Preparation method of artificial double-site bionic multi-enzyme catalyst and lignin degradation application of artificial double-site bionic multi-enzyme catalyst

Abstract

The invention discloses an artificial double-site bionic multienzyme method for preparing monophenol compounds by degrading lignin, and belongs to the technical field of bionic enzymes. The method is inspired by white-rot fungi in nature to realize the synergistic degradation process of lignin by secreting laccase and peroxidase, and the construction of double-site bionic multienzyme and the application research of the double-site bionic multienzyme in the synergistic degradation of lignin are developed. Specifically, a high-activity single-site metal organic framework bionic laccase is firstly synthesized, and then a double-site bionic multienzyme catalyst is constructed by reducing and loading Pd nanoparticles with peroxidase activity. The catalyst has high laccase and peroxidase activity at the same time, and can efficiently degrade lignin model substances and real lignin under mild conditions to generate monophenol compounds. The preparation method disclosed by the invention is low in cost and simple and convenient to operate, and provides important technical support and practical value for wide application of the biomimetic enzyme in the field of catalytic degradation of lignin.

Description

Technical Field

[0001] The invention belongs to the technical field of bionic enzymes, and in particular relates to a preparation method of a double-site bionic enzyme and an application thereof in lignin degradation. Background Art

[0002] With the over-exploitation and use of fossil energy, global energy issues are becoming increasingly prominent. In recent years, more and more research has focused on the development and utilization of biomass resources to replace traditional fossil energy. As the world's most abundant natural aromatic polymer, lignin has become an extremely attractive biomass resource due to its recycling and green environmental protection characteristics. However, currently more than 90% of industrial lignin is only used for inefficient direct combustion. Therefore, improving the high-value utilization of lignin is of great significance to alleviating environmental pressure and resource shortage problems. Degrading lignin into single aromatic ring compounds for the preparation of chemicals or liquid fuels is an important way to achieve high-value utilization.

[0003] To achieve the effective depolymerization of lignin from macromolecules to small molecules, the internal inter-unit bonds must be broken. However, due to the high stability of carbon-carbon bonds and ether bonds in lignin, and the repolymerization phenomenon that easily occurs during the degradation process, the degradation of lignin faces great challenges. At present, the degradation technology of lignin mainly includes methods such as hydrogenolysis and pyrolysis. Although these technologies have achieved certain results in the yield of single benzene rings, their reaction conditions are harsh (such as high temperature and high pressure or hydrogen atmosphere), and there are many limitations in industrial applications. Therefore, the development of catalytic methods for achieving efficient degradation of lignin under mild conditions (low temperature, normal pressure) has broad application prospects.

[0004] As a biocatalyst with the advantages of mild reaction conditions and high catalytic efficiency, enzymes can degrade lignin to prepare monoaromatic compounds under mild conditions, which has become an attractive research direction. In nature, the degradation of lignin mainly depends on the multi-enzyme synergistic system secreted by white rot fungi (including laccase, lignin peroxidase and manganese peroxidase). Studies have shown that compared with single enzymes, the synergistic effect of laccase, lignin peroxidase and manganese peroxidase can significantly improve the degradation efficiency of lignin. However, although some researchers have made some progress in using natural enzymes to degrade lignin (such as Liu et al. in ACS Sustainable Chemistry & Engineering, Vol. 7, 2019, and Pham et al. in Biotechnology for Biofuels, 2021), the current effect is still not ideal (monophenol yield <1%, see Li et al. in RSC Advances, Vol. 13, 2023). At the same time, natural biological enzymes are complex in expression and purification, sensitive to the environment and easy to inactivate, difficult to recycle, and their single active site has limited effect on complex lignin macromolecules, which further limits their practical application. Therefore, it is urgent to develop an artificial dual-site biomimetic multi-enzyme active catalyst that can efficiently break the bonds between lignin units under mild conditions and produce single aromatic ring compounds. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the present invention aims to provide a method for preparing an artificial dual-site bionic multi-enzyme active catalyst and its application in efficiently catalyzing the degradation of lignin to prepare monophenolic compounds under mild conditions. The bionic catalyst has the advantages of simple synthesis process, no loss of the catalyst in the product and recyclable use, mild catalytic reaction conditions, and the main degradation products are aromatic aldehydes and aromatic acids with high yields, which provides an effective method for realizing high-value utilization of lignin from the perspective of bionic enzymes under mild conditions.

