Dye-decolorizing peroxidase mutants and uses thereof

Abstract

The application discloses a dye-decolorizing peroxidase mutant and application thereof, and a novel dye-decolorizing peroxidase GA1DyPB screened from Rhodococcus globerulus GA1 is used as a starting enzyme, computer-aided semi-rational design enzyme engineering is used for site-directed modification and saturation mutation modification, and comparative analysis is conducted on the enzymatic properties of the mutant and the original enzyme, so as to provide a theoretical basis and technical support for subsequent directional mutation and catalytic performance improvement of the dye-decolorizing peroxidase. Finally, lignin is used as a substrate to conduct long-time lignin enzymatic depolymerization reaction for 48 hours, and the ability of the mutant in the lignin depolymerization is verified.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a dye decolorizing peroxidase mutant and its application in lignin depolymerization. Background Technology

[0002] Dye decolorizing peroxidases (DyPs) belong to one of the six families of peroxidases. They were named after their unique ability to decolorize azo and anthraquinone dyes. The discovery of certain DyPs in lignin-degrading fungi found in specific forest ecosystems has inspired research into their potential to oxidize phenolic and non-phenolic aromatic compounds, including lignin. Studies using lignin model compounds and various DyPs have shown that DyPs can depolymerize lignin by targeting and oxidizing common chemical bonds in lignin. In addition to lignin model compounds, there are reports of DyPs degrading industrial lignin, laying the foundation for downstream applications of lignin depolymerization. Compared to commercially available lignin-degrading enzymes such as laccase, natural DyPs have slightly lower degradation efficiency, but they possess advantages such as broader substrate applicability, stronger thermal stability in some cases, and the ability to initiate oxidation reactions using H₂O₂ as an electron acceptor, making them promising candidates for lignin degradation. However, many natural enzymes exhibit low thermal stability, which is a significant disadvantage in prolonged lignin depolymerization reactions and subsequent fermentation experiments. Besides external optimization of the system and conditions, protein engineering to modify enzymes is currently a reliable technique in enzyme engineering strategies.

[0003] Protein engineering, as a robust technological strategy, aims to construct enzyme variants suitable for novel biotechnological applications. In most applications, rational design and functional modification based on the structure-function relationship of enzymes can effectively improve their core catalytic performance, such as catalytic activity, specificity, and stability. In the field of biochemical engineering, the targeted modification of enzymes through rational design has become a research hotspot and important development direction of common interest to both academia and industry.

[0004] Currently, research on the modification of DyP enzymes has expanded from its single application in dye decolorization to the fields of multifunctional biocatalysis and biorefining. The latest research focuses on heme pocket engineering and substrate-specific modification. Through site-directed mutagenesis of key residues within the heme pocket, the activity spectrum of DyP enzymes has been significantly broadened, endowing them with catalytic capabilities for high-molecular-weight lignin and phenolic compounds, while exhibiting good stability and high efficiency under alkaline conditions. There is an urgent need in this field to develop a highly active and stable dye decolorization peroxidase to achieve efficient lignin degradation and high-value utilization of lignin. Summary of the Invention

[0005] The objective of this invention is to obtain a mutant of a dye decolorizing peroxidase and to use it for the depolymerization of lignin.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] An isolated or purified dye decolorizing peroxidase derived from Rhodococcus biphenylivorans GA1, with the amino acid sequence shown in SEQ ID NO:1, exhibits lignin depolymerization activity.

[0008] A dye decolorizing peroxidase mutant, wherein the mutant is obtained by performing a D210R site-directed mutation on the amino acid sequence of wild-type dye decolorizing peroxidase shown in SEQ ID NO:1; that is, the aspartic acid (D) at position 210 in the amino acid sequence of wild-type dye decolorizing peroxidase is mutated to arginine (R).

[0009] The dye decolorizing peroxidase mutant exhibits higher thermal stability and stronger catalytic ability for lignin depolymerization, enabling it to catalyze at a faster rate.

[0010] The gene encoding the above-mentioned dye decolorization peroxidase mutant has the nucleotide sequence shown in SEQ ID NO:2.

[0011] A vector containing the gene of the dye decolorizing peroxidase mutant described above.

[0012] Preferably, the vector described in this invention is the pET28a plasmid.

[0013] A host cell containing the aforementioned vector.

[0014] Preferably, the host cell of the present invention is Escherichia coli BL21(DE3).

