A lignin peroxidase mutant based on rational design, a preparation method of enzyme preparation, and application in straw degradation

Abstract

The present application belongs to the field of biotechnology and biomass conversion, and particularly relates to a lignin peroxidase mutant based on semi-rational design, a preparation method of enzyme preparation and application in straw degradation. The present application evaluates the three-dimensional model of wild-type lignin peroxidase, screens potential mutation hotspots by combining molecular docking technology, and then obtains high-activity lignin peroxidase mutants by three rounds of mutation of alanine scanning, saturation mutation and combination mutation. After analyzing the mechanism of enzyme activity improvement by molecular dynamics simulation, the preferred high-activity mutants are optimized according to the codon bias of Pichia pastoris, integrated into the expression vector pPIC9K of Pichia pastoris, and the secretion expression of the mutants is realized. The mutants show significantly improved catalytic efficiency in straw degradation. The present application has important significance in promoting the green and efficient development of the feed industry while achieving outstanding degradation effect.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and biomass conversion, specifically relating to a lignin peroxidase mutant based on semi-rational design modification, a method for preparing the enzyme preparation, and its application in straw degradation. Background Technology

[0002] Lignin peroxidase (LiP), a key enzyme in the lignocellulose degradation system, plays an irreplaceable role in the depolymerization and oxidation of lignin. Lignin is a complex and recalcitrant aromatic polymer in lignocellulose, and its three-dimensional network structure severely hinders the enzymatic hydrolysis of cellulose and hemicellulose. LiP, through its unique oxidation mechanism, can catalyze the breaking of C-C and CO bonds in lignin, generating low-molecular-weight aromatic compounds. This effectively overcomes the lignin barrier, creating conditions for the efficient action of cellulase and hemicellulase. This enzyme not only selectively removes lignin, significantly improving the accessibility of cellulase and reducing the conversion cost of biomass, but also catalyzes the directed conversion of lignin-derived aromatic compounds, providing a new pathway for the green synthesis of bio-based chemicals.

[0003] Despite the significant application value of LiP, its industrialization is still constrained by two key factors: insufficient enzyme activity and low production efficiency. Natural hosts have significant limitations in enzyme production—white-rot fungi that efficiently produce LiP not only grow slowly, but their enzyme production is also induced and regulated by lignin, making it difficult to steadily increase yield. Furthermore, heterologous expression systems also face serious challenges: in E. coli expression, LiP mostly exists in the form of inactive inclusion bodies, making operation complex, and the endotoxicity of this system increases the complexity of downstream applications. While soluble expression can be achieved in fungal expression systems, the resulting enzyme activity still does not meet industrial requirements.

[0004] Semi-rational enzyme design, by combining partial structural information, computational simulation, and experimental screening, can efficiently optimize enzyme performance and has broad application prospects in industry, medicine, and environmental protection. Semi-rational enzyme design combines the advantages of rational design and directed evolution. Based on partial structural information and catalytic mechanisms, it uses molecular docking and molecular dynamics simulations to determine key amino acids at the active site and substrate binding site. Diverse mutations are introduced at these key sites to construct a mutant library, and high-throughput screening techniques are used to select mutants with improved performance. Finally, multiple rounds of mutation and screening are performed on the selected mutants for further optimization. The advantage of this strategy is that computational simulation reduces the number of mutants requiring experimental verification, and the combination of structural information and experimental screening improves the optimization success rate. Summary of the Invention

[0005] Given the widespread demand for lignin peroxidase in industrial applications and the limitations of existing enzyme preparation production technologies and directed evolution screening technologies, this invention develops a highly efficient heterologous expression and modification strategy. Utilizing the PelB signal peptide of the *E. coli* pET-22b vector, this invention successfully achieved the secretory expression of LiP into the periplasmic space. Based on the three-dimensional structural model of wild-type LiP, potential catalytic hotspot sites were identified using molecular docking technology. A three-stage strategy of alanine scanning, saturation mutagenesis, and combinatorial mutagenesis was employed to ultimately obtain a highly active LiP mutant. Molecular dynamics simulations revealed the molecular mechanism of enhanced enzyme activity. The optimal mutant gene sequence was then optimized according to the codons of *Pichia pastoris* and integrated into the pPIC9K expression system, achieving highly efficient secretory expression of the mutant.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0007] L4 mutant was obtained by semi-rational design based on Escherichia coli exocrine secretion and a three-stage strategy of alanine scanning, saturation mutagenesis and combinatorial mutagenesis. Subsequently, the gene sequence of L4 mutant was optimized according to the codons of Pichia pastoris and expressed in Pichia pastoris to obtain a highly active mutant.

[0008] The mutant is the LiP-P4 mutant, whose amino acid sequence is shown in SEQ ID NO.6 and whose nucleotide sequence is shown in SEQ ID NO.5. The LiP-P4 mutant contains mutations at the following three amino acid sites: histidine (His) at position 38 is mutated to alanine (Ala), threonine (Thr) at position 267 is mutated to asparagine (Asn), and glutamic acid (Glu) at position 320 is mutated to alanine (Ala).

[0009] The preparation steps of the LiP-P4 mutant are as follows:

[0010] 1. The lignin peroxidase (LiP) gene sequence (GenBank: X15599) from *Phanerochaete chrysospora* was optimized according to the codon bias of *Escherichia coli* to obtain the gene encoding LiP. Its nucleotide sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2. The protein encoded by the LiP gene is denoted as B1.

[0011] 2. Using the gene encoding LiP described in step 1 as a template, PCR was performed using primer P1 to amplify the LiP gene fragment. The amplified LiP gene fragment was double-digested with restriction endonucleases NcoI and XhoI, and ligated with the linearized fragment of the E. coli expression vector pET-22b digested with the same restriction enzymes. The ligation product was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the recombinant plasmid DH5α-pET-22b-LiP. Subsequently, the recombinant plasmid DH5α-pET-22b-LiP was transformed into E. coli BL21(DE3) competent cells to obtain a recombinant lignin peroxidase engineered bacterium that can be used for periplasmic space expression, denoted as E. coli BL21(DE3)-pET-22b-LiP.

[0012] Preferably, the P1 primer sequences are: P1-F: TGGCCATGGCACCGAATCTGGAT, P1-R: GTGCTCGAGATTCGGTGACGGC.

[0013] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 10 min.

[0014] 3. The recombinant lignin peroxidase engineered bacteria *E. coli* BL21(DE3)-pET-22b-LiP obtained in step 2 was inoculated into LB medium containing ampicillin sodium. After the first culture, a culture solution was obtained. This culture solution was then inoculated into LB liquid medium containing ampicillin sodium for a second culture. When the OD600 reached 0.6–0.8, a soluble inducer was added to continue induction culture. After the culture was completed, the bacterial cell pellet was collected by centrifugation and resuspended in a disruption buffer containing lysozyme to obtain a resuspension. After incubation, the pellet was centrifuged again, and the supernatant was collected. This supernatant is the lignin peroxidase enzyme preparation, denoted as E-LiP.

[0015] Preferably, in step 3, the inoculum size of the recombinant lignin peroxidase engineered bacteria inoculated into LB medium containing ampicillin sodium is 1-5%, wherein the concentration of ampicillin sodium in the LB medium is 0.1-0.2 mg / mL. The inoculum size of the culture medium inoculated into LB liquid medium containing ampicillin sodium is 1-5% (v / v). The components of the lysozyme-containing lysis buffer are: 0.5 mg / mL lysozyme, 200 mM Tris, 500 mM sucrose, 1 mM EDTA, pH 8.0. The conditions for the first culture are 30-40℃, 160-200 rpm for 10-16 h. The conditions for the second culture are 30-40℃, 160-200 rpm for 2-6 h. The amount of soluble inducer added is 1-5% of the culture medium volume. After adding the soluble inducer, the culture conditions are 12-20℃ for 8-16 hours. The composition of the soluble inducer is: 2.5% Triton X-100, 2.5% Tween 80, 10% ethanol, 5% glycerol, 50mM CaCl2, and 3mM IPTG. The bacterial cell pellet is resuspended in a lysozyme-containing disruption buffer at a ratio of 0.5-1g:2.5-5mL. The incubation conditions are: 30-40℃ for 30-45 minutes.

