A method for assisted degradation of lignin using visible light

Abstract

The application provides a method for degrading lignin with visible light assistance, and the method comprises the following steps: degrading lignin in herbaceous plant straw with manganese peroxidase under visible light conditions.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, this invention relates to a method for degrading lignin using visible light-assisted degradation. Background Technology

[0002] Lignin is the world's second largest biomass energy source, second only to cellulose in abundance. It plays a crucial role not only in plant growth and development but also holds great potential for human biomass energy utilization. The presence of lignin in the straw of food crops such as corn, wheat, and rice makes them difficult to degrade. Overall, from 1981 to 2020, the amount of crop straw resources in my country showed an increasing trend, especially in the last 15 years, where the growth rate was significantly higher than in the previous 20 years. This means that finding green, environmentally friendly, and efficient methods for degrading lignin is particularly important.

[0003] Lignin is a substance found in plant fiber raw materials, playing a binder role in the papermaking process. Without removing lignin, the fiber raw material cannot disperse into individual fibers, affecting paper formation. Currently, lignin removal is primarily achieved through chemical methods. To remove lignin, large quantities of chemicals such as sodium hydroxide, sodium sulfide, and sodium sulfite are used during the wood chip cooking process to obtain cellulose-based pulp, which is then further processed. This deligninization process is a common method in the papermaking industry, but it generates a large amount of chemical waste, severely impacting the natural environment.

[0004] Therefore, there is an urgent need in this field to explore a low-cost, green and environmentally friendly method for degrading lignin. Summary of the Invention

[0005] The purpose of this invention is to provide a method for degrading lignin using visible light.

[0006] A first aspect of the present invention provides a method for lignin degradation using visible light-assisted degradation, the method comprising: degrading lignin in herbaceous plant straw using manganese peroxidase under visible light conditions.

[0007] In one or more embodiments, the method includes: adding a solution containing manganese peroxidase to an aqueous solution of herbaceous plant straw under visible light conditions, thereby utilizing manganese peroxidase to degrade lignin in the herbaceous plant straw.

[0008] In one or more embodiments, in the method, under visible light conditions, herbaceous plant straw can be photocatalyzed to produce H2O2, and the generated H2O2 catalyzes manganese peroxidase to degrade lignin in the herbaceous plant straw.

[0009] In one or more embodiments, the method does not require the addition of H2O2 or substances containing H2O2.

[0010] In one or more embodiments, the reaction temperature of the method is 20–45°C, more preferably 20–40°C, 22–38°C, 26–35°C or 28–32°C.

[0011] In one or more embodiments, the reaction time of the method is more than 3 days, more preferably 4 days, 5 days or 6 days.

[0012] In one or more embodiments, the herbaceous plant straw includes reeds, corn stalks, wheat straw, rice straw, sorghum stalks, cotton stalks, rapeseed stalks, and miscanthus.

[0013] In one or more embodiments, the herbaceous plant straw is the cell wall residue of destarched herbaceous plant straw.

[0014] In one or more embodiments, the destarched herbaceous plant straw cell wall residue is obtained by treating herbaceous plant straw with enzymes or organic solvents.

[0015] In one or more embodiments, the enzymes include α-amylase and amylopectin; and / or the organic solvents include acetone, chloroform, methanol, and DMSO.

[0016] In one or more embodiments, the organic solvent is added two, more than two, or more than three times.

[0017] In one or more embodiments, the herbaceous plant straw is first dissolved in ethanol, centrifuged, and then a solution containing chloroform and methanol is added at a ratio of chloroform:methanol = 1:1 and reacted for a period of time. After centrifugation, acetone is added to dissolve the residual organic solvent. The acetone is then removed by evaporation, centrifugation, etc., and then mixed with 90% DMSO. After removing the DMSO, the straw powder is washed again with 70% ethanol and acetone to obtain destarched herbaceous plant straw cell wall residue powder.

[0018] In one or more embodiments, the aqueous solution of the herbaceous plant straw is an aqueous solution of destarched herbaceous plant straw cell wall residue.

[0019] In one or more embodiments, the concentration of the aqueous solution of destarched herbaceous plant straw cell wall residue is 0.1–100 mg / mL, more preferably 1–50 mg / mL, 1–30 mg / mL, 1–20 mg / mL, 1–10 mg / mL or 1–5 mg / mL.

