A method for degrading lignin by mechanical chemical enzyme coupling

Abstract

The present invention discloses a method for mechanochemical enzyme-coupled lignin degradation. The specific operation process is to place lignin powder, a solid-phase reagent, and zirconium oxide beads in a polytetrafluoroethylene ball mill, perform a co-grinding reaction in the ball mill, add an enzyme after ball milling, add a solvent for stirring, and reflux reaction. After the reaction, the mixture is dissolved in water and extracted with ethyl acetate. The insoluble residue is centrifuged, washed with ethyl acetate, and vacuum dried for detection and analysis. The present invention has the advantages of a short process route, low reagent usage, simple operation, low cost, low pollution, low molecular weight of the obtained product, and good degradation efficiency. It is a lignin degradation method with good promotion and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of lignin degradation, and particularly relates to a method for degrading lignin by mechanical chemical enzyme coupling. Background Art

[0002] Lignin is an important component of wood cellulose besides hemicellulose and cellulose. Although the research and application of hemicellulose and cellulose are very mature and extensive, the utilization of lignin, a biological resource with an aromatic structure, has been neglected. The main reason for the above problems is that, compared with cellulose connected by sugar chains, lignin is a stable composite phenolic polymer with saturated CO and CC bonds as the main connecting bonds. Its reaction activity is significantly lower than that of hemicellulose and cellulose. In addition, lignin also has certain antibacterial effects and serves as a barrier for plants to inhibit external invasion. Therefore, the degradation and utilization of lignin is more difficult than that of cellulose, requiring more complex chemical reactions and specific degradation microorganisms, with high costs and relatively low efficiency.

[0003] Research has found that while plant lignin is diverse, the primary reactive link within its macromolecules is the β-O-4 bond, formed by a CO bond. This bond accounts for over 45% of the total lignin content and is key to its chemical degradation. Existing processes are all based on the cleavage of lignin's β-O-4 bond, requiring the use of strong oxidants or reducing agents in combination with metal catalysts and ligands to achieve efficient lignin degradation.

[0004] For example, Crestini et al. (Crestini C, Pro P, Neri V, et al. Methyltrioxorhenium: a new catalyst for the activation of hydrogen peroxide to the oxidation of lignin and lignin model compounds [J]. Bioorganic & Medicinal Chemistry, 2005, 13 (7): 2569-2578.) used methyltrioxorhenium (MeReO3, MTO) as a catalyst to achieve side chain oxidation and aromatic ring cleavage of lignin by air oxidation. Analysis showed that the alkyl side chains of lignin underwent oxidative cleavage under these conditions, and lignin was directly degraded into vanillin, 2,4-hydroxy-3-methoxybenzoic acid, mucilagenol and p-benzoquinone derivatives. Wang et al. (Wang N, et al., Efficient oxidative cleavage of lignin CC model compound using MOF-derived Cobalt / Nickel sulfide heterostructures[J]. Fuel, 2022, 320:123993-.) used a metal-organic framework as a precursor and trimellitic acid as a ligand to synthesize a Ni / C-10 metal-based catalyst. The catalyst catalyzed the oxidation of lignin dimers and lignin β-O-4 bonds at 160°C to produce high-value-added chemicals such as benzoic acid and phenol. Furthermore, the Ni / C-10 catalyst exhibited excellent desulfurization ability in the oxidative depolymerization of lignin sulfonates. Lu et al. (Lu X, Clément R, Lu R. Selective C–CBond Cleavage in Diols and Lignin Models: High-Throughput Screening of Metal Oxide-Anchored Vanadium in Mesoporous Silica[J]. Catalysts, 2021, 11.) synthesized a supported V(Al / Ti / Zr / Ce)-MCM-41 composite material as a catalyst and found that it can effectively oxidize and crack the C–C bonds in lignin model (1,2-diphenyl-2-methoxyethanol) and polyols, providing a new heterogeneous catalytic oxidation pathway for lignin. Although the above methods have high selectivity and mild reaction conditions, most catalysts or ligands are relatively expensive, resulting in excessively high costs for lignin degradation.In recent years, the research and application of lignin degradation based on bio-enzymatic technology has gradually increased. Peroxidases, phenol oxidases and laccases used for lignin metabolism in nature have been found to be able to directly degrade lignin. For example, Majeke et al. (Majeke BM, Collard FX, Tyhoda L, et al. The synergistic application of quinone reductase and ligninperoxidase for the deconstruction of industrial (technical) lignins and analysis of the degraded lignin products [J]. Bioresource Technology, 2021, 319.) found that although lignin peroxidase can efficiently degrade lignin, the degradation process is accompanied by the production of phenoxy radicals and quinone intermediates, which are randomly coupled to difficult-to-degrade polymers. Therefore, the additional addition of quinone oxidase can limit the repolymerization of lignin caused by lignin peroxidase. The synergistic application of the two enzymes reduced the molecular weight of lignin by more than 31%, and the product was mainly monomeric phenol. Zhang et al. (Zhang YH P. Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges [J]. Biotechnology Advances, 2015, 33(7): 1467-1483.) studied the degradation of corn straw lignin using laccase, lignin peroxidase, and manganese peroxidase, achieving a lignin degradation rate of 25.79%. The degradation destroyed the macromolecular structure of lignin, including the benzene ring structure, and produced a large number of acidic compounds.

