Method for modifying and cracking lignin through bacterial laccase / mediator system
By combining a bacterial laccase/mediator system with a NiMoS/P-Al2O3 catalyst, the problem of poor catalytic contact of fungal laccase under alkaline conditions was solved, and efficient lignin depolymerization and preparation of monophenolic substances were achieved.
Patent Information
- Application Number
- CN202511304205.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2026-01-13
AI Technical Summary
In existing technologies, fungal laccases exhibit good functionality at low pH and low temperature, but under alkaline conditions, the contact between macromolecular lignin and enzyme catalysis is poor, resulting in low Ca-OH oxidation levels. Furthermore, the chemical oxidation process easily causes lignin recombination, making efficient depolymerization difficult.
Lignin was enzymatically modified using a bacterial laccase/mediator system, and then lignin oil was obtained by hydrogen catalytic cracking in a NiMoS/P-Al2O3 catalyst and a high-temperature and high-pressure reactor.
It improves the depolymerization efficiency of lignin, reduces the degree of polymerization and molecular weight of macromolecules, enhances alkali resistance, and improves the efficiency of enzyme catalytic reaction, thus realizing the efficient preparation of monophenolic substances.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of lignin resource utilization, specifically to a method for modifying and cleaving lignin using a bacterial laccase / mediator system. Background Technology
[0002] Lignin, a complex macromolecular compound formed by phenylpropane units linked by various bonds, is the second largest renewable biomass resource in the plant kingdom and has been a subject of close research attention in recent years. A highly efficient strategy for lignin depolymerization is to preferentially oxidize the Ca-OH on the lignin side chain to Ca=O followed by hydrogenation depolymerization. However, the oxidation process using chemical reagents inevitably leads to lignin repolymerization. In the field of biotechnology, microbial laccases have been widely used and researched in the modification and degradation of lignin due to their advantages such as oxidizing phenolic and non-phenolic lignin-related compounds and producing environmentally friendly products. Fungal laccases can achieve oxidative modification of macromolecular lignin Ca-OH without inducing lignin repolymerization, and have a significant advantage in reducing the degree of lignin polymerization. However, fungal laccases generally exhibit better functionality at low pH and low temperature, while macromolecular lignin only dissolves under alkaline conditions. Therefore, the contact between macromolecular lignin and enzyme catalysis is poor during fungal laccase catalysis, resulting in a lower degree of oxidation of macromolecular lignin Ca-OH. In contrast, bacteria have advantages such as rich biodiversity and good environmental adaptability. Furthermore, highly stable bacterial laccases can function over a wider pH range and at higher temperatures. The enzymes they secrete are easily manipulated for gene and protein expression, and their expression levels, stability, and catalytic performance can be improved through directed evolution. Summary of the Invention In view of the shortcomings of the prior art, the technical problem solved by the present invention is to provide a method for modifying and cleaving lignin with a bacterial laccase / mediator system that has good treatment effect. This method for modifying and cleaving lignin with a bacterial laccase / mediator system can effectively utilize waste gas containing chemical components and achieve safe emission standards.
[0003] To address the aforementioned technical problems, this invention provides a method for modifying and cleaving lignin using a bacterial laccase / mediator system. This method can oxidize the Ca-OH on the lignin side chain to C=O and also reduce the bond energy of adjacent C-C bonds in lignin, which is beneficial for promoting the catalytic cleavage of lignin.
[0004] The method for modifying and lysing lignin using a bacterial laccase / mediator system provided by this invention comprises the following steps: S01. Enzymatic modification of groundwood lignin macromolecules by bacterial laccase and mediator to obtain enzyme-modified lignin; SO2, using NiMoS / P-Al2O3 as a catalyst, the enzyme-modified lignin, the catalyst and methanol solvent are placed in a high-temperature and high-pressure reactor, and then an appropriate amount of hydrogen is introduced to catalyze the cleavage of lignin. S03. Take out the mixture after the reaction, separate the solid and liquid phases by centrifugation, and obtain the lignin oil product by vacuum distillation and vacuum drying; S04. Extract the lignin oil product with an appropriate amount of dichloromethane to obtain lignin monophenol products.
[0005] As a preferred embodiment of the above technical solution, the method for modifying and lysing lignin using a bacterial laccase / mediator system provided by the present invention further includes some or all of the following technical features: As an improvement to the above technical solution, in step S01, the bacterial laccase is an engineered bacterium that is heterologously expressed using gene knockout technology. Four different types of bacterial laccases were obtained: wild-type laccase WT, randomly mutated wild-type laccase X33-WT, site-directed mutant laccase 495, and randomly mutated laccase X33-255; the mediator is the mediator HBT.