[0020] In one or more embodiments, the solution containing manganese peroxidase further contains manganese sulfate solution and / or malonic acid / sodium malonate buffer; preferably, the solution containing manganese peroxidase comprises: manganese peroxidase, manganese sulfate and malonic acid / sodium malonate buffer.

[0021] In one or more embodiments, the concentration of manganese peroxidase is 0.01–1 U / mL, more preferably 0.01–0.8 U / mL, 0.01–0.6 U / mL, 0.01–0.5 U / mL, 0.01–0.1 U / mL or 0.05–0.1 U / mL.

[0022] In one or more embodiments, the concentrations of malonic acid and sodium malonate are 10–500 mM, more preferably 20–400 mM, 30–300 mM, 50–200 mM or 100–150 mM.

[0023] In one or more embodiments, the concentration of manganese sulfate is 0.1–50 mM, more preferably 0.8–45 mM, 0.5–40 mM, 1–30 mM, 1–20 mM or 1–10 mM.

[0024] In one or more embodiments, the manganese peroxidase is selected from the group consisting of:

[0025] (i) A protein having the amino acid sequence shown in SEQ ID NO:1;

[0026] (ii) A protein derived from (i) having the lignin-degrading function, formed by substituting, deleting, or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:1; or

[0027] (iii) A protein with ≥90% homology to the amino acid sequence shown in SEQ ID NO:1, and having the lignin-degrading function.

[0028] In one or more embodiments, the wavelength range of the visible light is 400-700 nm.

[0029] In one or more embodiments, the method further includes: determining the lignin content; preferably, the method for determining the lignin content includes: acetyl bromide method, sulfuric acid method, acid washing fiber method, acetyl bromide method, and ultraviolet spectrophotometry.

[0030] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0031] Figure 1Schematic diagram of the reaction mechanism of 10-acetyl-3,7-dihydroxyphenazine with hydrogen peroxide and horseradish peroxidase (HRP).

[0032] Figure 2 Kinetic detection results of H2O2 production from corn stalk powder by photocatalysis. The concentration of H2O2 was detected by 10-acetyl-3,7-dihydroxyphenazine. H2O2 formation rate = H2O2 concentration (μM) / weight of corn stalk powder (mg).

[0033] Figure 3 A schematic diagram illustrating the principle of lignin peroxidase degradation of lignin.

[0034] Figure 4 A schematic diagram illustrating the reaction principle of H2O2-catalyzed manganese peroxidase degradation of lignin in herbaceous plant straw. The components include RH—phenol, organic acids, nitro aromatic compounds, chlorinated aromatic compounds, and methoxy aromatic compounds; R·—RH-formed free radicals; and [P+]—porphyrin P- free radicals. Detailed Implementation

[0035] Through in-depth research, the inventors discovered that visible light can catalyze the production of H2O2 from corn stalk powder. This H2O2 then assists manganese peroxidase in degrading lignin without the need for additional H2O2. By cleverly utilizing lignin to catalyze the production of H2O2, and further utilizing H2O2 to assist manganese peroxidase in degrading lignin, the problem of environmental pollution caused by the addition of photocatalysts in the catalytic degradation of lignin is solved. This not only saves raw materials but is also green and environmentally friendly.

[0036] Methods for lignin degradation assisted by visible light

[0037] This invention provides a method for degrading lignin using visible light-assisted degradation, the method comprising: degrading lignin in herbaceous plant straw using manganese peroxidase under visible light conditions.

[0038] In this invention, the wavelength range of the visible light can be 400-700nm.

[0039] In this invention, the herbaceous plant straw includes reeds, corn stalks, wheat straw, rice straw, sorghum stalks, cotton stalks, rapeseed stalks, and miscanthus. Herbaceous plant straw typically contains lignin, starch, cellulose, and hemicellulose, with lignin primarily located in the cell walls. The lignin in herbaceous plant straw typically has the following structure:

[0040]

[0041] The lignin structure in herbaceous plant straw is significantly different from that of industrial lignin sulfate lignin (CAS No.: 8068-05-01). Industrial lignin has many complex chemical bonds added during industrial synthesis, such as carbon-carbon bond polymerization under acidic conditions that does not exist in natural lignin, and the addition of SH and other structures that are not present in the lignin of herbaceous plant straw.

