Lytic polysaccharide monooxygenase mutants and their use in degrading straw

Abstract

The application provides a lytic polysaccharide monooxygenase mutant and application thereof in degradation of straw, and belongs to the technical field of enzyme engineering and biomass conversion. The application performs homologous modeling and molecular docking on MtC1LPMO to identify key neutral residues in the active center range of the substrate binding surface, and obtains the lytic polysaccharide monooxygenase mutant through steps of virtual screening, site-directed mutagenesis, enzyme activity verification and molecular dynamics simulation analysis. The amino acid sequence of the lytic polysaccharide monooxygenase mutant is shown in SEQ ID No: 4 or SEQ ID No: 6, the catalytic activity of the mutant is 151% and 190% of the wild type respectively, and the mutant has good application in degradation of lignocellulose. The application further constructs an immobilized enzyme preparation of the spore surface-displayed lytic polysaccharide monooxygenase mutant, which can be directly used after feeding without complex purification, and the spore has strong stress resistance and can be repeatedly used, thereby significantly reducing the cost of industrial application.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and biomass conversion technology, specifically relating to a polysaccharide cleavage monooxygenase mutant and its application in the degradation of straw. Background Technology

[0002] Lignocellulose is the most abundant renewable biomass resource on Earth, and its efficient saccharification is the core of the second-generation bioenergy industry. However, the highly ordered crystalline regions of cellulose severely hinder the hydrolysis efficiency of conventional cellulase systems, becoming a key rate-limiting step in biomass conversion. Lignocellulose monooxygenase (LPMO) is a copper-dependent oxidase that can destroy the crystalline regions of cellulose through oxidative cleavage, improving the accessibility of cellulase and serving as an important coenzyme for lignocellulose saccharification. The catalytic process of LPMO involves the activation of molecular oxygen by the copper active site and the oxidative cleavage of glycosidic bonds. This process is jointly regulated by the local electrostatic environment of the active site, the substrate binding mode, and the electron transfer efficiency. However, the catalytic efficiency of wild-type LPMO still has considerable room for improvement: the local electrostatic potential distribution of its active site and the surface charge characteristics of the substrate binding surface have not been directionally optimized, limiting the surface adsorption and stabilization efficiency of the substrate polysaccharide chain, thus affecting the catalytic turnover rate. In addition, free LPMO generally suffers from difficulties in recovery, poor stability, and high purification costs in industrial applications, severely restricting its large-scale application.

[0003] Currently, LPMO molecular modification research has made some progress, including rational design optimization of substrate-binding region surface conformation based on crystal structure, copper coordination environment stability modification, and substrate-binding module fusion. However, LPMO molecular modification is limited by the dependence of the LPMO catalytic system on copper ions, reducing agents, and specific reaction conditions, which makes it difficult to establish high-throughput directed evolution screening systems for LPMO, and related reports are still relatively limited. Existing structure-based modification strategies mostly focus on the geometric matching of active sites, hydrogen bond networks, and optimization of hydrophobic interactions. Although strategies to introduce charged residues through site-directed or random mutations to change the enzyme surface charge characteristics or active site microenvironment have been reported in glycoside hydrolases and lyases, these strategies mostly target the overall surface charge distribution of the enzyme, optimal pH, or solvent tolerance, or are based on random screening rather than rational design based on electrostatic potential calculations. In addition, there are no systematic research reports on the rational design of targeting the local electric field of the LPMO active site and regulating the local electrostatic environment by introducing negative charges to improve catalytic efficiency. Therefore, it is necessary to rationally design and develop a polysaccharide cleavage monooxygenase mutant and its enzyme preparation with high catalytic activity, good stability, and recyclability, and to use it for the degradation of lignocellulose. Summary of the Invention

[0004] To address the shortcomings of existing LPMO rational design strategies, which are mostly limited to geometric configuration optimization, insufficient regulation of the electrostatic microenvironment of the active site, and limited downstream applications, this invention provides a polysaccharide monooxygenase mutant and its application in straw degradation. This invention proposes a negative charge introduction design strategy by using homology modeling and molecular docking identification of key neutral residues within the active site range of the substrate binding surface of MtC1LPMO. This strategy involves directionally mutating neutral residues near the active site into negatively charged glutamic acid and aspartic acid, thereby altering the local electrostatic potential of the active site and thus regulating substrate binding affinity and catalytic reaction energy barriers. Through virtual screening, site-directed mutagenesis, enzyme activity verification, and molecular dynamics simulation analysis, a polysaccharide monooxygenase mutant with significantly enhanced catalytic activity was obtained. The amino acid sequence of the polysaccharide monooxygenase mutant is shown in SEQ ID No:4 or SEQ ID. As shown in No:6, the catalytic activity of the mutant is 151% and 190% of that of the wild type, respectively, and it has good application in the degradation of lignocellulose. The present invention also constructs an immobilized enzyme preparation that displays a polysaccharide monooxygenase mutant on the surface of the spore. It can be directly fed into the substrate without complicated purification. Moreover, the spores are highly resistant to stress and can be reused, which significantly reduces the cost of industrial application.