[0015] The method for preparing the dye decolorizing peroxidase mutant includes the following steps:

[0016] (i) Under suitable expression conditions, the above-mentioned host cells are cultured to express the dye decolorizing peroxidase mutant.

[0017] (ii) The expression product is isolated to obtain the dye decolorizing peroxidase mutant.

[0018] Application of the dye decolorizing peroxidase mutant in lignin depolymerization.

[0019] The application described involves adding purified dye decolorizing peroxidase to a reaction system containing lignin.

[0020] The lignin concentration is 100-1000 mg / L, and the dye decolorizing peroxidase concentration is 30-100 mg / L.

[0021] Preferably, a pH 4 disodium hydrogen phosphate-citric acid buffer solution is added to the reaction system to mix the system and bring it up to the appropriate total volume, with the total volume of the reaction system being 50-100 mL;

[0022] In the reaction system, alkali lignin is selected, and the concentration range of alkali lignin is 500±10mg / L;

[0023] The purified protein mass in the reaction system ranged from 3 to 5 mg.

[0024] Hydrogen peroxide solution was added to the reaction system to make the enzyme concentration: H2O2 concentration 20 μg / mL: 1.5 mM, which was used to start the reaction. After 48 hours of reaction, 5-10 mL of 4 M sodium hydroxide solution was added to terminate the reaction and complete the lignin depolymerization process.

[0025] A method for in vitro identification of the activity and enzymatic properties of dye decolorizing peroxidase.

[0026] The present invention uses the ABTS method to determine enzyme activity, comprising: mixing the dye-decolorized peroxidase mutant with ABTS at a final concentration of 5 mM in a disodium hydrogen phosphate-citrate buffer at pH 4.

[0027] A 5M ABTS solution and a 1M H2O2 solution were prepared in advance.

[0028] The reaction was carried out at 37℃. H2O2 was added to initiate the reaction at a ratio of 20 μg / mL protein to 1.5 mM H2O2. After waiting 10 minutes, an equal volume of acetonitrile was added to terminate the reaction. After centrifugation, the change in absorbance at 420 nm was measured using a spectrophotometer. Enzyme activity was calculated based on the change.

[0029] A method for in vitro identification of the activity of dye decolorizing peroxidase in lignin depolymerization.

[0030] The present invention uses a spectrophotometer to determine enzyme activity, comprising: mixing 3-5 mg of the dye decolorizing peroxidase mutant with alkali lignin at a final concentration of 500 mg / L in a pH 4 disodium hydrogen phosphate-citric acid buffer, with a total reaction volume of 50-100 mL.

[0031] Prepare a 50 g / L alkali lignin mother liquor, a 4 M sodium hydroxide solution, and a 1 M H2O2 solution in advance.

[0032] The reaction was carried out at 37℃. A 1M H2O2 solution was added to initiate the reaction at a ratio of 20 μg / mL protein to a final concentration of 1.5 mM H2O2. After waiting 48 hours, an appropriate volume of 4 M sodium hydroxide solution was added to adjust the pH of the reaction solution to 8, simultaneously terminating the reaction. After centrifugation, the absorbance of the reaction solution at 280 nm was measured using a spectrophotometer.

[0033] The amount of lignin consumed is calculated based on the light absorption value.

[0034] Beneficial effects:

[0035] This invention proposes a novel dye decolorizing peroxidase mutant that enhances the efficiency of enzymatic depolymerization of lignin. The mutant dye decolorizing peroxidase exhibits 34.88% higher activity and 38.20% improved thermal stability compared to the original enzyme. The depolymerized lignin can be directly used as a carbon source for the strain, providing a new pathway for the high-value utilization of lignin. Attached Figure Description

[0036] Figure 1 Three-dimensional structure diagram of GA1DyPB protein;

[0037] Figure 2 Three-dimensional structure diagram of guaiacolylglycerol-β-guaiacolylpropyl ether;

[0038] Figure 3 Dynamic simulation results of the complex at 25℃ and 37℃, where A: RMSD results and B: RMSF results;

[0039] Figure 4 FoldX alanine scan results;

[0040] Figure 5 ESM-scan saturation mutation simulation results;

[0041] Figure 6 ESM-scan and FoldX combined score results;

[0042] Figure 7 Preliminary screening of the thermal stability of the crude enzyme solution of the mutant;

[0043] Figure 8 Enzymatic properties determination of mutant pure enzyme solution: A: Enzyme activity determination; B: Residual enzyme activity determination after 1 h at 37℃; C: Tm value determination.