[0005] Compared to chemical degradation, enzymatic degradation of lignin is less polluting, but high-performance peroxidases are expensive, while inexpensive laccases used alone have weak oxidative capacity, require longer reaction times, and generally produce poor results. In short, the key to lignin resource utilization currently lies in finding an efficient, convenient, and low-cost degradation technology. Combining the advantages of biodegradation and chemical degradation technologies and employing novel reaction optimization techniques could be a breakthrough.

[0006] Mechanochemistry, as a reaction optimization technology, has gained favor in the industrial sector due to its unique reaction mechanism, higher yields and selectivity, and the convenience of solvent-free or low-solvent reactions. Many metal-catalyzed reactions that originally required prolonged high temperatures and large amounts of high-boiling-point organic solvents can now be carried out efficiently at room temperature, achieving even higher yields and selectivities. It can even utilize raw materials that are unavailable in solution reactions. Especially for chemical reactions between poorly soluble macromolecules, mechanochemistry can directly promote various reactions such as complexation, neutralization, degradation, and grafting in the solid phase of macromolecules, regardless of solvent selection. It has currently found widespread application in the processing of polymers such as cellulose, pectin, lignin, cyclodextrins, and cucurbiturils. There are reports on the mechanochemical oxidation and degradation of lignin using DDQ as an oxidant. The mechanical energy provided by ball milling generates various stresses and shear points in the reaction system, which break down larger solid particles into smaller ones, increase the specific surface area, and promote the conversion of lignin into valuable aromatics. However, the current mechanochemical degradation of lignin is still based on chemical reactions and still requires the use of DDQ, NaNO2, and perchlorate (Su W, Sun C, Zheng L, et al. Mechanochemical Cleavage of Lignin Models and lignin via Oxidation and Subsequent Base-Catalyzed Strategy[J]. Green Chemistry, 2020(11). Lancefield CS, Ojo OS, Tran F, et al. Isolation of functionalized phenolic monomers throughselective oxidation and CO bond cleavage of the beta-O-4 linkages in lignin[J]. Angew Chem Int Ed Engl, 2015, 54(1): 258-262. NGUYEN JD, MATSUURA BS and STEPHENSON C RA photochemical strategy for lignin degradation at roomtemperature[J]. J Am Chem Soc, 2014, 136(4): 1218-1221.) as chemical oxidants, but there are still certain issues with greenness and safety. Therefore, it would be a good direction to fully learn from the mechanism of mechanochemistry to promote solid-phase reaction between polymers and introduce cheap commercial laccase as a green oxidant to achieve efficient degradation and resource utilization of lignin. Summary of the Invention

[0007] The present invention aims to overcome the problems of large chemical reagent usage, high extraction temperature, long extraction time, and complex process in existing lignin degradation processes, and to provide a simple, efficient, and environmentally friendly method for lignin degradation via mechanochemical-enzymatic coupling. Leveraging the advantages of mechanochemistry in promoting polymer reactions, the method utilizes mechanochemical pretreatment to activate the CO bonds of lignin and the laccase oxidation center with a weak oxidant, achieving the goal of oxidative degradation of lignin, overcoming the challenges of laccase's weak oxidizing ability and low lignin solubility.