[0006] As an improvement to the above technical solution, step S01 specifically involves mixing ground wood lignin and glycine-sodium hydroxide buffer solution evenly, then adding reaction mediator HBT, then adding enzyme solution containing bacterial laccase, and placing the mixture in an air shaker at a constant temperature and speed to start the enzyme treatment reaction. After the reaction was completed, the reaction mixture was removed, the solid and liquid phases were separated by centrifugation, the aqueous phase was removed, the precipitate was washed, and the mixture was freeze-dried to obtain enzyme-modified lignin. The final concentration of the groundwood lignin is 0.01–0.1 mg / mL; the final concentration of the glycine-sodium hydroxide buffer is 10–100 mM with a pH of 7.0–12.0; the amount of bacterial laccase is 0–2000 U / g substrate; and the final concentration of the reaction mediator HBT is 1–5 mM.
[0007] As an improvement to the above technical solution, the shaking speed of the air shaker is 50-500 rpm, the air shaker temperature is 20-50℃, and the reaction time is 1-5 days.
[0008] As an improvement to the above technical solution, in step S02, the NiMoS / P-Al2O3 catalyst is prepared by a two-step initial wet impregnation method, specifically as follows: Step 1 Impregnation: Nickel nitrate hexahydrate and diammonium hydrogen phosphate are dissolved separately and added to the pseudoboehmite, followed by high-temperature roasting. The second step involves impregnation, in which the dissolved ammonium tetrathiomolybdate is loaded a second time and calcined twice at high temperatures under nitrogen and hydrogen to obtain the NiMoS / P-Al2O3 catalyst.
[0009] As an improvement to the above technical solution, the atomic molar ratio of molybdenum, nickel, and phosphorus in the impregnation is 7:3:0-4. As an improvement to the above technical solution, the muffle furnace calcination temperature in the first impregnation step is 300-800℃ for 2-5 hours; the second impregnation step involves calcination in a tube furnace: calcination at 200-500℃ for 2-5 hours in a nitrogen atmosphere; and calcination at 300-800℃ for 2-5 hours in a hydrogen atmosphere.
[0010] As an improvement to the above technical solution, in step S02, the catalytic cracking temperature is 100℃~250℃, the time is 2h~10h, and the hydrogen pressure is 1~5MPa.
[0011] As an improvement to the above technical solution, the catalytic cracking reaction process in step S02 is as follows:
[0012] As an improvement to the above technical solution, the amount of NiMoS / P-Al2O3 catalyst used is 10-50% of the enzyme-modified lignin, and the amount of solvent used is 0.5-2 times the amount of enzyme-modified lignin.
[0013] As an improvement to the above technical solution, in step S03, the centrifugation time is 5-15 min, the rotation speed is 3000-10000 rpm; the temperature of vacuum distillation is 30-80℃; and the temperature of vacuum drying oven is 40-80℃.
[0014] As an improvement to the above technical solution, in step S04, the amount of dichloromethane required for extracting the lignin oil product is 1-5 ml; the mass of the added ethyl vanillin internal standard is 0.5-2.5 mg.
[0015] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: The lignin modified by oxidation using a laccase / mediator system achieved oxidation of Ca-OH on the lignin side chains and partial breakage of carbon-carbon bonds. Simultaneously, the degree of polymerization and molecular weight of the lignin macromolecules decreased accordingly. Furthermore, through mutagenesis modification, its alkali resistance was further enhanced, maintaining high enzyme activity while improving the contact between alkali lignin and the enzyme catalyst, thereby increasing the efficiency of the enzyme catalytic reaction. Then, lignin oil was obtained by catalytic cleavage of lignin using a NiMoS / P-Al2O3 catalyst. This combined bacterial multifunctional alkali-resistant laccase and hydrogenation depolymerization technology catalyzes the depolymerization of lignin to prepare monophenolic substances, significantly improving the yield of lignin depolymerization.
[0016] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, the following detailed description is provided in conjunction with preferred embodiments. Attached Figure Description
[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings of the embodiments will be briefly described below.
[0018] Figure 1(a) shows the lignin depolymerization catalyzed by different laccases at pH=7; Figure 1(b) shows the lignin depolymerization catalyzed by different laccases at pH=9.6; Figure 1(c) shows the lignin depolymerization catalyzed by different laccases at pH=10.6. Detailed Implementation
[0019] The following detailed description of specific embodiments of the present invention is part of this specification. The principles of the present invention are illustrated through examples, and other aspects, features and advantages of the present invention will become apparent from this detailed description.