[0005] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0006] This invention first provides a polysaccharide cleavage monooxygenase mutant, which is based on SEQ ID No:2 and undergoes any one or more of the following mutations:

[0007] (a) Mutate the alanine residue at position 144 to glutamic acid;

[0008] (b) Mutate the asparagine residue at position 114 to glutamic acid.

[0009] Preferably, the amino acid sequence of the polysaccharide monooxygenase mutant is as shown in SEQ ID No:4 or SEQ ID No:6.

[0010] The present invention also provides biological materials, said biological materials including any one of polynucleotides, expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria;

[0011] The polynucleotide encodes the polysaccharide cleavage monooxygenase mutant;

[0012] The expression cassette contains the polynucleotide;

[0013] The recombinant vector carries the gene or carries the expression cassette;

[0014] The recombinant cells or bacteria carry the gene or expression cassette, or contain the recombinant vector, or are capable of expressing the polysaccharide monooxygenase mutant.

[0015] Preferably, the nucleotide sequence of the polynucleotide includes SEQ ID No:3, SEQ ID No:5, or their degenerate sequences.

[0016] Preferably, the recombinant vector comprises the Escherichia coli-Bacillus subtilis shuttle expression vector pHS-cotB and a polynucleotide encoding the cleavage polysaccharide monooxygenase mutant; the polynucleotide is fused with the CotB spore coat anchoring domain for expression.

[0017] Preferably, the recombinant cells or recombinant bacteria include engineered bacteria displayed on the surface of spores.

[0018] The present invention also provides an enzyme preparation comprising the polysaccharide cleavage monooxygenase mutant.

[0019] Preferably, the enzyme preparation comprises an immobilized enzyme preparation displaying a polysaccharide monooxygenase mutant on the spore surface;

[0020] The method for preparing the immobilized enzyme preparation includes: fermentation using recombinant cells or recombinant bacteria from the biomaterial.

[0021] The present invention also provides the application of the cleavage polysaccharide monooxygenase mutant, or the biomaterial, or the enzyme preparation in the degradation of lignocellulose.

[0022] Preferably, the source of lignocellulose includes wheat straw.

[0023] The present invention also provides a method for degrading lignocellulose, the method comprising: adding the polysaccharide monooxygenase mutant, or the biomaterial, or the enzyme preparation to lignocellulose.

[0024] Preferably, the method includes:

[0025] The substrate particles containing lignocellulose, immobilized enzyme preparations, mixed enzyme systems, electron donor ascorbic acid, and Cu were used. 2+ The mixture was used to form a reaction system, and the water content of the reaction system was controlled. The degradation was carried out at 37℃ for 72 h.

[0026] The mixed enzyme system comprises a mixture of mannanase, glucanase, acid cellulase, cellobiase, and xylanase in a mass ratio of 1:1:1:1:1.

[0027] Preferably, the water content of the reaction system is controlled at 65%, and the degradation is carried out at 37°C for 72 hours.

[0028] Based on the mass of substrate particles containing lignocellulose, the immobilized enzyme preparation was added at 20% (v / m, mL / g), the mixed enzyme system was added at 0.3% (m / m), the final ascorbic acid concentration was 4 mM, and Cu... 2+ The final concentration was 0.075 mM.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) Significantly enhanced catalytic activity: The enzyme activity of the polysaccharide monooxygenase mutant A144E described in this invention reaches 190% of that of the wild type, and the polysaccharide monooxygenase mutant N114E reaches 151% of that of the wild type, breaking through the catalytic efficiency bottleneck of wild-type LPMO.

[0031] (2) In-depth elucidation of the mechanism: 100 ns molecular dynamics simulation and free energy morphology analysis show that, while maintaining the overall folding stability of the protein, the mutation reshapes the local electrostatic environment of the substrate binding surface through a trade-off mechanism between intramolecular hydrogen bond sacrifice and intermolecular polar interaction gain, increasing the conformational flexibility of functional regions and broadening the conformational sampling space, which is beneficial to substrate induction fit and stabilization. The wild-type protein maintains about 8 intramolecular hydrogen bonds, while A144E and N114E are reduced to about 5, a decrease of nearly 40%, indicating that the mutation sacrifices local rigidity for conformational freedom. Free energy landscape analysis shows that compared with the deep and concentrated energy basin of the wild type, the free energy landscape of the mutant is significantly broadened and shallowed, the lowest energy point shifts to higher RMSD, and the conformational entropy increases.

