by incubation, centrifugation, washing and lyophilization. The pH of the disodium hydrogen phosphate-citric acid buffer solution is 2-6; The volume concentration of the glutaraldehyde solution is 50%. The concentration of the laccase was 0.28 mg / mL.

[0012] Furthermore, the method for preparing the immobilized laccase is characterized by comprising the following steps: Zinc-doped graphite carbon nitride material was added to disodium hydrogen phosphate-citric acid buffer solution and sonicated to obtain a silicon-zinc-doped graphite carbon nitride dispersion. Zinc-doped graphite carbon nitride dispersion, glutaraldehyde, and laccase were mixed, incubated, centrifuged, washed, and dried to obtain immobilized laccase. The incubation conditions are 30℃~40℃, 180rpm / min, 70min~370min.

[0013] Application of zinc-doped graphite carbon nitride materials or immobilized laccase in the degradation of methylene blue.

[0014] Furthermore, the immobilized laccase is added to a methylene blue solution for the degradation of methylene blue; wherein, based on the mass of laccase, 20 mg of immobilized laccase is added to every 100 mL of methylene blue solution, the concentration of the methylene blue solution is 10 mg / L to 30 mg / L, the pH during degradation is 2 to 11, and the temperature during degradation is 20℃ to 50℃.

[0015] The principle of this invention: Among catalytic materials, graphitic carbon nitride (g-C3N4) has become a highly promising non-metallic photocatalyst due to its simple synthesis, stable structure, environmental friendliness, and suitable band gap. Its large specific surface area and excellent electron transport capabilities support long-term photocatalytic activity in water treatment. The nanoscale cavities formed by its layered structure can load metal nanoparticles or other catalytic components, thereby preparing high-performance composite materials. However, the practical application efficiency of pristine g-C3N4 is limited due to its slow charge generation and migration rates. This invention improves the application efficiency of g-C3N4 through zinc doping and silanization reactions.

[0016] In addition, laccase precisely extracts electrons from dye molecules through a redox reaction mediated by copper ions at its active center, generating unstable free radical intermediates. These active intermediates then undergo non-enzymatic polymerization or ring-opening reactions, disrupting the conjugated chromophore system of the dye and achieving rapid decolorization and deep degradation of MB.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a zinc-doped graphitic carbon nitride material for loading laccase. The material is prepared by mixing zinc salt, urea, and deionized water in a ratio of (0.04 g ~ 0.08 g): 8 g: 20 mL, drying, and calcining under an inert atmosphere to obtain a solid material. The solid material is then silanized to obtain the zinc-doped graphitic carbon nitride material. Graphitic carbon nitride is modified using metal doping to synthesize a hierarchical porous zinc-doped graphitic carbon nitride with a surface filled with mesopores. Laccase is then loaded onto this hierarchical porous zinc-doped graphitic carbon nitride. Experiments using UV-Vis diffuse reflectance spectroscopy, photoluminescence spectroscopy, photocurrent response density, and electrochemical impedance spectroscopy show an improvement in its photoresponse and electron transfer capabilities, demonstrating that the zinc-doped graphitic carbon nitride has superior photocatalytic activity. The specific surface area of ​​the zinc-doped graphitic carbon nitride is calculated to be 239.3 m². 2 / g, while the specific surface area of ​​g-C3N4 material is 113.28m². 2 / g, the former is 2.11 times that of the latter, which greatly improves the enzyme carrying capacity of zinc-doped graphite carbon nitride; when the doping amount of zinc chloride is 1%, the enzyme carrying capacity of zinc-doped graphite carbon nitride is 36.6mg / g, which is 45.8% higher than the enzyme carrying capacity of g-C3N4 of 25.1mg / g.

[0018] (2) The immobilized laccase prepared in this invention is used as a photo-enzyme catalyst to degrade methylene blue MB. After 60 min of light irradiation, its degradation rate is 89.8%, which is higher than the degradation rate of zinc-doped graphite nitride and laccase alone. The immobilized laccase is a composite material of zinc-doped graphite nitride loaded with laccase.

[0019] (3) The immobilized laccase provided by the present invention exhibits better enzyme activity than free laccase over a wider range of pH and temperature, and maintains effective MB degradation effect under different pH, temperature and dye concentrations.

