Manganese-doped MOF (Metal Organic Framework) derived nano laccase as well as preparation method and application thereof
Manganese-doped ZIF-8 derivatives were prepared by aqueous-phase synthesis and low-temperature calcination, which solved the problems of complexity and insufficient substrate affinity in the synthesis of MOF-based laccases and achieved efficient and stable nanozyme catalytic performance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN NORMAL UNIVERSITY
- Filing Date
- 2026-04-09
- Publication Date
- 2026-05-12
AI Technical Summary
Existing methods for synthesizing MOF-based laccases are complex, costly, and have low yields. They also lack sufficient affinity for substrates, making it difficult to meet the needs of industrial-scale mass production. Furthermore, their stability and catalytic efficiency need to be improved.
ZIF-8 substrate material was prepared by aqueous synthesis, and manganese was introduced by metal impregnation. The resulting manganese-doped ZIF-8 derivative was obtained by low-temperature calcination and acid washing in an oxygen atmosphere.
MOF-derived nanozymes with high stability and high catalytic activity have been developed, exhibiting excellent structural stability and environmental tolerance, higher catalytic efficiency than natural enzymes, and are suitable for catalyzing a variety of organic pollutants.
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Figure CN122006703A_ABST
Abstract
Description
Technical Field
[0001] This invention mainly relates to the technical field of nanozyme catalysis, specifically a manganese-doped MOF-derived nanolaccase, its preparation method, and its application. Background Technology
[0002] Laccase is a redox enzyme with a broad substrate range and excellent catalytic efficiency. It catalyzes the oxidation of various organic pollutants such as phenols and aromatic amines by molecular oxygen, producing only water and exhibiting outstanding green catalytic performance. However, natural laccase has many shortcomings in practical applications: poor stability, high production cost, and difficulty in recycling. Compared with natural enzymes, nanozymes have superior structural stability and environmental tolerance, playing an important role in the field of catalysis as a substitute for biological enzymes.
[0003] In the research of various emerging nanozyme structures, enzymes based on metal-organic frameworks (MOFs) have driven the development and innovation of artificially simulated enzyme systems. Compared with natural enzymes, MOF-based enzymes possess precisely simulated catalytic centers and highly dispersed catalytic sites, which is the main reason why simulated enzymes have significantly improved substrate affinity and catalytic efficiency compared to natural enzymes. In addition, MOF-based enzymes also have the advantages of low cost, simple preparation, high stability, and high durability.
[0004] Currently reported synthesis of MOF-based laccases mostly employs a solvothermal method, requiring high temperature and pressure, expensive organic ligands, and is characterized by complex processes, low yields, poor batch stability, and high post-processing costs (solvent removal, activation) of MOFs, making it difficult to meet the needs of industrial-scale mass production. Moreover, although they exhibit high activity, their affinity for substrates is insufficient, requiring high substrate concentrations to function effectively. Summary of the Invention
[0005] Based on this, this invention synthesizes the zeolite imidazole ester framework-8 using an aqueous phase synthesis method, introducing manganese into the ZIF-8 structure using potassium permanganate as a precursor. Manganese-doped ZIF-8 derivatives are obtained by low-temperature air calcination. The synthesis conditions are mild, and the method is green and pollution-free. This material exhibits high laccase-like activity, providing a manganese-doped MOF-derived nanolaccase, its preparation method, and its applications, thereby addressing the technical problems mentioned in the background section.
[0006] To achieve the above objectives, the present invention provides the following technical solution: A manganese-doped MOF-derived nanolaccase was obtained by derivatization of a precursor via low-temperature calcination in an oxygen atmosphere using ZIF-8 synthesized in an aqueous phase as a base. The aqueous phase synthesis refers to the use of ultrapure water as the solvent during the synthesis process. ZIF-8 is a nanolaccase derived from Zn... 2+ A zeolite-like topological MOF material formed by self-assembly with 2-methylimidazole; derived by low-temperature calcination of precursors in an oxygen atmosphere.
[0007] Preferably, the precursor introduces manganese element by metal impregnation, and the raw materials used for metal impregnation are potassium permanganate and ZIF-8, with a mass ratio of 0 to 0.5.
[0008] Preferably, the low-temperature calcination method uses a tube furnace with an air atmosphere, a temperature of 200-300℃, a holding time of 1-3 hours, and a heating rate of 1-5℃ / min.
[0009] Preferably, the low-temperature calcination temperature is 250°C, the holding time is 2 hours, and the heating rate is 2°C / min.