[0020] This invention discloses a method for modifying and lysing lignin using a bacterial laccase / mediator system, comprising the following steps: enzymatic modification of groundwood lignin macromolecules using wild-type laccase WT, randomly mutant wild-type laccase X33-WT, site-directed mutant laccase 495, and randomly mutant laccase X33-255 with mediator HBT; the modified lignin, NiMoS / P-Al2O3 catalyst, and methanol solution are then placed in a mechanically stirred reactor, and an appropriate amount of hydrogen gas is introduced before the reactor is assembled for reaction; after the reaction, the reaction solution is removed and centrifuged; an appropriate amount of tetrahydrofuran extraction catalyst is added, and the extract is added to the supernatant for vacuum distillation and vacuum drying to obtain lignin oil; finally, the oil is dissolved in dichloromethane, an internal standard is added, and quantitative analysis is performed by GC-MS. The advantages of this invention lie in the fact that, through laccase / mediator system modification, lignin exhibits oxidation of Ca-OH on its side chains and partial breakage of carbon-carbon bonds. Simultaneously, the degree of polymerization and molecular weight of the lignin macromolecules are correspondingly reduced. Furthermore, through mutagenesis modification, its alkali resistance is further enhanced, maintaining high enzyme activity while improving the contact between the ground wood lignin and the enzyme catalyst, thereby increasing the efficiency of the enzyme catalytic reaction. Then, lignin oil is obtained by catalytic cleavage of lignin with a NiMoS / P-Al2O3 catalyst. This bacterial multifunctional alkali-resistant laccase combined with hydrogenation depolymerization technology catalyzes the depolymerization of lignin to prepare monophenolic substances, significantly improving the yield of lignin depolymerization.
[0021] Example 1 A method for modifying and lysing lignin using a bacterial laccase / mediator system, comprising the following steps: 1) Take an appropriate amount of ground wood lignin and disperse it evenly in a glycine-sodium hydroxide buffer solution with a pH of 7. Then add the reaction mediator HBT, and finally add a blank sample with an enzyme activity of 0 U / g and four different laccase enzyme solutions with an enzyme activity of 1000 U / g. Place the mixture in an air shaker and start the enzyme treatment reaction at a constant temperature and speed. The shaking speed of the air shaker is 220 rpm, the temperature is 37℃, and the reaction time is 3 days. Take out the reaction mixture, separate the solid and liquid phases, remove the aqueous phase, wash the precipitate, and freeze-dry to obtain enzyme-treated lignin. 2) Take 25 mg of enzyme-modified lignin sample, 10 mg of NiMoS / P-Al2O3 catalyst, and 20 mL of methanol and put them into a mechanically stirred reactor. Add an appropriate amount of nitrogen to the reactor to replace the air inside, then add an appropriate amount of hydrogen to replace it, and then replace the air inside the reactor with hydrogen before introducing H2 at 22 MPa. React at 230℃ for 4 h.
[0022] 3) Remove the reaction vessel and place it in an ice bath to cool rapidly to room temperature. Centrifuge the reaction mixture in the vessel, wash the precipitate with an appropriate amount of tetrahydrofuran until the washing liquid is colorless, combine the washing liquid with the supernatant obtained by centrifugation, remove the solvent by rotary evaporation at 50℃, and then dry thoroughly in a vacuum drying oven at 60℃ to obtain lignin oil. Extract the depolymerization products in the lignin oil with dichloromethane 3-5 times, combine the extracts, centrifuge, take the supernatant, add 200 μL of ethyl vanillin (5 mg / mL) as an internal standard, take 1 mL in a chromatographic vial, and analyze it by gas chromatography-mass spectrometry (GC / MS). The results are shown in Figure 1(a). Example 2 A method for modifying and lysing lignin using a bacterial laccase / mediator system, comprising the following steps: 1) Take an appropriate amount of ground wood lignin and disperse it evenly in a glycine-sodium hydroxide buffer solution with a pH of 9.6. Then add the reaction mediator HBT, and finally add a blank sample with an enzyme activity of 0 U / g and four different laccase enzyme solutions with an enzyme activity of 1000 U / g. Place the mixture in an air shaker and start the enzyme treatment reaction at a constant temperature and speed. The shaking speed of the air shaker is 220 rpm, the temperature is 37℃, and the reaction time is 3 days. Take out the reaction mixture, separate the solid and liquid phases, remove the aqueous phase, wash the precipitate, freeze-dry, and obtain the enzyme-treated lignin. 2) Take 25 mg of enzyme-oxidized lignin sample, 10 mg of NiMoS / P-Al2O3 catalyst, and 20 mL of methanol and put them into a mechanically stirred reactor. Add an appropriate amount of nitrogen to the reactor to replace the air inside, then add an appropriate amount of hydrogen to replace it, and then replace the air inside the reactor with hydrogen before introducing H2 at 22 MPa. React at 230℃ for 4 h.