[0020] (4) The immobilized laccase prepared in this invention can achieve a high MB degradation rate when used as a photo-enzyme catalyst for synergistic catalytic reaction, thus it has great industrial application potential in photocatalysis and biocatalysis. Attached Figure Description

[0021] Figure 1 In the image, (a) is a scanning electron microscope image of ZCN-1% prepared in Example 2; (b) is a transmission electron microscope image of ZCN-1% prepared in Example 2.

[0022] Figure 2 In the image, (a) is a scanning electron microscope image of g-C3N4 prepared in Comparative Example 1; (b) is a transmission electron microscope image of g-C3N4 prepared in Comparative Example 1.

[0023] Figure 3 In the figure, (a) is the EDS elemental distribution diagram of ZCN-1% prepared in Example 2; (b) is the XRD pattern of zinc-doped graphitic carbon nitride prepared in Comparative Examples 1, 3, 4, 1 and 2.

[0024] Figure 4 The UV-Vis diffuse reflectance spectra of g-C3N4 prepared in Comparative Example 1 and ZCN-1% prepared in Example 2 are shown.

[0025] Figure 5 Photoluminescence spectra of g-C3N4 prepared in Comparative Example 1 and ZCN-1% prepared in Example 2.

[0026] Figure 6 Electrochemical impedance spectroscopy for g-C3N4 prepared in Comparative Example 1 and ZCN-1% prepared in Example 2.

[0027] Figure 7 Photocurrent response diagrams of g-C3N4 prepared in Comparative Example 1 and ZCN-1% prepared in Example 2.

[0028] Figure 8 EDS elemental distribution map of ZCN-1% / LC prepared in Example 3.

[0029] Figure 9 The XRD patterns are of ZCN-1% and ZCN-1% / LC prepared in Examples 2 and 3.

[0030] Figure 10The graph shows the enzyme loading and activity of ZCN-1% / LC obtained in Examples 3 to 8 under different incubation times.

[0031] Figure 11 The graph shows the enzyme loading and enzyme activity of ZCN-1% / LC obtained in Examples 3, 9 to 16 under different buffer pH conditions.

[0032] Figure 12 The graph shows the enzyme loading and activity of ZCN-1% / LC prepared in Examples 3, 17 to 21 under different amounts of glutaraldehyde.

[0033] Figure 13 The graph shows the enzyme loading and enzyme activity of ZCN-1% / LC prepared in Examples 3 and 22-27 under different enzyme addition amounts.

[0034] Figure 14 Temperature and pH stability of laccase in ZCN-1% / LC prepared in Example 3.

[0035] Figure 15 In the figures, (a) shows the test results of the degradation of methylene blue MB by ZCN-1% / LC in Example 3 at different pH values; (b) shows the test results of the degradation of methylene blue MB by different dye concentrations; (c) shows the test results of the degradation of methylene blue MB at different temperatures; (d) shows the MB removal rate of ZCN-1% in Example 3 and ZCN-1% / LC in Example 2 at different pH conditions after 90 min; (e) shows the MB removal rate of ZCN-1% in Example 3 and ZCN-1% / LC in Example 2 at different MB concentrations after 90 min; and (f) shows the MB removal rate of ZCN-1% in Example 3 and ZCN-1% / LC in Example 2 at different temperatures after 90 min.

[0036] Figure 16 In the figure, (a) shows the free radical quenching results of ZCN-1% prepared in Example 3; (b) shows the EPR experimental results of ZCN-1% prepared in Example 3. Detailed Implementation

[0037] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods. Specific explanations are needed as follows:

[0038] Specific surface area, abbreviated as BET; graphite carbon nitride, abbreviated as g-C3N4; zinc-doped graphite carbon nitride, abbreviated as ZCN; zinc-doped graphite carbon nitride loaded with laccase, abbreviated as immobilized laccase; 3-aminopropyltriethoxysilane, abbreviated as APTES; laccase, abbreviated as LC, purchased from Xiasheng (Beijing) Biotechnology Co., Ltd., catalog number FDG-2264, unit is 0.28mg / mL; disodium hydrogen phosphate-citrate buffer, abbreviated as PBS buffer; EDS represents elemental analysis; XRD represents X-ray diffraction analysis; methylene blue, abbreviated as MB.

