Spinning engineering nano-enzyme and application thereof in lignin conversion and adhesive preparation

Abstract

The invention belongs to the technical field of crossing of nano-enzymes, biomass catalytic conversion and green materials, and particularly relates to a spin engineering nano-enzyme and application thereof in lignin conversion and adhesive preparation. The spin engineering nano-enzyme is marked as COHBLO, and is a bionic nano-enzyme with natural laccase-like multi-copper center active sites, which is constructed on the basis of a two-dimensional COHB nano-sheet through cooperative regulation and control of acetate ligand exchange and L-ascorbic acid reduction. The nano-enzyme can be used for selectively cracking beta-O-4 ether bonds in lignin to generate active fragments rich in phenolic hydroxyl groups, and then the active fragments react with epoxy monomers to prepare the high-performance lignin epoxy adhesive. The adhesive has excellent permeability and adhesive performance, completely does not release formaldehyde, has comprehensive performance obviously better than that of commercial phenolic resin and epoxy resin, and provides an innovative technical scheme for realizing efficient utilization of biomass resources and development of green adhesives.

Description

TECHNICAL FIELD

[0001] The present application belongs to the cross technical field of nanomaterials, biomimetic catalysis and biomass refining, and particularly relates to a spin engineering nanoscale enzyme and its application in lignin conversion and adhesive preparation. BACKGROUND

[0002] Lignin, as the core structural component of plant cell walls, is the most abundant renewable aromatic polymer in nature, with an annual renewable amount of about 150 billion tons. Its complex amorphous three-dimensional network structure is formed by the connection of phenylpropane units through various C–O–C bonds and C–C bonds. Among them, C–O–C bonds mainly include β–O–4, α–O–4 and 4–O–5 types, while C–C bonds include β–5, β–β and 5–5 forms. Among these connection modes, β–O–4 ether bonds account for the highest proportion, about 45% to 60%. This unique structural characteristic makes lignin an ideal raw material for replacing petrochemical phenolic substances and for preparing bio-based chemicals and green adhesives. The rich aromatic rings and active functional groups such as phenolic hydroxyl and alcoholic hydroxyl in lignin molecules make it have the potential to participate in the construction of epoxy crosslinking network, and theoretically can realize the complete replacement of phenol.

[0003] However, the inherent structural complexity and chemical inertness of lignin pose severe challenges to its high-value utilization. The current mainstream lignin controlled depolymerization technologies, including thermal chemical methods such as hydrolysis and pyrolysis, and chemical oxidation methods, generally have problems such as harsh reaction conditions, high energy consumption and environmental unfriendliness, especially poor selectivity. These methods often cause random breaking and severe condensation side reactions of lignin, generating complex product mixtures with wide molecular weight distribution and unclear functional groups, which are difficult to meet the requirements of raw material uniformity for high-performance material synthesis.

[0004] Based on the biological catalytic degradation technology of natural enzymes, such as the application of laccase, lignin peroxidase and manganese peroxidase, has attracted widespread attention because it can selectively break the key linkages in lignin, especially the β–O–4 bond, under mild conditions such as room temperature, normal pressure and near neutral pH. Laccase, as a multi-copper center-containing oxidoreductase, its active center usually contains a T1 copper site responsible for substrate oxidation and electron transfer, a T2 copper site and two T3 double copper sites, among which T2 and T3 together form a trinuclear copper cluster responsible for the reduction activation of oxygen. This multi-copper center cooperates through different spin states of copper ions to efficiently reduce oxygen to water with four electrons, and simultaneously oxidizes the substrate. However, natural laccase faces insurmountable bottlenecks in industrial application, such as poor stability, sensitivity to temperature, pH, organic solvents and inhibitors, easy deactivation, high production cost, complex fermentation extraction process and difficulty in recycling and reuse.

[0005] Since the first report in 2007, nanoscale enzymes, a kind of functional nanomaterials with enzymatic catalytic activity, have provided a new way to break through the limitations of natural enzymes. They have the advantages of high catalytic activity of natural enzymes, high stability of nanomaterials, easy scale-up preparation and controllability. In recent years, developing nanoscale enzymes with laccase mimetic activity for lignin degradation has become a new research direction. However, the catalytic efficiency of most existing laccase nanoscale enzymes is still far lower than that of natural laccase, and the selectivity control of lignin degradation process is insufficient, the fundamental reason is that the essence of the multi-copper active center of natural laccase, i.e. the synergistic catalytic mechanism between different spin state copper ions, has not been accurately simulated from the electronic structure level. SUMMARY

[0006] In view of the problems of poor stability, difficult recovery, high cost of existing natural laccase in practical application, and insufficient performance of traditional lignin-based adhesives, the application provides a spin engineering nanoscale enzyme and its application in lignin conversion and adhesive preparation. The application realizes the whole chain integration from controllable depolymerization of lignin to preparation of high-performance adhesives, and provides a complete technical solution for high-value utilization of biomass resources.