[0006] The concept of the present invention is based on the following points: In nature, white rot fungi degrade lignin by secreting laccase and peroxidase, which inspired the construction of the dual-site biomimetic enzyme and its application in the synergistic degradation of lignin. The active center of natural laccase is composed of Cu 2+ / Cu 1+ The primary coordination microenvironment of histidine, cysteine ​​residues and hydroxyl groups plays a role. Based on this, we used multivalent cerium (Ce 4+ / Ce 3+) nanomaterials and their redox reactivity are used to simulate the active center of natural laccase, and non-natural carboxylic acid structures (such as pyrazole and thiophene) are used to regulate the activity of bionic laccase, so as to efficiently assemble them into metal-organic framework bionic laccase. Pd nanoparticles have been shown to have strong peroxidase activity, and metal-organic frameworks, as excellent carriers of metal nanoparticles, can further enhance the catalytic performance. Accordingly, the present invention successfully constructed an artificial dual-site bionic enzyme catalyst with high laccase and peroxidase activities by reduction-loading Pd nanoparticles on metal-organic framework bionic laccase, and explored its bionic enzyme properties and its application in lignin degradation (such as Figure 1 This provides theoretical and practical references for the development of bionic multi-enzyme catalysts.

[0007] The artificial dual-site bionic multi-enzyme design can efficiently imitate the functions of natural laccase and peroxidase. Its catalytic process only produces oxidation products and water, avoiding secondary pollution, which is in line with the concept of green catalysis. In addition, compared with the single-site bionic laccase, the artificial dual-site bionic multi-enzyme not only has higher laccase activity, but also exhibits significant peroxidase activity. At 50°C and aqueous conditions, the artificial dual-site bionic multi-enzyme catalyst can achieve a nearly 100% degradation rate of the lignin model compound 2-phenoxy-1-phenylethan-1-one, and the yields of benzoic acid and phenol reach 82.4% and 97.6%, respectively. For real lignin, the monophenol yield of the catalyst can reach 15.6%.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for preparing an artificial dual-site bionic multi-enzyme catalyst, characterized in that it comprises the following steps:

[0010] (1) mixing ammonium cerium nitrate, a biomimetic ligand and a modulator, heating and stirring to react, collecting the precipitate by suction filtration and washing, and drying to obtain a single-site metal organic framework biomimetic enzyme Ce-MOF;

[0011] The bionic ligand is one or more of 3,5-pyrazoledicarboxylic acid, 2,5-thiophenedicarboxylic acid, and 2,5-furandicarboxylic acid; the molar ratio of the bionic ligand to ammonium cerium nitrate is (1-5):1; the molar ratio of the modulator to the bionic ligand is (5-40):1;

[0012] (2) The Ce-MOF obtained in step (1) was ultrasonically dispersed in a mixed solution of ethanol and water, and Pd 2+ , continue ultrasonic dispersion, then add NaBH4 for stirring, filter and wash, and obtain Pd@Ce-MOF dual-site biomimetic multi-enzyme;

[0013] The loading amount of Pd in ​​the Pd@Ce-MOF dual-site bionic multi-enzyme is 1.2 wt% to 5.0 wt%.

[0014] Preferably, the biomimetic ligand is 3,5-pyrazoledicarboxylic acid or 2,5-thiophenedicarboxylic acid, and the molar ratio of the biomimetic ligand to ammonium cerium nitrate is (1.3-1.6): 1. The ligands selected in the present invention are 3,5-pyrazoledicarboxylic acid or 2,5-thiophenedicarboxylic acid, which are similar to histidine or cysteine ​​of laccase in the coordination environment, so that the synthesized MOF has a typical cubic octahedral reo topological structure.

[0015] Preferably, the modulator is one or more of formic acid, acetic acid or benzoic acid, and the molar ratio of the modulator to the biomimetic ligand is (10-15):1.

[0016] Preferably, the reaction temperature in step (1) is 80-110° C., and the reaction time is 10-25 minutes. The suction filtration and washing step uses a sand core funnel and a 0.45 μm organic membrane, and N,N-dimethylformamide and acetone are used to wash alternately three times. The washed solid needs to be soaked in acetone for 12 hours. The vacuum drying conditions are preferably dried at 50° C. and -0.1 MPa for 12 hours.

[0017] Preferably, the mass ratio of ethanol to water in the mixed solution in step (2) is 1.5:1; 2+ The molar ratio of Pd to NaBH4 is 1:(1-8); 2+ The mass feed ratio of Ce-MOF is (1.25~10):100.