[0039] Example 1 A method for preparing a solid material, the specific steps of which are as follows: 0.04 g of zinc chloride and 8 g of urea were added to 20 mL of deionized water and ultrasonically dispersed at 30 °C for 20 min. The mixture was then vacuum dried in an oven at 70 °C for 24 h to obtain a solid precipitate. The dried solid precipitate was ground for 15 min to obtain a solid powder, which was then placed in an alumina crucible and heated to 550 °C at a rate of 2.5 °C / min under a nitrogen atmosphere and held at this temperature for 4 h. After the holding period, the mixture was allowed to cool naturally to 30 °C to obtain a solid material, labeled as ZCN-0.5% powder.

[0040] Example 2 A method for preparing zinc-doped graphitic carbon nitride material for loading laccase, the specific steps are the same as in Example 1, except that: the amount of zinc chloride added is 0.08g, and a solid material is obtained, labeled as ZCN-1% powder.

[0041] Example 3 A method for preparing immobilized laccase includes the following steps: S1. 100 mg of ZCN-1% powder from Example 2 was ultrasonically dispersed in 20 mL of ethanol at 30 °C, and 2 mL of APTES was added. Silanization was carried out by magnetic stirring and water bath heating for 12 h at 70 °C and magnetic stirring speed of 800 rpm / min. Subsequently, the mixture was centrifuged at 8000 rpm / min for 3 min, and the precipitate was collected and freeze-dried at -40 °C for 2 h to obtain the silanized ZCN-1% powder, i.e., zinc-doped graphitic carbon nitride material.

[0042] S2. Disperse 20 mg of silanized ZCN-1% powder ultrasonically in 3.92 mL of PBS buffer with a pH of 3. Add 0.08 mL of 50% glutaraldehyde solution and incubate in a gas-phase shaker at 30 °C and 180 rpm for 10 min. Add 4 mL of laccase and incubate at 40 °C and 180 rpm for 4 h. After incubation, centrifuge at 8000 rpm for 3 min, wash with PBS buffer, and freeze-dry at -40 °C for 2 h to obtain immobilized laccase, labeled as ZCN-1% / LC.

[0043] Example 4 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH value of PBS buffer is 4, and after adding laccase, it is incubated at 40°C and 180 rpm / min for 1 h.

[0044] Example 5 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH value of PBS buffer is 4, and after adding laccase, it is incubated at 40°C and 180 rpm / min for 2 h.

[0045] Example 6 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH value of PBS buffer is 4, and after adding laccase, it is incubated at 40°C and 180 rpm / min for 3 h.

[0046] Example 7 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH value of PBS buffer is 4, and after adding laccase, it is incubated at 40°C and 180 rpm / min for 5 h.

[0047] Example 8 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH value of PBS buffer is 4, and after adding laccase, it is incubated at 40°C and 180 rpm / min for 6 h.

[0048] Example 9 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH of the PBS buffer is 2.

[0049] Example 10 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH of the PBS buffer is 2.5.

[0050] Example 11 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH of the PBS buffer is 3.

[0051] Example 12 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH of the PBS buffer is 3.5.

[0052] Example 13 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH of the PBS buffer is 4.5.

[0053] Example 14 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH of the PBS buffer is 5.

[0054] Example 15 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH of the PBS buffer is 5.5.

[0055] Example 16 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the pH of the PBS buffer is 6.

[0056] Example 17 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the amount of 50% glutaraldehyde solution added is 0.04 mL.

[0057] Example 18 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the amount of 50% glutaraldehyde solution added is 0.16 mL.

[0058] Example 19 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the amount of 50% glutaraldehyde solution added is 0.24 mL.

[0059] Example 20 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the amount of 50% glutaraldehyde solution added is 0.32 mL.

[0060] Example 21 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the amount of 50% glutaraldehyde solution added is 0.64 mL.

[0061] Example 22 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the volume of PBS buffer is 5.52 mL and the volume of laccase is 2.4 mL.

[0062] Example 23 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the volume of PBS buffer is 4.72 mL and the volume of laccase is 3.2 mL.

[0063] Example 24 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the volume of PBS buffer is 3.12 mL and the volume of laccase is 4.8 mL.

[0064] Example 25 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the volume of PBS buffer is 2.32 mL and the volume of laccase is 5.6 mL.

[0065] Example 26 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the volume of PBS buffer is 1.52 mL and the volume of laccase is 6.4 mL.

[0066] Example 27 A method for preparing immobilized laccase, the specific steps are the same as in Example 3, except that in step S2, the volume of PBS buffer is 0.72 mL and the volume of laccase is 7.2 mL.