[0007] To achieve the above object, the application adopts the following technical scheme: In a first aspect, the application provides a two-dimensional copper-based MOF nanoscale enzyme (COHBLO), which is prepared by reacting Cu2O cubes with terephthalic acid ligands in a polar solvent. The interface of the Cu2O cubes is etched by coordination, and the released Cu + is oxidized to Cu 2+ by dissolved oxygen, and then coordinates with terephthalic acid ligands to grow into two-dimensional COHB nanosheets, wherein the spin state of the copper active site is 5.5-5.6 μB; then the two-dimensional COHB nanosheets are subjected to acetate ligand exchange and L-ascorbic acid reduction treatment in sequence to construct a nanoscale enzyme with a laccase-like catalytic center with multiple spin states, i.e. the spin engineering nanoscale enzyme.

[0008] In view of the deficiencies of existing laccase nanoscale enzymes in activity and selectivity, and the core problem of poor stability of natural laccase, the application proposes a rational design strategy based on spin state regulation. By deeply analyzing the coordination microenvironment and electron transfer path of the multi-copper active center of natural laccase, we innovatively use oxidation-reduction treatment and ligand exchange engineering to successfully construct copper active sites with different spin states in two-dimensional copper-based MOF, and combined with characterization and activity test, we reveal the volcano-type structure-activity relationship between laccase-like activity and copper spin state, and accordingly synthesize the excellent COHBLO nanoscale enzyme, and finally realize the whole chain technology integration from controllable depolymerization of lignin to preparation of high-performance adhesives.

[0009] In a second aspect, the present application provides a preparation method of the spin-engineered nanoscale enzyme, comprising the following steps: S1, synthesizing Cu2O cubes by a precipitation method, dispersing in anhydrous ethanol to obtain a Cu2O ethanol suspension; S2, dissolving phthalic acid in a mixed solvent of anhydrous ethanol and a polar solvent, then adding the Cu2O ethanol suspension to react until the color of the solution gradually changes from orange red to blue, to obtain two-dimensional COHB nanosheets; S3, performing ligand exchange on the two-dimensional COHB nanosheets by using copper acetate to obtain COHB-OAc; S4, performing reduction treatment on the COHB-OAc by using L-ascorbic acid to obtain the spin-engineered nanoscale enzyme.

[0010] Further, in S2, the reaction condition is 25℃ for 3.5-4.5 hours.

[0011] Further, in S3, the step of performing ligand exchange on the two-dimensional COHB nanosheets by using copper acetate is as follows: mixing the two-dimensional COHB nanosheets and copper acetate in a polar solvent, reacting at 60℃ for 23-25 hours, and the addition concentration of copper acetate is 2.4-2.6 mg·mL -1 ; The step of performing reduction treatment on the COHB-OAc by using L-ascorbic acid is as follows: mixing the COHB-OAc and L-ascorbic acid in water, reacting at 60℃ under light-proof condition for 5.5-6.5 hours, and the addition concentration of L-ascorbic acid is 0.35-0.45· mg·ml -1 ; The polar solvent is N,N-dimethylformamide.

[0012] In a third aspect, the present application provides an application of the spin-engineered nanoscale enzyme in lignin directional degradation.

[0013] The application comprises: in a neutral buffer system, at a suitable temperature range, using the nanoscale enzyme to selectively catalyze the cleavage of the β-O-4 ether bond in lignin to directionally generate low molecular weight lignin fragments with significantly improved content of phenolic hydroxyl groups.

[0014] In a third aspect, the present application provides a preparation method of phenolated lignin, comprising: using the spin-engineered nanoscale enzyme to treat lignin raw materials in a buffer solution to selectively break the β-O-4 ether bond, so as to realize the phenolation of lignin, and to prepare phenolated lignin with significantly improved content of phenolic hydroxyl groups.

[0015] Specifically, lignin raw material and the two-dimensional copper-based MOF nanozyme are dispersed in a neutral buffer solution at a ratio of 5:1 and reacted at 37°C with shaking for 72 hours. After centrifugation, the nanozyme is separated and recovered, and the supernatant is freeze-dried to obtain the phenolic lignin. The number average molecular weight of the phenolic lignin is controlled within 300~500 Da, and the phenolic hydroxyl content is significantly increased.

[0016] Fourthly, the present invention provides a phenolic lignin prepared by the above-described preparation method. Fifthly, the present invention provides a method for preparing a bio-based epoxy adhesive, comprising the following steps: The phenolic lignin is cross-linked with a polyepoxy group cross-linking agent (glycerol triglycidyl ether) under an inert gas atmosphere to form a thermosetting adhesive with a three-dimensional cross-linked structure. After the reaction, volatile components are removed by vacuum distillation. The adhesive is completely formaldehyde-free and suitable for bonding and processing wood materials.

[0017] In a sixth aspect, the present invention provides a bio-based epoxy adhesive, which is prepared by the above-described preparation method.