[0044] Figure 9 The concentration of residual lignin in the depolymerized lignin reaction solution;

[0045] Figure 10RMSD of original GA1DyPB and mutant D210R at 25℃(A) and 37℃(B);

[0046] Figure 11 RMSF of original GA1DyPB and mutant D210R at 25℃(A) and 37℃(B);

[0047] Figure 12 Analysis of molecular docking results of the original GA1DyPB;

[0048] Figure 13 Analysis of molecular docking results of mutant D210R. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto. Furthermore, the embodiments described are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0050] The culture medium used in the examples is as follows:

[0051] LB solid medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 20 g / L agar powder, and the remainder is water.

[0052] LB medium: sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L, the remainder being water.

[0053] The preparation method is as follows: Weigh 1 g of sodium chloride, 1 g of peptone, and 0.5 g of yeast powder, and dissolve them in 100 mL of distilled water. Add 2 g of agar powder to the LB solid medium, shake well, and sterilize at 115℃ for 20 min.

[0054] The culture conditions for the strain are as follows:

[0055] Seed culture: Inoculate the -80℃ frozen bacterial culture from the preservation tube into 5 mL of liquid LB medium at an inoculation rate of 1%. The antibiotic is kanamycin sulfate at a final concentration of 50 mg / L. Incubate overnight at 37℃ and 200 r / min.

[0056] Enrichment culture: Inoculate the overnight seed culture into LB flasks for expansion culture at an inoculum volume of 1%. The antibiotic is kanamycin sulfate, with a final concentration of 50 mg / L. Incubate at 37°C and 200 r / min for 2-3 h until OD reaches 50%. 600 It reaches 0.6-0.8.

[0057] Enzyme-induced expression: 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added as an inducer, the induction temperature was about 18℃, and the induction time was about 12 h.

[0058] The purification steps of the dye decolorizing peroxidase in the examples are as follows:

[0059] The crude enzyme solution was purified by removing impurities using a nickel-NTA column, followed by gradient elution with elution buffers containing different concentrations of imidazole. The gradient was achieved by adding 40 ml of 30 mM imidazole, 5 ml of 100 mM imidazole, 15 ml of 300 mM imidazole, and 10 ml of 500 mM imidazole in that order, until the dye-decolorizing peroxidase was completely concentrated in the 500 mM imidazole eluent. The purified eluent was then dialyzed overnight in the same buffer using a 14 kDa dialysis bag to remove imidazole. The purified dye-decolorizing peroxidase was obtained.

[0060] The formulation of the external aid additive in the embodiment is as follows:

[0061] Disodium hydrogen phosphate mother liquor: Weigh 2.84 g ± 0.01 g of anhydrous disodium hydrogen phosphate, add 100 mL of deionized water to make the concentration of the mother liquor 0.2 M, and filter it through an organic phase filter membrane for later use.

[0062] Citric acid mother liquor: Weigh 3.84 g ± 0.01 g of anhydrous citric acid, add 100 mL of deionized water to make the concentration of the mother liquor 0.2 M, and filter it through an organic phase filter membrane for later use.

[0063] pH 4 disodium hydrogen phosphate-citric acid buffer: Mix 6.8 mL of disodium hydrogen phosphate stock solution with 93.2 mL of citric acid stock solution to obtain 100 mL of pH 4 disodium hydrogen phosphate-citric acid buffer solution, and filter it through an organic phase filter membrane for later use.

[0064] ABTS stock solution: Weigh 225±0.01g ABTS, add 100mL of deionized water to make the stock solution concentration 5 M, and filter it through an organic phase filter membrane for later use.

[0065] H2O2 mother liquor: Take 10.21 mL of 30% H2O2 solution, add deionized water, and make up to 100 mL in a 100 mL volumetric flask to make the mother liquor concentration 1 M. Filter through an organic phase filter membrane for later use.

[0066] Sodium hydroxide mother liquor: Weigh 16 g ± 0.01 g of anhydrous sodium hydroxide and add it to a beaker containing about 60–70 mL of deionized water while stirring. After cooling to room temperature, transfer it to a 100 mL volumetric flask and make up to 100 mL to make the concentration of the mother liquor 4 M. Filter it through an organic phase filter membrane for later use.

[0067] Lignin mother liquor: Weigh 5.0±0.01g of lignin, add 100mL of deionized water to make the concentration of mother liquor 50g / L, and filter through an organic phase filter membrane for later use.