[0042] In this invention, the herbaceous plant stalks can be cell wall residues of herbaceous plant stalks. The terms "cell wall residues of herbaceous plant stalks" and "cell wall residues of destarched herbaceous plant stalks" are used interchangeably, generally referring to residues containing only plant cell walls, such as lignin, cellulose, and hemicellulose, after removing starch and soluble small molecules (e.g., phenolic substances, flavonoids) from the herbaceous plant stalks.

[0043] In the method described in this invention, cell wall residue powder from herbaceous plant straw can be dissolved in water, and a solution containing manganese peroxidase can be added under visible light conditions to degrade lignin in the herbaceous plant straw. Specifically, the concentration of the aqueous solution of cell wall residue from herbaceous plant straw can be 0.1–100 mg / mL, preferably 1–50 mg / mL, 1–30 mg / mL, 1–20 mg / mL, 1–10 mg / mL, or 1–5 mg / mL, for example, 2 mg / mL.

[0044] In the method described in this invention, acetone, chloroform, and methanol can be used to remove soluble small molecules from herbaceous plant straw, and DMSO can be used to remove starch from the herbaceous plant straw. An exemplary method includes: first, dissolving the herbaceous plant straw in ethanol, centrifuging, and then adding a solution containing chloroform and methanol at a chloroform:methanol ratio of 1:1 and reacting for a period of time (e.g., 1 hour). After centrifugation, acetone is added, and the acetone is removed by evaporation, centrifugation, etc. The starch is further dissolved using dimethyl sulfoxide (DMSO) or destarched using α-amylase and amylopectin. Organic solvents (acetone, chloroform, methanol, DMSO, etc.) can be added multiple times (e.g., 2 times, more than 2 times, 3 times or more) to enhance the removal effect of soluble small molecules and starch from the herbaceous plant straw.

[0045] In this invention, the term "manganese peroxidase" or "MnP" refers to a protein or mutant thereof with the sequence shown in SEQ ID NO:1 that has manganese peroxidase activity. The mutant refers to a protein that has mutated corresponding to the wild-type manganese peroxidase protein. Specifically, the mutation includes (but is not limited to): deletion, insertion, and / or substitution of several amino acids (e.g., 1, 2, 3). Any protein with high homology (e.g., 80% or higher; preferably 90% or higher, more preferably 95%, 98%, 99% or higher) to the MnP specifically referenced in this invention and having the same function as said MnP (e.g., the function of degrading lignin) is also included in this invention. Generally, the mutant also has the same or similar enzymatic activity as wild-type manganese peroxidase, for example, enzymatic activity for degrading lignin. In this invention, the manganese peroxidase includes manganese peroxidase isolated from organisms (e.g., but not limited to white-rot fungi), as well as artificially synthesized or commercially available manganese peroxidase.

[0046] In this invention, the solution containing manganese peroxidase may further include manganese sulfate solution and / or malonic acid / sodium malonate buffer. Exemplarily, the solution containing manganese peroxidase includes: manganese peroxidase, manganese sulfate, and malonic acid / sodium malonate buffer. In this solution, the concentration of manganese peroxidase can be 0.01–1 U / mL, for example, 0.01–0.8 U / mL, 0.01–0.6 U / mL, 0.01–0.5 U / mL, 0.01–0.1 U / mL, or 0.05–0.1 U / mL. In this reaction system, the concentrations of malonic acid and sodium malonate can be 10–500 mM, for example, 20–400 mM, 30–300 mM, 50–200 mM, or 100–150 mM. The concentration of manganese sulfate in the solution can be 0.1–50 mM, for example 0.8–45 mM, 0.5–40 mM, 1–30 mM, 1–20 mM or 1–10 mM.

[0047] Typically, the suitable temperature for manganese peroxidase to degrade lignin is 20–45°C, preferably 20–40°C, 22–38°C, 26–35°C, or 28–32°C, such as 30°C. The reaction time for manganese peroxidase to degrade lignin can be more than 3 days, such as 4 days, 5 days, 6 days, or longer.