[0016] 4. Using the three-dimensional structure of the B1 protein from step 1 (from...) https: / / swiss The three-dimensional structures of model.expasy.org and ABTS (from http: / / pubchem.ncbi.nlm.nih.gov) were used for molecular docking using AutoDockVina software. By analyzing the frequently occurring interacting residues in the high-scoring (low-energy) complex, seven potential interaction sites were identified: His38, Gln133, His247, Asn265, Thr267, Asn304, and Glu320.

[0017] 5. Using the recombinant plasmid DH5α-pET-22b-PcLiP described in step 2 as a template, a one-step whole plasmid PCR technique was used to mutate the seven potential action sites (His38, Gln133, His247, Asn265, Thr267, Asn304 and Glu320) in step 4 to alanine (Ala) to construct single-point mutants;

[0018] Engineered bacteria were constructed based on single-point mutants, and crude enzyme solutions of each mutant were prepared according to the method described in step 3. Then, using the enzyme activity of E-LiP as a 100% baseline, dominant mutants with the highest activity were screened. H38A (198%), E320A (182%) and T267A (235%) were found to be dominant mutants, with T267A having the highest activity.

[0019] 6. Using Thr267 obtained in step 5 as the mutation site, a one-step whole-plasmid PCR method was used for saturation mutagenesis. The whole-plasmid PCR product with the saturation mutation at position 267 was amplified using P3 primers. Then, the whole-plasmid PCR product was treated with the restriction endonuclease DpnI. After selectively digesting the original template, the DpnI-treated whole-plasmid PCR product was transformed into E. coli DH5α competent cells. Plasmid was extracted to obtain the mutant plasmid DH5α-pET-22b-LiP-T267K. The mutant plasmid was transformed into E. coli BL21(DE3) competent cells to obtain a recombinant lignin peroxidase mutant strain library, denoted as E. coli BL21(DE3)-pET-22b-LiP-T267K.

[0020] The preferred P3 primer sequence for one-step whole plasmid PCR is:

[0021] P3-F: CAGTCATTTGTTAATAATCAGNNKAAACTGCAGGAAG,

[0022] P3-R:GCAGTTTMNNCTGATTATTAACAAATGACTGCCATTCGC.

[0023] The whole plasmid PCR system contained 1 μL of DH5α-pET-22b-LiP plasmid as template, 1 μL of each primer, 7 μL of ddH2O, and 10 μL of 2x Primer Star Max.

[0024] Whole plasmid PCR program: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 80 s, 30 cycles; 72℃ extension for 10 min.

[0025] 7. Based on the recombinant lignin peroxidase mutant strain library (E.coli BL21(DE3)-pET-22b-LiP-T267K) constructed in step 6, the following high-throughput screening process was used:

[0026] In a sterile laminar flow hood, a single-clonal mutant strain of E. coli BL21(DE3)-pET-22b-LiP-T267K was inoculated into a 96-well plate containing ampicillin sodium in LB broth, designated as plate A. After the first culture, the culture was transferred to another 96-well plate containing ampicillin sodium in LB broth, designated as plate B. Plate B underwent a second culture. After culture, N positive integer positions were randomly selected in plate B, and bacterial culture was taken from each of the N positions to measure OD600, thus determining the growth status of each mutant. When the OD600 of each well was in the range of 0.6–0.8 and the difference in OD600 values ​​was within 0.1, a soluble inducer was added to plate B for induction culture.

[0027] After induction culture, the supernatant was discarded by centrifugation and the precipitate was collected. The precipitate was resuspended in a disruption buffer containing lysozyme to obtain a suspension. After incubation, a suspension of mutant recombinant protein was obtained. The suspension of mutant recombinant protein was centrifuged to obtain the supernatant, which was transferred to a new shallow 96-well plate, designated as plate C, for enzyme activity assay. The obtained supernatant was the mutant recombinant protein. After enzyme activity assay, a highly active mutant, T267Q (275%), was screened out.

[0028] Preferably, in step (7), the final concentration of ampicillin sodium in plates A and B is 0.1-0.2 mg / mL. The first culture is carried out at 30-40℃ and 160-200 rpm in a constant temperature shaking incubator for 8-16 h. The second culture is carried out at 30-40℃ and 160-200 rpm in a constant temperature shaking incubator for 4-8 h. N≥3, preferably 8. The induction culture is carried out at 12-20℃ and 160-200 rpm in a low temperature constant temperature shaking incubator for 8-16 h.

[0029] The inoculum size for transferring from plate A to plate B is 1%–5% (v / v), and the amount of soluble inducer added is 1%–5% of the culture medium volume in plate B.

[0030] Centrifugation was performed at 4000 rpm for 15–20 min. The lysozyme concentration in the lysis buffer was 0.5 mg / mL. The lysis buffer consisted of 200 mM Tris, 500 mM sucrose, 1 mM EDTA, and pH 8.0. Incubation was carried out in a constant temperature water bath at 30–40 °C for 30–45 min.

[0031] 8. Based on the three high-quality mutation sites H38A, E320A obtained in step 5 and T267Q obtained in step 7, perform combined mutations;

[0032] S1. Site-directed mutagenesis was performed using one-step full plasmid PCR to mutate threonine (Thr, T) at position 267 to glutamine (Gln, Q). The full plasmid PCR product of T267Q was amplified using primer P4. The PCR product was then treated with the restriction endonuclease DpnI. After selective digestion of the original template, the DpnI-treated (mutated) PCR product was transformed into E. coli DH5α competent cells. Plasmid was extracted to obtain the mutant plasmid DH5α-pET-22b-LiP-T267Q.

[0033] The preferred P4 primer sequence for one-step whole plasmid PCR is:

[0034] P4-F:TCATTTGTTAATAATCAGCAGAAACTGCAGGAAGATT;

[0035] P4-R:CTGATTATTAACAAATGACTGCCATTCGCATGCT.

[0036] The whole plasmid PCR system contained 1 μL of DH5α-pET-22b-LiP plasmid as a template, 1 μL of each primer, 7 μL of ddH2O, and 10 μL of 2 x Primer Star Max.

[0037] Whole plasmid PCR program: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 80 s, 30 cycles; 72℃ extension for 10 min.

[0038] S2. Using the DH5α-pET-22b-LiP-T267Q recombinant plasmid constructed in S1 of step 8 as a template, add the H38A mutation primer and amplify the T267Q-H38A full plasmid PCR product using full plasmid PCR. Then, treat the above PCR product with the restriction endonuclease DpnI. After selectively digesting the original template, transform the DpnI-treated (mutant) PCR product into E. coli DH5α competent cells, extract the plasmid, and obtain the mutant plasmid DH5α-pET-22b-LiP-T267Q-H38A recombinant plasmid.

[0039] S3. Using the recombinant plasmid DH5α-pET-22b-LiP-T267Q-H38A constructed in step S2 of step 8 as a template, a one-step full-plasmid PCR was performed using the E320A mutant primer to amplify the full-plasmid PCR product T267Q-H38A-E320A. Then, the PCR product was treated with the restriction endonuclease DpnI. After selectively digesting the original template, the DpnI-treated (mutant) PCR product was transformed into E. coli DH5α competent cells. Plasmids were extracted to obtain the mutant plasmid DH5α-pET-22b-LiP-H38A-T267Q-E320A. The lignin peroxidase mutant gene fragment carried by this plasmid is designated LiP-L4, and its nucleotide sequence is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4. The recombinant mutant plasmid was transformed into E. coli. In BL21(DE3) competent cells, a recombinant lignin peroxidase mutant engineered bacterium, E. coli BL21(DE3)-pET-22b-LiP-H38A-T267Q-E320A, was constructed and designated as the L4 mutant.

[0040] 9. The L4 mutant gene encoded in step 8 was optimized according to the codon preference of Pichia pastoris to obtain the gene encoding LiP-P4, which was then ligated into the pPIC9K vector and transformed into E. coli DH5α competent cells. The plasmid was then extracted to obtain the recombinant plasmid DH5α-pPIC9K-LiP-P4; wherein the amino acid sequence of LiP-P4 is shown in SEQ ID NO.6.

[0041] The present invention also relates to a gene encoding the lignin peroxidase mutant, the nucleotide sequence of which is shown in SEQ ID NO.5; and a recombinant vector carrying the gene.

[0042] The present invention also relates to a recombinant engineered bacterium expressing a lignin peroxidase mutant, wherein the recombinant engineered bacterium comprises the recombinant vector described above.