[0018] In a seventh aspect, the present invention provides the application of the bio-based epoxy adhesive in wood bonding or wood-based panel preparation, wherein the adhesive is cured by hot pressing.

[0019] This invention provides the application of the bio-based epoxy adhesive in wood processing.

[0020] Through the above technical solution, the present invention achieves the following beneficial effects: This invention, for the first time, successfully simulates the synergistic catalytic mechanism of multiple copper centers in natural laccase by precisely controlling the spin state of copper active sites in a two-dimensional copper-based MOF. The COHBLO nanozyme developed based on this method exhibits superior catalytic performance, with a catalytic efficiency significantly higher than that of natural laccase, breaking through the technical bottleneck of low catalytic efficiency in traditional nanozymes.

[0021] The nanozyme exhibits excellent stability, maintaining good activity over a wide pH range, at high temperatures and with high salt concentrations. It can be recycled multiple times without significant performance degradation, which is significantly superior to natural laccase and other reported nanozyme materials.

[0022] This technology enables the complete chain transformation from lignin to high-performance adhesives. Through the selective catalysis of COHBLO nanozymes, lignin is directionally degraded into active fragments with high phenolic hydroxyl content and concentrated molecular weight distribution. Based on this, a bio-based epoxy adhesive exhibits superior bonding performance, with mechanical properties significantly better than commercial products. Moreover, the entire process is formaldehyde-free, providing a complete technical solution for the development of green, high-performance wood adhesives.

[0023] The complete technical system constructed by this invention, from lignin degradation to adhesive preparation, has the characteristics of renewable raw materials, green process, and high-performance products. It has great social benefits, economic benefits and industrial application prospects in the fields of wood processing and artificial board manufacturing. Attached Figure Description

[0024] Figure 1 Synthesis and morphological characterization of COHB-based nanozymes. a) TEM image of COHB. b) AFM image and thickness measurement of COHB. c) TEM images of COHB-LA, COHB-H2O2, COHB-CF3SO3, and COHB-OAc. d) AFM image and thickness measurement of COHB-LA, COHB-H2O2, COHB-CF3SO3, and COHB-OAc.

[0025] Figure 2 Electronic structure and spin state analysis of COHB-based nanozymes. a) XRD pattern of the COHB-based nanozyme. b) BMPO / •O2 of COHBLO. - EPR spectra and DMPO / •OH EPR spectra. c shows the Cu 2p XPS spectra of COHB, COHB-LA, COHB-H2O2, COHB-CF3SO3, and COHB-OAc. d shows the fitted magnetic susceptibility versus temperature relationship of COHB, COHB-LA, COHB-H2O2, COHB-CF3SO3, and COHB-OAc based on the Curie-Weiss law. e shows the Gibbs free energy curves of the laccase reaction on the surface of COHB-based nanozymes.

[0026] Figure 3 The laccase activity of COHB nanozymes is regulated by spin state. a) shows the laccase-like activities of different concentrations of COHB, COHB-LA, COHB-H2O2, COHB-CF3SO3, and COHB-OAc. b) shows the Michaelis-Menten kinetics of COHB-based nanozymes.

[0027] Figure 4 This study analyzes the catalytic mechanism of laccase. a) shows the laccase-like activities of different concentrations of COHBLO and natural laccase. b) shows the Michaelis-Menten kinetic curves of COHBLO and natural laccase. c) shows the effects of pH, temperature, and NaCl on the activities of COHBLO and natural laccase. d) shows SEM images of lignin (top) and lignin treated with COHBLO (bottom). e) shows the 3D fluorescence profiles of lignin (top) and lignin treated with COHBLO after 7 days (bottom). f) shows the quantification of VG and GG-derived reaction products at different time points during COHBLO incubation.

[0028] Figure 5 This study investigates the preparation and structure of lignin adhesives, as well as the performance and stability of CTLA. a) Fourier transform infrared absorption spectra of lignin adhesives and lignin (COHBLO) adhesives. b) Zeta potentials of lignin, lignin (COHBLO), LNA, and CTLA. c) Mass spectrometric changes of lignin adhesives and CTLA. d) 1H-13C NMR spectra of LA and CTLA. e) Effects of different adhesives on lap shear strength. f) Lap shear strength of CTLA, phenolic resin, and epoxy resin after immersion in different solvents for 24 hours. g) Lap shear strength of CTLA, phenolic resin, and epoxy resin after treatment at different temperatures. h) SEM images of the control, phenolic resin, epoxy resin, and CTLA as adhesives on wood panels after the lap shear test. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0030] Example 1: Preparation, screening and performance evaluation of COHBLO nanozymes To develop nanozyme materials with highly efficient laccase-like activity, this embodiment synthesized a series of two-dimensional copper-based MOF nanozymes via a template-guided solvothermal method. Cu₂O cubes were synthesized using a precipitation method. The synthesis steps are as follows: 17.0 mg of copper chloride dihydrate and 100 mg of polyvinylpyrrolidone were dissolved in 40 mL of deionized water and stirred on a magnetic stirrer. Then, the solution was added at 30 μL / s⁻¹. -1 2.5 mL of a 0.2 M sodium hydroxide aqueous solution was slowly added dropwise at a rate of [missing value]. After stirring for 5 minutes, the solution was added in 10 μL increments [missing value]. -1 2.5 mL of ascorbic acid aqueous solution (0.1 M) was added dropwise at a rate of [missing value]. The reaction was carried out at room temperature for 5 minutes. The resulting yellow precipitate was collected by centrifugation, washed twice with ethanol, and redispersed in 10 mL of anhydrous ethanol to obtain a Cu₂O ethanol suspension.