[0048] After the lignin degradation reaction is completed, the lignin content can be determined using methods such as the acetyl bromide method (Klason method), acid detergent fiber (ADF) method, acetyl bromide (AB) method, and ultraviolet spectrophotometry (UV) to analyze the lignin degradation status. These methods are all within the scope of protection of this invention.

[0049] This invention is the first to discover that, in the method described, under visible light conditions, herbaceous plant straw can be photocatalyzed to produce H2O2, which in turn catalyzes manganese peroxidase to degrade lignin in the herbaceous plant straw. Therefore, no additional H2O2 or substances containing H2O2 needs to be added during this process.

[0050] Specifically, under visible light conditions, herbaceous plant straw can be photocatalyzed to produce H2O2. The mechanism includes: herbaceous plant straw forming H2O2 through O2 reduction and H2O oxidation under visible light. The generated H2O2 can be detected using methods such as titration, spectrophotometry, and enzyme sensors. For example, using hydrogen peroxide and 10-acetyl-3,7-dihydroxyphenazine as reactants, in the presence of horseradish peroxidase (HRP), resorufin is generated. Since resorufin produces strong red fluorescence, the absorbance value of resorufin can be used to quantitatively reflect the generation of H2O2.

[0051] The mechanism by which the generated H2O2 catalyzes the degradation of lignin in herbaceous plant straw by manganese peroxidase includes: the oxygen atom in the H2O2 molecule has strong electronegativity; the two electrons on the heme iron in the MnP molecule first migrate to the oxygen-oxygen bond, and MnP is oxidized to a reactive oxygen-rich complex of tetravalent iron porphyrin (MnP compound 1). Then, Mn... 2+ Losing an electron, MnP compound 1 is reduced to MnP compound 2, simultaneously opening the lignin benzene ring and completing the degradation of lignin. Figure 4 ).

[0052] The advantages of this invention include:

[0053] This invention provides a new low-cost, green and environmentally friendly method for degrading lignin in corn stalks. Under visible light conditions, manganese peroxidase is used to degrade lignin in herbaceous plant stalks without the need for additional H2O2, which not only saves raw materials but is also green and environmentally friendly.

[0054] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0055] Experimental materials

[0056] Manganese peroxidase (catalog number: 803057-10MG), manufacturer: Sigma-Aldrich;

[0057] Manganese sulfate (product number: 10013418), manufacturer: Sinopharm;

[0058] Malonic acid (product number: 23281E), manufacturer: Adamas-beta;

[0059] Sodium malonate (product number: MB2622_25G), manufacturer: meilunbio;

[0060] 10-Acetyl-3,7-Dihydroxyphenazine (Catalog No.: ST010-25mg), Manufacturer: Beyotime Biotechnology Co., Ltd.

[0061] Horseradish peroxidase (product number: MB6017_25MG / unit), manufacturer: meilunbio;

[0062] Hydrogen peroxide standard (item number: 10011218), manufacturer: Sinopharm Group;

[0063] Lignin peroxidase (product number: 42613-30-9), manufacturer: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0064] Dosage is shown in Tables 1, 2, and 3 below:

[0065] Table 1.10-Acetyl-3,7-dihydroxyphenazine premixed solution system for H2O2 detection

[0066]

[0067] Table 2. Reaction system for manganese peroxidase degradation of corn straw

[0068]

[0069] Table 3. Reaction system for lignin peroxidase degradation of corn straw

[0070]

[0071]

[0072] Experimental methods

[0073] 1. Preparation of corn stalk cell wall residue (AIR)

[0074] 1) Dry the corn stalk sample in an oven at 40℃, grind it into fine powder using a ball mill, and pass it through a 60-mesh sieve;

[0075] 2) Add 50 mg of sample to a 2 mL screw-cap tube, add 1.5 mL of 70% ethanol, treat at 65℃ for 1 h, centrifuge at 10,000 rpm for 10 min, discard the supernatant, repeat 3 times; add 1.5 mL of chloroform / methanol (1:1) solution, suspend the precipitate and shake for 1 h. Centrifuge at 10,000 rpm for 10 min, discard the supernatant, repeat 3 times;

[0076] 3) Add 1.5 mL of acetone, vortex to suspend, centrifuge at 10,000 rpm for 10 min, and remove the supernatant. Evaporate to dryness at 45°C on a hot plate. Add 1.5 mL of 90% DMSO to the precipitate for starch removal, vortex, and shake overnight at at least 50 rpm on a rotary mixer. The next day, centrifuge to remove the supernatant, add another 1.5 mL of 90% DMSO, vortex, centrifuge, and remove the supernatant.