[0010] Preferably, the process also includes an acid pickling step, wherein the pickling temperature is 50-70℃, the pickling time is 0.5-2h, and the acid is sulfuric acid.
[0011] Preferably, the pickling process is carried out at a temperature of 60°C for 1 hour.
[0012] A method for preparing manganese-doped MOF-derived nanolaccase is provided, comprising the following steps: Step 1: Synthesize ZIF-8 using an aqueous phase synthesis method; Step 2: Introduce manganese into ZIF-8 using a metal impregnation method to obtain the precursor; Step 3: Calcining the precursor at low temperature; Step 4: The calcined material is acid-washed to obtain manganese-doped ZIF-derived nanozymes.
[0013] Preferably, the aqueous synthesis method specifically involves adding zinc nitrate hexahydrate and 2-methylimidazole to ultrapure water, stirring, centrifuging, and then vacuum drying to obtain ZIF-8.
[0014] Preferably, the metal impregnation method specifically involves: adding ZIF-8 to ultrapure water and stirring, then adding potassium permanganate, stirring, centrifuging, and vacuum drying to obtain the precursor.
[0015] In summary, the present invention has the following main beneficial effects: This invention uses ultrapure water as a solvent to prepare ZIF-8 as a substrate material. Manganese is introduced through metal impregnation, and then the material is transformed into a porous carbon material by low-temperature calcination and acid washing, using ZIF as a sacrificial template. This process retains the morphology and pore structure of ZIF while exhibiting higher stability. Attached Figure Description
[0016] Figure 1 A scanning electron microscope schematic diagram of the manganese-doped ZIF-derived nanozyme of the present invention; Figure 2 This is an XRD test image of the present invention; Figure 3 The results of evaluating the laccase-like activity of the present invention were obtained by measuring its absorbance at λ=510nm. Figure 4 The relative activity was determined at a wavelength of 510 nm for the purposes of this invention. Figure 1 ; Figure 5 The relative activity was determined at a wavelength of 510 nm for the purposes of this invention. Figure 2 ; Figure 6 Steady-state kinetics test of manganese-doped ZIF-derived nanozymes of the present invention Figure 1 ; Figure 7 Steady-state kinetics test of manganese-doped ZIF-derived nanozymes of the present invention Figure 2 ; Figure 8 This is a graph showing the specific activity of the manganese-doped ZIF-derived nanozyme of the present invention. Figure 9 The catalytic mechanism results of the manganese-doped ZIF-derived nanozyme of the present invention; Figure 10 The results of placing the manganese-doped ZIF-derived nanozyme and natural laccase at different pH values for 7 hours are shown in Figure 1. Figure 11 The results of placing the manganese-doped ZIF-derived nanozyme and natural laccase at different pH values for 7 hours are shown in Figure 2. Figure 12 This is a graph illustrating the effect of ionic strength on catalytic activity in this invention. Figure 13 This is a graph showing the catalytic activity of the present invention at different ethanol concentrations; Figure 14 The peak area of 2,4-dichlorophenol at concentrations of 20-200 μg / ml is used in this invention. Figure 15 The results of adding the manganese-doped ZIF-derived nanozyme of the present invention to a phosphate buffer containing 2,4-dichlorophenol at a final concentration of 400 μg / ml. Detailed Implementation
[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
[0018] A manganese-doped MOF-derived nanolaccase was obtained by derivatization of a precursor via low-temperature calcination in an oxygen atmosphere using ZIF-8 synthesized in an aqueous phase as a substrate. The aqueous phase synthesis refers to the use of ultrapure water as the solvent during the synthesis process. ZIF-8 is a nanolaccase derived from Zn... 2+ A zeolite-like topological MOF material formed by self-assembly with 2-methylimidazole; derived by low-temperature calcination of precursors in an oxygen atmosphere.
[0019] The precursor introduces manganese through a metal impregnation method. The raw materials used for metal impregnation are potassium permanganate and ZIF-8, with a mass ratio of 0 to 0.5.
[0020] The low-temperature calcination method uses a tube furnace with an air atmosphere, a temperature of 200-300℃, a holding time of 1-3 hours, and a heating rate of 1-5℃ / min.
[0021] The low-temperature calcination temperature was 250℃, the holding time was 2h, and the heating rate was 2℃ / min.
[0022] It also includes an acid pickling process, during which the temperature is 50-70℃, the pickling time is 0.5-2h, and the acid used is sulfuric acid.
[0023] The pickling temperature is 60℃ and the pickling time is 1 hour.