[0032] (3) The display system has outstanding advantages: This invention uses Bacillus subtilis spore surface display technology to anchor mutant LPMO to the outer layer of the spore shell. It can be directly fed into the system without complicated purification. Moreover, the spores are highly resistant to stress and can be reused, which significantly reduces the cost of industrial applications.

[0033] (4) Clear application prospects: The engineered bacteria and mixed enzyme system displayed on the spore surface of the present invention can be used synergistically for biological pretreatment of lignocellulose substrates such as wheat straw, thereby improving the degradation rate of cellulose and making it suitable for second-generation bioenergy and biorefining industrial scenarios. Attached Figure Description

[0034] Figure 1 This is a two-dimensional interaction diagram of cellohexasaccharide docking with wild-type MtC1LPMO molecules; the amino acid residue numbers in the diagram are based on the full-length protein sequence including the signal peptide (original numbering for homology modeling), and there is a 17-position offset from the mature peptide sequence number shown in SEQ ID No:2, that is: the number in the diagram − 17 = the mature peptide sequence number.

[0035] Figure 2This is an electrophoresis image of colony PCR verification of mutant transformants; in the image, 1-9 represent colony PCR verification bands of different mutants, 1 is G29D; 2 is G29E; 3 is A150D; 4 is A150E; 5 is N114D; 6 is N114E; 7 is A114D; 8 is A114E; 9 is I66D.

[0036] Figure 3 This is a comparison chart of relative enzyme activity assays for site-directed mutant strains.

[0037] Figure 4 The RMSD curves are obtained from 100 ns molecular dynamics simulations of the wild-type and mutant protein-ligand complexes.

[0038] Figure 5 RMSF fluctuation diagrams for the simulated process of wild-type and mutant enzyme-substrate complexes.

[0039] Figure 6 The curves show the changes in the radius of gyration of wild-type and mutant proteins.

[0040] Figure 7 The curves show the changes in the number of hydrogen bonds within the molecules of wild-type and mutant molecules.

[0041] Figure 8 The curves show the changes in solvent-accessible surface area of ​​wild-type and mutant proteins.

[0042] Figure 9 A comparison of the free energy landscape of wild type (A) and mutants N114E (B) and A144E (C).

[0043] Figure 10 The electrostatic potential distribution of the surface of wild-type (A) and mutant proteins A144E (B) and N114E (C) is shown.

[0044] Figure 11 Western blot validation image of engineered bacteria displayed on the surface of Bacillus subtilis spores; in the image, lane 1 is WB800N-pHS-cotB empty vector; lane 2 is A144E; lane 3 is N114E.

[0045] Figure 12 This image shows the results of engineered bacteria catalyzing the degradation of wheat straw on the surface of spores. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental materials used in the following embodiments can be obtained commercially. Unless otherwise specified, the present invention employs existing technology in this field.

[0047] In the following examples, the enzyme activity assay method is as follows: 1 mL of reaction system contains 860 μL of 116 mM pH 6.0 phosphate buffer, 100 μL of 10 mM 2,6-DMP solution, 20 μL of 5 mM H2O2 stock solution, and 20 μL of enzyme. The enzyme activity (ε) is calculated by measuring the change in absorbance before and after 300 s of reaction at 469 nm. 469 = 53200 L·mol -1 ·cm -1 0.5 μM CuSO4 was used as a blank control. One unit of enzyme activity is defined as the production of 1 μmol of oxidation product per minute under the reaction conditions.

[0048] Example 1: Homology modeling, molecular docking and virtual screening of MtC1LPMO

[0049] Homology modeling of wild-type MtC1LPMO was performed using the SWISS-MODEL server to obtain a high-quality three-dimensional structural model. The nucleotide sequence of the wild-type MtC1LPMO is shown in SEQ ID No:1, and the amino acid sequence is shown in SEQ ID No:2.