[0031] Two-dimensional MOFs were synthesized using a template conversion method. First, 83.0 mg of phthalic acid (H₂BDC) was dissolved in a mixed solvent of 5 mL anhydrous ethanol and 5 mL DMF. Then, 10 mL of a pre-prepared Cu₂O ethanol suspension was added. The reaction was allowed to stand at room temperature for 4 hours. During the reaction, the solution color gradually changed from orange-red to blue, indicating successful formation of the complex Cu(OH)₂BDC nanosheets. After the reaction, the reaction mixture was transferred to a 50 mL centrifuge tube and centrifuged at 8000 rpm for 15 minutes at 4 °C. The supernatant was carefully removed. The precipitate was washed three times with methanol, each time under the same centrifugation conditions. Finally, the obtained blue precipitate was placed in a vacuum drying oven and dried at 60 °C for 12 hours to obtain basic Cu(OH)₂BDC nanosheet powder, i.e., basic COHB nanozyme, or simply COHB.

[0032] To further optimize material properties, we systematically post-processed and modified the basic COHB nanozyme. The following four methods were used: ① Synthesis of COHB-LA: 50.0 mg of COHB was dispersed in 50 mL of a 0.4 mg / mL solution. -1 ① Synthesis of COHB-H2O2: 50.0 mg COHB was added to 50 mL of 5% H2O2 aqueous solution and stirred at 60°C for 6 hours. The product was collected by centrifugation and washed three times with deionized water. ② Synthesis of COHB-OAc: 50.0 mg COHB was added to 20 mL glass vials containing 9.0 mL of DMF. Then 1.0 mL of 2.5 mg∙mL⁻¹ HMF was added. -1 A DMF solution of copper acetate was used. The mixture was allowed to stand at 60°C for 24 hours. The product was collected by centrifugation, washed three times with DMF, and then washed with acetone. ④ Synthesis of COHB-CF3SO3: 50.0 mg of COHB was added to a 20 mL glass vial containing 8.7 mL of DMF. Then 1.3 mL of a 2.2 mg / mL solution was added. -1 A DMF solution of copper trifluoromethanesulfonate was prepared. The mixture was allowed to stand at 60°C for 24 hours. The product was collected by centrifugation, washed three times with DMF, and finally washed with acetone.

[0033] The structure of COHB was systematically analyzed using various characterization methods. Transmission electron microscopy (TEM) revealed that the material maintains a complete two-dimensional square nanosheet morphology, with edge lengths distributed in the range of 380-420 nm, a smooth and flat surface, and sharp edges. Figure 1a). Atomic force microscopy (AFM) measured its thickness to be approximately 2 nm, confirming the successful construction of the ultrathin two-dimensional structure. Figure 1 b). Morphological differences were observed among the four modified COHB variants. COHB-H2O2 and COHB-LA retained the sharp, straight, square edges of the original COHB and exhibited a highly ordered morphology. In contrast, the edges of the ligand-exchanged COHB-OAc and COHB-CF3SO3 showed slight serrations or irregularities. Figure 1 c). Atomic force microscopy thickness analysis further confirmed that all post-processed samples maintained a stable thickness of approximately 2 nm. Figure 1 d) indicates that chemical modification only caused the reorganization of the surface microstructure without destroying the layered integrity of the two-dimensional framework.

[0034] X-ray diffraction (XRD) patterns showed characteristic diffraction peaks at 2θ = 9.2°, 18.5°, and 26.3°, which perfectly matched the simulated COHB crystal structure. Figure 2 a). A strong ·OH radical signal was observed in the electron paramagnetic resonance (EPR) spectrum at g = 2.004, and a significant O2·⁻ signal was detected at g = 2.035, indicating that the material has a high efficiency in generating reactive oxygen species. Figure 2 b). In-depth spin-state characterization revealed the material's excellent electronic structure properties. X-ray photoelectron spectroscopy (XPS) showed that the binding energies of Cu 2p3 / 2 and Cu 2p1 / 2 in COHB were at 933.8 eV and 953.7 eV, respectively. + / Cu 2+ The ratio reached 0.483. COHB-H2O2 showed an increase in Cu. 2+ The content of Cu was reduced, while COHB-LA, COHB-OAc, and COHB-CF3SO3 showed enhanced Cu content. + Signal.( Figure 2 c). Variable-temperature magnetic susceptibility testing showed that COHB, COHB-LA, COHB-H2O2, and COHB-OAc materials maintained stable paramagnetism in the temperature range of 50-300 K. The effective magnetic moments obtained by fitting with the Curie-Weiss law were 5.56 μB, 1.72 μB, 4.78 μB, and 2.55 μB, respectively. In contrast, COHB-CF3SO3 exhibited a lower χ² value. m The value shows almost no temperature dependence, indicating significant diamagnetism, and the fitted effective magnetic moment is 0 μB ( Figure 2 d).

