A method for preparing laccase immobilized in FeMn-MOF material

Abstract

This invention belongs to the field of bioengineering materials technology, specifically providing a method for preparing FeMn-MOF material-immobilized laccase. This method uses manganese chloride tetrahydrate and ferric chloride hexahydrate as metal salts, terephthalic acid as an organic ligand, and N,N-dimethylacetamide as a solvent. FeMn-MOF material is prepared using a solvothermal method, and then the laccase is immobilized using glutaraldehyde cross-linking to obtain FeMn-MOF@Lac. Compared to free laccase, the immobilized enzyme obtained by this method maintains higher and more stable catalytic activity over a wider temperature and pH range, while exhibiting excellent storage stability and reusability. Furthermore, in terms of application, this immobilized enzyme can efficiently degrade tetracycline, providing a green and efficient biocatalytic solution for the treatment of antibiotic residues in water bodies, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering materials technology, specifically relating to a method for preparing laccase immobilized on FeMn-MOF material. Background Technology

[0002] With the widespread use of antibiotics in medicine and aquaculture, the pollution of aquatic environments caused by their residues has become a serious ecological and health problem. Due to their stable chemical structure, antibiotics are difficult to effectively degrade using traditional methods, tend to persist in the environment, and can induce the development of resistance genes. Compared with traditional physicochemical methods, enzymatic degradation has potential advantages such as mild conditions, high selectivity, and no secondary pollution, and is considered a green remediation approach. Laccase, as a multi-copper oxidase, can catalyze the oxidation of various aromatic compounds and shows good potential in degrading phenols, dyes, and pharmaceutical pollutants, providing a possible biocatalytic tool for the conversion and removal of antibiotics. However, natural laccase faces significant bottlenecks in practical applications: its protein structure is fragile, extremely sensitive to environmental temperature and pH fluctuations, and easily inactivated under complex aquatic conditions. These shortcomings severely restrict its engineering applications. To overcome these limitations, enzyme immobilization technology has emerged, which involves immobilizing enzymes on insoluble carriers to improve enzyme stability and reusability. The core of this technology lies in finding high-performance carrier materials. An ideal carrier needs to have a high specific surface area to load sufficient enzymes, good biocompatibility to maintain enzyme activity, and a stable structure to withstand the usage environment.

[0003] In recent years, metal-organic frameworks (MOFs) have become cutting-edge carriers for immobilized enzyme research due to their ultra-high specific surface area, highly ordered pore structure, and tunable chemical properties. MOFs can immobilize enzymes through physical or chemical means, and their regular channels provide a unique confined protective microenvironment for enzyme molecules, thereby significantly enhancing the enzyme's tolerance to adverse external conditions. However, the performance of conventional single-metal MOF materials in laccase immobilization still has room for improvement. On the one hand, some MOF materials lack long-term chemical stability in aqueous solutions; on the other hand, their single metal center may have limited synergistic effect with the laccase catalytic system, making it difficult to broaden the enzyme's applicable conditions while significantly improving the degradation efficiency for specific pollutants.

[0004] The design of bimetallic MOF-based supports offers new insights into performance enhancement. These materials, through synergistic effects between metals, can not only enhance the stability of the framework but also potentially modulate the electronic structure and surface properties of the material, thereby creating a more favorable interfacial environment for laccase immobilization and catalytic function. For example, iron-based MOFs possess certain peroxide-mimicking enzyme activity, while manganese is frequently involved in redox reactions. Theoretically, their combination can construct a multifunctional catalytic microenvironment that synergizes with the oxidative catalysis of laccase, potentially improving both support stability and the degradation performance of the composite material. Therefore, developing immobilized laccase materials based on specific bimetallic MOF systems has become a promising research direction. However, how to securely immobilize laccase on bimetallic MOF supports using a simple and efficient preparation process, and enable it to exhibit catalytic activity, environmental tolerance, and operational stability far exceeding that of free enzymes in the degradation of antibiotics (such as tetracycline), remains a technical challenge that requires further exploration and resolution. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for preparing laccase immobilized in FeMn-MOF material, which has the advantages of simple preparation method, significant degradation effect and good stability.

[0006] One aspect of this invention provides a method for preparing laccase immobilized on FeMn-MOF material, comprising the following steps: S1. Dissolve ferric chloride hexahydrate and manganese chloride tetrahydrate in N,N-dimethylacetamide to obtain mixture A; dissolve terephthalic acid in N,N-dimethylacetamide to obtain mixture B; Mixture A and mixture B are mixed to obtain homogeneous mixture C. Isopropanol is added to mixture C, and then the mixture is transferred to a high-pressure reactor for reaction. After the reaction is completed, the product is washed with deionized water and anhydrous ethanol and dried to obtain FeMn-MOF powder. S2. The FeMn-MOF powder obtained in step S1 is treated with glutaraldehyde and then washed with deionized water; laccase solution is added to the material and the reaction is continued to obtain immobilized laccase FeMn-MOF@Lac.