[0024] Example 1 It should be noted that, in this embodiment, a manganese-doped ZIF-derived nanozyme was prepared, and the preparation method includes the following steps: A two-step synthesis method was adopted. First, the aqueous phase ZIF-8 was synthesized: 2.95 g of zinc nitrate hexahydrate and 6.5 g of 2-methylimidazole were added to 200 mL of ultrapure water and stirred for 40 min. After repeated centrifugation of the ultrapure water (9000 r / min), it was dried under vacuum at 60 °C for 12 h to obtain the aqueous phase ZIF-8.
[0025] In the second step, 0.1 g of ZIF-8 was added to 60 mL of ultrapure water and stirred. While stirring, 0.05 g of KMnO4 was added and stirred for 5 h. The mixture was then centrifuged and washed with ultrapure water. After vacuum drying at 60 °C for 12 h, the precursor was obtained. The precursor was calcined in air at 250 °C for 2 h at a speed of 2 °C / min to obtain the material. The material was then acid-washed with 1 M sulfuric acid at 60 °C for 1 h until neutral, yielding the product.
[0026] Test Example 1 This test example characterizes the properties of the manganese-doped ZIF-derived nanozyme obtained in Example 1: (1) The manganese-doped ZIF-derived nanozyme prepared in Example 1 was analyzed by scanning electron microscopy (SEM), and the results are as follows: Figure 1 As shown; XRD testing results are as follows Figure 2 As shown, the sample exhibited diffraction peaks at 19.05°, 38.667°, and 44.95°. The diffraction peak at 19.05° corresponds to the (111) crystal plane of MnO2, the diffraction peak at 38.667° corresponds to the (222) crystal plane of MnO2, and the diffraction peak at 44.95° corresponds to the (400) crystal plane of MnO2.
[0027] (2) Determination of catalytic activity: 150 μL of manganese-doped ZIF-derived nanozyme (50 μg / mL) was mixed with 2,4-DP (100 μg / mL) and 4-AP (100 μg / mL) and reacted in a NaH2PO4-Na2HPO4 buffer solution at pH 6.8 for 10 min. The total reaction volume was 3 mL, and the reaction temperature was 25 °C. After the reaction, the suspension was centrifuged at 11000 rpm for 3 min, the supernatant was collected, and its absorbance was measured at λ=510 nm to evaluate the laccase-like activity. The results are as follows: Figure 3 As shown.
[0028] (3) The catalytic activity of manganese-doped ZIF-derived nanozymes under different temperature conditions was tested. The test temperature was adjusted by connecting a water bath system to a UV / Vis spectrometer. The total reaction volume was set to 3 mL, which contained 100 μg / mL of 2,4-DP solution, 100 μg / mL of 4-AP solution, and 50 μg / mL of 4-AP solution. The absorbance change was measured at 510 nm wavelength over 10 min. The relative activity was calculated using the maximum absorbance value as a reference. The temperature was adjusted to 20℃, 25℃, 30℃, 40℃, 50℃, 65℃, and 80℃.
[0029] The results are as follows Figure 4 As shown, the absorbance value first increases and then decreases with increasing temperature, reaching a peak at 40℃. Therefore, the manganese-doped ZIF-derived nanozyme exhibits the highest catalytic activity at 40℃.
[0030] (4) The catalytic activity of manganese-doped ZIF-derived nanozymes under different pH conditions was tested. Buffer systems with pH values of 4, 5, 6, 6.8, 7, 8, 9, and 10 were prepared using a mixed buffer solution of 0.2 M disodium hydrogen phosphate and 0.2 M sodium dihydrogen phosphate. The total reaction volume was set to 3 mL, containing 100 μg / mL of 2,4-DP solution, 100 μg / mL of 4-AP solution, and 50 μg / mL of [unspecified solution]. The absorbance change was measured at 510 nm wavelength over 10 min. The relative activity was calculated using the maximum absorbance value as a reference.
[0031] The results are as follows Figure 5 As shown, the absorbance value first increases and then decreases with increasing pH, reaching a peak at pH 6.8. Therefore, the manganese-doped ZIF-derived nanozyme exhibits the highest catalytic activity at pH 6.8. Steady-state kinetics of manganese-doped ZIF-derived nanozymes were performed. Different concentrations of 2,4-DP (10, 20, 40, 60, 80, and 100 µg / mL) were mixed with 4-AP (150 µg / mL) and 50 µg / mL of manganese-doped ZIF-derived nanozymes. The pH of the solution was controlled at 6.8, and the total reaction volume was 3 mL. The reaction process was recorded in time-driven mode using the kinetics module of a UV-Vis spectrometer to obtain catalytic kinetic curves. Based on the initial linear range of the catalytic kinetic curves, the initial reaction rates at different substrate concentrations were calculated and fitted using the Michaelis-Menten equation. Finally, the Michaelis constant (Km) and the maximum reaction rate (Vmax) with 2,4-DP as the substrate were calculated using the following formulas.