[0018] Preferably, in step (2), Pd 2+ It is one or more of PdCl2, H2PdCl4, Pd(OAc)2; the ultrasonic dispersion step uses water bath ultrasound with a power of 200W and a duration of 15 minutes; the stirring temperature is room temperature, the rotation speed is 400-800rpm, and the duration is 8 to 16 hours; the suction filtration and washing step again uses a sand core funnel and a 0.45μm organic membrane, and washes alternately with water and ethanol three times; the vacuum drying conditions are 50°C, -0.1MPa, and drying is performed for 10 to 12 hours.

[0019] The artificial double-site bionic multi-enzyme prepared by the method of the invention.

[0020] The invention discloses an application of the artificial dual-site bionic multi-enzyme in degrading lignin.

[0021] Preferably, 50 mg of lignin is added with 5-30 mg of the artificial dual-site biomimetic multi-enzyme according to claim 7, 2.5 ml of 3.0 wt% NaOH aqueous solution, 0.3 ml of H2O, and 0.2 ml of H2O2, and the mixture is sealed and placed in an oil bath pot to react at 60°C for 16 hours.

[0022] Preferably, the lignin is one or more of birch lignin, poplar lignin, and pine lignin.

[0023] The present invention also provides the use of an artificial dual-site bionic multi-enzyme catalyst in catalyzing phenolic substances and the results thereof: preferably, the phenolic substances include one of trichlorophenol, dichlorophenol, 4-chlorophenol, dimethoxyphenol, naphthol and hydroquinone, preferably 4-chlorophenol. The catalyst of the present invention conforms to the Michaelis-Menten equation when catalyzing and oxidizing phenolic substances, exhibits catalytic activity superior to that of natural laccase and single-site bionic laccase, and can catalyze a variety of phenolic substances.

[0024] The present invention further provides the application and activity evaluation of an artificial dual-site bionic multi-enzyme catalyst in catalytic oxidation of a peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB): when catalyzing TMB, the catalyst conforms to the classical Michaelis-Menten equation, exhibits a catalytic activity similar to that of pure reduced Pd NP, and far exceeds that of a single-site bionic enzyme.

[0025] The catalyst of the present invention can also be used to catalyze the degradation of lignin model compound 2-phenoxy-1-phenylethan-1-one: the preferred degradation conditions are 50°C, 6 hours, a catalyst-to-substrate ratio of 1:1, and an H2O2 dosage of 600mM. The catalyst degrades 2-phenoxy-1-phenylethan-1-one, can achieve a degradation rate of nearly 100%, and obtain a high product yield, far exceeding a single-site bionic enzyme, and is suitable for lignin degradation to prepare monophenolic compounds.

[0026] The present invention also relates to the application of the catalyst in the degradation of real birch lignin: the preferred degradation conditions are 60°C, 50mg birch, 20mg catalyst, 600mM H2O2 dosage, and 16h reaction time. In the degradation of real birch lignin, the catalyst can achieve a monophenol yield of 15.6%, which is significantly higher than that of a single-site bionic enzyme, and is suitable for the preparation of monophenol compounds by degradation of real lignin under mild conditions.

[0027] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0028] (1) Inspiration from multi-enzyme synergistic effect and bionic design: The present invention is inspired by the natural multi-enzyme synergistic degradation of lignin in nature. 4+ / Ce 3+The biomimetic ligand 3,5-pyrazoledicarboxylic acid was used to successfully synthesize a metal organic framework biomimetic laccase with high laccase activity, and Pd nanoparticles with peroxidase activity were efficiently loaded and reduced on it, and finally an artificial dual-site biomimetic multienzyme was prepared. This artificial multienzyme not only has the functions of natural laccase and peroxidase, but also has high activity and can be recycled, making it suitable for practical applications.

[0029] (2) Significant synergistic enhancement effect: The artificial dual-site biomimetic multi-enzyme Pd@Ce-PZDC prepared by the present invention exhibits an internal dual-site synergistic enhancement activity effect. Its kinetic analysis of 2,4-dichlorophenol (2,4-DP) shows that the catalytic efficiency V max / K m The activity of the catalyst was enhanced by 2.98 and 1.54 times that of natural laccase and single-site biomimetic laccase Ce-PZDC, respectively. In addition, the catalyst also showed a significant activity enhancement effect on other phenolic substrates.