[0007] Furthermore, the ratio of ferric chloride hexahydrate, manganese chloride tetrahydrate, and N,N-dimethylacetamide used in step S1 is (2.525) mmol : 5 mmol : 50 ml.

[0008] Furthermore, the ratio of terephthalic acid, N,N-dimethylacetamide and isopropanol used in step S1 is (6.7-20) mmol : 25 ml : 45 ml.

[0009] Furthermore, in step S1, the reaction temperature is 120-180℃ and the reaction time is 312 h.

[0010] Furthermore, the concentration of glutaraldehyde in step S2 is 0.1%-5%, and the treatment time with glutaraldehyde is 1-5 hours.

[0011] Furthermore, the concentration of the laccase solution in step S2 is 0.2-5.0 mg / ml, and the immobilization reaction time is 2-16 h.

[0012] Another aspect of the present invention provides a FeMn-MOF material-immobilized laccase.

[0013] Another aspect of the present invention provides the application of FeMn-MOF material-immobilized laccase in the preparation of antibiotic degradation products.

[0014] Furthermore, the antibiotic is a tetracycline antibiotic.

[0015] Furthermore, the tetracycline antibiotic is tetracycline hydrochloride.

[0016] In summary, the present invention has the following beneficial effects: 1. The present invention successfully constructed a FeMn-MOF support material with stable structure and uniform morphology, such as... Figure 1 As shown, the synthesized FeMn-MOF exhibits a spindle-shaped structure; Figure 2 XRD patterns confirmed that it has good crystallinity; Figure 3 FTIR results showed that the iron and manganese bimetals were successfully coordinated with the organic ligands; Figure 4 BET analysis showed that the material has a rich pore structure and a specific surface area of ​​38.04 m². 2 / g. These results demonstrate that the present invention successfully constructed a FeMn-MOF vector with well-defined physicochemical characteristics, providing a solid foundation for the efficient immobilization of subsequent enzymes.

[0017] 2. This invention achieves efficient immobilization of laccase on a FeMn-MOF support via glutaraldehyde cross-linking. The resulting immobilized enzyme exhibits stable and significantly improved catalytic performance and environmental tolerance. Figure 5 , 6 As shown, compared with the free enzyme, the optimal reaction temperature of the immobilized enzyme increased from 50°C to 60°C, and it still maintained 82.73% of its relative activity at 70°C. Regarding pH tolerance, the immobilized enzyme retained 96.87% of its activity at pH 3, and 75.59% and 56.08% of its activity at pH 6 and 7, respectively, all significantly better than the free enzyme. Furthermore, Figure 7 , 8Stability tests showed that the immobilized enzyme retained 57.14% of its activity after treatment at 55°C for 6 h, and its relative activity reached 85.64% after storage at room temperature for 30 days, proving that the immobilization strategy of the present invention effectively protects the activity of laccase.

[0018] 3. The immobilized laccase FeMn-MOF@Lac prepared in this invention exhibits excellent overall performance and demonstrates good potential for industrial application in antibiotic pollution control. Regarding reusability, for example… Figure 9 As shown, the immobilized enzyme retained 77.08% of its initial activity after 10 consecutive cycles, demonstrating good operational stability. Regarding application performance, such as... Figure 10 As shown, FeMn-MOF@Lac exhibits highly efficient catalytic degradation of tetracycline hydrochloride, achieving a degradation rate of up to 93.40% for tetracycline hydrochloride at an initial concentration of 100 mg / L within 30 h of reaction, significantly superior to free laccase. This immobilized enzyme system combines excellent reusability with high catalytic performance, providing a green and efficient biocatalytic technology solution for the removal of antibiotic residues in water. Attached Figure Description