[0023] 3) Remove the reaction vessel and place it in an ice bath to cool rapidly to room temperature. Centrifuge the reaction mixture in the vessel, wash the precipitate with an appropriate amount of tetrahydrofuran until the washing liquid is colorless, combine the washing liquid with the supernatant obtained by centrifugation, remove the solvent by rotary evaporation at 50℃, and then dry thoroughly in a vacuum drying oven at 60℃ to obtain lignin oil. Extract the depolymerization products in the lignin oil with dichloromethane 3-5 times, combine the extracts, centrifuge, take the supernatant, add 200 μL of ethyl vanillin (5 mg / mL) as an internal standard, take 1 mL in a chromatographic vial, and analyze it by gas chromatography-mass spectrometry (GC / MS). The results are shown in Figure 1(b). Example 3 A method for modifying and lysing lignin using a bacterial laccase / mediator system, comprising the following steps: 1) Take an appropriate amount of ground wood lignin and disperse it evenly in a glycine-sodium hydroxide buffer solution with a pH of 10.6. Then add the reaction mediator HBT, and finally add a blank sample with an enzyme activity of 0 U / g and four different laccase enzyme solutions with an enzyme activity of 1000 U / g. Place the mixture in an air shaker and start the enzyme treatment reaction at a constant temperature and speed. The shaking speed of the air shaker is 220 rpm, the temperature is 37℃, and the reaction time is 3 days. Take out the reaction mixture, separate the solid and liquid phases, remove the aqueous phase, wash the precipitate, freeze-dry, and obtain the enzyme-treated lignin. 2) Take 25 mg of enzyme-oxidized lignin sample, 10 mg of NiMoS / P-Al2O3 catalyst, and 20 mL of methanol and put them into a mechanically stirred reactor. Add an appropriate amount of nitrogen to the reactor to replace the air inside, then add an appropriate amount of hydrogen to replace it, and then replace the air inside the reactor with hydrogen before introducing H2 at 22 MPa. React at 230℃ for 4 h.
[0024] 3) Remove the reaction vessel and place it in an ice bath to cool rapidly to room temperature. Centrifuge the reaction mixture in the vessel, wash the precipitate with an appropriate amount of tetrahydrofuran until the washing liquid is colorless, combine the washing liquid with the supernatant obtained by centrifugation, remove the solvent by rotary evaporation at 50℃, and then dry thoroughly in a vacuum drying oven at 60℃ to obtain lignin oil. Extract the depolymerization products in the lignin oil with dichloromethane 3-5 times, combine the extracts, centrifuge, take the supernatant, add 200 μL of ethyl vanillin (5 mg / mL) as an internal standard, take 1 mL in a chromatographic vial, and analyze it by gas chromatography-mass spectrometry (GC / MS). The results are shown in Figure 1(c). Figure 1 shows the lignin depolymerization catalyzed by different laccases under different pH conditions. In Figure 1(a) pH=7, Figure 1(b) pH=9.6, and Figure 1(c) pH=10.6; WT is wild-type laccase, 495 is a site-directed mutant laccase, X33-255 is a random mutant laccase, and X33-WT is a randomly mutated wild-type laccase.
[0025] As shown in Figure 1, when the solution system was at pH 7, the lignin oil yield of all four bacterial laccases was lower than that of the control group, while the monomer yield did not change significantly. This may be because lignin underwent recombination during the enzymatic reaction. With further increases in pH, the lignin oil yield of mutant laccases 495 and X33-255 did not decrease significantly compared to wild-type laccases WT and X33-WT, indicating that the mutant laccases had improved tolerance to strong alkalis.
[0026] All the raw materials listed in this invention, as well as the upper and lower limits and ranges of the raw materials and the upper and lower limits and ranges of the process parameters (such as temperature, time, etc.), can realize this invention. Examples are not listed one by one here.
[0027] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.