[0077] 4) Add 1 mL of 70% ethanol and wash 6 times, then vortex and centrifuge to remove the supernatant;

[0078] 5) Add 1 mL of acetone, centrifuge at 10,000 rpm for 10 min, discard the supernatant, and repeat 3 times. The last time, evaporate to dryness on a hot plate at 45°C. The remaining material is the destarched corn stalk cell wall residue (AIR).

[0079] 2. Detection of H2O2 generated from photocatalytic corn stalk powder

[0080] 1) Weigh 2 mg of destarched corn stalk cell wall residue (AIR) powder and dissolve it in 1 mL of H2O. Catalyze the solution under visible light (400-700 nm wavelength) for 30, 60, 90, 120, 150, and 180 min, respectively. The control group was protected from light by aluminum foil. Detect the H2O2 content.

[0081] 2) 10-Acetyl-3,7-dihydroxyphenazine is a fluorescent probe highly sensitive to hydrogen peroxide and peroxidase. In the presence of peroxidases such as horseradish peroxidase (HRP) and myeloperoxidase (MPO), 10-acetyl-3,7-dihydroxyphenazine reacts with H2O2 in a 1:1 ratio to produce a strong red fluorescent substance, resorufin. The reaction principle is described in [link to reaction protocol]. Figure 1The maximum excitation wavelength of halogen is 571 nm, the maximum emission wavelength is 585 nm, and it has strong visible light absorption at the excitation wavelength, so the absorbance can be detected by A570.

[0082] 3) Preparation of premixed solution: 50 μL of 10-acetyl-3,7-dihydroxyphenazine (9.719 mM); 100 μL of horseradish peroxidase (10 U / mL); 4.85 mL of H2O, as shown in Table 1;

[0083] 4) Standard setup: H2O2 concentration: 0, 1, 2, 3, 4, 5 μM, dissolved in H2O. Using known concentrations of H2O2 standard, add 100 μL each of the premixed solution to a 96-well black plate. After incubating at 37℃ for 20 min, detect the fluorescence values ​​at excitation and emission wavelengths of 530 nm and 590 nm, respectively, to plot the standard curve.

[0084] 5) Take 100 μL each of the test solution and the premixed solution into a 96-well black plate, incubate at 37℃ for 20 min, and then detect the fluorescence values ​​at excitation and emission wavelengths of 530 nm and 590 nm, respectively. Obtain the concentration of H2O2 in the test solution according to the standard curve.

[0085] 3. Photocatalytic degradation of lignin by manganese peroxidase assisted by corn stalk powder

[0086] 1) Weigh 10 mg of destarched corn stalk cell wall residue (AIR) powder into a 2 mL screw cap tube and perform 3 biological replicates. Add 965 μL of malonic acid / sodium malonic acid buffer (100 mL, pH=4.5), 10 μL of manganese sulfate (final concentration 1 mM), and 25 μL of manganese peroxidase solution (final concentration 0.05 U / mL) to each of the three replicates. The reaction system is shown in Table 2.

[0087] 2) Catalyze at 30℃ under visible light for 4 days, terminate the reaction at 100℃, centrifuge at 10000rpm for 10min and discard the supernatant; add 1mL H2O to wash, repeat the above steps three times; add 1mL acetone to wash, repeat the above steps three times, evaporate to dryness on a hot table at 45℃ and then detect the lignin content.

[0088] 3) Weigh approximately 2 mg of manganese peroxidase-treated powder directly into a 2 mL EP tube, leaving one empty tube as a blank control (subsequent operations are the same as for the sample tube), rinse the tube wall with 250 μL of acetone, collect all the powder at the bottom of the tube, and evaporate the acetone to dryness.