[0030] (3) High peroxidase activity: The artificial dual-site biomimetic multi-enzyme Pd@Ce-PZDC also has high peroxidase activity. Its catalytic activity towards 3,3,5,5-tetramethylbenzidine (TMB) is comparable to that of pure Pd nanoparticles and much higher than that of the single-site biomimetic enzyme Ce-PZDC, which lays a good foundation for the practical application of this catalyst.

[0031] (4) Highly efficient degradation of lignin model substances: Under mild conditions (50°C, aqueous phase), the artificial dual-site bionic multi-enzyme Pd@Ce-PZDC of the present invention can efficiently catalyze the degradation of non-phenolic lignin model substances, with a degradation rate close to 100%, and the yields of phenol and benzoic acid reaching 97.6% and 82.4%, respectively, showing excellent lignin degradation activity.

[0032] (5) Application of real lignin degradation: The artificial dual-site biomimetic multi-enzyme Pd@Ce-PZDC of the present invention can catalyze the degradation of real lignin under mild conditions (60°C, aqueous phase), and the main products are lignin structural unit compounds such as syringaldehyde, vanillin and p-hydroxybenzoic acid, with a yield of about 15.6%. This discovery provides a promising strategy for the degradation of lignin under mild conditions.

[0033] The novel artificial dual-site bionic multi-enzyme prepared by the bionic strategy of the present invention has the characteristics of high efficiency and universality, provides an important reference for the creation of novel dual-site bionic enzymes, and provides example verification for the application of dual-site bionic enzymes in the field of lignin degradation, which has important reference value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a bionic idea diagram of the artificial dual-site bionic enzyme catalyst of the present invention.

[0035] Figure 2 The XRD diagrams of Example 1 and Comparative Example 1 are shown in FIG.

[0036] Figure 3 It is a comparison chart of oxidase activity and peroxidase activity of Examples 1-2 and Comparative Examples 1, 3, and 4.

[0037] Figure 4 The activity comparison chart of Example 1 and Comparative Examples 1 and 5 on classic phenolic substrates.

[0038] Figure 5 The oxidase kinetics of Example 1 and Comparative Examples 1 and 5.

[0039] Figure 6 The peroxidase kinetics of Example 1 and Comparative Examples 1 and 2 are shown.

[0040] Figure 7 This is a gas chromatogram of the catalytic degradation of the lignin model substance 2-phenoxy-1-phenylethan-1-one in Example 1.

[0041] Figure 8 The gas chromatograms of the catalytic degradation of real lignin in Example 1 and Comparative Example 1 are shown. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with specific implementation modes and drawings, but the implementation modes of the present invention are not limited thereto.

[0043] The methods described in the present invention are all conventional methods unless otherwise specified, and the raw materials described can be obtained from public commercial channels unless otherwise specified.

[0044] Example 1: Preparation of 5.0wt% Pd@Ce-PZDC

[0045] Synthesis of single-site biomimetic laccase Ce-PZDC: Weigh 548 mg (1 mmol) of ammonium cerium nitrate and dissolve it in 2 ml of deionized water, and take 234 mg (1.5 mmol) of 3,5-pyrazoledicarboxylic acid and dissolve it in 6 ml of N,N-dimethylformamide (DMF), and add 0.5 ml (13.3 mmol) of formic acid as a modulator. After mixing the above solutions, place them in a 25 ml pressure-resistant glass bottle and seal them. Stir and react in a 100 ° C oil bath for 15 minutes, and then cool to room temperature. After the reaction is completed, wash it with 6 ml of DMF and acetone alternately by suction filtration for 3 times, and collect the precipitate. Place the precipitate in 6 ml of acetone and let it stand for 12 hours to replace the residual DMF. Finally, collect the precipitate by suction filtration and dry it in a vacuum oven at 50 ° C and -0.1 MPa for 12 hours to obtain a single-site biomimetic laccase Ce-PZDC.

[0046] Loading of Pd nanoparticles and preparation of dual-site bionic multi-enzyme: Take 145mg Ce-PZDC, add it to a mixed solution of 10ml water and 25ml ethanol, and ultrasonicate for 15 minutes to ensure uniform dispersion. Then add 12.8mg PdCl2 and continue ultrasonication for 15 minutes to make PdCl2 evenly dispersed on Ce-PZDC. Then, add 3ml NaBH4 solution with a concentration of 2mg / ml, and stir magnetically at 600rpm for 12 hours at room temperature (25℃) to reduce PdCl2 to Pd nanoparticles. After the reaction is completed, wash twice with 10ml water and ethanol alternately, collect the precipitate, and dry it in a vacuum oven at 50℃ and -0.1MPa for 12 hours to obtain dual-site bionic multi-enzyme 5.0wt% Pd@Ce-PZDC.