Claims
1. A method for modifying and lysing lignin using a bacterial laccase / mediator system, characterized in that, It includes the following steps: S01. Enzymatic modification of groundwood lignin macromolecules by adding bacterial laccase and mediator HBT to obtain enzyme-modified lignin. SO2, using NiMoS / P-Al2O3 as a catalyst, the enzyme-modified lignin, the catalyst and methanol solvent are placed in a high-temperature and high-pressure reactor, and then an appropriate amount of hydrogen is introduced to catalyze the cleavage of lignin. S03. Take out the mixture after the reaction, separate the solid and liquid phases by centrifugation, and obtain the lignin oil product by vacuum distillation and vacuum drying; S04. The lignin oil product was extracted with an appropriate amount of dichloromethane, and after adding ethyl vanillin as an internal standard, the lignin monophenol products were obtained by quantitative analysis by GC-MS.
2. The method for modifying and lysing lignin using a bacterial laccase / mediator system as described in claim 1, characterized in that: In step S01, the bacterial laccase is selected from at least one of wild-type laccase WT, randomly mutated wild-type laccase X33-WT, site-directed mutant laccase 495, and randomly mutated laccase X33-255; the mediator is the mediator HBT.
3. The method for modifying and lysing lignin using a bacterial laccase / mediator system as described in claim 1 or 2, characterized in that: Specifically, step S01 involves mixing groundwood lignin and glycine-sodium hydroxide buffer solution evenly, then adding reaction mediator HBT, then adding enzyme solution containing bacterial laccase, and placing the mixture in an air shaker at a constant temperature and speed to begin the enzyme treatment reaction. After the reaction was completed, the reaction mixture was removed, the solid and liquid phases were separated by centrifugation, the aqueous phase was removed, the precipitate was washed, and the mixture was freeze-dried to obtain enzyme-modified lignin. The final concentration of the groundwood lignin is 0.01–0.1 mg / mL; the final concentration of the glycine-sodium hydroxide buffer is 10–100 mM with a pH of 7.0–12.0; the amount of bacterial laccase is 0–2000 U / g substrate; and the final concentration of the reaction mediator HBT is 1–5 mM.
4. The method for modifying and lysing lignin using a bacterial laccase / mediator system as described in claim 3, characterized in that: The shaking speed of the air shaker is 50-500 rpm, the air shaker temperature is 20-50℃, and the reaction time is 1-5 days.
5. The method for modifying and lysing lignin using a bacterial laccase / mediator system as described in claim 1, characterized in that, In step S02, the NiMoS / P-Al2O3 catalyst is prepared by a two-step initial wet impregnation method, specifically as follows: Step 1 Impregnation: Nickel nitrate hexahydrate and diammonium hydrogen phosphate are dissolved separately and added to the pseudoboehmite, followed by high-temperature roasting. The second step involves impregnation, in which the dissolved ammonium tetrathiomolybdate is loaded a second time and calcined twice at high temperatures under nitrogen and hydrogen to obtain the NiMoS / P-Al2O3 catalyst.
6. The method for modifying and lysing lignin using a bacterial laccase / mediator system as described in claim 5, characterized in that: The atomic molar ratio of the impregnated molybdenum, nickel and phosphorus is 7:3:0 to 4.
7. The method for modifying and lysing lignin using a bacterial laccase / mediator system as described in claim 5, characterized in that: In the first impregnation step, the muffle furnace calcination temperature is 300-800℃ for 2-5 hours; in the second impregnation step, calcination is carried out in a tube furnace: calcination at 200-500℃ for 2-5 hours in a nitrogen atmosphere; and calcination at 300-800℃ for 2-5 hours in a hydrogen atmosphere.
8. The method for modifying and lysing lignin using a bacterial laccase / mediator system as described in claim 1, characterized in that: In step S02, the catalytic cracking temperature is 100℃~250℃, the time is 2h~10h, and the hydrogen pressure is 1~5MPa.
9. The method for modifying and lysing lignin using a bacterial laccase / mediator system as described in claim 1, characterized in that: The amount of NiMoS / P-Al2O3 catalyst used is 10-50% of the enzyme-modified lignin, and the amount of solvent used is 0.5-2 times the amount of enzyme-modified lignin.
10. The method for modifying and lysing lignin using a bacterial laccase / mediator system as described in claim 1, characterized in that: In step S03, the centrifugation time is 5-15 min, the rotation speed is 3000-10000 rpm; the temperature of vacuum distillation is 30-80℃; the temperature of vacuum drying oven is 40-80℃; in step S04, the amount of dichloromethane required for extracting lignin oil is 1-5 ml; the mass of the added ethyl vanillin internal standard is 0.5-2.5 mg.