[0067] Example 28 The 20 mg immobilized laccase ZCN-1% / LC obtained in Example 3 was placed in 100 mL of methylene blue solution with a concentration of 20 mg / L. The solution was magnetically stirred at 30 °C and 650 rpm for 30 min under light-protected conditions until adsorption equilibrium was reached. The concentration of the dye at this time was measured by a UV spectrophotometer.

[0068] After adsorption equilibrium was reached, the methylene blue solution was irradiated under a 300W xenon lamp. Every 15 minutes, 4 ml of the filtered methylene blue solution was taken and the concentration of methylene blue was recorded using a UV spectrophotometer.

[0069] Example 29 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as those in Example 28, except that the pH of the methylene blue solution is 2.

[0070] Example 30 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as those in Example 28, except that the pH of the methylene blue solution is 5.

[0071] Example 31 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as those in Example 28, except that the pH of the methylene blue solution is 7.

[0072] Example 32 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as those in Example 28, except that the pH of the methylene blue solution is 9.

[0073] Example 33 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as those in Example 28, except that the pH of the methylene blue solution is 11.

[0074] Example 34 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as in Example 28, except that the concentration of the methylene blue solution is 10 mg / L.

[0075] Example 35 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as those in Example 28, except that the concentration of the methylene blue solution is 30 mg / L.

[0076] Example 36 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as those in Example 28, except that the temperature of the methylene blue solution is 20°C.

[0077] Example 37 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as those in Example 28, except that the temperature of the methylene blue solution is 40°C.

[0078] Example 38 A method for immobilized laccase to degrade methylene blue, the specific steps of which are the same as those in Example 28, except that the temperature of the methylene blue solution is 50°C.

[0079] Comparative Example 1 A method for preparing graphite carbon nitride material, the specific steps of which are as follows: 8g of urea was ground and placed in an alumina crucible. The crucible was heated to 550℃ in a tube furnace at a rate of 2.5℃ / min and held at that temperature for 4 hours. After the holding period, the mixture was allowed to cool naturally to room temperature to obtain graphitic carbon nitride material, labeled as g-C3N4 powder.

[0080] Comparative Example 2 A method for preparing laccase-loaded graphitic carbon nitride, comprising the following steps: S1. Take 100 mg of g-C3N4 powder from Comparative Example 1, and ultrasonically disperse it in 20 mL of ethanol. Add 2 mL of LAPTES to the ethanol. Perform silanization by magnetic stirring and water bath heating for 12 h at 70 °C and magnetic stirring speed of 800 rpm / min. Then centrifuge and freeze-dry to obtain silanized g-C3N4 powder.

[0081] S2. Disperse 20 mg of silanized g-C3N4 powder sonically in 3.92 mL of PBS buffer (pH 3). Add 0.08 mL of 50% glutaraldehyde solution and incubate in a gas-phase shaker at 30 °C and 180 rpm for 10 min. Add 4 mL of laccase and incubate at 40 °C and 180 rpm for 4 h. After incubation, centrifuge, wash, and lyophilize to obtain laccase-loaded graphitic carbon nitride, labeled g-C3N4 / LC.

[0082] Comparative Example 3 A method for preparing a solid material, the specific steps of which are the same as in Example 1, except that the amount of zinc chloride added is 0.16g, and a solid material is obtained, which is labeled as ZCN-2% powder.

[0083] Comparative Example 4 A method for preparing a solid material, the specific steps of which are the same as in Example 1, except that: the amount of zinc chloride added is 0.40g, and a solid material is obtained, which is labeled as ZCN-5% powder.

[0084] The following experiments were conducted on the above materials: Experiment 1: Characterization of the solid material of Example 2 and the graphitic carbon nitride material of Comparative Example 1 The ZCN-1% prepared in Example 2 was observed by scanning electron microscopy and transmission electron microscopy, and the results are as follows: Figure 1(a) shows a scanning electron microscope image of ZCN-1%; and (b) shows a transmission electron microscope image of ZCN-1%.

[0085] The g-C3N4 powder prepared in Comparative Example 1 was observed by scanning electron microscopy and transmission electron microscopy, and the results are as follows: Figure 2 Image (a) shows a scanning electron microscope image of g-C3N4, and image (b) shows a transmission electron microscope image of g-C3N4.