[0047] Example 2: Preparation of 2.5wt% Pd@Ce-PZDC

[0048] The single-site biomimetic laccase Ce-PZDC was synthesized by referring to step 1 of Example 1. Subsequently, the process was carried out according to step 2 of Example 1, except that 6.3 mg PdCl2 was added to load Pd nanoparticles at a lower ratio, thereby obtaining 2.5 wt% Pd@Ce-PZDC.

[0049] Comparative Example 1: Synthesis of Ce-PZDC

[0050] Referring to step 1 of Example 1, a single-site biomimetic laccase Ce-PZDC was synthesized, but without loading Pd nanoparticles.

[0051] Comparative Example 2: Synthesis of Pd Nanoparticles

[0052] Weigh 12.8 mg PdCl2 and dissolve it in a mixed solution of 10 ml water and 25 ml ethanol, and ultrasonicate for 15 minutes to ensure uniform dispersion. Then add 3 ml NaBH4 solution with a concentration of 2 mg / ml, and stir magnetically at 600 rpm for 12 hours at room temperature (25°C) to reduce PdCl2 to Pd nanoparticles. After the reaction is completed, wash it twice with 10 ml water and ethanol alternately, collect the precipitate, and dry it in a vacuum oven at 50°C and -0.1 MPa for 12 hours. After drying, grind the product into powder to obtain Pd nanoparticles.

[0053] Comparative Example 3: Synthesis of 0wt% Pd@Ce-PZDC

[0054] The single-site biomimetic laccase Ce-PZDC was synthesized with reference to step 1 of Example 1. Subsequently, the Pd nanoparticle loading step was performed according to step 1 of Example 1, but without adding PdCl2, only NaBH4 was added for treatment to obtain 0wt% Pd@Ce-PZDC.

[0055] Comparative Example 4: Synthesis of 1.25wt% Pd@Ce-PZDC

[0056] Ce-PZDC was synthesized by referring to step 1 of Example 1. Subsequently, according to step 2 of Example 1, only 3.1 mg PdCl2 was added to load Pd nanoparticles at a lower ratio to obtain 1.25 wt% Pd@Ce-PZDC.

[0057] Comparative Example 5: Natural Laccase

[0058] Natural laccase was purchased from Yuanye Biotechnology. It is a refined enzyme with an enzyme activity of >0.5U / mg.

[0059] Example Effect Description

[0060] Figure 1 The bionic design concept of the artificial dual-site bionic enzyme catalyst of the present invention is shown.

[0061] Figure 2 The XRD diagrams of Example 1 and Comparative Example 1 show that both have good crystallinity, and Pd loading does not affect the crystal structure of Ce-PZDC. At the same time, no diffraction peaks corresponding to Pd NPs are observed, indicating that Pd nanoparticles are effectively dispersed on MOF. In terms of the distribution and size of Pd, the Pd of the present invention is uniformly distributed on Ce-MOF, and the size is very small, about 5nm, which is at a relatively good level.

[0062] Figure 3 The following is a comparison chart of the oxidase (OXD) activity and peroxidase (POD) activity of Example 1-2 and Comparative Examples 1, 3, and 4. The results show that as the Pd loading increases, the OXD and POD activities are enhanced. When the Pd loading reaches 2.5wt% and above (Examples 1-2), the catalytic activity is significantly improved, while at 0wt% and 1.25wt% loading, the OXD and POD activities are not significantly improved, and even decrease, which may be related to the change in Ce valence caused by NaBH4 treatment. When the loading is 5wt%, the catalytic activity is optimal.

[0063] Figure 4 The catalytic activity comparison diagram of Example 1 and Comparative Examples 1 and 5 on classic phenolic substrates shows that the catalytic activity of 5.0wt% Pd@Ce-PZDC dual-site biomimetic multienzyme on phenolic compounds is better than that of natural laccase and single-site biomimetic laccase Ce-PZDC, especially for 4-chlorophenol and 2,4-DP, showing its application prospect in the treatment of phenolic pollutants.