[0089] 4) Slowly add 100 μL of freshly prepared acetyl bromide solution (25%, acetyl bromide is soluble in glacial acetic acid) along the tube wall. React at 50°C for 2 hours. Continue the reaction for another hour, vortexing once every 15 minutes. Cool to room temperature on ice, add 400 μL of NaOH (2M) and 70 μL of freshly prepared 0.5M hydroxylamine hydrochloride, and vortex to mix.

[0090] 5) Add glacial acetic acid to each tube precisely to 2 mL, invert several times to mix, and centrifuge at 10,000 rpm for 2 min;

[0091] 6) Pipette 200 μL of the solution prepared in step 5) into a UV-specific 96-well plate (Costar 3635UV-plate, www.corning.com), and read the ABS value at 280 nm using a microplate reader. 280nm Each sample was measured in triplicate. The %ABSL (acetyl bromide soluble lignin) content was calculated using the following formula:

[0092]

[0093] Wherein, coefficient (Coeff): 17.747. 0.539 cm is the height of 200 μL of solution, i.e., the UV path length; this value is not fixed and varies slightly between different 96-well plate models. Weight is the precise value of 2 mg corn stalk (AIR). %ABSL multiplied by 10 is the lignin concentration value (μg / mg AIR).

[0094] Example 1: Photocatalytic generation of hydrogen peroxide from corn stalk powder

[0095] Corn stalks were used as plant samples, dried in an oven at 40℃, ground into a fine powder using a ball mill, and passed through a 60-mesh sieve. 50 mg of the sample was added to a 2 mL screw-cap tube, followed by 1.5 mL of 70% ethanol. The mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was discarded. This process was repeated three times. Then, 1.5 mL of chloroform / methanol (1:1) solution was added to suspend the precipitate, and the mixture was shaken for 1 h. The mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was discarded. Finally, 1.5 mL of acetone was added to vortex the precipitate, and the mixture was centrifuged at 10,000 rpm for 10 min to remove the supernatant. The precipitate was evaporated to dryness at 45℃ on a hot plate. 1.5 mL of 90% DMSO was added to the precipitate to remove starch, and the mixture was vortexed and shaken overnight at at least 50 rpm on a rotary mixer. The next day, the supernatant was removed by centrifugation, and 1.5 mL of 90% DMSO was added. The mixture was then vortexed and centrifuged again to remove the supernatant. The mixture was washed six times with 1 mL of 70% ethanol, vortexed, and the supernatant was removed. 1 mL of acetone was added, and the mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was discarded, and this process was repeated three times. The mixture was then evaporated to dryness at 45°C on a hot plate. The remaining material was powdered remnants of the destarched corn stalk cell walls.

[0096] Weigh 2 mg of destarched corn stalk cell wall residue powder and dissolve it in 1 mL of H2O. Catalyze the solution under visible light (400-700 nm wavelength) for 30, 60, 90, 120, 150, and 180 min, respectively. The control group was protected from light by aluminum foil. The H2O2 content was then measured. 10-Acetyl-3,7-dihydroxyphenazine is a fluorescent probe highly sensitive to hydrogen peroxide and peroxidase. In the presence of horseradish peroxidase (HRP), 10-acetyl-3,7-dihydroxyphenazine reacts with H2O2 in a 1:1 ratio to produce a strong red fluorescent substance, resorufin. The reaction principle is described in [link to reaction description]. Figure 1 The maximum excitation wavelength of the test halogen is 571 nm, and the maximum emission wavelength is 585 nm. It also exhibits strong visible light absorption at the excitation wavelength, and its absorbance can be detected using an A570 microscope. The premixed solution is prepared as follows: 50 μL of 10-acetyl-3,7-dihydroxyphenazine (9.719 mM); 100 μL of horseradish peroxidase (10 U / mL); and 4.85 mL of H2O (see Table 1). Standards are prepared by dissolving H2O at concentrations of 0, 1, 2, 3, 4, and 5 μM in H2O. 100 μL each of the test solution and the premixed solution are added to a 96-well black plate. After incubating at 37°C for 20 min, the fluorescence values ​​at excitation and emission wavelengths of 530 nm and 590 nm, respectively, are measured.