[0019] Figure 1 SEM images of FeMn-MOF and FeMn-MOF@Lac materials prepared in Example 1 (a and b are SEM images of FeMn-MOF, and c and d are SEM images of FeMn-MOF@Lac). Figure 2 The XRD patterns of the FeMn-MOF and FeMn-MOF@Lac materials prepared in Example 1 are shown below. Figure 3 The images show the FTIR spectra of the FeMn-MOF and FeMn-MOF@Lac materials prepared in Example 1. Figure 4 The BET plots of the FeMn-MOF@Lac material prepared in Example 1 are shown in Figure 1 (a is the nitrogen adsorption-desorption curve, and b is the pore size distribution). Figure 5 The graph shows the effect of reaction temperature on the enzyme activity of Lac and FeMn-MOF@Lac in Example 3. Figure 6 The figure shows the effect of reaction pH on the enzyme activity of Lac and FeMn-MOF@Lac in Example 4. Figure 7 The graph shows the results of verifying the thermal stability of Lac and FeMn-MOF@Lac in Example 5; Figure 8 This is a graph showing the results of the storage stability verification of Lac and FeMn-MOF@Lac in Example 6; Figure 9 This is a graph showing the results of the repeated use stability verification of FeMn-MOF@Lac in Example 7; Figure 10 The graph shows the degradation performance of tetracycline hydrochloride by Lac and FeMn-MOF@Lac in Example 8. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all 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.

[0021] Example 1: Synthesis of FeMn-MOF: 25 ml of a terephthalic acid (10 mmol) N,N-dimethylacetamide solution was mixed with 50 ml of a ferric chloride hexahydrate (15 mmol) and a manganese chloride tetrahydrate (5 mmol) N,N-dimethylacetamide solution. After stirring for 30 min, 45 ml of isopropanol was added, and the mixture was poured into a 200 ml dry Teflon container and thoroughly mixed. The container was placed in a stainless steel autoclave and heated in an oven at 120 °C for 9 h, then allowed to cool naturally to room temperature. The precipitate was collected by centrifugation at 8000 rpm for 5 min, washed three times with water and ethanol, and then dried at 60 °C for 12 h to obtain FeMn-MOF powder.

[0022] Preparation method of FeMn-MOF@Lac: Add 1% glutaraldehyde solution to FeMn-MOF and stir at room temperature for 4 h. Centrifuge at 6000 rpm for 10 min. Wash three times with deionized water. Add 2 mg / ml laccase to the material and shake at 150 rpm for 12 h at room temperature. Centrifuge to separate, collect the precipitate, and freeze-dry to obtain the immobilized enzyme FeMn-MOF@Lac.

[0023] The microstructure of the prepared FeMn-MOF and FeMn-MOF@Lac was observed using scanning electron microscopy, and the results are as follows: Figure 1 As shown. Figure 1 As shown in a and b, FeMn-MOF exhibits a spindle-shaped structure with a smooth surface, crystal lengths of 800–900 nm, and widths of 150–300 nm. After enzyme immobilization (e.g., Figure 1The morphology of FeMn-MOF@Lac (c and d) was basically unchanged compared with FeMn-MOF, indicating that the immobilization of laccase had little effect on the morphology of the FeMn-MOF vector.

[0024] X-ray diffraction was used to test FeMn-MOF and FeMn-MOF@Lac, and the results are as follows: Figure 2 As shown. Diffraction peaks were observed at 9.1°, 10.6°, and 21.4° on the sample, which are attributed to the (200), (110), and (220) crystal planes of Fe-MOF. The prepared FeMn-MOF and FeMn-MOF@Lac exhibited similar XRD peaks to Fe-MOF, indicating that the addition of Mn and the loading of laccase did not have a significant impact on the MOF framework structure.

[0025] FeMn-MOF and FeMn-MOF@Lac were tested using Fourier transform infrared spectroscopy, and the results are as follows: Figure 3 As shown. In the spectrum of FeMn-MOF, the peak value is located at 555 cm⁻¹. -1 The characteristic peak at this location can be attributed to the stretching vibration of the metal-oxygen bond, confirming that a metal oxide cluster was formed by the coordination of the carboxyl group of terephthalic acid with the metal node. 752 cm⁻¹ -1 The characteristic peak at 1390 cm⁻¹ originates from the out-of-plane bending vibration of the CH bond in the benzene ring. -1 and 1589 cm -1 The characteristic peaks at these locations correspond to the carboxyl group (-COO). - Symmetric and asymmetric stretching vibrations. Furthermore, 1695 cm... -1 The characteristic peak at 2900-3400 cm⁻¹ originates from the stretching vibration of C=O in the benzene ring. -1 The broad absorption band within the range is related to the OH stretching vibration and coordinated hydroxyl groups of adsorbed water molecules in the sample. After the immobilized enzyme forms FeMn-MOF@Lac, the metal-oxygen bond vibration peak increases from 555 cm⁻¹. -1 Blue shifted to 551 cm -1 This indicates that laccase molecules interact with metal nodes through coordination, altering the local electronic environment of the metal cluster. (1650 cm⁻¹) -1 and 1540 cm -1 The broadening of the nearby spectral bands can be attributed to the introduction of the protein amide I and amide II bands.