[0086] It can be seen that with the addition of zinc chloride, ZCN-1% exhibits more porous structure. Transmission electron microscopy shows that the surface of ZCN-1% becomes rougher, the specific surface area increases, more active sites are exposed, and the enzyme carrying capacity and catalytic ability of the material are also improved.

[0087] Experiment 2: EDS elemental scanning and XRD characterization To investigate the effect of zinc incorporation on g-C3N4, EDS elemental scanning was performed on the ZCN-1% prepared in Example 2, and XRD characterization was performed on the solid materials prepared in Examples 1, 2, 3, and 4.

[0088] The results are as follows Figure 3 As shown in (a), the successful incorporation of zinc demonstrates the successful synthesis of the material. Figure 3 (b) shows that a typical characteristic peak of graphitic carbon nitride appears at 27.4°, but the addition of excessive zinc chloride will destroy the crystal structure of the material, such as the solid materials in Comparative Examples 3 and 4.

[0089] Experiment 3: Further characterization of the solid material of Example 2 and the graphitic carbon nitride of Comparative Example 1. To further verify that zinc doping improves the photocatalytic ability of the material, the ZCN-1% obtained in Example 2 and the graphitic carbon nitride obtained in Comparative Example 1 were characterized by ultraviolet-visible diffuse reflectance spectroscopy, photoluminescence spectroscopy, photocurrent response density characterization using transient photocurrent response (TPR) technology, and electrochemical impedance spectroscopy.

[0090] The results are as follows Figure 4 The image shows the UV-Vis diffuse reflectance spectrum of ZCN-1% prepared in Example 2, indicating that all samples exhibit typical semiconductor absorption characteristics in the 200–500 nm wavelength range. g-C3N4 shows a characteristic absorption edge at 450 nm, which is related to its limited visible light absorption capability. After zinc doping, the absorption edge of ZCN-1% undergoes a redshift, and the band gap energy decreases to 2.88 eV, indicating a significant enhancement in its light absorption capability.

[0091] The results are as follows Figure 5The image shows the photoluminescence spectra of ZCN-1% prepared in Example 2 and g-C3N4 prepared in Comparative Example 1. g-C3N4 exhibits a broad emission peak near 450 nm, indicating insufficient charge separation efficiency and rapid recombination of photogenerated carriers. In contrast, the photoluminescence intensity of ZCN-1% is significantly reduced, indicating an extended lifetime for photogenerated electrons and holes. This performance improvement is attributed to zinc doping, which promotes efficient charge separation and suppresses electron-hole recombination, thereby significantly enhancing photocatalytic performance.

[0092] By employing transient photocurrent response (TPR) technology and performing multiple switching cycle tests at 40-second intervals, the results are as follows: Figure 6 and Figure 7 Electrochemical impedance spectroscopy (EIS) was used to thoroughly evaluate the separation and transport behavior of photogenerated electrons and holes. Both ZCN-1% and g-C3N4 exhibited stable and reproducible photocurrent responses under visible light irradiation. The charge generation and migration rates of pristine g-C3N4 were slow. When the light source was turned off, the photocurrent decayed rapidly, confirming the material's excellent photoresponse characteristics. In the synthesized samples, the transient photocurrent intensity was ranked as ZCN-1% > g-C3N4, indicating that ZCN-1% can separate and transport photogenerated carriers and holes more quickly and efficiently than pristine g-C3N4. Furthermore, EIS tests were performed in the open-circuit potential range of 100 kHz to 10 mHz to further investigate the charge transport process in the photocatalyst. Compared with pristine g-C3N4, the charge transfer resistance of ZCN-1% was significantly reduced, confirming that the internal electron transfer efficiency of ZCN-1% was improved.

[0093] Experiment 4 Laccase Distribution Detection To investigate the distribution of the enzyme after immobilization on the material, EDS analysis was performed on the ZCN-1% / LC prepared in Example 3. The results are as follows: Figure 8 As shown, since laccase is a copper-containing oxidase, the presence of copper indicates that laccase was successfully loaded onto the material surface. XRD results are as follows. Figure 9 As shown, the crystal structure of ZCN-1% remained unchanged after enzyme loading, demonstrating the stability of the material during the immobilization of laccase.