[0068] Enzyme activity was determined using the ABTS method, which detects enzyme activity by measuring changes in absorbance at 420 nm. The amount of ABTS required to oxidize 1 μmol per minute was defined as one unit of activity, U. Specific enzyme activity was calculated using the following formula: ,in This indicates the change in absorbance. molar extinction coefficient (M) -1 ×cm -1 Where b is the thickness of the cuvette (cm), t is the reaction time (min), V1 is the volume of the reaction system (mL), V2 is the volume of the crude enzyme solution (mL), and n is the dilution factor. The molar extinction coefficient of ABTS is 𝜀. 420 =36000 M -1 ×cm -1 .

[0069] The enzyme activity assay system is shown in Table 1.

[0070] Table 1

[0071] Composition Volume μL Dye decolorization peroxidase 20 μg 5M ABTS 1 <![CDATA[1M H2O2]]> 1.5 pH 4 disodium hydrogen-citrate buffer Topping up to 1 mL

[0072] Determination of lignin concentration using ultraviolet spectrophotometry. Centrifuge 1 mL of the reaction solution at 12000 rpm for 1 min, wash twice with deionized water to remove sparingly soluble flocculent matter, and measure the OD using a spectrophotometer. 280 The formula for calculating lignin concentration is as follows:

[0073] Lignin (mg / L) =

[0074] Where: A-OD 280 Change value.

[0075] The system for determining the ability to depolymerize lignin is shown in Table 2.

[0076] Table 2

[0077] Composition Volume μL Dye decolorization peroxidase 1000 μg 50 g / L Alkali Lignin Solution 500 <![CDATA[1M H2O2 solution]]> 75 pH 4 disodium hydrogen-citrate buffer Add to 50 mL

[0078] Example 1

[0079] Construction of the original dye decolorizing peroxidase GA1DyPB expression vector and host strain.

[0080] The vector pET-28a(+) is 5369 bp in length, with a T7 promoter, kanamycin sulfate resistance, and a His purification tag. pET-28a(+) was linearized using restriction endonucleases Nde I and Hind III. Successful linearization was confirmed by agarose gel electrophoresis and followed by purification and recovery. Subsequently, using GA1-F and GA1-R primers and the genome of *Rhodococcus biphenyl* GA1 as a template, PCR was performed and the target gene GA1DyPB was recovered via gel electrophoresis. Finally, recombinant pET-28a(+)-GA1Dyp was obtained through one-step cloning. This was then transfected into *E. coli* BL21(DE3) cells, and colony PCR was performed using primers GA1YZ-F and GA1YZ-R for verification. The expected bands confirmed the successful construction of pET-28a(+)-GA1DypB.

[0081] The sequence of GA1DyPB is shown in SEQ ID NO:1.

[0082] The primer sequences used are shown in Table 3.

[0083] Table 3: Primers used in this study

[0084] Primers Genotype GA1-F 5' CCTGGTGCCGCGCGGCAGCCATATGGTT CGTGCACAACCGATACTGAC 3' GA1-R 5' GGTGCTCGAGTGCGGCCGCAAGCTTCAGGGTCGAACTCCTTCGC 3' GA1YZ-F 5' ATGGTTCGTGCACAACCGATAC 3' GA1YZ-R 5' CAGGGTCGAACTCCTTCGC 3'

[0085] Example 2

[0086] Computer-aided semi-rational design for targeted modification of enzyme engineering

[0087] (1) Protein model prediction and ligand model acquisition:

[0088] The GA1DyPB protein sequence was input into AlpHaFold3 (https: / / alphafoldserver.com / login) for homology modeling, and a 3D protein model was exported, as shown below. Figure 1 As shown. Locate the molecular ligand file for guaiacol-β-guaiacol-propyl ether on the PDB website (https: / / pubchem.ncbi.nlm.nih.gov / ) and export the 3D model, as shown. Figure 2 As shown.