[0097] H2O2 detection results are as follows Figure 2 As shown, the H2O2 content also shows an increasing trend with the increase of photocatalytic time.

[0098] Example 2: Reaction conditions for manganese peroxidase degradation of corn straw

[0099] 10 mg of destarched corn stalk cell wall residue powder was placed in a 2 mL screw-cap tube for three biological replicates. 965 μL of malonic acid / sodium malonate buffer (100 mL, pH 4.5), 10 μL of manganese sulfate (final concentration 1 mM), and 25 μL of manganese peroxidase solution (final concentration 0.05 U / mL) were added to each replicate. The reaction system is shown in Table 2. Catalysis was performed at 30℃ under visible light for 4 days.

[0100] Example 3: Reaction conditions for lignin peroxidase degradation of corn straw

[0101] 10 mg of destarched corn stalk cell wall residue powder was placed in a 2 mL screw-cap tube for three biological replicates. 975 μL of malonic acid / sodium malonate buffer (100 mL, pH 4.5) and 25 μL of lignin peroxidase solution (final concentration 0.025 U / mL) were added to each replicate. The reaction system is shown in Table 3. Catalysis was performed at 30℃ under visible light for 4 days.

[0102] Example 4: Detection of lignin content in corn stalks before and after degradation by manganese peroxidase and lignin peroxidase.

[0103] The corn stalks were degraded using manganese peroxidase according to the method of Example 2, or using lignin peroxidase according to the method of Example 3.

[0104] After the degradation of corn stalks was completed, the reaction was terminated by reacting at 100℃ for 10 min, centrifuging at 10000 rpm for 10 min, and discarding the supernatant. 1 mL of H2O was added for washing, and the above steps were repeated three times. 1 mL of acetone was added for washing, and the above steps were repeated three times. The mixture was then evaporated to dryness on a hot plate at 45℃ for lignin content detection. Approximately 2 mg of corn stalk powder treated with manganese peroxidase or lignin peroxidase was weighed and placed in a 2 mL centrifuge tube, leaving one empty tube as a blank control (subsequent operations were the same as for the sample tube). The tube wall was rinsed with 250 μL of acetone, and all the powder was collected at the bottom of the tube. The acetone was then evaporated to dryness. 100 μL of freshly prepared acetyl bromide solution (25%, acetyl bromide dissolved in glacial acetic acid) was slowly added along the tube wall. The reaction was carried out at 50℃ for 2 h. The reaction was continued for another h, with vortexing every 15 min during this period. Cool to room temperature on ice, add 400 μL of 2M NaOH and 70 μL of freshly prepared 0.5M hydroxylamine hydrochloride, and vortex to mix. Make up to 2 mL in each tube with glacial acetic acid, and invert several times to mix. Centrifuge at 10,000 rpm for 2 min; pipette 200 μL of the solution into a UV-specific 96-well plate (Costar 3635UV-plate, www.corning.com), and read the ABS value at 280 nm using a microplate reader. Perform three replicates for each sample, and use one-way ANOVA for statistical analysis. Calculate the %ABSL (acetyl bromide soluble lignin) content according to the following formula:

[0105]

[0106] The coefficient is 17.747. 0.539 cm is the height of a 200 μL solution, i.e., the UV path length; this value is not fixed and varies slightly depending on the type of 96-well plate. Weight is 2 mg, the precise value of the destarched corn stalk powder. %ABSL multiplied by 10 gives the concentration value (μg / mg destarched corn stalk powder).

[0107] Table 4 shows a comparison of the effects of manganese peroxidase and lignin peroxidase on the degradation of corn stalks. The results show that manganese peroxidase is more efficient at degrading lignin than lignin peroxidase.

[0108] Table 4. Comparison of the degradation effects of manganese peroxidase and lignin peroxidase on corn straw.

[0109]

[0110] Data are expressed as mean ± standard error of mean. The Student's t-test was used to analyze the data. ns indicates no difference, and **p<0.05 indicates a significant difference.