[0035] To evaluate the catalytic activity of the materials, the activity of the nanozymes laccase and the determination of enzyme kinetic parameters were performed. The specific methods are as follows: A 50 mM MES buffer solution at pH 6.8 was prepared as the reaction medium and filtered through a 0.22 μm microporous membrane for sterilization. Each nanozyme sample (COHB, COHB-LA, COHB-H2O2, COHB-CF3SO3, and COHB-OAc) was accurately weighed and prepared into 1 mg / mL solutions using the above buffer solution. -1 The working concentration. Using 2,4-dichlorophenol (2,4-DP) as the substrate, prepare 1 mg / mL solution with MES buffer. -1 The substrate working solution was prepared by mixing 20 μL of nanozyme working solution with 20 μL of substrate working solution and 140 μL of MES buffer, and reacting precisely in a 37°C water bath for 30 minutes. Immediately after the reaction, 20 μL of freshly prepared 1 mg / mL solution was added. -1 4-Aminoantipyrine (4-AP) chromogenic reagent was used, and the mixture was allowed to stand at room temperature in the dark for 10 minutes. The absorbance was measured at 510 nm using a UV-Vis spectrophotometer. During the enzyme kinetic parameter determination, the nanozyme concentration was kept constant, and the 2,4-DP substrate concentration gradient was set to 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1.2, 1.8, 2.4, and 3.0 mM. The initial reaction rate was measured at each concentration. The maximum reaction rate was calculated using nonlinear fitting of the Michaelis-Menten equation. V max ) and Michaelis constant ( K M All experiments were performed in triplicate, and the results are expressed as mean ± standard deviation.

[0036] Through system performance screening experiments ( Figure 3 a) Experimental data showed that, at a concentration of 20 μg / mL... -1 At the final concentration of the materials, COHB-CF3SO3 exhibited the lowest catalytic activity, with an absorbance of only 0.50; the basic COHB material showed baseline activity, with an absorbance of 0.76; COHB-H2O2 showed moderate to high catalytic activity, with an absorbance of 1.03; while COHB-OAc and COHB-LA showed significantly enhanced catalytic performance, with absorbances of 1.23 and 1.74, respectively, representing improvements of 61.8% and 128.9% compared to the basic material.

[0037] In-depth enzymatic kinetic studies further confirmed the superior performance of COHB-LA. Figure 3 b). Within the substrate concentration range of 0.05–2.5 mM, this nanozyme follows a typical Michaelis-Menten kinetic model, and its maximum reaction rate was calculated through nonlinear fitting.V max The value is 8.18 × 10 -4 mM s -1 Michaelis constant ( K M The concentration was 0.19 mM. Compared with other nanozymes, COHB-LA not only has the highest catalytic efficiency but also exhibits the best substrate affinity, laying a solid foundation for its application in lignin degradation.

[0038] Table 1: Parameters of the Michaelis-Menten kinetic model for different nanozymes Density functional theory calculations show that the d-band center of the material shifts upward by 0.35 eV, significantly enhancing the adsorption and activation capacity of oxygen molecules. Figure 2 e). These characterization results collectively confirm that we have successfully constructed laccase-inspired nanomaterials with superior catalytic performance through precise spin state modulation.

[0039] Example 2: Optimized preparation and structural characterization of COHBLO nanozymes Based on previous screening results, we developed a composite modification strategy to prepare high-performance COHBLO nanozymes. The specific steps are as follows: 50.0 mg of COHB was added to a 20 mL glass vial containing 9.0 mL of DMF. Then, 1.0 mL of a 2.5 mg·mL⁻¹ solution was added. -1 A DMF solution of Cu(CH3COO)2·4H2O was prepared. The mixture was allowed to stand at 60 °C for 24 hours to complete the acetate ligand exchange process. The product was collected by centrifugation, washed three times with DMF, and then washed with acetone to obtain the COHB-OAc precursor. 50.0 mg of the COHB-OAc precursor was reacted with 0.4 mg·mL⁻¹ of DMF. -1 Ascorbic acid was mixed in 10 mL of deionized water and reacted with continuous stirring at 60 °C for 6 hours for reduction treatment. The product was collected by centrifugation, washed three times with deionized water, and dried in a vacuum drying oven at 60 °C for 24 hours to obtain the dark blue COHBLO nanozyme final product.