[0043] The steps are as follows: using Pichia pastoris GS115 competent cells as the host cell, the recombinant vector is introduced into Pichia pastoris GS115 competent cells by electroporation to obtain recombinant lignin peroxidase recombinant engineered bacteria that can be used for exocrine expression of Pichia pastoris.

[0044] This invention also relates to the application of the aforementioned mutants, genes, or recombinant vectors in the preparation of highly active enzyme preparations.

[0045] The present invention also provides a method for preparing a highly active enzyme preparation, which involves fermentation culture based on the recombinant engineered bacteria, and collection of the extracellular culture medium to obtain the highly active enzyme preparation.

[0046] The specific steps are as follows: Inoculate the recombinant engineered bacteria into YPD medium at an inoculation rate of 1-5%, and culture at 25-30℃ and 180 rpm for 16-24 hours. After centrifugation, collect the bacterial cell pellet and transfer it to BMGY liquid medium at an inoculation rate of 1-5% for expansion culture. After the OD600 reaches 2-6, centrifuge to collect the bacterial cell pellet. Wash the pellet with ddH2O and resuspend it in BMMY medium to obtain the resuspension. The ratio of bacterial cell pellet to BMMY medium is 1g:50mL.

[0047] Methanol was added to the resuspended solution for induction culture, with a final methanol concentration of 0.5-1%. Methanol was added every 24 hours to maintain the concentration at 0.5-1%, and the culture was induced for a total of 84-120 hours. After induction, the supernatant was collected to obtain the enzyme preparation.

[0048] Finally, the present invention also relates to the application of enzyme preparations in straw degradation, specifically for the degradation of alkali lignin or straw lignin.

[0049] S1. Application of mutant enzyme preparations for the degradation of alkali lignin:

[0050] Using alkali lignin as a substrate, enzyme preparation, BR buffer (pH 3-4), and H2O2 are added to form a reaction system, and the degradation of alkali lignin can be achieved after the reaction.

[0051] The enzyme preparation was added at 25% (V / V) of the total volume of the reaction system, i.e., the volume ratio of enzyme preparation to reaction system was 1:4. In the reaction system, the concentration of BR buffer was 25-100 mM, the concentration of alkali lignin was 0.8-1.2 g / L, and the concentration of H2O2 was 2-4 mM. The reaction temperature was 30-40℃, and the reaction time was 18-36 h.

[0052] S2. Applications of enzyme preparations for degrading straw lignin:

[0053] Using wheat straw as raw material, enzyme preparations are added to form a mixed system in conjunction with laccase, glucose oxidase, and cellulase. By controlling the moisture content of the mixed system and treating it under certain temperature conditions, lignin in wheat straw can be degraded.

[0054] Preferably, based on the mass of wheat straw: the amount of enzyme preparation added is 5-30% (v / m, ml / g), that is, the ratio of enzyme preparation to wheat straw is 5-30 ml: 100 g; the amount of laccase added is 1-5%; the amount of glucose oxidase added is 1-5%; the amount of cellulase system added is 0.4-2%, wherein the cellulase system is composed of a mass mixture of glucanase, acid cellulase, xylanase, mannanase and cellobiase; the moisture content of the mixture is controlled at 40%-60%, the temperature is 30-40℃, and the treatment time is 70-120 h.

[0055] The advantages and technical effects of this invention are:

[0056] 1. This invention provides a method for producing a soluble lignin peroxidase enzyme preparation, which utilizes the secretion expression system of Escherichia coli to secrete lignin peroxidase into the periplasmic space to obtain a highly active enzyme preparation.

[0057] 2. This invention is based on a semi-rational design strategy, identifying potential mutation hotspots through molecular docking and simplifying the screening process by combining a mutant library; it utilizes ABTS. + A colorimetric method was established based on absorbance changes at 420 nm to rapidly screen highly active mutants.

[0058] 3. The combined mutant sites (H38A / T267Q / E320A) obtained by screening were introduced into Pichia pastoris for exocrine expression. The prepared enzyme preparation significantly improved the degradation efficiency of alkali lignin and wheat straw lignin.

[0059] 4. The enzyme preparation method provided by this invention is low in cost and simple in process. The developed LiP high-efficiency expression and modification technology provides a new solution for the degradation of lignin resources. It can solve the technical problem that lignin peroxidase is difficult to produce on a large scale in industrial applications. It is of great significance to promote the green and sustainable development of the feed industry and has broad potential for industrial application. Attached Figure Description

[0060] Figure 1 Comparison of the original and optimized sequences of lignin peroxidase.

[0061] Figure 2 PCR amplification of the LiP gene fragment.

[0062] Figure 3 The LiP gene fragment and pET-22b vector were digested with double enzymes.

[0063] Figure 4 Specificity verification of E. coli BL21(DE3)-pET-22b-LiP recombinant strain.

[0064] Figure 5 Global quality score of QMEANDisCo for building B1 protein models using Swiss-model.

[0065] Figure 6 QMEAN Z scores and comparison charts for the B1 protein model.

[0066] Figure 7 The B1 protein model was evaluated, including (a) analysis of non-bonded interactions between amino acid residues; (b) compatibility analysis between the model and the amino acid sequence; and (c) Laplace plot analysis.

[0067] Figure 8 Molecular docking between ABTS and B1 protein.

[0068] Figure 9 The first round of alanine scanning was used to detect mutant activity.

[0069] Figure 10 Second round of saturated mutant enzyme activity retest.

[0070] Figure 11 Third round of combined mutant enzyme activity detection.

[0071] Figure 12 : Curve showing the change of RMSD of protein-ligand complex over time.

[0072] Figure 13 : The SASA time change curve of protein during the protein-ligand complexation simulation.

[0073] Figure 14 : This refers to the change in the protein's gyration radius during the protein-ligand complexation process.

[0074] Figure 15 The root mean square fluctuation of the enzyme-substrate complex simulation process.

[0075] Figure 16 Changes in the number of hydrogen bonds during the enzyme-substrate complex simulation process.

[0076] Figure 17 : Verification of transformant specificity of Pichia pastoris recombinant strain.

[0077] Figure 18 Identification of P1 and P4 expression in Pichia pastoris.

[0078] Figure 19 P1 and P4 degrade alkali lignin. Detailed Implementation

[0079] The following embodiments further illustrate the present invention. Those skilled in the art can understand the basic features of the present invention based on the above description and embodiments, and can make adaptive modifications and adjustments without departing from the spirit and scope of the present invention to adapt to different application scenarios and conditions. Unless otherwise stated, the experimental materials used in the following embodiments are all commercially available. Unless otherwise specified, the technologies used in the present invention are all prior art.

[0080] Reagent and material specifications:

[0081] Laccase, glucose oxidase, glucanase, acid cellulase, xylanase, and cellobiase are common feed-grade commercial enzyme preparations, which are available on the market and were purchased from Xiasheng (Ningxia) Biotechnology Development Co., Ltd.

[0082] ABTS was purchased from Shanghai Myriel Co., Ltd., and alkali lignin was purchased from Maclean Biochemical Technology Co., Ltd.

[0083] LB medium components: 0.5% yeast extract, 1% tryptone, 0.5% sodium chloride.

[0084] YPD medium components: 1% yeast extract, 2% peptone, 2% glucose, adjusted to pH 5.8; solid YPD medium is supplemented with an additional 2% agar.

[0085] MD plates: 1.34% YNB, 2% glucose, 2% agar.

[0086] 1.34% YNB: Weigh 1.34g of YNB and dissolve it in 100mL of distilled water.

[0087] BMGY medium components: 1% yeast extract, 2% peptone, 1.34% YNB, 10% 1M pH 6.0 phosphate buffer, 1% glycerol.

[0088] BMMY medium components: 1% yeast extract, 2% peptone, 1.34% YNB, 10% 1M pH 6.0 phosphate buffer, 1% methanol.

[0089] Example 1: Semi-rational design of lignin peroxidase mutants;

[0090] Construction of recombinant lignin peroxidase bacteria:

[0091] The lignin peroxidase (LiP) gene sequence (GenBank: X15599) from *Phanerochaete chrysospora* was optimized according to the codon bias of *Escherichia coli* to obtain the gene encoding LiP. Its nucleotide sequence is shown in SEQ ID NO.1, and the corresponding amino acid sequence is shown in SEQ ID NO.2. Figure 1 This is a sequence alignment diagram of the original and codon-optimized sequences of the lignin peroxidase (LiP) gene; the protein encoded by the LiP gene is denoted as B1.