[0008] The specific technical solutions are as follows:

[0009] A method for mechanochemical enzyme coupling degradation of lignin comprises the following steps: placing lignin powder, a solid phase reagent and zirconium oxide beads in a polytetrafluoroethylene ball mill, performing a co-grinding reaction in the ball mill, adding an enzyme after ball milling, adding a solvent for stirring, and performing a reflux reaction. After the reaction is completed, the lignin is dissolved in water, and ethyl acetate is added for extraction. After centrifugation, the insoluble residue is washed with ethyl acetate, and the residue is vacuum dried and tested and analyzed.

[0010] Furthermore, the lignin powder is alkali-extracted lignin.

[0011] Furthermore, the solid-phase reagent is a co-oxidant or a mixture of a co-oxidant and an oxidant, the co-oxidant is selected from copper oxide, cuprous oxide, copper hydroxide, copper acetate, copper sulfate, copper chloride or copper bromide, and the oxidant is selected from sodium bromide or sodium chloride. When the solid-phase reagent is a mixture of a co-oxidant and an oxidant, the mass ratio of the oxidant to the co-oxidant is 6:1 to 8:1.

[0012] Furthermore, the mass ratio of the solid phase reagent to the lignin is 5 to 12:10, preferably 9 to 12:10.

[0013] Furthermore, the grinding speed of the grinder is 100-500 rpm, preferably 400 rpm, 30 minutes of grinding is one cycle, and 5 minutes of rest is performed, and a total of 6-12 cycles, preferably 10 cycles, are performed.

[0014] Furthermore, the enzyme is laccase, and the mass ratio of laccase to lignin is 1:400-67, preferably 1:100-67.

[0015] Furthermore, the solvent is ethanol, n-hexane, acetone, acetonitrile or ethyl acetate, preferably acetone.

[0016] Furthermore, the reflux reaction time is 90 to 300 min, preferably 180 min.

[0017] The beneficial effects of the present invention are:

[0018] 1) The lignin is degraded by combining mechanical ball milling reaction with enzyme, which is simple to operate, has a short process route and a short degradation time;

[0019] 2) Compared with traditional methods, mechanical ball milling can greatly reduce the amount of chemical reagents used in lignin degradation, lower production costs, and alleviate environmental pollution;

[0020] 3) The lignin product obtained by the method of the present invention has low molecular weight and high product value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the degradation rate of lignin obtained through Examples 1-9;

[0022] Figure 2 The effect of different solid phase reagent addition amounts on the degradation rate;

[0023] Figure 3 The effect of different ball mill speeds on the degradation rate;

[0024] Figure 4 The effect of different ball milling cycles on the degradation rate;

[0025] Figure 5 The effect of different laccase addition amounts on the degradation rate;

[0026] Figure 6 The effect of different reaction solvents on the degradation rate;

[0027] Figure 7 The effect of different reflux times on degradation rate;

[0028] Figure 8 GC-FID diagram of birch lignin degradation products;

[0029] Figure 9 GC-FID diagram of rice straw lignin degradation products. DETAILED DESCRIPTION

[0030] The technical solutions of the present invention are further described below through specific examples, but the protection scope of the present invention is not limited thereto.