[0040] To quantitatively characterize the catalytic advantages of the COHBLO nanozyme compared to natural laccase, a comparison of their catalytic activities and catalytic kinetics was conducted. Figure 4 a, 4b). Catalytic kinetic parameter measurements showed that COHBLO nanozyme possesses substrate affinity comparable to natural laccase, while its maximum reaction rate is approximately 71 times that of natural laccase, nearly two orders of magnitude higher. Under various conditions, including a wide pH range, temperature variations, and high ionic strength, COHBLO exhibits significantly higher stability than natural laccase. Figure 4 c).

[0041] Table 2: Parameters of the Michaelis-Menten kinetic model of COHBLO and natural laccase Example 3: Preparation and Characterization of Phenolic Lignin This embodiment details the complete process flow and structural characterization of phenolic lignin prepared using COHBLO nanozymes. Alkali lignin was used as the raw material, and the COHBLO nanozyme was prepared according to the method in Example 2. The buffer system was a 50 mM MES buffer solution containing 2% DMSO at pH 6.5. 10 g of dealkalized lignin raw material and 2 g of COHBLO nanozyme were accurately weighed and placed in a 250 mL Erlenmeyer flask. 100 mL of MES buffer solution was added to form a uniform dispersion system. The mixture was placed in a constant temperature shaking incubator and reacted continuously at 37°C and 150 rpm for 72 hours. During the reaction, samples were taken periodically to monitor the reaction progress, and changes in the characteristic absorption peak of lignin at 280 nm were measured using a UV-Vis spectrophotometer. After the reaction, the reaction solution was centrifuged at 10,000 rpm for 15 minutes at 4°C to achieve effective separation and recovery of the nanozyme. The collected supernatant was filtered through a 0.22 μm microporous membrane, frozen at -80℃ for 12 hours, and then dried in a freeze dryer for 48 hours to obtain a dark brown powdery phenolic lignin product. Three parallel experiments showed that the average yield of the product reached 85.2% ± 2.3%, with good reproducibility.

[0042] The morphology of raw material lignin and phenolic lignin was systematically characterized by scanning electron microscopy. Figure 4 d). The raw lignin exhibits a regular spherical structure with uniform particle size distribution, a smooth and dense surface, and an average diameter of approximately 70 μm. After treatment with COHBLO nanozymes, the spherical structure of the lignin completely dissociates, transforming into an irregular fragmented morphology with obvious tearing and delamination at the edges. The surface roughness increases significantly, forming numerous nanoscale porous structures. This significant change in morphology directly demonstrates the effective depolymerization and structural reorganization of lignin macromolecules.

[0043] Further analysis of the molecular characteristics of degradation products using 3D fluorescence spectroscopy. Figure 4e). The raw lignin showed only weak fluorescence signals in the excitation wavelength region of 250-300 nm and the emission wavelength region of 300-400 nm, indicating that its aromatic structure was relatively dense and its functional group activity was low. Phenolic lignin treated with COHBLO showed significantly enhanced fluorescence intensity in the same region, especially with strong fluorescence peaks near the excitation wavelength of 280 nm and the emission wavelength of 350 nm. This corresponds to small molecule aromatic compounds containing abundant phenolic and alcoholic hydroxyl groups, such as lignin, vanillic acid, and syringic acid. Furthermore, the weak signal observed near the excitation wavelength of 350 nm and the emission wavelength of 500 nm indicates that a small amount of highly condensed aromatic byproducts were generated during the degradation process, but their content was far lower than that of the main products.

[0044] The degradation process of the β-O-4 model compound resveratrol glycerol-β-guaiacol (VG) was monitored by high performance liquid chromatography. Figure 4 f). Under the action of COHBLO nanozyme, VG rapidly degraded within 30 minutes, and β-O-4 cleavage products such as guaiacol and vanillin accumulated rapidly, with the conversion rate reaching over 85% at 30 minutes. To verify the reaction selectivity, a control experiment was conducted using guaiacol-glycerol-β-guaiacol ether (GG), which has low reactivity, as a substrate. Figure 4 f). The results showed that COHBLO could still generate the same cleavage products in the GG system, but the reaction rate was reduced by about 2.1 times compared with the VG system, which fully demonstrates the high specificity of nanozymes for β-O-4 bond cleavage.

[0045] Example 4: Construction and performance evaluation of bio-based epoxy adhesives This embodiment systematically introduces the preparation process and comprehensive performance evaluation of a bio-based epoxy adhesive based on phenolic lignin. 10 g of phenolic lignin prepared in Example 3 was dissolved in 90 mL of 0.2 wt% sodium hydroxide solution to prepare a 10 wt% lignin solution. 10 g of glycerol triglycidyl ether was added to this lignin solution, and the mixture was continuously stirred for 60 minutes in an oil bath at 120°C under nitrogen protection. The reaction system was equipped with a mechanical stirring and temperature control system to ensure thorough mixing of the reactants and precise temperature control. The viscosity change of the system was monitored during the reaction, and the reaction progress was tracked by measuring the epoxy value. After the reaction, volatile components were removed by vacuum distillation at 60°C and 0.1 MPa using a rotary evaporator (RE-52AA, Shanghai Yarong) to obtain a dark brown, high-viscosity bio-based epoxy adhesive (denoted as CTLA). During the preparation process, the rheological properties of the adhesive were controlled by adjusting the solid content to ensure suitable processing performance and storage stability.