[0092] Using the gene encoding LiP as a template, PCR was performed using primer P1 to amplify the LiP gene fragment, and the results are as follows. Figure 2 As shown. The amplified LiP gene fragment was double-digested using restriction endonucleases NcoI and XhoI. Figure 3 Lane 1 of the swim buds, and the linearized expression vector pET-22b of E. coli digested with the same enzymes ( Figure 3 The ligation product was transferred to E. coli DH5α competent cells via lane 2, and the plasmid was extracted to obtain the recombinant plasmid DH5α-pET-22b-LiP. Subsequently, the recombinant plasmid DH5α-pET-22b-LiP was transformed into E. coli BL21(DE3) competent cells, and the specificity of the resulting single bacteria was verified using primer P2. The results are as follows: Figure 4 As shown. Figure 4 The left image shows the plate diagram of E. coli BL21(DE3) transformed with the recombinant plasmid, and lanes 1-4 in the right image are PCR verification diagrams of the recombinant bacteria, both of which are as expected. A recombinant lignin peroxidase engineered bacterium suitable for periplasmic spatial expression was successfully obtained, denoted as E. coli BL21(DE3)-pET-22b-LiP.

[0093] The sequences of the P1 primers are: P1-F: TGGCCATGGCACCGAATCTGGAT, P1-R: GTGCTCGAGATTCGGTGACGGC.

[0094] The sequences of the P2 primers are: P2-F: CTCGATCCCGCGAAATTAATACG; P2-R: CAAGGGGTTATGCTAGTTATTGC.

[0095] Molecular docking identifies potential mutation sites

[0096] The B1 protein was modeled in three dimensions using the Swiss-Model online server (https: / / swissmodel.expasy.org / ). For example... Figure 5 As shown, the model validation parameters indicate that the Global Model Quality Assessment Index (GMQE) reaches 0.97 (confidence interval 0-1), close to the theoretical optimum, indicating high model reliability. The Quality Assessment Discreteness Consistency Score (QMEANDis Co) is 0.88±0.05 (ideal range 0.7-1.0), showing a high degree of agreement between the model topology and the template. Figure 6The standardized quality score (QMEANZ-score) was -0.07 (-4.0 to 0.0), and statistics showed that the model parameter distribution was within the standard deviation range (|Z| < 1). Overall validation results indicate that the three-dimensional model of the B1 protein possesses atomic-level structural accuracy.

[0097] The structure model of the B1 protein was systematically validated using the SAVESv6.1 integrated structure validation platform (Structure Validation Server): First, the ERRAT structure validation tool was used to validate the structure of the protein's three-dimensional model. Statistical analysis was performed on atom type-specific pairings within the spatial scale (short-range non-bonding interaction threshold) to quantitatively analyze the distribution frequency of van der Waals contacts and electrostatic interactions. A score greater than 85 indicated a relatively good result. The results are as follows: Figure 7 As shown in (a), the overall quality factor of the model is 89.286, significantly higher than the recommended threshold of 85, indicating that the surface photoatom stacking is reasonable. Subsequently, the Verify 3D module was used to evaluate the compatibility of the three-dimensional structure-primary sequence. Quantitative analysis using the three-dimensional contour method showed that 90.6% of the residues had a 3D / 1D compatibility score greater than or equal to 0.1. Figure 7 (b)) This achieves the standard for a high-quality model. Finally, Ramachandran region validation of the psi-phi dihedral distribution was performed using Ramachandran statistical analysis of the PROCHECK module. The data showed that 90.9% of the residues were located in the red core allowed region, and 9.1% of the residues were distributed in the yellow additional allowed region. Figure 7 (c) indicates that the model is good. In summary, the above validation results show that the constructed B1 protein model has a reasonable conformation and good stereochemical parameters, and can be used for molecular docking.

[0098] Finally, molecular docking between the ABTS substrate and the B1 protein was performed using AutoDockVina software. Figure 8 By analyzing frequently occurring interacting residues in high-scoring (low-energy) complexes, seven potential interaction sites were identified: His38, Gln133, His247, Asn265, Thr267, Asn304, and Glu320.

[0099] The first round of alanine scanning identified mutation hotspots.

[0100] Using the DH5α-pET-22b-LiP recombinant plasmid as a template, a one-step whole-plasmid PCR method was employed to mutate His38, Gln133, His247, Asn265, Thr267, Asn304, and Glu320 to alanine. Whole-plasmid PCR was then performed using primers for each alanine mutation (Table 1) to amplify the whole-plasmid PCR products. The PCR products were then treated with the restriction endonuclease DpnI. After selectively digesting the original template plasmid, the DpnI-treated (mutant) PCR products were transformed into E. coli DH5α competent cells. Plasmids were extracted to obtain the corresponding mutant plasmids DH5α-pET-22b-LiP-H38A, DH5α-pET-22b-LiP-Q133A, DH5α-pET-22b-LiP-H247, DH5α-pET-22b-LiP-N265A, pET-22b-LiP-T267A, DH5α-pET-22b-LiP-N304A, and DH5α-pET-22b-LiP-E320A.

[0101] The mutant plasmid was then transformed into E. coli BL21(DE3) competent cells to construct recombinant lignin peroxidase engineered bacteria: E. coli BL21(DE3)-pET-22b-LiP-H38A, E. coli BL21(DE3)-pET-22b-LiP-Q133A, E. coli BL21(DE3)-pET-22b-LiP-H247A, E. coli BL21(DE3)-pET-22b-LiP-N265A, E. coli BL21(DE3)-pET-22b-LiP-T267A, E. coli BL21(DE3)-pET-22b-LiP-N304A, and E. coli BL21(DE3)-pET-22b-LiP-E320A.

[0102] Table 1. Primer sequences for mutants

[0103]

[0104]

[0105] The PCR reaction system consisted of: 1 μL DH5α-pET-22b-LiP recombinant plasmid, 1 μL each of forward and reverse primers, 7 μL ddH2O, and 10 μL 2 x PrimeSTAR Max Premix. The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 80 s, 30 cycles; 72℃ extension for 10 min.

[0106] Recombinant lignin peroxidase engineered bacteria (including E. coli BL21(DE3)-pET-22b-LiP-H38A, E. coli BL21(DE3)-pET-22b-LiP-Q133A, E. coli BL21(DE3)-pET-22b-LiP-H247A, E. coli BL21(DE3)-pET-22b-LiP-N265A, E. coli BL21(DE3)-pET-22b-LiP-T267A, E. coli BL21(DE3)-pET-22b-LiP-N304A, E. coli BL21(DE3)-pET-22b-LiP-E320A, and E. coli) BL21(DE3)-pET-22b-LiP was inoculated at 1% into 5 mL of LB medium containing 0.1 mg / mL ampicillin sodium and cultured overnight at 37°C and 180 rpm for 12 h to obtain the culture medium. The culture medium was then inoculated at 1% into 100 mL of LB liquid medium containing 0.1 mg / mL ampicillin sodium. When the OD600 reached 0.8, 1% of a soluble inducer (components: 2.5% Triton X-100, 2.5% Tween 80, 10% ethanol, 5% glycerol, 50 mM CaCl2, 3 mM IPTG) was added, and the culture was continued at 37°C and 180 rpm for 6 h. After culturing, the bacterial pellet was collected by centrifugation.

[0107] 1 g of bacterial cell precipitate was resuspended in 5 mL of disruption buffer (200 mM Tris, 500 mM sucrose, 1 mM EDTA, pH 8.0) containing lysozyme (0.5 mg / mL) to obtain a suspension. After incubation at 37 °C for 40 min, the suspension was centrifuged again, and the supernatant was collected. The resulting recombinant lignin peroxidase enzyme preparations were labeled as enzyme preparation H38A, enzyme preparation Q133A, enzyme preparation H247A, enzyme preparation N256A, enzyme preparation T267A, enzyme preparation N304A, enzyme preparation E320A, and enzyme preparation E-LiP, respectively.

[0108] Enzyme activity assay: The enzyme activities of recombinant proteins H38A, Q133A, H247A, N265A, T267A, N304A, and E320A were detected using the ABTS (2,2-azino-di-3-ethylbenzothiazoline-6-sulfonic acid) method via microplate reader. The activity of mutants was assessed using E-LiP activity as 100% (LiP for short), and three mutants with significantly increased enzyme activity were obtained. The relative enzyme activities of mutants H38A, T267A, and E320A were increased by 0.98, 1.35, and 0.82 times, respectively. The results are shown in [Figure number missing]. Figure 9As shown. In the next round, the Thr267 site, which has the highest enzyme activity, will be used as the target for saturation mutation in the second round.