[0031] Method for determining lignin molecular weight:

[0032] The weight-average molecular weight (Mw) of the sample was determined by gel permeation chromatography. The measurement was performed on a Shimadzu instrument equipped with a refractive index (RI) detector and a Waters Styragel 5E series column, using THF as the mobile phase and narrow-distribution polymethyl methacrylate (PMMA) as the standard sample. The flow rate was 0.3 mL / min and the temperature was set to 40°C. Since birch and straw lignin are insoluble in THF, the lignin needs to be acetylated first. Acetylation method: Under the condition of 10 mg of birch (or straw) lignin, lignin is acetylated with a mixed solvent of 1 mL of acetic anhydride and 1 mL of pyridine at room temperature for 48 hours. After completion, the acetic anhydride and pyridine in the acetylated lignin are removed in a vacuum oven at 40°C for 24 hours, and then the acetylated birch (or straw) lignin is dissolved in THF for GPC analysis.

[0033] Lignin degradation rate determination method:

[0034] The degradation rate of lignin was calculated by 2D-HSQC NMR spectroscopy. 2D-HSQC NMR spectroscopy was measured on a 600 MHz Bruker instrument. A 20 mg sample was weighed and dissolved in d6-DMSO solvent at 300 K. 1 H, 13 C-HSQC experiments were performed using the standard Bruker Plus sequence "hsqcedetgpsisp2.3" with 4 scans, 10 pulse widths, an acquisition time of 0.2 s, and a relaxation delay of 1.5 s. Conversions were calculated from the Aα integral relative to the methoxy integral, assuming that the methoxy group content remained constant during the reaction.

[0035] Analytical method for organic soluble products of lignin degradation:

[0036] The organic-soluble degradation products of lignin were dissolved in ethyl acetate and characterized by GC-FID and GC-MS chromatography. GC-MS was performed on an Agilent 7890B / 5977B instrument equipped with an HP-5 capillary mass spectrometer column. Analytical conditions included an inlet temperature of 260°C, helium carrier gas, a flow rate of 1 mL / min, a hold at 60°C for 5 minutes, followed by heating to 260°C at a rate of 10°C / min, and then a hold at 260°C for 5 minutes. Gas chromatography was performed on a GC-9720 system using an SH-RTX-1 capillary column and a flame ionization detector (FID). 1,3,5-Trimethylbenzene was used as the internal standard. The GC-FID temperature program was identical to the GC-MS temperature program.

[0037] Example 1

[0038] To a 250 mL polytetrafluoroethylene ball mill, add 10 g of lignin powder (birch or rice straw), 10 g of copper oxide, and six 14 mm diameter zirconium oxide beads. Mill at 300 rpm for 10 cycles of 30 min each, followed by a 5 min rest. After milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After completion of the reaction, dissolve in water and extract with ethyl acetate (3 × 10 mL). The insoluble residue is centrifuged and washed with ethyl acetate (3 × 10 mL). The residue is dried under vacuum and analyzed by HSQC and GPC.

[0039] In this example, the molecular weight of the birch lignin after degradation is 6408 Da, and the degradation rate of birch lignin is 28%; the molecular weight of the rice straw lignin is 9346 Da, and the degradation rate of the rice straw lignin is 17%.

[0040] Example 2

[0041] To a 250 mL polytetrafluoroethylene ball mill, add 10 g of lignin powder (birch or rice straw), 10 g of cuprous oxide, and six 14 mm diameter zirconium oxide beads. Mill at 300 rpm for 10 cycles of 30 min each, followed by a 5-min rest. After milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After completion of the reaction, dissolve in water and extract with ethyl acetate (3 × 10 mL). The insoluble residue is centrifuged and washed with ethyl acetate (3 × 10 mL). The residue is dried under vacuum and analyzed by HSQC and GPC.

[0042] The molecular weight of the birch lignin after degradation in this example is 6947 Da, and the degradation rate of birch lignin is 11%. The molecular weight of the rice straw lignin is 9557 Da, and the degradation rate of rice straw lignin is 8%.