[0046] The adhesive curing process was systematically characterized using Fourier transform infrared spectroscopy. Figure 5a). Raw material lignin is 1650-1800 cm⁻¹ -1 The absence of a distinct absorption peak in this region indicates a lack of oxidizable active sites. After COHBLO treatment, a clear C=O stretching vibration peak appeared in the same region, directly demonstrating the introduction of carboxyl and ketone functional groups through the oxidation ring-opening of the aromatic unit and the cleavage of the aliphatic side chain. After phenolation treatment, the peak at 3400 cm⁻¹... -1 The OH stretching vibration peak at the hydroxyl group was significantly enhanced, while the carbonyl peak completely disappeared, confirming that the oxidation product was effectively captured by the phenolic hydroxyl group and formed a phenolic lignin structure. These changes collectively confirm the successful ring-opening addition reaction between the epoxy group and the phenolic hydroxyl group, forming a stable COC covalent bond.

[0047] Zeta potential analysis showed that ( Figure 5 (b) The potential of the raw lignin was -22.6 mV, which decreased to -25.6 mV after COHBLO oxidation. This is due to the enhanced ionization by the newly formed carbonyl and carboxyl groups. The physically mixed sample (LNA) showed an intermediate potential of -23.5 mV, while CTLA reached the most negative potential of -28.1 mV, a decrease of 4.6 mV compared to LNA. This conclusively demonstrates the covalent bonding between the epoxy groups and the aromatic ring framework of the phenolic lignin. The optimized surface charge properties significantly enhanced the dispersion stability of the colloid in aqueous systems and improved the interfacial bonding efficiency with positively charged substrates.

[0048] Mass spectrometry analysis provides molecular-level evidence for the structural characteristics of the adhesive. Figure 5 c). LNA showed only a weak ionic signal in the low molecular weight region (<500 Da), indicating limited depolymerization of the natural lignin structure. In contrast, CTLA showed a significantly enhanced signal intensity within the same mass range, with the main fragment peaks corresponding to lignin-derived oligomers, including m / z 168.042 (vanillic acid) and m / z 198.053 (syringic acid). This significant difference directly demonstrates that COHBLO pretreatment mediates the oxidative cleavage of β-O-4 bonds and other unstable bonds in the lignin macromolecular network, generating abundant phenolic end-group oligomeric fragments.

[0049] High-resolution nuclear magnetic resonance spectra provide direct evidence of the adhesive's chemical structure. Figure 5d). The characteristic resonance signal at δC 50.68 / δH 3.08 is attributed to the methoxymethyl group (-OCH3), the characteristic signal at δC 44.31 / δH 2.71 corresponds to the methylene proton (-CH2-), and the methine proton signal coincides with the lignin methoxy signal at δC 50.68 / δH 3.08. Most importantly, the newly emerging methoxy signal at δC 78.00 / δH 3.70 provides conclusive evidence for GTE ring-opening and grafting onto the lignin side chain. These spectroscopic features collectively demonstrate that COHBLO pretreatment effectively cleaves the natural lignin framework, enriches the phenolic hydroxyl content, optimizes molecular weight and solubility properties, and ultimately achieves efficient grafting of GTE onto the modified lignin structure.

[0050] CTLA was applied to test the bonding performance of birch veneer. Figure 5 e). The bonding performance test used a size of 10 × 4 × 0.1 cm. 3 Using birch veneer as the substrate, adhesive was evenly applied to the veneer surface using a precision applicator, covering an area of ​​1 cm². 2 The coating concentration is approximately 150 gm -2 The glued veneers were stacked and hot-pressed in a hot press at 140℃ and 0.5 MPa for 20 minutes to complete the curing process. Tensile shear strength was determined using a universal testing machine according to GB / T 9846-2015 standard, with the test speed set to 2 mm / min. -1 Five valid replicates were tested for each group of samples, and the average value was taken. Solvent resistance testing involved immersing the cured bonded specimens in solvents such as water, artificial seawater, tetrahydrofuran, ethyl acetate, acetone, and ethanol. After immersion at room temperature for 24 hours, the specimens were removed, the surface liquid was wiped dry with filter paper, and the tensile shear strength was immediately measured. The strength retention rate was calculated as: (strength after immersion / initial strength) × 100%. Tensile shear strength testing showed that CTLA achieved a shear strength of 15.13 MPa under dry conditions and retained 11.38 MPa under wet conditions (24 hours of immersion in water at room temperature), which is superior to commercial phenolic resin (PF, 14.96 MPa dry, 11.33 MPa wet) and epoxy resin (Epoxy, 11.96 MPa dry, 7.96 MPa wet). Environmental adaptability testing showed (…). Figure 5 f), CTLA retains over 90% of its bond strength after immersion in representative solvents such as water, artificial seawater, tetrahydrofuran, ethyl acetate, acetone, and ethanol for 24 hours, demonstrating excellent solvent resistance. Thermal stability tests show ( Figure 5 (g) CTLA exhibits stable bonding performance in a temperature range of -20℃ to 100℃, with no obvious thermal decomposition or performance degradation, ensuring its long-term reliability under wide temperature conditions.