[0109] Each of the above catalytic reactions was performed in triplicate, with two replicates. The reaction system contained 1.25 mM ABTS, 25 mM BR buffer, 100 μL of enzyme preparation, and finally, H2O2 (final concentration 2 mM) was added to initiate the reaction. The free radical cations (ABTS) generated by the oxidation of ABTS within 10 min were detected using a microplate reader at 420 nm. + Enzyme activity is quantified by changes in absorbance. If the absorbance change increases rapidly, the enzyme solution should be diluted 2-5 times and measured again, and all mean values ​​should be between 0.2 and 0.8.

[0110] The method for calculating enzyme activity is as follows:

[0111] ΔA: Change in absorbance of ABTS oxidation products in the reaction system (usually detected at 420 nm).

[0112] ε: ABTS cation radical (ABTS) + The molar extinction coefficient (unit: L·mol) -1 ·cm -1 ), commonly used value: ε 420 nm = 3.6 × 10 4 L·mol -1 ·cm -1 ;

[0113] Vtotal: Total volume of the reaction system (unit: L).

[0114] t: Reaction time (unit: min).

[0115] V_enzyme: Volume of enzyme solution added (unit: L).

[0116] D: Enzyme solution dilution factor (if the original enzyme solution is diluted before use).

[0117] The second round of saturation mutations of key amino acids

[0118] According to the alanine scanning results, the enzyme activity was significantly enhanced after Thr267 was mutated to Ala, indicating that the amino acid at position 267 is a key residue for substrate binding. To further explore the function of the dominant site (Thr267) in the initial screening process, a mutant library for targeted screening was constructed using a saturation mutagenesis strategy. P3 primers were designed: (P3-F: CAGTCATTTGTTAATAATCAGNNKAAACTGCAGGAAG, P3-R: GCAGTTTMNNCTGATTATTAACAAATGACTGCCATTCGC). Using DH5α-pET-22b-LiP as a template, a saturation mutant library of Thr267 was constructed by whole-plasmid PCR. Then, the full-plasmid PCR product of the Thr267 saturated mutant library was treated with the restriction endonuclease DpnI. After selective digestion of the original template plasmid, the DpnI-treated (mutant) PCR product was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the mutant plasmid library DH5α-pET-22b-LiP-T267K. The mutant plasmid library was transformed into E. coli BL21(DE3) competent cells to construct the mutant strain library E. coli BL21(DE3)-pET-22b-LiP-T267K.

[0119] High-throughput screening was performed on the saturated mutant strain library *E. coli* BL21(DE3)-pET-22b-LiP-T267K for Thr267. The specific steps were as follows: In a sterile laminar flow hood, single clones of the mutant library were picked from the plates and transferred to 96-well A plates (each well containing 800 μL of LB broth containing 0.1 mg / mL ampicillin sodium). The plates were incubated at 30°C and 180 rpm for 12 h. 1% of the liquid culture volume from the 96-well A plates was transferred to 96-well B plates (each well containing 800 μL of LB broth containing 0.1 mg / mL ampicillin sodium). The plates were incubated at 37°C and 180 rpm for 8 h. Eight wells were randomly selected, and 200 μL of bacterial culture was collected to measure OD600 and evaluate the growth status of each mutant. If the OD600 values ​​of each well are basically the same (defined as an OD600 value difference ≤ 0.1, OD600 ≈ 0.8), add 1% soluble inducer to plate B and induce culture for 10 h in a low-temperature constant temperature shaking incubator at 16℃ and 180 rpm. Add 15% glycerol to the remaining culture medium in plate A and store the mutant library at -80℃.

[0120] After induction culture, the cells were centrifuged at 4,000 rpm for 15 min using a benchtop low-speed centrifuge (2×2×96-well rotor), and the supernatant was discarded to collect the cell pellet. 200 μL of a disruption buffer containing 0.5 mg / mL lysozyme (200 mM Tris, 500 mM sucrose, 1 mM EDTA, pH 8.0) was added to each well of the pellet to resuspend it and obtain a suspension. The suspension was incubated in a 40℃ water bath for 30 min. After incubation, centrifugation was performed, and 100 μL of the supernatant was transferred to a new shallow-bottom 96-well plate (plate C) using a multi-channel pipette. This supernatant is the crude enzyme solution of the mutant, used for enzyme activity assay. After multiple rounds of screening, a mutant with high catalytic activity, T267Q (275%), was selected.

[0121] Enzyme activity assay: Add 25 mM BR buffer and 1.25 mM MABTS to each well of plate C containing the crude mutant enzyme solution, and finally add 2 mM H2O2. Immediately detect the change in absorbance at 420 nm within 10 min using a microplate reader. The mutant activity is assessed using E-PcLiP activity as 100% (abbreviated as LiP). Figure 10 The results showed that the activity of the site mutant B5 was significantly higher than that of E-LiP, reaching a maximum of 275 ± 4.2%, while the activity of B11 was the lowest, only 13 ± 5.1% of that of E-LiP. Subsequently, the specificity of mutant B5 was verified using P2-F and P2-R primers, and sequencing was performed. Comparison of the sequencing results revealed that the activity fluctuation was due to an amino acid mutation. The B5 mutant was a mutation at position 267, where threonine was replaced with asparagine.

[0122] Third round of combined mutations

[0123] To further enhance the catalytic activity of the mutant enzyme, the superior single-point mutants (H38A, T267Q, and E320A) obtained from the first two rounds of screening were combined by stacking enzyme activities. First, using the DH5α-pET-22b-LiP recombinant plasmid as a template, a mutation primer P4 was designed to introduce the mutation at the T267Q site. Whole-plasmid PCR amplification was then performed to obtain the amplification product. Next, the whole-plasmid PCR amplification product was treated with the restriction endonuclease DpnI. After selectively digesting the original template plasmid, the DpnI-treated (mutant-containing) PCR product was transformed into E. coli DH5α competent cells. Plasmid extraction yielded the DH5α-pET-22b-LiP-T267Q mutant plasmid. Using the DH5α-pET-22b-LiP-T267Q mutant plasmid as a template, H38A mutant primers were added, and the full-plasmid PCR products of T267Q-H38A were amplified separately. Then, the above PCR products were treated with the restriction endonuclease DpnI. After selectively digesting the original template plasmid, the DpnI-treated (mutant) PCR products were transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the mutant plasmid DH5α-pET-22b-LiP-T267Q-H38A recombinant plasmid.

[0124] Finally, using the recombinant plasmid DH5α-pET-22b-LiP-T267Q-H38A as a template, a one-step full-plasmid PCR was performed using the E320A mutant primers to amplify the full-plasmid PCR product T267Q-H38A-E320A. The PCR product was then treated with the restriction endonuclease DpnI to selectively digest the original template. The DpnI-treated (mutant) PCR product was transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the mutant plasmid DH5α-pET-22b-LiP-H38A-T267Q-E320A. The lignin peroxidase mutant gene fragment carried by this plasmid is designated LiP-L4, and its nucleotide sequence is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4.

[0125] The whole plasmid PCR system contained 1 μL of plasmid as template, 1 μL each of forward and reverse primers F and R, 7 μL of dd H2O, and 10 μL of 2 x Primer Star Max; the sequences of the P4 primers are as follows:

[0126] F:TCATTTGTTAATAATCAGCAGAAACTGCAGGAAGATT;

[0127] R: CTGATTATTAACAAATGACTGCCATTCGCATGCT.

[0128] Whole plasmid PCR program: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 80 s, 30 cycles; 72℃ final extension for 10 min.