[0043] Example 3

[0044] To a 250 mL polytetrafluoroethylene ball mill, add 10 g of lignin powder (birch or rice straw), 10 g of copper hydroxide, and six 14 mm diameter zirconium oxide beads. Mill at 300 rpm for 10 cycles of 30 min each, followed by a 5-min rest. After milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After completion of the reaction, dissolve in water and extract with ethyl acetate (3 × 10 mL). The insoluble residue is centrifuged and washed with ethyl acetate (3 × 10 mL). The residue is dried under vacuum and analyzed by HSQC and GPC.

[0045] The molecular weight of the birch lignin after degradation in this example is 6322 Da, and the degradation rate of birch lignin is 27%. The molecular weight of the rice straw lignin is 9147 Da, and the degradation rate of rice straw lignin is 15%.

[0046] Example 4

[0047] To a 250 mL polytetrafluoroethylene ball mill, add 10 g of lignin powder (birch or rice straw), 10 g of copper acetate, and six 14 mm diameter zirconium oxide beads. Mill at 300 rpm for 10 cycles of 30 min each, followed by a 5 min rest. After milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After completion of the reaction, dissolve in water and extract with ethyl acetate (3 × 10 mL). The insoluble residue is centrifuged and washed with ethyl acetate (3 × 10 mL). The residue is dried under vacuum and analyzed by HSQC and GPC.

[0048] The molecular weight of the birch lignin after degradation in this example is 4379 Da, and the degradation rate of birch lignin is 37%. The molecular weight of the rice straw lignin is 8184 Da, and the degradation rate of rice straw lignin is 20%.

[0049] Example 5

[0050] To a 250 mL polytetrafluoroethylene ball mill, add 10 g of lignin powder (birch or rice straw), 10 g of copper sulfate, and six 14 mm diameter zirconium oxide beads. Mill at 300 rpm for 10 cycles of 30 min each, followed by a 5-min rest. After milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After completion of the reaction, dissolve in water and extract with ethyl acetate (3 × 10 mL). The insoluble residue is centrifuged and washed with ethyl acetate (3 × 10 mL). The residue is dried under vacuum and analyzed by HSQC and GPC.

[0051] The molecular weight of the birch lignin after degradation in this example is 4659 Da, and the degradation rate of birch lignin is 32%. The molecular weight of the rice straw lignin is 8280 Da, and the degradation rate of rice straw lignin is 19%.

[0052] Example 6

[0053] To a 250 mL polytetrafluoroethylene ball mill, add 10 g of lignin powder (birch or rice straw), 10 g of copper chloride, and six 14 mm diameter zirconium oxide beads. Mill at 300 rpm for 10 cycles of 30 min each, followed by a 5-min rest. After milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After completion of the reaction, dissolve in water and extract with ethyl acetate (3 × 10 mL). The insoluble residue is centrifuged and washed with ethyl acetate (3 × 10 mL). The residue is dried under vacuum and analyzed by HSQC and GPC.

[0054] The molecular weight of the birch lignin after degradation in this example is 3971 Da, and the degradation rate of birch lignin is 45%. The molecular weight of rice straw lignin is 7963 Da, and the degradation rate of rice straw lignin is 24%.

[0055] Example 7

[0056] To a 250 mL polytetrafluoroethylene ball mill, add 10 g of lignin powder (birch or rice straw), 10 g of copper bromide, and six 14 mm diameter zirconium oxide beads. Mill at 300 rpm for 10 cycles of 30 min each, followed by a 5 min rest. After milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After completion of the reaction, dissolve in water and extract with ethyl acetate (3 × 10 mL). The insoluble residue is centrifuged and washed with ethyl acetate (3 × 10 mL). The residue is dried under vacuum and analyzed by HSQC and GPC.

[0057] The molecular weight of the birch lignin after degradation in this example is 3589 Da, and the degradation rate of birch lignin is 55%. The molecular weight of rice straw lignin is 7670 Da, and the degradation rate of rice straw lignin is 33%.