[0032] The results are as follows Figure 6 , 7 As shown, Vmax = 80.52 μM / min and Km = 0.1856 mM indicate that the catalytic ability and substrate affinity of this material are both higher than those of natural laccase.
[0033] (6) The specific activity of manganese-doped ZIF-derived nanozymes was determined. Different concentrations of manganese-doped ZIF-derived nanozymes, 0.052 g / L catechol solution and 0.2 M NaH2PO4-Na2HPO4 buffer solution were placed in 3 mL centrifuge tubes and mixed evenly. The change of absorbance of the reaction system at the characteristic wavelength of 410 nm was monitored in real time under time scan mode, and the specific activity of the material was calculated.
[0034] The results are as follows Figure 8 As shown, the specific activity of the manganese-doped ZIF-derived nanozyme is 17.5 U / mg, which greatly exceeds the activity of most nanozymes reported to date, demonstrating excellent catalytic performance.
[0035] (7) The catalytic mechanism of manganese-doped ZIF-derived nanozymes was briefly determined. Several different free radical scavengers were selected, including thiourea (TH) to scavenge hydroxyl radicals, superoxide anions, and singlet oxygen, tryptophan to scavenge singlet oxygen, isopropanol to scavenge hydroxyl radicals, and p-benzoquinone to selectively scavenge superoxide anions. The total volume of the reaction system was controlled at 3 mL. Under the conditions of 25 °C and pH 6.8, the concentration of manganese-doped ZIF-derived nanozymes was 50 µg / mL, and the concentrations of 2,4-DP and 4-AP were both 100 µg / mL. A series of free radical scavengers of different concentrations were added, and the absorbance at 510 nm was monitored after 10 min of reaction.
[0036] The results are as follows Figure 9 As shown, singlet oxygen plays a major role in the catalytic process of manganese-doped ZIF-derived nanozymes.
[0037] (8) The catalytic stability of manganese-doped ZIF-derived nanozymes was compared with that of natural Yunzhi laccase.
[0038] (a) To compare pH storage stability, manganese-doped ZIF-derived nanozymes and natural laccase were placed at different pH values for 7 hours before being added to the reaction system. The pH storage stability of manganese-doped ZIF-derived nanozymes and natural laccase was compared by measuring the colorimetric reactions of 2,4-DP and 4-AP within 10 minutes. Figure 10 It was found that natural laccase exhibits optimal activity at a pH close to 8, with activity significantly decreasing under strongly acidic or alkaline conditions. Manganese-doped ZIF-derived nanozymes, however, maintain high activity under both strongly acidic and alkaline conditions, and their catalytic performance is almost unaffected under strongly acidic conditions, demonstrating excellent pH stability.
[0039] (b) To compare temperature storage stability, manganese-doped ZIF-derived nanozymes and natural laccase were incubated at different temperatures for 45 min. The relative catalytic activity after treatment at different temperatures was determined, using the activity at 25°C as a baseline. Figure 11 As shown, the activity of natural laccase decreases sharply with increasing temperature; above 60°C, natural laccase is almost completely inactivated, while manganese-doped ZIF-derived nanozymes maintain high activity in the range of 0–80°C. This material exhibits superior temperature stability compared to natural laccase.
[0040] (c) The effect of ionic strength on catalytic activity was evaluated by adding different concentrations (0–500 mM) of NaCl solution during the reaction. Figure 12 As shown, the activity of natural laccase decreased significantly with increasing NaCl concentration, while manganese-doped ZIF-derived nanozymes maintained stable catalytic activity.
[0041] (d) The storage stability of the organic solvents was studied, and the catalytic activities of both were determined at different ethanol concentrations (0–100%). Figure 13 As shown, the activity of natural laccase gradually decreases with increasing ethanol concentration, and it is completely inactivated under 50% ethanol conditions. In contrast, Mn / NC nanozymes exhibit stronger tolerance under the same conditions.
[0042] Test Example 2 This test example uses the manganese-doped ZIF-derived nanozyme prepared in Example 1 to perform a 2,4-dichlorophenol degradation experiment. The test methods include the following: (1) 2,4-Dichlorophenol standard curve: The peak area of 2,4-dichlorophenol at concentrations of 20~200 μg / ml was tested under the same conditions as in (2). Figure 14 The results show that 2,4-dichlorophenol exhibits a good linear relationship with peak area in the concentration range of 20–200 μg / ml.