[0129] The recombinant mutant plasmid was transformed into E. coli BL21(DE3) competent cells to construct the recombinant lignin peroxidase mutant strain E. coli BL21(DE3)-pET-22b-LiP-H38A-T267Q-E320A. The constructed recombinant lignin peroxidase mutant strain E. coli BL21(DE3)-pET-22b-LiP-H38A-T267Q-E320A was inoculated at a 1% inoculum into 5 mL LB medium containing 0.1 mg / mL ampicillin sodium and cultured at 37℃ and 180 rpm for 12 h to obtain the culture medium. The culture medium was then inoculated at a 1% inoculum into 100 mL of LB liquid medium containing 0.1 mg / mL ampicillin sodium and cultured at 37 °C and 180 rpm. When the OD600 reached 0.8, 1% soluble inducer (2.5% Triton X-100, 2.5% Tween 80, 10% ethanol, 5% glycerol, 50 mM CaCl2, 3 mM IPTG) was added and cultured at 16 °C for 12 h. After culture, the bacterial pellet was collected by centrifugation. 1 g of the bacterial pellet was resuspended in 5 mL of disruption buffer (200 mM Tris, 500 mM sucrose, 1 mM EDTA, pH 8.0) containing lysozyme (0.5 mg / mL). The resuspended solution was incubated at 37 °C for 40 min and then centrifuged again. The supernatant was collected, and the resulting recombinant lignin peroxidase enzyme preparation was labeled as enzyme preparation H38A-T267Q-E320A.

[0130] Enzyme activity assay: The enzyme activity of the H38A-T267Q-E320A recombinant protein was detected using the ABTS (2,2-azido-di-3-ethylbenzothiazoline-6-sulfonic acid) method via microplate reader. The activity of the mutant was measured using E-LiP activity as 100% (LiP for short). Figure 11 As shown, H38A-T267Q-E320A exhibited the highest activity compared to LiP, reaching 391% + 10.5%, and was designated as L4. The nucleotide sequence of L4 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0131] Example 2: Molecular Dynamics Simulation

[0132] Molecular dynamics simulations were used to explore the conformational equilibrium mechanism of proteins and analyze the impact of mutations on protein structural dynamics. A protein-solvent system was established based on the CHARMMM all-atom force field. After pretreatment, GROMACS was used to perform 100 ns molecular dynamics simulations. Root mean square deviation (RMSD) was used to assess structural stability, and root mean square fluctuation (RMSF) was used to assess the flexibility of the regions. Solvent-accessible surface area (SASA) was calculated to analyze molecular interactions, and spatial topological analysis was performed using radius of gyration (Rg). Theoretically, this verified that the L4 mutation site enhances enzyme activity. WT represents wild-type B1 protein. Specific results are as follows:

[0133] By comparing the RMSD convergence thresholds of wild-type (WT) and mutant systems, the regulatory role of mutations on protein structural stability can be revealed. Figure 12 As shown, the average RMSD values ​​of the wild-type WT and mutant L4 protein-ligand complexes were 0.2935±0.0422 and 0.2362±0.0187 nm, respectively, both reaching equilibrium after 80 ns, indicating that the entire simulation process was stable and reliable. The mutant L4 exhibited a lower RMSD value, suggesting enhanced structural stability after mutation.

[0134] In the gmx SASA module, SASA values ​​specific to the whole protein and functional domains were recorded at 10 ps intervals to assess the conformational stability of the protein and ligands. Figure 13 As shown, there was no significant difference in SASA values ​​between the wild-type WT and mutant L4 protein ligand groups, indicating that protein-ligand binding remained stable in both systems.

[0135] The radius of cyclicity (Rg) is used as the core evaluation index for the dynamic fluctuations of protein tertiary structure to study changes in functional conformation. Figure 14 As shown, there was no significant difference in the Rg value of the protein in the wild-type WT and mutant L4 complex, indicating that the mutation did not affect the tightness of the protein structure and that the protein ligand binding was stable.

[0136] The dynamic flexibility profile of proteins was analyzed using root mean square fluctuation (RMSF) to elucidate the conformational changes induced by mutations. Figure 15 As shown, the average RMSF values ​​of proteins in the wild-type WT and mutant L4 complex were 0.1130 nm and 0.1006 nm, respectively, indicating that the L4 mutant binds more stably to small molecules.

[0137] The interaction mechanism between proteins and ligands is explored by analyzing hydrogen bonding. Figure 16As shown, the maximum number of hydrogen bonds between the protein and ligand in the wild-type WT and mutant L4 complexes were 8 and 6, respectively, and the average number of hydrogen bonds were 1.14 and 0.74, respectively, indicating that hydrogen bond interactions exist in both systems.

[0138] The binding free energy of the protein-ligand complex was quantitatively analyzed using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. Table 2 shows that the binding free energies of wild-type WT and mutant L4 in the protein-ligand complex system were -41.473 kJ / mol and -73.578 kJ / mol, respectively, both negative. This indicates that mutant L4 binds more stably to small molecules, and its main interaction energy is van der Waals force.

[0139] Table 2. Analysis of protein ligand MMPBSA

[0140]

[0141] Example 3: Preparation method of recombinant lignin peroxidase enzyme preparation expressed by Pichia pastoris;

[0142] Construction of recombinant plasmids of recombinant lignin peroxidase mutant

[0143] The L4 mutant was optimized according to the codon preference of Pichia pastoris and secreted in Pichia pastoris to prepare an enzyme preparation for application. The L4 mutant gene from Example 1 was optimized according to the codon preference of Pichia pastoris to obtain the gene encoding LiP-P4, which was ligated into the pPIC9K vector, transformed into E. coli DH5α competent cells, and the plasmid was extracted to obtain the recombinant plasmid DH5α-pPIC9K-LiP-P4; the nucleotide sequence of LiP-P4 is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0144] Construction and expression identification of recombinant lignin peroxidase engineered bacteria

[0145] The recombinant plasmids DH5α-pPIC9K-LiP and DH5α-pPIC9K-LiP-P4 were digested with the restriction endonuclease SacI, and then electroporated into Pichia pastoris GS115 competent cells at 1.5 kV. Specificity was verified as follows: Figure 17 As shown, lane 1 represents LiP, and lane 2 represents LiP-P4. These results indicate the successful construction of recombinant lignin peroxidase engineered strains P. pastoris GS115-pPIC9K-LiP and P. pastoris GS115-pPIC9K-LiP-P4, which can be used for exocrine expression in Pichia pastoris.

[0146] The recombinant lignin peroxidase engineered bacteria P. pastoris GS115-pPIC9K-LiP and P. pastoris GS115-pPIC9K-LiP-P4 were inoculated at 1% in 10 mL of LYPD medium and cultured at 30 °C with shaking at 180 rpm for 24 h to obtain the first culture medium. Subsequently, the first culture medium was inoculated at 1% in 50 mL of BMGY liquid medium for expansion culture until the OD600 reached 4 to obtain the second culture medium. Then, the cells were collected by centrifugation at 4000 rpm for 10 min, washed twice with ddH2O, and the cell pellet was resuspended in BMGY medium to obtain the resuspension. The ratio of the amount of BMGY medium used to resuspend the cell pellet was 1 g: 50 mL.

[0147] Methanol (100% concentration) was added to the resuspension for induction culture, with a final methanol concentration of 1%. 100% methanol was added every 24 hours to maintain a final methanol concentration of 1%, for a total induction culture of 96 hours. After induction, the supernatant was collected, and enzyme preparations secreted by Pichia pastoris were obtained, designated as mutant enzyme preparations P1 and P4, respectively. Expression verification of P1 and P4 is as follows... Figure 18 As shown, lane 1 (P1) and lane 2 (P4) show distinct bands at around 50 kDa, while the fermentation supernatant of lane 3 (control P. pastoris GS115-pPIC9K, stored in our laboratory) shows no bands, indicating that P1 and P4 proteins were successfully expressed.

[0148] Example 4: Preparation of lignin peroxidase enzyme preparation for alkali lignin degradation;

[0149] Based on the recombinant engineered bacteria P. pastoris GS115-pPIC9K-LiP and P. pastoris GS115-pPIC9K-LiP-P4 obtained in Example 3, recombinant lignin peroxidase preparations were prepared, respectively denoted as mutant enzyme preparations P1 and P4, for the degradation of alkali lignin. The steps are as follows:

[0150] Using alkali lignin as a substrate, a mutant enzyme preparation P4, BR buffer (pH 3), and H2O2 were added to form a reaction system, which degraded alkali lignin after the reaction. The mutant enzyme preparation P4 was added at 25% (V / V) of the total reaction system volume, i.e., the volume ratio of mutant enzyme preparation P4 to the reaction system was 1:4. The concentrations of the BR buffer, alkali lignin, and H2O2 in the reaction system were 25 mM, 1 g / L, and 2 mM. The reaction temperature was 37℃, and the reaction time was 24 h. The control group (P1) was prepared in the same manner, except that mutant enzyme preparation P4 was replaced with mutant enzyme preparation P1.