[0051] Microscopic morphology analysis of the bonding interface was performed using scanning electron microscopy. Figure 5 h). The blank control group showed typical brittle fracture characteristics of wood with a smooth fracture surface. Commercial adhesive systems (PF and epoxy resin) exhibited a mixed failure mode, including partial tearing of wood fibers, adhesive layer failure, and interfacial debonding. In contrast, CTLA-bonded specimens mainly showed wood matrix failure, with a highly textured fracture surface containing a large number of forcibly extracted and broken wood fiber bundles. The degree of wood matrix failure induced by CTLA was equal to or greater than that induced by high-performance commercial phenolic resin, indicating that its bond strength was close to or exceeded the intrinsic strength of the wood itself. Further microscopic observation showed that CTLA formed a dense three-dimensional network of mechanical anchor points within the wood structure. Fracture path analysis confirmed that the failure mainly occurred in the solid wood cell walls, exhibiting a cohesive failure mode with a large number of fiber bundle breaks and vessel wall failures, while adhesive layer failure or interfacial debonding was minimal. These observations provide direct evidence for the superior interfacial bond strength of CTLA.

[0052] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0053] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A spin-engineered nanozyme, characterized in that, Using Cu₂O cubes as templates, terephthalic acid ligands are reacted in a polar solvent, resulting in interfacial coordination etching of the Cu₂O cubes and the release of Cu. + Oxidized to Cu by dissolved oxygen 2+ Subsequently, it coordinates with terephthalic acid ligands and is epitaxially grown into two-dimensional COHB nanosheets, wherein the spin state of the copper active site is 5.5~5.6 μB; then the two-dimensional COHB nanosheets are subjected to acetate ligand exchange and L-ascorbic acid reduction treatment in sequence to construct a nanozyme with a laccase-like catalytic center having multiple spin states, namely the spin-engineered nanozyme.

2. The method for preparing the spin-engineered nanozyme according to claim 1, characterized in that, Includes the following steps: S1. Cu2O cubes were synthesized by precipitation and dispersed in anhydrous ethanol to obtain a Cu2O ethanol suspension. S2. Diphthalic acid is dissolved in a mixture of anhydrous ethanol and a polar solvent, and then Cu2O ethanol suspension is added to react until the solution color gradually changes from orange-red to blue, thus obtaining two-dimensional COHB nanosheets. S3. Copper acetate was used to perform ligand exchange on two-dimensional COHB nanosheets to obtain COHB-OAc; S4. COHB-OAc is reduced using L-ascorbic acid to obtain the spin-engineered nanozyme.

3. The method for preparing spin-engineered nanozymes according to claim 2, characterized in that, In S2, the reaction conditions are 25°C for 3.5 to 4.5 hours.

4. The method for preparing spin-engineered nanozymes according to claim 2, characterized in that, In S3, the steps for ligand exchange of two-dimensional COHB nanosheets using copper acetate are as follows: Two-dimensional COHB nanosheets and copper acetate were mixed in a polar solvent and reacted at 60°C for 23–25 hours. The concentration of copper acetate added was 2.4–2.6 mg∙mL. -1 ; The steps for reducing COHB-OAc using L-ascorbic acid are as follows: COHB-OAc and L-ascorbic acid were mixed in water and reacted at 60°C in the dark for 5.5–6.5 hours. The concentration of L-ascorbic acid added was 0.35–0.45 mg∙ml. -1 ; The polar solvent is N,N-dimethylformamide.

5. The application of the spin-engineered nanozyme of claim 1 in the directional degradation of lignin.

6. A method for preparing phenolic lignin, characterized in that, include: Using the spin-engineered nanozyme described in claim 1, lignin raw materials are treated in a buffer solution to selectively break β-O-4 ether bonds, thereby achieving phenolization of lignin and obtaining phenolized lignin with significantly increased phenolic hydroxyl content.

7. A phenolic lignin, characterized in that, It is prepared by the preparation method described in claim 6.

8. A method for preparing a bio-based epoxy adhesive, characterized in that, Includes the following steps: The phenolic lignin of claim 7 is crosslinked with glycerol triglycidyl ether under inert gas protection to form a thermosetting adhesive. After the reaction is completed, the volatile components are removed by vacuum distillation.

9. A bio-based epoxy adhesive, characterized in that, It is prepared by the preparation method described in claim 8.

10. The application of the bio-based epoxy adhesive of claim 9 in wood bonding or wood-based panel preparation, characterized in that, The adhesive is cured by hot pressing.

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