[0089] (2) Prediction of active pockets and molecular dynamics simulations:

[0090] The active pocket of GA1DyPB was simulated using D3Pockets (https: / / www.d3pharma.com / D3Pocket / page_upload.php), a software developed by Professors Weiliang Zhu and Zhijian Xu. After locating the active pocket, substrate-protein molecular docking experiments were conducted with the assistance of Autodock Vina software. Following the results of the molecular docking experiments, molecular dynamics simulations of the GA1DyPB-GGE complex were performed at 25℃ and 37℃ using MD simulations. The root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of the complex at different temperatures were obtained, as shown below. Figure 3 As shown, the RMSF of GA1DyPB fluctuates significantly at positions 144-169 and 183-225 at 25℃ and 37℃, respectively, representing potential sites. Visual analysis of the 3D protein model of GA1DyPB was performed in PyMOL to locate the loop region within the 144-169 and 183-225 position ranges. The ultimately identified mutant amino acid position was in the 198-216 range.

[0091] (3) Simulated saturation mutation analysis:

[0092] Next, FoldX was used to scan all sites within the interval for alanine, and the results are presented in the form of a heatmap, as shown below. Figure 4 As shown, the difference in unfolding free energy (ΔG) between blue and red represents the difference in protein unfolding free energy before and after the mutation. The redder the color, the smaller the unfolding free energy of the protein after the mutation, and the more likely it is to be a positive mutation. Subsequently, the same site was analyzed using the ESM-scan tool to perform virtual saturation mutation analysis. The results are presented in the form of a heatmap, as shown below. Figure 5 As shown in the figure, similar to the alanine scan results, the redder the color, the higher the score, and the more likely it is a positive mutation. Because the accuracy of alanine scanning differs from that of the ESM-scan tool, a comprehensive scoring method is used to determine mutation points. The final score is obtained by subtracting 60% of the Frodx simulation score from 40% of the ESM-Scan simulation score. A higher score indicates a greater likelihood of a positive mutation. The specific scoring results are presented in the form of a heatmap as shown below. Figure 6 As shown. The final mutants are I198F, T201I, T201L, T201F, T201R, K202L, D204W, D204F, D204L, D204R, D209L, D209P, D210P, D210R, K213L, K213I, K213R, and A215P.

[0093] (4) Site-directed mutagenesis primer design:

[0094] First, primers were designed in the manner of 5'-15-21 bp reverse complementary region + to 15 bp non-complementary region -3', and the plasmid pET-28a(+)-GA1DypB was amplified by reverse PCR.

[0095] (5) Template plasmid digestion:

[0096] The amplification product contains the original template plasmid. To prevent false positive transformants after transformation, it must be digested with Dpn I before recombination to remove the methylated template plasmid. The amplification product and Dpn I are gently mixed by pipetting, briefly centrifuged to collect the residue at the bottom of the tube, and incubated at 37°C for 1-2 h. The amplification product is then recovered. The Dpn I digestion system is shown in Table 4.

[0097] Table 4

[0098] Composition Volume μL Dpn I 1 Amplification products 40-50

[0099] (6) One-step cloning of circularized plasmids:

[0100] The digested amplification product was circularized using a one-step cloning method.

[0101] For single-fragment homologous recombination reactions: Optimal cloning vector amount = [0.02 × number of base pairs in cloning vector] ng (0.03 pmol) Optimal insert amount = [0.04 × number of base pairs in insert] ng (0.06 pmol)

[0102] For multi-fragment homologous recombination reactions: Optimal amount of cloning vector used = [0.02 × number of base pairs in cloning vector] ng (0.03 pmol) Optimal amount of each fragment used = [0.02 × number of base pairs in each fragment] ng (0.03 pmol)

[0103] The one-step cloning reaction system is shown in Table 5.

[0104] Table 5

[0105] Composition Volume μL Linearized carrier X ng n inserted segments <![CDATA[Y1+Y2….Y N of]]> 2×CE Mix 5 <![CDATA[ddH2O]]> Add to 20 μL

[0106] For single-fragment recombination reactions, 50°C for 5 min, then wait to cool to 4°C or immediately place on ice to cool.

[0107] For 2-3 fragment recombination reactions, 50°C for 15 min, then wait to cool to 4°C or immediately place on ice to cool.

[0108] Subsequently, the mutant was transformed into E. coli BL21(DE3) cells to obtain E. coli BL21(DE3) cells for expressing the mutant.