[0043] (2) The manganese-doped ZIF-derived nanozyme prepared in Example 1 (final concentration 50 μg / ml) was added to a phosphate buffer solution containing 2,4-dichlorophenol at a final concentration of 400 μg / ml. The resulting 3 ml mixture was stirred at room temperature for 10 min (samples were taken at 0, 2, 4, 6, 8, and 10 min). The reaction mixture was filtered through a membrane to recover residual 2,4-dichlorophenol, which was then analyzed by high-performance liquid chromatography (HPLC). The experiment was performed using a Waters 2489 series chromatograph. The chromatographic column used was an Eclipse Plus C18 (5 μm particle size, 4.6 mm inner diameter, and 250 mm length). The mobile phase was a mixture of methanol and water (80:20 v / v), with isocratic elution mode and a flow rate of 1 mL / min. The column temperature was controlled at 30 °C, and the UV detector was used for signal acquisition at a wavelength of 286 nm. Figure 15 As shown, 2,4-dichlorophenol was completely degraded within 10 minutes.
[0044] Effects of this invention: The manganese-doped ZIF-derived nanozyme prepared in this invention exhibits high laccase activity. In Example 1, the specific activity was calculated by measuring the absorbance changes after mixing the material with catechol (0.052 g / ml, 125 μL) at different final concentrations (30, 60, 90, 120, 150, 180 μg / mL). The calculated enzyme activity reached 17.5 U / mg, significantly higher than the activity of natural laccase (0.5 U). Such high laccase-like activity can improve detection sensitivity, accelerate reaction rate, and enhance signal response. Kinetic tests of the material in Example 1 yielded Vmax = 80.52 μM / min and Km = 0.1856 mM, indicating that the catalytic ability and substrate affinity of this material are both higher than those of natural laccase.
[0045] The above embodiments are merely illustrative of the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solutions based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.
Claims
1. A manganese-doped MOF-derived nanolaccase, characterized in that, ZIF-8 synthesized in an aqueous phase was obtained by derivatization of a precursor at low temperature under an oxygen atmosphere; the aqueous phase synthesis refers to the use of ultrapure water as the solvent in the synthesis process; ZIF-8 is a derivative of Zn 2+ The zeolite-like topological MOF material formed by self-assembly with 2-methylimidazole; derived by low-temperature calcination of precursors in an oxygen atmosphere.
2. The manganese-doped MOF-derived nanolaccase according to claim 1, characterized in that, The precursor introduces manganese through a metal impregnation method. The raw materials used for metal impregnation are potassium permanganate and ZIF-8, with a mass ratio of 0 to 0.
5.
3. The manganese-doped MOF-derived nanolaccase according to claim 1, characterized in that, The low-temperature calcination method uses a tube furnace with an air atmosphere, a temperature of 200-300℃, a holding time of 1-3 hours, and a heating rate of 1-5℃ / min.
4. The manganese-doped MOF-derived nanolaccase according to claim 1, characterized in that, The low-temperature calcination temperature is 250℃, the holding time is 2h, and the heating rate is 2℃ / min.
5. The manganese-doped MOF-derived nanolaccase according to claim 1, characterized in that, It also includes an acid pickling process, during which the temperature is 50-70℃, the pickling time is 0.5-2h, and the acid used is sulfuric acid.
6. The manganese-doped MOF-derived nanolaccase according to claim 5, characterized in that, The pickling process is carried out at a temperature of 60°C for 1 hour.
7. The method for preparing manganese-doped MOF-derived nanolaccase according to claims 1-6 is characterized in that, Includes the following steps: Step 1: Synthesize ZIF-8 using an aqueous phase synthesis method; Step 2: Introduce manganese into ZIF-8 using a metal impregnation method to obtain the precursor; Step 3: Calcining the precursor at low temperature; Step 4: The calcined material is acid-washed to obtain manganese-doped ZIF-derived nanozymes.
8. The method for preparing manganese-doped MOF-derived nanolaccase according to claim 7, characterized in that, The aqueous phase synthesis method specifically involves adding zinc nitrate hexahydrate and 2-methylimidazole to ultrapure water, stirring, centrifuging, and then vacuum drying to obtain ZIF-8.
9. The method for preparing manganese-doped MOF-derived nanolaccase according to claim 7, characterized in that, The metal impregnation method specifically involves adding ZIF-8 to ultrapure water and stirring, then adding potassium permanganate, stirring, centrifuging, and vacuum drying to obtain the precursor.