[0026] The nitrogen adsorption-desorption isotherms and pore size distribution of FeMn-MOF were detected using a specific surface area and pore size analyzer. The results are as follows: Figure 4 As shown. From Figure 4As can be seen from graph a, FeMn-MOF exhibits typical type IV isotherm characteristics, accompanied by an H3 type hysteresis loop, indicating the presence of a slit-like mesoporous structure in the material. The specific surface area of ​​the sample is 38.04 m². 2 / g, total pore volume is 0.122cm³ 3 / g, with an average pore size of 17.17 nm. The pore size distribution was calculated using the BJH model ( Figure 4 (b) The prepared material is mainly mesoporous with a small number of micropores, which is beneficial to improving the accessibility of adsorption sites, promoting the diffusion and transport of substances, and providing a good structural basis for the performance of its related properties.

[0027] Example 2: This example provides the determination of laccase activity, the method of which is as follows: Add 0.5 ml of free laccase or 100 mg of immobilized laccase FeMn-MOF@Lac to 0.2 M acetate-sodium acetate buffer (pH=4.5) and 1 mM ABTS. After reacting at room temperature for 5 minutes, measure the absorbance at 420 nm using a UV spectrophotometer. Filter the supernatant when collecting the immobilized enzyme.

[0028] Example 3: Free laccase and FeMn-MOF@Lac were incubated at 20-70℃ for 10 min, and the enzyme activity of laccase was measured. The highest activity was taken as 100%, and the relative activities of immobilized laccase and free enzyme at different temperatures were calculated.

[0029] like Figure 5 As shown, the free enzyme reaches its peak activity at 40°C, but its activity decreases sharply with increasing temperature, retaining only 15.46% of its activity at 70°C. In contrast, the optimal reaction temperature of FeMn-MOF@Lac shifts from 40°C to 50°C, and it exhibits excellent tolerance in the high-temperature range, maintaining 82.73% activity at 70°C. This is partly because the immobilized carrier effectively rigidifies the enzyme molecules through covalent cross-linking, limiting excessive stretching and inactivation of the enzyme conformation at high temperatures; and partly because the microenvironment effect of the carrier may buffer against the drastic impact of external temperature.

[0030] Example 4: The enzyme activities of free laccase and FeMn-MOF@Lac were measured under pH 3-7 (acetic acid-sodium acetate buffer), and the highest activity was taken as 100%. The relative activities of immobilized laccase and free laccase at different pH were calculated.

[0031] Figure 6As can be seen, the relative activities of both enzymes reached a peak of 100% at pH=4, indicating that the immobilization process did not change the optimal pH conditions of the enzymes. However, the free enzymes are highly sensitive to pH, and their activity drops sharply after deviating from the optimal pH. FeMn-MOF@Lac, on the other hand, exhibits a wider adaptation window, maintaining an activity of 56.08% even at pH=7. For practical applications, immobilized enzymes expand the applicability of enzymes, enabling them to maintain high activity even under conditions of large pH fluctuations.

[0032] Example 5: Free laccase and FeMn-MOF@Lac were stored in a water bath at 55°C. A group of samples was taken out every 1 h, cooled to room temperature, and their remaining enzyme activity was measured. The highest activity was taken as 100% to detect the thermal stability of laccase.

[0033] Figure 7 The results showed that the activity of the free enzyme rapidly decreased to 55.56% after incubation at 55°C for 1 h, and then continued to decline, with a residual activity of only 9.76% after 6 h. This indicates that the free enzyme is sensitive to high temperatures and is prone to heat-induced conformational collapse and inactivation. In contrast, the thermal inactivation curve of FeMn-MOF@Lac was significantly flatter, and even after 6 h of heat treatment, its relative activity remained as high as 57.14%. In summary, FeMn-MOF@Lac exhibits superior thermal stability under high-temperature conditions and can maintain high enzyme activity for a longer period of time, which has important practical application value for industrial processes that need to operate in high-temperature environments.

[0034] Example 6: Free laccase and FeMn-MOF@Lac were stored at room temperature. A group of samples was taken out every 5 days to determine their remaining enzyme activity, and the highest activity was taken as 100% to test the storage stability of laccase.

[0035] like Figure 8 As shown, the free enzyme exhibited rapid activity decay during storage, with only 16.36% residual activity remaining after 30 days. This indicates that the free enzyme is prone to slow conformational relaxation, autolysis, or microbial degradation at room temperature, leading to inactivation. In contrast, the immobilized enzyme demonstrated excellent storage stability, maintaining a relative activity of 85.64% even after 30 days of long-term storage. This demonstrates that FeMn-MOF@Lac, as an enzyme preparation, possesses the potential for long-term preservation, significantly reducing storage and transportation costs.