[0058] Example 8

[0059] To a 250 mL polytetrafluoroethylene ball mill, add 10 g of lignin powder (birch or rice straw), 1.3 g of copper bromide, 8.7 g of sodium bromide, and six 14 mm diameter zirconium oxide beads. Mill at 300 rpm for 10 cycles of 30 min each, followed by a 5 min rest. After milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After completion of the reaction, dissolve in water and extract with ethyl acetate (3 × 10 mL). The insoluble residue is centrifuged and washed with ethyl acetate (3 × 10 mL). The residue is dried under vacuum and analyzed by HSQC and GPC.

[0060] The molecular weight of the birch lignin after degradation in this example is 3487 Da, and the degradation rate of birch lignin is 56%. The molecular weight of rice straw lignin is 7421 Da, and the degradation rate of rice straw lignin is 33%.

[0061] Example 9

[0062] To a 250 mL polytetrafluoroethylene ball mill, add 10 g of lignin powder (birch or rice straw), 1.3 g of copper bromide, 8.7 g of sodium chloride, and six 14 mm diameter zirconium oxide beads. Mill at 300 rpm for 10 cycles of 30 min each, followed by a 5 min rest. After milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After completion of the reaction, dissolve in water and extract with ethyl acetate (3 × 10 mL). The insoluble residue is centrifuged and washed with ethyl acetate (3 × 10 mL). The residue is dried under vacuum and analyzed by HSQC and GPC.

[0063] The molecular weight of the birch lignin after degradation in this example is 3751 Da, and the degradation rate of birch lignin is 48%. The molecular weight of the rice straw lignin is 7745 Da, and the degradation rate of rice straw lignin is 28%.

[0064] like Figure 1 As shown in the experimental results, Cu(II) salts have the best catalytic effect on this reaction, with CuBr2 showing the strongest catalytic ability, achieving a degradation rate of 55% for birch lignin and 33% for rice straw lignin. Furthermore, when using non-halogen copper salts, the conversion rate of the raw materials is not high, indicating that the halogen actually participates in the reaction. However, the amount of CuBr2 used alone is too high, which does not conform to the concept of green chemistry. Therefore, low-cost, high-halogen-content halogen salts were further used as halogen sources to reduce costs and improve efficiency. Experimental results show that using NaBr as the halogen source can achieve higher degradation rates of CuBr2 under catalytic conditions, and the mass ratio of NaBr to CuBr2 is controlled to be 6:1 to 8:1.

[0065] Example 10 Preferred mass ratio of solid phase reagent to lignin

[0066] Add 10 g of lignin powder (birch lignin or straw lignin) to a 250 mL polytetrafluoroethylene ball mill, control the mass ratio of solid phase reagent (copper bromide: sodium bromide = 1:7) to lignin to 5:10, 6:10, 7:10, 8:10, 9:10, 10:10, 11:10, 12:10, and 6 zirconia beads with a diameter of 14 mm, grind and react at 300 rpm, 30 minutes as a cycle, 5 minutes of rest, and grind for 10 cycles. After ball milling, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 minutes. After the reaction, dissolve in water and extract with ethyl acetate (3×10 mL); the insoluble residue is centrifuged and washed with ethyl acetate (3×10 mL). The residue is dried under vacuum and analyzed by HSQC. Figure 2 As shown, the test results show that if the amount of solid phase reagent added is too little, the lignin cannot be fully oxidized and degraded, and the degradation effect is poor; if the amount of solid phase reagent added is too much, the reagent will be wasted, so the preferred mass ratio of solid phase reagent to lignin is 9:10 to 12:10.

[0067] Example 11 Optimal ball mill speed

[0068] Add 10 g of lignin powder (birch lignin or rice straw lignin), 1.3 g of copper bromide, 8.7 g of sodium bromide, and 6 14 mm diameter zirconium oxide beads to a 250 mL polytetrafluoroethylene ball mill. After mixing evenly, place the ball mill in a planetary ball mill. Control the speed of the ball mill to 100, 150, 200, 250, 300, 350, 400, 450, and 500 rpm for 10 cycles. After the ball milling is completed, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 min. After the reaction is completed, dissolve in water and extract with ethyl acetate (3×10 mL); the insoluble residue is centrifuged and washed with ethyl acetate (3×10 mL). The residue is dried under vacuum and analyzed by HSQC. Figure 3 As shown, the test results show that if the rotation speed is too low, the grinding and dispersion are insufficient, resulting in poor degradation effect; if the grinding rate is too high, the heat release of the system increases, causing partial deactivation of the solid phase catalyst and reduced degradation efficiency, so the preferred rotation speed is 400 rpm.