[0094] Experiment 5 Enzyme Loading and Enzyme Activity Detection The enzyme loading and activity of the ZCN-1% / LC prepared in Examples 3-8 were determined by Coomassie Brilliant Blue assay and ABTS spectrophotometry, respectively. The results are as follows: Figure 10 As shown, with the increase of incubation time, the enzyme load and enzyme activity also increase relatively. When the incubation time is 4 hours, the enzyme load and laccase activity reach a relatively high value.

[0095] The enzyme loading and enzyme activity test results of the ZCN-1% / LC prepared in Examples 3, 9-16 are as follows: Figure 11 As shown, excessively high pH levels can decrease enzyme loading and laccase activity. At pH 3, the material achieves both high enzyme loading and activity simultaneously; at this pH, ZCN-1% has an enzyme loading of 42.6 mg / g and a relative enzyme activity of 95.1%. Therefore, the optimal pH for the buffer solution during immobilization is 3.

[0096] The enzyme loading and enzyme activity test results of the ZCN-1% / LC prepared in Examples 3, 17-21 are as follows: Figure 12 As shown, excessive glutaraldehyde will decrease laccase activity. When the optimal addition amount of glutaraldehyde is 0.5% of the total volume, the material can simultaneously achieve high enzyme loading and enzyme activity. At this point, the enzyme loading of ZCN-1% is 46.1 mg / g, and the relative enzyme activity is the highest in the group. Therefore, the optimal addition amount of glutaraldehyde during immobilization is 0.5% of the total volume, where the total volume is the sum of the volumes of glutaraldehyde, PBS buffer, and laccase.

[0097] like Figure 13 The figures show the enzyme loading and activity test results of ZCN-1% / LC prepared in Examples 3 and 22-27 at different enzyme addition amounts. The results show that higher laccase concentrations resulted in higher enzyme loading, reaching a peak of 48.25 mg / g at 0.9 mL / mL. At this high concentration, laccase better covered the carrier surface. However, further increases in enzyme concentration may lead to enzyme aggregation and steric hindrance, which could limit substrate contact and cause a decrease in activity. Therefore, the optimal immobilized laccase concentration was determined to be 0.5 mL / mL.

[0098] Experiment 6 Stability test after loading laccase The immobilized laccase prepared in Example 3 was incubated in 10 mL of pH 2-6 in a shaker at 10℃-60℃ for 2 h. The pH stability and temperature stability of ZCN-1% / LC prepared in Example 3 were then tested.

[0099] The results are as follows Figure 14 As shown, the laccase activity in ZCN-1% / LC is less affected by temperature and pH, indicating that the laccase is protected by the material and its temperature and pH tolerance is improved.

[0100] Experiment 7 Degradation of Methylene Blue Test Examples 28-38 illustrate the degradation of methylene blue using ZCN-1% / LC. The concentration of MB was calculated by measuring the absorbance of the dye using a UV spectrophotometer. The degradation efficiency of methylene blue MB by ZCN-1% / LC in Example 3 was evaluated under different pH values, dye concentrations, and temperatures by detecting the concentration of methylene blue.

[0101] The results are as follows Figure 15 As shown in the figure, laccase exhibits good activity under acidic conditions, thus achieving a good photo-enzyme similarity effect. This results in the ZCN-1% / LC prepared in Example 3 having a better degradation effect than the ZCN-1 prepared in Example 2. Under different dye concentrations and temperatures, ZCN-1% / LC maintains better catalytic performance than the ZCN-1% catalyst, which further confirms its good degradation ability. The immobilized laccase was used to degrade pollutant MB at a concentration of 10~30 mg / L and a pH of 2~11.

[0102] Experiment 8 Free Radical Quenching Experiment like Figure 16 As shown in (a), the main reactive species generated by ZCN-1% / LC is superoxide radical, as indicated by EPR testing. Figure 16 As shown in (b), a significant superoxide radical signal was generated under light conditions, further illustrating that superoxide radicals are the main active species.

[0103] The specific surface area, pore volume, and pore volume of g-C3N4 prepared in Comparative Example 1, ZCN-1% prepared in Example 2, and ZCN-1% / LC prepared in Example 3 were tested. The results are shown in Table 1. The specific surface area of ​​g-C3N4 was 113.28 m². 2 / g, while the specific surface area of ​​ZCN-1% is 239.3m². 2 / g, after modification, the specific surface area of ​​the material increased by 1.11 times. Furthermore, the pore volume of ZCN-1% increased from 0.81 cm⁻¹ after enzyme loading. 3 / g became 0.06cm 3 The / g value is likely due to the enzyme entering the pores, altering the surface environment of the material, and thus reducing the pore volume. This further demonstrates the success of enzyme loading on the material surface.