[0109] (7) Analysis of mutation results:

[0110] Preliminary screening of thermostability changes was performed using crude enzyme solutions of mutants, and the results are as follows: Figure 7 As shown, most mutants exhibited decreased thermostability, while the thermostability of four mutants—T201F, T201L, T201I, and D210R—was improved compared to the original GA1DyPB. Further screening was conducted using purified enzyme solutions of the four mutants. The enzyme activities of the four mutants and the original GA1DyPB, the residual enzyme activity after retention at 37°C for 1 h, and the Tm value parameters were measured, as shown below. Figure 8 As shown in the results, the thermostability of the three mutants other than T201L was improved compared with the original GA1DyPB, specifically reflected in the increase of residual enzyme activity and Tm value. Among them, T201L and D210R showed significant improvements in thermostability, with increases of 50.1% and 38.2%, respectively. Although the thermostability of T201L was improved, its enzyme activity decreased significantly, while D210R showed improved thermostability and enzyme activity increased by 36.2% compared with the original GA1DyPB.

[0111] Example 3

[0112] Comparison of long-term response performance of mutants:

[0113] Industrial biocatalysis processes typically require enzyme catalysts to possess both high catalytic activity and good structural stability. However, enzymes generally face a trade-off between activity and thermal stability, limiting their practical applications. This study compares the enzyme activity of the mutant D210R under long-term reaction conditions with that of the original enzyme.

[0114] Using the original GA1DyPB and the mutant D210R, the reaction was carried out under optimal conditions for 48 h, and the lignin depolymerization ability of each enzyme was determined. The reaction temperature was 37℃, and the reaction system is shown in Table 6.

[0115] Table 6

[0116] Composition Volume μL 50 g / L alkali lignin solution 500 Original GA1DyPB or mutant D210R 1000 μg <![CDATA[1M H2O2 solution]]> 75 pH 4 disodium hydrogen-citrate buffer Add to 50 mL

[0117] It was observed that, except for the protein-free control, all enzymes depolymerized lignin to produce flocculent substances, which proves that both the original GA1DyPB and the mutant D210R have lignin depolymerization activity under long-term reaction.

[0118] (2) After removing the flocculent material, the lignin concentration in the remaining reaction solution was measured, and the results are as follows: Figure 9Under long-term reaction conditions of 48 h, the residual concentration of lignin in the reaction solution of mutant D210R was lower. The enzyme activity of depolymerizing 1 mg of lignin per hour was defined as 1 U. The enzyme activity of the original GA1DyPB was 0.46 U / mg, and the enzyme activity of mutant D210R was 0.62 U / mg, which was 34.88% higher than that of the original enzyme.

[0119] Example 4

[0120] Mechanism analysis of the enhanced thermostability and enzyme activity of mutants:

[0121] (1) The stability of the protein-ligand complex at different temperatures was studied using MD simulations:

[0122] Using guaiacol-glycerol-β-guaiacol-propyl ether as a small molecule ligand, the root mean square deviation and root mean square fluctuation of the original GA1DyPB and the mutant D210R at two different temperature environments of 25℃ and 37℃ were obtained by MD simulation. Figure 10 , Figure 11 As shown in the figure, at 25℃, the mutant D210R stabilized after 10 ns, while the original GA1DyPB stabilized after 30 ns. At 37℃, the mutant D210R stabilized after 30 ns, while the original GA1DyPB stabilized after 60 ns. This indicates that the mutant D210R stabilized its conformation more quickly at different temperatures. At different temperatures, the RMSF fluctuation of the original GA1DyPB was significantly greater than that of the mutant D210R, indicating that the mutant D210R underwent smaller conformational changes, had a smaller flexibility range, and was more stable at different temperatures. Combined analysis of RMSD and RMSF data shows that the mutant D210R underwent smaller conformational changes at different temperatures, and its conformation was more stable and its overall structure more rigid at all temperatures. This explains the improved thermal stability of the mutant D210R.

[0123] (2) Analysis of the enzyme activity enhancement mechanism using molecular docking results:

[0124] The molecular docking results using guaiacol-glycerol-β-guaiacol-propyl ether as a small molecule ligand were analyzed. Figure 12 The molecular docking results of the original GA1DyPB are presented. Figure 13The molecular docking results of the mutant D210R are presented. Comparison of the molecular docking results reveals that the position of one hydrogen bond between the mutant D210R and the substrate has changed, indicating that the binding mode between the mutant and the substrate has changed compared to the original enzyme. Although some affinity for the substrate is lost, the conformation of the enzyme-small molecule ligand complex is more precise, which is more conducive to the reaction. Simultaneously, the hydrogen bond lengths between the mutant D210R and the substrate at sites VAL-144, VAL-219, and ARG-236 are smaller, meaning shorter hydrogen bonds and significantly enhanced bond energies. This indicates a more stable binding of the substrate to the enzyme's active site and higher affinity. Shorter and stronger hydrogen bonds allow for precise substrate positioning within the catalytic pocket, effectively lowering the activation barrier of the enzymatic reaction, while optimizing the enzyme's catalytic conformation and reducing structural fluctuations. This more stable interaction accelerates the substrate conversion rate, ultimately directly enhancing the catalytic activity of the mutant D210R.