[0036] Example 7: Since free laccase is a liquid and mixes with acetate buffer, it cannot be recycled after use. Using ABTS as the reaction substrate, the immobilized laccase was cyclically catalyzed 10 times, and its enzyme activity was measured. After washing the immobilized laccase twice with buffer, a new batch of ABTS reaction solution was added to start the next batch of reaction to test the reusability stability of the immobilized laccase.

[0037] Figure 9 The results showed that after 10 cycles, the relative activity of the immobilized laccase FeMn-MOF@Lac remained at 77.08%, indicating that the immobilization treatment effectively enhanced the enzyme structure and reduced enzyme protein shedding and inactivation during the reaction process. The prepared immobilized laccase could still maintain most of its catalytic activity after repeated use, showing good stability and making it suitable for industrial catalytic scenarios of continuous conversion.

[0038] Example 8: Application of FeMn-MOF@Lac prepared in Example 1 in the catalytic degradation of tetracycline hydrochloride. The specific method is as follows: Free enzyme, FeMn-MOF material, and immobilized laccase FeMn-MOF@Lac were placed in a 100 mg / L tetracycline hydrochloride solution and reacted with shaking under suitable conditions. Samples were taken at 1, 4, 8, 12, 24, and 30 h, filtered through a 0.22 μm filter membrane, and analyzed by high performance liquid chromatography to determine the residual amount of tetracycline hydrochloride and calculate the degradation rate.

[0039] like Figure 10 As shown, the self-degradation rate of the blank control group was only 9.23% after 30 h, while the degradation rate of free laccase after 30 h was 51.93%. In contrast, the removal rate of the immobilized enzyme FeMn-MOF@Lac system reached as high as 93.40% after 30 h, significantly superior to that of free laccase. This result indicates that FeMn-MOF carrier immobilization of laccase effectively improves enzyme stability and catalytic efficiency, overcoming the shortcomings of free laccase such as easy inactivation and short activity duration, and providing more stable and durable degradation performance.

[0040] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing laccase immobilized in FeMn-MOF material, characterized in that, Includes the following steps: S1. Dissolve ferric chloride hexahydrate and manganese chloride tetrahydrate in N,N-dimethylacetamide to obtain mixture A; Terephthalic acid was dissolved in N,N-dimethylacetamide to obtain mixture B; Mixture A and mixture B are mixed to obtain homogeneous mixture C. Isopropanol is added to mixture C, and then the mixture is transferred to a high-pressure reactor for reaction. After the reaction is completed, the product is washed with deionized water and anhydrous ethanol and dried to obtain FeMn-MOF powder. S2. The FeMn-MOF powder obtained in step S1 is treated with glutaraldehyde and then washed with deionized water; laccase solution is added to the material and the reaction is continued to obtain immobilized laccase FeMn-MOF@Lac.

2. The method for preparing laccase immobilized in FeMn-MOF material according to claim 1, characterized in that, The ratio of ferric chloride hexahydrate, manganese chloride tetrahydrate and N,N-dimethylacetamide used in step S1 is (2.525) mmol : 5 mmol : 50 ml.

3. The method for preparing laccase immobilized in FeMn-MOF material according to claim 1, characterized in that, The ratio of terephthalic acid, N,N-dimethylacetamide and isopropanol used in step S1 is (6.7-20) mmol : 25 ml : 45 ml.

4. The method for preparing laccase immobilized in FeMn-MOF material according to claim 1, characterized in that, In step S1, the reaction temperature is 120-180℃ and the reaction time is 312 h.

5. The method for preparing laccase immobilized using FeMn-MOF material according to claim 1, characterized in that, The concentration of glutaraldehyde in step S2 is 0.1%-5%, and the treatment time with glutaraldehyde is 1-5 h.

6. The method for preparing laccase immobilized using FeMn-MOF material according to claim 1, characterized in that, The concentration of the laccase solution in step S2 is 0.2-5.0 mg / ml, and the immobilization reaction time is 2-16 h.

7. A FeMn-MOF material immobilized with laccase prepared by any one of claims 1-6.

8. The application of FeMn-MOF material immobilized laccase according to claim 7 in the preparation of antibiotic degradation products.

9. The application according to claim 8, characterized in that, The antibiotic in question is a tetracycline antibiotic.

10. The application according to claim 9, characterized in that, The tetracycline antibiotic in question is tetracycline hydrochloride.

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