[0069] Example 12 Preferred ball milling cycle

[0070] Add 10 g of lignin powder (birch lignin or rice straw lignin), 1.3 g of copper bromide, 8.7 g of sodium bromide, and 6 14 mm diameter zirconium oxide beads to a 250 mL polytetrafluoroethylene ball mill. After mixing evenly, place the ball mill in a planetary ball mill. Control the speed of the ball mill to 400 rpm, and the time is 6, 7, 8, 9, 10, 11, and 12 cycles respectively. After the ball milling is completed, add laccase (100 mg, 0.01 g / g), stir in acetone, and reflux for 240 minutes. After the reaction is completed, dissolve in water and extract with ethyl acetate (3×10 mL); the insoluble residue is centrifuged and washed with ethyl acetate (3×10 mL). The residue is dried under vacuum and analyzed by HSQC. Figure 4 As shown in FIG, the test results show that when the ball milling time is short, the materials are not fully mixed and reacted in the ball mill, and the degradation rate is low; while when the ball milling time is too long, the degradation rate of the product does not increase, so the preferred ball milling time is 10 cycles.

[0071] Example 13 Preferred mass ratio of laccase to lignin

[0072] Add 10 g of lignin powder (birch lignin or straw lignin), 1.3 g of copper bromide, 8.7 g of sodium bromide, and 6 zirconia beads with a diameter of 14 mm to a 250 mL polytetrafluoroethylene ball mill. After mixing evenly, place the ball mill in a planetary ball mill. The ball milling speed is 400 rpm and the time is 10 cycles. After the ball milling is completed, the mass ratio of laccase to lignin is controlled to be 1:400, 1:200, 1:133, 1:100, 1:80, and 1:67, respectively. Stir in acetone and reflux for 240 minutes. After the reaction is completed, it is dissolved in water and the solution is extracted with ethyl acetate (3×10 mL); the insoluble residue is centrifuged and washed with ethyl acetate (3×10 mL). The residue is dried under vacuum and analyzed by HSQC. Figure 5 As shown, the test results show that the optimal mass ratio of laccase to lignin is 1:100 to 1:67.

[0073] Example 14 Preferred reaction solvent

[0074] Add 10 g of lignin powder (birch lignin or straw lignin), 1.3 g of copper bromide, 8.7 g of sodium bromide, and 6 14 mm diameter zirconium oxide beads to a 250 mL polytetrafluoroethylene ball mill. Mix well and place the ball mill into a planetary ball mill. Control the speed of the ball mill to 400 rpm for 10 cycles. After the ball milling is completed, add laccase (100 mg, 0.01 g / g) and stir in ethanol, n-hexane, acetone, acetonitrile, and ethyl acetate solvents, respectively. Reflux for 240 min. After the reaction is completed, dissolve in water and extract with ethyl acetate (3×10 mL); the insoluble residue is centrifuged and washed with ethyl acetate (3×10 mL). The residue is dried under vacuum and analyzed by HSQC. Figure 6 As shown, the experimental results show that because lignin does not have good solubility in organic solvents with high or low polarity, and acetone has moderate polarity and good solubility for lignin, when acetone is used as the reaction solvent, lignin has the highest degradation rate.