[0151] Degradation results as follows Figure 19 As shown, P4 has a better ability to degrade alkali lignin than P1, reaching 210% of P1, which is 1.1 times higher.

[0152] Example 5: Preparation of lignin peroxidase enzyme preparation for wheat straw pretreatment;

[0153] Based on the recombinant engineered bacteria P. pastoris GS115-pPIC9K-LiP and P. pastoris GS115-pPIC9K-LiP-P4 obtained in Example 3, recombinant lignin peroxidase preparations P1 or P4 were prepared for the degradation of lignin in wheat straw.

[0154] Using wheat straw as raw material, substances ①-⑧ were added to form a mixed system. The moisture content of the mixed system was controlled at 50%. After thorough mixing, the mixture was placed in a constant temperature and humidity incubator at 70% humidity for 120 hours at 37℃ for degradation. After degradation, the residual lignin content was determined using the filter bag method. The effect of LiP enzyme preparation on straw pretreatment was evaluated using the wheat straw lignin degradation rate as an indicator. The results are shown in Table 3.

[0155] After crushing the wheat straw, pass it through a 40-mesh sieve and place it in a dry environment. Weigh out 4g of wheat straw for each sample, and set up groups according to the weight of wheat straw as follows: ① Mixed enzyme; ② Mixed enzyme + 10% empty pack; ③ Mixed enzyme + 10% P1; ④ Mixed enzyme + 10% P4; ⑤ Mixed enzyme + 5% P4; ⑥ Mixed enzyme + 15% P4; ⑦ Mixed enzyme + 20% P4; ⑧ Mixed enzyme + 25% P4.

[0156] Note: Mixed enzyme composition: 3% laccase (10,000 U / g), 3% glucose oxidase (10,000 U / g), 0.2% glucanase (50,000 U / g), 0.2% xylanase (30,000 U / g), 0.2% acid cellulase (10,000 U / g), 0.2% cellobiase (10,000 U / g), 0.2% mannosase (50,000 U / g).

[0157] The empty load refers to the fermentation supernatant of P. pastoris GS115-pPIC9K.

[0158] Table 3 Lignin Degradation Rate

[0159] Group Lignin degradation rate ① 2.46％ ② 3.86％ ③ 9.26％ ④ 17.66％ ⑤ 12.68％ ⑥ 21.52％ ⑦ 26.84％ ⑧ 26.89％

[0160] The results showed that the enzyme preparation prepared in this invention can effectively degrade wheat straw lignin, and its degradation rate of wheat straw lignin is significantly improved compared with groups ① and ②.

[0161] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

[0162] sequence list

[0163] SEQ ID NO.1

[0164] LiP mature fragment nucleotide sequence (codon optimization based on E. coli)

[0165] GCACCGAATCTGGATAAACGTGTTGCGTGCCCGGATGGCGTTCATACCGCGAGCAATGCAGCGTGCTG

[0166] CGCGTGGTTTCCGGTTCTGGATGATATTCAGCAGAATCTGTTTCATGGTGGCCAGTGTGGTGCGGAAG

[0167] CGCATGAAGCGCTGCGTATTGGTTTTTCATGATAGCATTGCGATTAGCCCGAAACTGCAGAGCCAGGGT

[0168] AAATTTGGTGGTGGTGGCGCAGATGGTAGCATTATTACCTTTAGCAGCATTGAAACAACCTATCATCCG

[0169] AATATTGGTCTGGATGAAGTTGTGGCGATTCAGAAACCGTTTATTGCGAAACATGGCGTTACACGTGG

[0170] TGATTTTATTGCATTTGCAGGTGCGGTTGGCGTGTCAAATTGTCCGGGTGCGCCGCAGATGCAGTTTTT

[0171] TCTGGGCCGCCCGGAAGCGACACAGGCGGCACCTGATGGTCTGGTTCCGGAACCGTTTCATACCATTG

[0172] ATCAGGTTCTGGCACGTATGCTGGATGCGGGTGGCTTTGATGAAATTGAAACAGTTTGGCTGCTGAGC

[0173] GCACATTCAATTGCGGCGGCAAATGATGTGGACCCTACAATTTCAGGCCTGCCGTTTGATTCAACCCC

[0174] GGGCCAGTTTGATAGCCAGTTTTTTGTGGAAACCCAGCTGCGTGGTACAGCATTTCCGGGCAAAACCG

[0175] GCATTCAGGGCACAGTGATGAGCCCGCTGAAAGGTGAAATGCGCCTGCAGACCGATCATCTGTTTGC

[0176] ACGTGATAGCCGCACAGCATGCGAATGGCAGTCATTTGTTAATAATCAGACCAAACTGCAGGAAGATT

[0177] TTCAGTTTATTTTCACAGCACTGTCAACACTGGGCCATGATATGAATGCGATGACCGATTGTTCAGAAG

[0178] TGATTCCGGCACCGAAACCTGTTAATTTTGGCCCGAGCTTTTTTCCGGCAGGTAAAACCCATGCAGATA

[0179] TTGAACAGGCGTGTGCATCAACCCCGTTTCCGACACTGATTACCGCACCGGGTCCGAGCGCAAGCGT

[0180] TGCACGTATTCCGCCGCCGCCGTCACCGAATTAA

[0181] SEQ ID NO.2

[0182] Amino acid sequence encoded by mature LiP fragment

[0183] APNLDKRVACPDGVHTASNAACCAWFPVLDDIQQNLFHGGQCGAEAHEALRMVFHDSIAISPKLQSQGK

[0184] FGGGGADGSIITFSSIETTYHPNIGLDEVVAIQKPFIAKHGVTRGDFIAFAGAVGVSNCPGAPQMQFFLGRP

[0185] EATQAAPDGLVPEPFHTIDQVLARMLDAGGFDEIETVWLLSAHSIAAANDVDPTISGLPFDSTPGQFDSQF

[0186] FVETQLRGTAFPGKTGIQGTVMSPLKGEMRLQTDHLFARDSRTACEWQSFVNNQTKLQEDFQFIFTALSTLGHDMNAMTDCSEVIPAPKPVNFGPSFFPAGKTHADIEQACASTPFPTLITAPGPSASVARIPPPPSPN*SEQ ID NO.3:

[0187] Nucleotide sequence of L4

[0188] GCACCGAATCTGGATAAACGTGTTGCGTGCCCGGATGGCGTTCATACCGCGAGCAATGCAGCGTGCTG

[0189] CGCGTGGTTTCCGGTTCTGGATGATATTCAGCAGAATCTGTTTGCTGGTGGCCAGTGTGGTGCGGAAG

[0190] CGCATGAAGCGCTGCGTATGGTTTTTCATGATAGCATTGCGATTAGCCCGAAACTGCAGAGCCAGGGT

[0191] AAATTTGGTGGTGGTGGCGCAGATGGTAGCATTATTACCTTTAGCAGCATTGAAACAACCTATCATCCG

[0192] AATATTGGTCTGGATGAAGTTGTGGCGATTCAGAAACCGTTTATTGCGAAACATGGCGTTACACGTGG

[0193] TGATTTTATTGCATTTGCAGGTGCGGTTGGCGTGTCAAATTGTCCGGGTGCGCCGCAGATGCAGTTTTT

[0194] TCTGGGCCGCCCGGAAGCGACACAGGCGGCACCTGATGGTCTGGTTCCGGAACCGTTTCATACCATTG

[0195] ATCAGGTTCTGGCACGTATGCTGGATGCGGGTGGCTTTGATGAAATTGAAACAGTTTGGCTGCTGAGC

[0196] GCACATTCAATTGCGGCGGCAAATGATGTGGACCCTACAATTTCAGGCCTGCCGTTTGATTCAACCCC

[0197] GGGCCAGTTTGATAGCCAGTTTTTTGTGGAAACCCAGCTGCGTGGTACAGCATTTCCGGGCAAAACCG

[0198] GCATTCAGGGCACAGTGATGAGCCCGCTGAAAGGTGAAATGCGCCTGCAGACCGATCATCTGTTTGC

[0199] ACGTGATAGCCGCACAGCATGCGAATGGCAGTCATTTGTTAATAATCAGCAGAAACTGCAGGAAGATT

[0200] TTCAGTTTATTTTCACAGCACTGTCAACACTGGGCCATGATATGAATGCGATGACCGATTGTTCAGAAG

[0201] TGATTCCGGCACCGAAACCTGTTAATTTTGGCCCGAGCTTTTTTCCGGCAGGTAAAACCCATGCAGATA

[0202] TTGCACAGGCGTGTGCATCAACCCCGTTTCCGACACTGATTACCGCACCGGGTCCGAGCGCAAGCGTT

[0203] GCACGTATTCCGCCGCCGCCGTCACCGAATTAA

[0204] SEQ ID NO.4:

[0205] Amino acid sequence of L4

[0206] APNLDKRVACPDGVHTASNAACCAWFPVLDDIQQNLFAGGQCGAEAHEALRMVFHDSIAISPKLQSQGK

[0207] FGGGGADGSIITFSSIETTYHPNIGLDEVVAIQKPFIAKHGVTRGDFIAFAGAVGVSNCPGAPQMQFFLGRP

[0208] EATQAAPDGLVPEPFHTIDQVLARMLDAGGFDEIETVWLLSAHSIAAANDVDPTISGLPFDSTPGQFDSQF

[0209] FVETQLRGTAFPGKTGIQGTVMSPLKGEMRLQTDHLFARDSRTACEWQSFVNNQQKLQEDFQFIFTALSTLGHDMNAMTDCSEVIPAPKPVNFGPSFFPAGKTHADIAQACASTPFPTLITAPGPSASVARIPPPPSPN*SEQID NO.5

[0210] Nucleotide sequence of P4 (suitable for Pichia pastoris expression)

[0211] GCTCCAAATTTGGATAAGAGAGTTGCTTGTCCTGATGGTGTTCATACTGCTTCTAATGCTGCTTGTTGT

[0212] GCTTGGTTTCCAGTTTTGGATGATATTCAACAGAACTTGTTCGCCGGTGGTCAATGTGGTGCTGAAGCT

[0213] CATGAAGCTCTTAGAATGGTTTTCCATGATTCTATCGCTATCTCTCCAAAGTTGCAATCTCAAGGTAAAT

[0214] TCGGTGGTGGTGGTGCTGATGGTTCTATTATTACTTTCTCTTCCATCGAGACCACTTATCATCCTAACAT

[0215] TGGTTTGGATGAGGTTGTTGCTATTCAAAAGCCTTTCATTGCTAAGCATGGTGTTACTAGAGGTGACTT

[0216] TATTGCTTTCGCTGGTGCTGTTGGTGTTTCTAACTGTCCTGGTGCTCCTCAAATGCAATTCTTCTTGGGT

[0217] AGACCTGAAGCTACTCAAGCTGCTCCAGATGGTTTGGTTCCTGAACCATTTCATACTATTGACCAAGTT

[0218] CTGGCTAGAATGTTGGATGCTGGTGGTTTCGATGAAATTGAAACTGTTTGGTTGCTGTCTGCTCATTCT

[0219] ATTGCTGCTGCTAATGATGTTGATCCAACTATTTCTGGTCTGCCTTTTGATTCTACTCCTGGTCAATTCG

[0220] ATTCTCAATTTTTCGTTGAGACCCAATTGAGAGGTACTGCTTTTCCAGGTAAAACTGGTATTCAAGGTA

[0221] CTGTTATGTCCCCATTGAAAGGTGAAATGAGATTGCAAACTGACCATTTGTTCGCTAGAGATTCTAGAA

[0222] CTGCTTGTGAATGGCAATCTTTCGTTAATAACCAGCAGAAGCTGCAAGAAGATTTCCAATTCATTTTCA

[0223] CCGCTTTGTCTACTTTGGGTCATGATATGAATGCTATGACTGATTGTTCCGAAGTTATCCCTGCTCCTAA

[0224] GCCTGTTAACTTTGGTCCATCTTTCTTTCCAGCTGGTAAAACTCATGCTGATATTGCACAAGCCTGTGC

[0225] TTCTACTCCTTTCCCAACTTTGATTACTGCTCCAGGTCCATCTGCTTCTGTTGCTAGAATCCCTCCACCA

[0226] CCATCTCCTAATTAA

[0227] SEQ ID NO.6

[0228] Amino acid sequence of P4

[0229] APNLDKRVACPDGVHTASNAACCAWFPVLDDIQQNLFAGGQCGAEAHEALRMVFHDSIAISPKLQSQGK

[0230] FGGGGADGSIITFSSIETTYHPNIGLDEVVAIQKPFIAKHGVTRGDFIAFAGAVGVSNCPGAPQMQFFLGRP

[0231] EATQAAPDGLVPEPFHTIDQVLARMLDAGGFDEIETVWLLSAHSIAAANDVDPTISGLPFDSTPGQFDSQF

[0232] FVETQLRGTAFPGKTGIQGTVMSPLKGEMRLQTDHLFARDSRTACEWQSFVNNQQKLQEDFQFIFTALSTLGHDMNAMTDCSEVIPAPKPVNFGPSFFPAGKTHADIAQACASTPFPTLITAPGPSASVARIPPPPSPN*

Claims

1. A semi-rational design modified lignin peroxidase mutant, characterized in that, The lignin peroxidase mutant is a LiP-P4 mutant, and the amino acid sequence of the mutant is shown in SEQ ID NO.

6.

2. A gene encoding the lignin peroxidase mutant of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

5.

3. A recombinant vector carrying the gene of claim 2.

4. A recombinant engineered bacterium expressing a lignin peroxidase mutant, characterized in that, The recombinant engineered bacteria comprises the recombinant vector as described in claim 3.

5. The application of the mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant engineered bacteria of claim 4 in the preparation of highly active enzyme preparations.

6. A method for preparing a highly active enzyme preparation, characterized in that, The recombinant engineered bacteria described in claim 4 are fermented and cultured, and the extracellular culture medium is collected to obtain a highly active enzyme preparation.

7. The method for preparing the enzyme preparation according to claim 6, characterized in that, The specific steps are as follows: Inoculate the recombinant engineered bacteria into YPD medium at an inoculation rate of 1-5%, and culture at 25-30℃ and 180 rpm for 16-24 h with shaking. After centrifugation, collect the bacterial cell pellet and transfer it to BMGY liquid medium at an inoculation rate of 1-5% for expansion culture. After culturing until the OD600 reaches 2-6, centrifuge to collect the bacterial cell pellet. After washing with ddH2O, collect the washed bacterial cell pellet and resuspend it in BMMY medium to obtain the resuspension. The ratio of the amount of washed bacterial cell pellet to BMMY medium is 1 g: 50 mL. Methanol was added to the resuspended solution for induction culture, with a final methanol concentration of 0.5-1%. Methanol was added every 24 hours to maintain the concentration at 0.5-1%, and the induction culture was carried out for a total of 84-120 hours. After induction, the supernatant was collected to obtain the enzyme preparation.

8. Use of the enzyme preparation prepared according to claim 6 or 7 for the degradation of alkali lignin or straw lignin.

9. The use according to claim 8, characterized in that, The steps for degrading alkali lignin are as follows: Using alkali lignin as a substrate, an enzyme preparation, a BR buffer solution with a pH of 3-4, and H2O2 are added to form a reaction system, which can achieve the degradation of alkali lignin after the reaction. The amount of enzyme preparation added is 25% of the total volume of the reaction system, that is, the volume ratio of enzyme preparation to reaction system is 1:

4. The concentration of BR buffer solution in the reaction system is 25-100 mM, the concentration of alkali lignin is 0.8-1.2 g / L, and the concentration of H2O2 is 2-4 mM. The reaction temperature is 30-40℃, and the reaction time is 18-36 h.

10. The use according to claim 8, characterized in that, The steps for degrading straw lignin are as follows: Using wheat straw as raw material, adding enzyme preparations, and synergistically combining laccase, glucose oxidase, and cellulase to form a mixed system, controlling the moisture content of the mixed system, and treating it under certain temperature conditions can degrade lignin in wheat straw; Based on the mass of wheat straw: the ratio of enzyme preparation to wheat straw is 5~30ml:100g, the amount of laccase added is 1~5%; the amount of glucose oxidase added is 1~5%, and the amount of cellulase system added is 0.4~2%, wherein the cellulase system is composed of glucanase, acid cellulase, xylanase, mannanase and cellobiase mixed by mass; the water content of the mixed system is controlled at 40%~60%, the temperature is 30~40℃, and the treatment time is 70~120 h.

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