[0064] Figure 5The results in Table 1 show that the synthesized dual-site biomimetic multi-enzyme Pd@Ce-PZDC has a higher V max (3.34×10 -3 mM min -1 ) and smaller K m (0.34mM), its catalytic efficiency V max / K m The increase in catalytic activity may be attributed to the enrichment effect of Pd on surface oxygen and the oxidation of part of Pd to Pd by O2. 2+ , Pd 2+ Then oxidize Ce in the material 3 + , thereby accelerating the electron transfer in the system.

[0065] Table 1 Kinetic parameters of different enzyme catalysts for 2,4-DP

[0066]

[0067] Figure 6 The peroxidase kinetic analysis of Example 1 and Comparative Examples 1 and 2 is shown. The results show that at the same mass concentration, the dual-site biomimetic multi-enzyme Pd@Ce-PZDC and pure reduced Pd nanoparticles have similar V for the catalytic reaction of TMB and H2O2. max and K m , and is significantly better than the single-site biomimetic enzyme Ce-PZDC. This indicates that the synthesized dual-site biomimetic multi-enzyme Pd@Ce-PZDC has excellent pseudo-peroxidase activity.

[0068] Figure 7 The gas chromatogram of the degradation of the lignin model 2-phenoxy-1-phenylethan-1-one catalyzed by the dual-site biomimetic multienzyme Pd@Ce-PZDC in Example 1 is shown. The chromatogram clearly shows that the catalyst can effectively degrade the model to generate benzoic acid and phenol as the main products. Table 2 compares the degradation effects of Example 1, Example 2 and Comparative Example 1, Comparative Example 2, and Comparative Example 4 under the same degradation conditions.

[0069] The specific degradation experimental conditions are as follows: 0.05 mmol of the model substance and 10 mg of the catalyst are placed in a 15 ml pressure-resistant glass bottle, 2.5 ml of a 2.5 wt% NaOH aqueous solution, 0.3 ml of H2O and 0.2 ml of H2O2 are added, and the mixture is sealed and reacted in an oil bath at 50° C. for 6 hours. After the reaction is completed, the solution is acidified to pH 2 with HCl and extracted with ethyl acetate (5 ml each time, 3 times in total). After removing the ethyl acetate by rotary evaporation, the residue is dissolved with acetone and analyzed by gas chromatography using n-hexadecane as an internal standard.

[0070] The above experimental results show that the Pd@Ce-PZDC dual-site bionic multi-enzyme exhibits higher catalytic activity in degrading the lignin model, and the yields of the main products benzoic acid and phenol are significantly better than those of the single-site bionic enzyme and the comparative sample without Pd loading. This further verifies the potential application value of Pd@Ce-PZDC in the field of lignin degradation.

[0071] Table 2 Product yields of model degradation products using different catalysts

[0072]

[0073] Figure 8 The gas chromatograms of the degradation of real lignin catalyzed by Example 1 and Comparative Example 1 are shown. The results show that compared with the single-site bionic enzyme Ce-PZDC, the dual-site bionic multi-enzyme Pd@Ce-PZDC can generate a variety of monophenolic products when catalyzing the degradation of lignin, such as phenol, vanillin, p-hydroxybenzoic acid, and syringaldehyde. The yields of these products are detailed in Table 3.

[0074] The specific degradation conditions are as follows: 50 mg of birch lignin and 20 mg of Pd@Ce-PZDC catalyst are weighed and placed in a 25 ml pressure-resistant glass bottle, 2.5 ml of 3.0 wt% NaOH aqueous solution, 0.3 ml of H2O and 0.2 ml of H2O2 are added, and the mixture is sealed and reacted in an oil bath at 60 ° C for 16 hours. After the reaction is completed, the reaction solution is acidified to pH 2 using HCl and extracted with ethyl acetate (5 ml each time, 3 times in total). After removing the ethyl acetate by rotary evaporation, the residue is dissolved in acetone, and the product is analyzed by gas chromatography using n-hexadecane as an internal standard. It can be seen from the results of gas chromatography that Pd@Ce-PZDC shows significant advantages in catalyzing the degradation of real lignin, and the generated monophenolic compounds are rich in variety and high in yield. At present, most of the lignin degradation conditions reported in the literature usually need to be carried out at high temperature (above 170 ° C) and high pressure (O2, H2 atmosphere). The present invention is carried out at 60 ° C in water phase, indicating its application potential for efficient degradation of lignin under mild conditions.