[0075] Example 15 Preferred Solution Reaction Time

[0076] Add 10 g of lignin powder (birch lignin or rice straw lignin), 1.3 g of copper bromide, 8.7 g of sodium bromide, and 6 zirconia beads with a diameter of 14 mm to a 250 mL polytetrafluoroethylene ball mill. After mixing evenly, place the ball mill into a planetary ball mill. Control the speed of the ball mill to 400 rpm for 10 cycles. After the ball milling is completed, add laccase (100 mg, 0.01 g / g), stir in acetone solvent, and reflux for 90, 120, 150, 180, 210, 240, 270, and 300 min, respectively. After the reaction is completed, dissolve in water and extract with ethyl acetate (3×10 mL); the insoluble residue is centrifuged and washed with ethyl acetate (3×10 mL). The residue is dried under vacuum and analyzed by HSQC. Figure 7 As shown in the figure, the test results show that when the reaction time is short, the materials are not fully mixed and reacted in the solution, and the degradation rate is low; when the reaction time is too long, the degradation rate of the product does not increase significantly, so the preferred solution time is 180 minutes.

[0077] Example 16 Lignin degradation products

[0078] Add 10 g of lignin powder (birch lignin or rice straw lignin), 1.3 g of copper bromide, 8.7 g of sodium bromide, and 6 14 mm diameter zirconium oxide beads to a 250 mL polytetrafluoroethylene ball mill. After mixing evenly, place the ball mill in a planetary ball mill. Control the speed of the ball mill to 400 rpm and the time to 10 cycles. After the ball milling is completed. Add laccase (100 mg, 0.01 g / g), stir in acetone solvent, and reflux for 180 minutes. After the reaction is completed, dissolve in water and extract with ethyl acetate (3×10 mL); the insoluble residue is centrifuged and washed with ethyl acetate (3×10 mL). At the same time, the organic phases are combined, dried with anhydrous MgSO4, filtered, and GC-MS and GC-FID product analysis is performed using 1,3,5-trimethylbenzene as the internal standard. The results are as follows: Figure 8 and Figure 9 The degradation products obtained by analysis are shown below:

[0079] (A) Birch lignin degradation products

[0080]

[0081] (B) Rice straw lignin degradation products

[0082]

Claims

1. A method for degrading lignin by mechanical chemical enzyme coupling, characterized in that The method comprises the following steps: placing lignin powder, a solid phase reagent and zirconium oxide beads in a polytetrafluoroethylene ball mill, performing a co-grinding reaction in the ball mill, adding an enzyme after ball milling, adding a solvent for stirring, performing a reflux reaction, dissolving the lignin in water after the reaction is completed, adding ethyl acetate for extraction, centrifuging the insoluble residue, washing the residue with ethyl acetate, vacuum drying the residue and performing detection and analysis; The solid phase reagent is a co-oxidant or a mixture of a co-oxidant and an oxidant, the co-oxidant is selected from copper oxide, cuprous oxide, copper hydroxide, copper acetate, copper sulfate, copper chloride or copper bromide, and the oxidant is selected from sodium bromide or sodium chloride; The grinding speed of the grinder is 100-500 rpm, and the grinding cycle is 30 minutes, with a rest period of 5 minutes, and a total of 6-12 cycles; The enzyme is laccase.

2. The method for degrading lignin by mechanical chemical enzyme coupling according to claim 1, characterized in that The lignin powder is alkali-extracted lignin.

3. The method for degrading lignin by mechanical chemical enzyme coupling according to claim 1, characterized in that When the solid phase reagent is a mixture of a co-oxidant and an oxidant, the mass ratio of the oxidant to the co-oxidant is 6:1 to 8:

1.

4. The method for degrading lignin by mechanical chemical enzyme coupling according to claim 3, characterized in that The mass ratio of the solid phase reagent to the lignin is 5 to 12:

10.

5. The method for degrading lignin by mechanical chemical enzyme coupling according to claim 1, characterized in that The mass ratio of laccase to lignin is 1:400-67.

6. The method for degrading lignin by mechanical chemical enzyme coupling according to claim 1, characterized in that The solvent is ethanol, n-hexane, acetone, acetonitrile or ethyl acetate.

7. The method for degrading lignin by mechanical chemical enzyme coupling according to claim 1, characterized in that The reflux reaction time is 90 to 300 minutes.

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