[0111] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

[0112] sequence

[0113] Protein sequence of manganese peroxidase derived from white-rot fungus (Phanerochaete chrysosporium) (SEQ ID NO:1):

[0114] MAVCPDGTRVSHAACCAFIPLAQDLQETIFQNECGEDAHEVIRLTFHDAIAISRSQGPKAGGGADGSMLLFPTVEPNFSANNGIDDSVNNLIPFMQKHNTISAADLVQFAGAVALSNCPGAPRLEFLAGRPNKTIAAVDGLIPEPQDSVTKILQRFEDAGGFTPFEVVSLLASHSVARA DKVDQTIDAAPFDSTPFTFDTQVFLEVLLKGVGFPGSANNTGEVASPLPLGSGSDTGEMRLQSDFALAHDPRTACIWQGFVNEQAFMAASFRAAMSKLAVLGHNRNSLIDCSDVVPVPKPATGQPAMFPASTGPQDLELSCPSERFPTLTTQPGASQSLIAHCPDGSMSCPGVQFNGPA.

Claims

1. A method for lignin degradation assisted by visible light, characterized in that, The method includes: degrading lignin in herbaceous plant straw using manganese peroxidase under visible light conditions.

2. The method as described in claim 1, characterized in that, The method includes: adding a solution containing manganese peroxidase to an aqueous solution of herbaceous plant straw under visible light conditions, and using manganese peroxidase to degrade lignin in the herbaceous plant straw; Preferably, in the method, under visible light conditions, herbaceous plant straw can be photocatalyzed to produce H2O2, and the generated H2O2 catalyzes manganese peroxidase to degrade lignin in herbaceous plant straw; More preferably, the method does not require the addition of H2O2 or substances containing H2O2.

3. The method as described in claim 2, characterized in that, The reaction temperature of the method is 20–45°C, more preferably 20–40°C, 22–38°C, 26–35°C, or 28–32°C; and / or, The reaction time of the method is more than 3 days, more preferably 4 days, 5 days or 6 days.

4. The method according to any one of claims 1-3, characterized in that, The herbaceous plant straws include reeds, corn stalks, wheat straw, rice straw, sorghum stalks, cotton stalks, rapeseed stalks, and miscanthus.

5. The method as described in claim 4, characterized in that, The herbaceous plant straw is the cell wall residue of destarched herbaceous plant straw; Preferably, the destarched herbaceous plant straw cell wall residue is obtained by treating herbaceous plant straw with enzymes or organic solvents; More preferably, the enzymes include α-amylase and amylopectin; and / or, the organic solvents include acetone, chloroform, methanol, and DMSO.

6. The method according to any one of claims 2-5, characterized in that, The aqueous solution of the herbaceous plant straw is an aqueous solution of the cell wall residue of destarched herbaceous plant straw; Preferably, the concentration of the aqueous solution of destarched herbaceous plant straw cell wall residue is 0.1–100 mg / mL, more preferably 1–50 mg / mL, 1–30 mg / mL, 1–20 mg / mL, 1–10 mg / mL or 1–5 mg / mL.

7. The method according to any one of claims 2-6, characterized in that, The solution containing manganese peroxidase also contains manganese sulfate solution and / or malonic acid / sodium malonate buffer; preferably, the solution containing manganese peroxidase comprises: manganese peroxidase, manganese sulfate and malonic acid / sodium malonate buffer.

8. The method as described in claim 7, characterized in that, The concentration of manganese peroxidase is 0.01–1 U / mL, more preferably 0.01–0.8 U / mL, 0.01–0.6 U / mL, 0.01–0.5 U / mL, 0.01–0.1 U / mL or 0.05–0.1 U / mL; The concentrations of malonic acid and sodium malonate are 10–500 mM, more preferably 20–400 mM, 30–300 mM, 50–200 mM or 100–150 mM; The concentration of manganese sulfate is 0.1–50 mM, more preferably 0.8–45 mM, 0.5–40 mM, 1–30 mM, 1–20 mM or 1–10 mM.

9. The method according to any one of claims 1-8, characterized in that, The wavelength range of the visible light is 400-700nm.

10. The method according to any one of claims 1-9, characterized in that, The method further includes: determining the lignin content; preferably, the method for determining the lignin content includes: acetyl bromide method, sulfuric acid method, acid washing fiber method, acetyl bromide method, and ultraviolet spectrophotometry.

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