[0104] Table 1 Statistical analysis of material characterization data In summary, the present invention provides a method for preparing and applying zinc-doped graphitic carbon nitride (ZCN-1%) immobilized laccase. A stable ZCN-1% with photocatalytic properties was synthesized via elemental doping, and the laccase was then immobilized by cross-linking with porous ZCN-1%. The immobilized LC exhibits better pH adaptability and thermal stability. The ZCN-1% / LC prepared by this invention can achieve photo-enzyme synergistic catalysis under illumination. When used as a photocatalyst and enzyme biocatalyst, the ZCN-1% / LC prepared by this invention can achieve a high MB degradation rate, demonstrating significant industrial application potential in water treatment and synergistic catalysis.

[0105] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A zinc-doped graphitic carbon nitride material for loading laccase, characterized in that, Zinc salt, urea, and deionized water were mixed in a ratio of (0.04g~0.08g):8g:20mL, dried, and calcined under an inert atmosphere to obtain a solid material. The solid material was then silanized to obtain zinc-doped graphitic carbon nitride material.

2. The zinc-doped graphite carbon nitride material according to claim 1, characterized in that, The zinc salt is ZnCl2.

3. The method for preparing zinc-doped graphitic carbon nitride material according to claim 1, characterized in that, Includes the following steps: Urea and ZnCl2 were dissolved in deionized water and dried to obtain a solid precipitate. The ratio of zinc salt, urea and deionized water was (0.04g~0.08g): 8g: 20mL. The solid precipitate was ground to obtain a solid powder, which was then calcined at 550°C for 4 hours under an inert atmosphere and cooled to 30°C to obtain a solid material. Solid materials, alcohol, and 3-aminopropyltriethoxysilane were silanized in a ratio of 50 mg: 10 mL: 1 mL to obtain zinc-doped graphitic carbon nitride materials.

4. The preparation method according to claim 3, characterized in that, The calcination conditions are as follows: heating to 550°C at a rate of 2.5°C / min under a nitrogen atmosphere.

5. The preparation method according to claim 3, characterized in that, The specific steps of silanization are as follows: Zinc-doped graphite carbon nitride material was added to alcohol and ultrasonically dispersed. 3-aminopropyltriethoxysilane was added, stirred, and heated at 70°C for 12 hours to carry out silanization reaction. After centrifugation and drying, zinc-doped graphite carbon nitride material was obtained.

6. An immobilized laccase comprising the zinc-doped graphitic carbon nitride material of claim 1, characterized in that, The solution was prepared by mixing zinc-doped graphite carbon nitride material, disodium hydrogen phosphate-citrate buffer, glutaraldehyde solution and laccase in a ratio of 20 mg: (0.72 mL~5.52 mL): (0.04 mL~0.64 mL): (2.4 mL~7.2 mL), followed by incubation, centrifugation, washing and lyophilization. The pH of the disodium hydrogen phosphate-citric acid buffer solution is 2-6; The volume concentration of the glutaraldehyde solution is 50%. The concentration of the laccase was 0.28 mg / mL.

7. The method for preparing immobilized laccase according to claim 6, characterized in that, Includes the following steps: Zinc-doped graphite carbon nitride material was added to disodium hydrogen phosphate-citric acid buffer solution and sonicated to obtain a silicon-zinc-doped graphite carbon nitride dispersion. Zinc-doped graphite carbon nitride dispersion, glutaraldehyde, and laccase were mixed, incubated, centrifuged, washed, and dried to obtain immobilized laccase. The incubation conditions are 30℃~40℃, 180rpm / min, 70min~370min.

8. The application of the zinc-doped graphite carbon nitride material of claim 1 or the immobilized laccase of claim 4, characterized in that, Application in the degradation of methylene blue.

9. The application according to claim 8, characterized in that, The immobilized laccase was added to the methylene blue solution to degrade the methylene blue. Specifically, based on the mass of laccase, 20 mg of immobilized laccase is added to every 100 mL of methylene blue solution. The concentration of the methylene blue solution is 10 mg / L to 30 mg / L, the pH during degradation is 2 to 11, and the temperature during degradation is 20℃ to 50℃.

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