[0075] Table 3 Distribution of degradation products of real birch lignin catalyzed by Pd@Ce-PZDC

[0076]

[0077] The above embodiments are preferred embodiments of the present invention, but they do not limit the embodiments of the present invention. Any changes, modifications, substitutions, combinations or simplifications made without departing from the spirit and principle of the present invention shall be deemed as equivalent replacements and shall be included in the protection scope of the present invention.

Claims

1. A method for preparing an artificial dual-site biomimetic multi-enzyme catalyst, characterized in that: The following steps are involved: (1) mixing ammonium cerium nitrate, a biomimetic ligand and a modulator, heating and stirring to react, collecting the precipitate by suction filtration and washing, and drying to obtain a single-site metal organic framework biomimetic enzyme Ce-MOF; The bionic ligand is one or more of 3,5-pyrazoledicarboxylic acid, 2,5-thiophenedicarboxylic acid, and 2,5-furandicarboxylic acid; the molar ratio of the bionic ligand to ammonium cerium nitrate is (1-5):1; the molar ratio of the modulator to the bionic ligand is (5-40):1; (2) The Ce-MOF obtained in step (1) was ultrasonically dispersed in a mixed solution of ethanol and water, and Pd 2+ , continue ultrasonic dispersion, then add NaBH4 for stirring, filter and wash, and obtain Pd@Ce-MOF dual-site biomimetic multi-enzyme; The loading amount of Pd in ​​the Pd@Ce-MOF dual-site bionic multi-enzyme is 1.2 wt% to 5.0 wt%.

2. The method for preparing an artificial dual-site biomimetic multi-enzyme catalyst according to claim 1, characterized in that: The bionic ligand is 3,5-pyrazoledicarboxylic acid or 2,5-thiophenedicarboxylic acid, and the molar ratio of the bionic ligand to ammonium cerium nitrate is (1.3-1.6):

1.

3. The method for preparing an artificial dual-site biomimetic multi-enzyme catalyst according to claim 2, characterized in that: The modulator is one or more of formic acid, acetic acid or benzoic acid, and the molar ratio of the modulator to the biomimetic ligand is (10-15):

1.

4. The method for preparing an artificial dual-site biomimetic multi-enzyme catalyst according to any one of claims 1 to 3, characterized in that: The reaction temperature of step (1) is 80-110° C., and the reaction time is 10-25 minutes. The suction filtration and washing step uses a sand core funnel and a 0.45 μm organic membrane, and N,N-dimethylformamide and acetone are used to wash alternately three times. The washed solid needs to be immersed in acetone for 12 hours. The vacuum drying conditions are preferably dried at 50° C. and -0.1 MPa for 12 hours.

5. The method for preparing an artificial dual-site biomimetic multi-enzyme catalyst according to claim 1, characterized in that: The mass ratio of ethanol to water in the mixed solution in step (2) is 1.5:1; the Pd 2+ The molar ratio of Pd to NaBH4 is 1:(1-8); 2+ The mass feed ratio of Ce-MOF is (1.25~10):

100.

6. The method for preparing an artificial dual-site biomimetic multi-enzyme catalyst according to any one of claim 5, characterized in that: In step (2), Pd 2+ It is one or more of PdCl2, H2PdCl4, Pd(OAc)2; the ultrasonic dispersion step uses water bath ultrasound with a power of 200W and a duration of 15 minutes; the stirring temperature is room temperature, the rotation speed is 400-800rpm, and the duration is 8 to 16 hours; the suction filtration and washing step again uses a sand core funnel and a 0.45μm organic membrane, and is washed alternately with water and ethanol three times; the vacuum drying conditions are 50°C, -0.1MPa, and the drying is performed for 10 to 12 hours.

7. The artificial dual-site bionic multi-enzyme prepared by the method according to any one of claims 1 to 6.

8. Use of the artificial dual-site bionic multi-enzyme according to claim 7 in degrading lignin.

9. The use according to claim 8, characterized in that: Take 50 mg of lignin, add 5-30 mg of the artificial dual-site biomimetic multi-enzyme according to claim 7, 2.5 ml of 3.0 wt% NaOH aqueous solution, 0.3 ml of H2O, and 0.2 ml of H2O2, seal and place in an oil bath pot, and react at 60°C for 16 hours.

10. The use according to claim 9, characterized in that: The lignin is one or more of birch lignin, poplar lignin and pine lignin.

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