[0125] (3) Steady-state dynamics analysis of the mutant:

[0126] Using ABTS as a substrate, the Michaelis-Menten equation curve of mutant D210R was obtained, and the corresponding K was calculated by fitting the equation. M With k cat See Table 7 below for the K-values ​​of the fitted original GA1DyPB. M With k cat See Table 8 below.

[0127] Table 7: Steady-state kinetic data of mutant D210R using ABTS as a substrate

[0128] For K M determinations, 1.5 mM H2O2 was used as co-substrate.

[0129] Table 8: Steady-state dynamic analysis of GA1DyPB with ABTS as substrate

[0130] For K M determinations, 1.5 mM H2O2 was used as co-substrate.

[0131] The comparison shows that the K of mutant D210R M Rise, k cat Rise, k cat / K M Overall increase. Analysis of steady-state dynamics results, KM The increase indicates that the mutant D210R sacrifices some substrate binding affinity, which corresponds to the slight adjustment of hydrogen bond positions in the molecular docking results mentioned above. And k cat The increase indicates an enhanced maximum reaction rate, demonstrating that the D210R mutant exhibits improved catalytic ability and can catalyze at a faster rate. cat / K M The overall improvement indicates that the mutant D210R performs better overall than the original GA1DyPB. The final results show that although the mutant D210R loses some affinity for the substrate compared to the original GA1DyPB, it optimizes the spatial conformation of the active pocket, enhances catalytic activity, and increases the maximum rate of enzyme reaction, thus achieving superior overall performance.

[0132] SEQ ID NO:1:MVRAQPILTPLTEAAIFLVVTVDEGAEDTVRDLLEDLSGLRRSVGFRIPEGGLSVVTGIGSDMWDRLFDGPRPAGLHPFVPLDGGRHQAPSTPGDLLFHLRASTMDLCFELAAKINDRLRGAARVVDETHGFRYFERRDLLGFVDGTENPEDDEAVEAALVSGEDPDFAGGSYVV VQKYLHDLASWNSLTVEEQERAIGRTKLDDIELDDETKPANSHVALNVIVDENGVEQQIVRANMPFGSFGADEFGTYFIGYSATPEVTEQMLRNMFLGSPPGNTDRILDFSTAVTGSLFFCPSLEFLEDLPPSPAEIPSHEDPSASTAGRPADGSLGVGALRRSSTLKLAAALEHHHHHH*

[0133] SEQ ID NO:2:

[0134]

Claims

1. A dye decolorizing peroxidase mutant, characterized in that, The mutant was obtained by D210R site-directed mutagenesis based on the amino acid sequence of the wild-type dye decolorizing peroxidase shown in SEQ ID NO:

1.

2. The gene encoding the dye decolorizing peroxidase mutant of claim 1.

3. A carrier, characterized in that, The vector contains the gene of the dye decolorizing peroxidase mutant as described in claim 2.

4. A host cell, characterized in that, The host cell contains the vector as described in claim 3.

5. A method for preparing the dye decolorizing peroxidase mutant of claim 1, characterized in that, Including the following steps: (a) Under suitable expression conditions, the host cells of claim 4 are cultured to express the dye decolorizing peroxidase mutant. (b) The expression product is isolated to obtain the dye decolorizing peroxidase mutant.

6. The application of the dye decolorizing peroxidase mutant of claim 1 in lignin depolymerization.

7. The application according to claim 6, characterized in that, Dye decolorizing peroxidase was added to a reaction system containing lignin and mixed in a disodium hydrogen phosphate-citrate buffer solution for the reaction.

8. The application according to claim 7, characterized in that, The lignin concentration is 100-1000 mg / L, and the dye decolorizing peroxidase concentration is 10-100 mg / L.

9. The application according to claim 7, characterized in that, The reaction system also includes H2O2, with an enzyme concentration of 20 μg / mL and an H2O2 concentration of 1.5 mM.

10. The application according to claim 7, characterized in that, The reaction system had a pH of 4, a temperature of 37°C, and a reaction time of 24–72 hours.

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