[0050] SEQ ID No:1

[0051] CATTAATACATTACCAAGAGTTGGTACTGGTTCTGATTGGCAACATGTTAGAAGAGCTGATAATTGGCAAAATAATGGTTTTGTTGGTGACGTTAATTCTGAACAAATTAGATGTTTCCAGGCTACACCAGCTGGTGCTCAAGATGTTTATACTGTTCAAGCTGGTTCTACTGTTACATATCATGCTAATCCATCTATATATCACCCAGGTCCAATGCAATTTTATTTGGCTAGAGTTCCAGATGGTCAAGATGTTAAATCTTGGACAGGTGAAGGTGCTGTTTGGTTTAAAGTTTATGAAGAACAACCACAATTCGGTGCTCAATTAACATGGCCATCTAATGGTAAATCTTCTTTTGAAGTTCCAATCCCATCTTGTATTAGAGCTGGTAATTATTTGTTGAGAGCTGAACATATTGCTTTGCATGTTGCTCAATCTCAAGGTGGTGCTCAATTTTATATTTCTTGTGCTCAATTGCAGGTTACTGGTGGTGGTTCTACTGAACCATCTCAAAAAGTTTCTTTTCCAGGTGCTTATAAATCTACTGACCCAGGTATTTTGATTAATATTAACTACCCAGTCCCAACTTCTTATCAAAATCCAGGTCCAGCTGTTTTTAGATGTCTCGAG

[0052] SEQ ID No:2

[0053] HYTLPRVGTGSDWQHVRRADNWQNNGFVGDVNSEQIRCFQATPAGAQDVYTVQAGSTVTYHANPSIYHPGPMQFYLARVPDGQDVKSWTGEGAVWFKVYEEQPQFGAQLTWPSNGKSSFEVPIPSCIRAGNYLLRAEHIALHVAQSQGGAQFYISCAQLQVTGGGSTEPSQKVSFPGAYKSTDPGILININYPVPTSYQNPGPAVFRCLE

[0054] Using the three-dimensional structure of cellohexasaccharide downloaded from the PubChem database as a ligand, molecular docking was performed using the molecular simulation software Discoverystudio 2019. Before docking, the protein underwent pretreatment including dehydration, hydrogenation, and CHARMm force field optimization; then, potential binding sites for the cellohexasaccharide ligand were identified and docking was performed. Non-bonded interactions were analyzed after docking (docking results are shown in Figure 1). Figure 1 As shown in the figure, TYR67 forms a conventional hydrogen bond with the substrate, playing a key role in substrate localization; GLN 151 exhibits unfavorable donor-donor interactions with the substrate, which may hinder stable substrate binding; HIS 1, HIS 68, and HIS 142 constitute a copper-coordinated histidine scaffold; PHE 27, GLY29, VAL 143, PRO 64, SER65, GLN147, and GLY148 residues are distributed around the substrate, interacting with the substrate through van der Waals forces or C-H bonds.

[0055] Using amino acid residues within the cellohexasaccharide binding site as candidate mutation sites, and excluding HIS1, HIS68, and HIS142 which directly participate in copper coordination, and PHE 27 which may cause steric hindrance, eight mutation targets were identified: G29, S65, I66, Y67, Q147, N114, A144, and A150. Among these, although I66, N114, A144, and A150 did not have specific interaction types marked in the docking two-dimensional planar diagram, their side chains are all located within the substrate binding surface and are polar, neutral, or hydrophobic residues, making them suitable as mutation targets for introducing negative charge. Although Y67 forms conventional hydrogen bonds with the substrate, it was also included in the mutation library as a probe site at the strategy boundary to examine the tolerance of the negative charge introduction strategy at key substrate anchoring sites. Although the amide side chain of GLN151 exhibits unfavorable donor-donor interactions with the substrate, it may also participate in the maintenance of the local hydrogen bond network at the active site. Given that the net effect of negative charge introduction on this site is difficult to predict, and the strategy at this stage focuses on neutral residues with clearly defined electrostatic effects, it was not included in the mutant library.

[0056] In summary, this embodiment, based on SWISS-MODEL homology modeling and Discovery Studio 2019 molecular docking, elucidated the key non-bonded interactions between cellohexasaccharide and MtC1LPMO after docking, clarifying the spatial distribution and action patterns of the copper coordination center histidine scaffold (HIS1, HIS68, HIS142), substrate anchoring site (TYR67), and surrounding residues (PHE27, GLY29, SER65, etc.). Furthermore, based on the rational design strategy of "local electrostatic microenvironment regulation of the active site," eight neutral residue targets suitable for negative charge introduction (G29, S65, I66, Y67, Q147, N114, A144, A150) were screened and identified, providing structural basis for subsequent site-directed mutant construction and enzyme activity verification.

[0057] Example 2: Site-directed mutagenesis, induced expression, and enzyme activity verification

[0058] Based on the virtual screening results of Example 1, this example designs site-directed mutagenesis primers for G29D, G29E, S65D, S65E, Y67D, Y67E, Q147D, Q147E, I66D, I66E, A150D, A150E, A144D, A144E, N114D, and N114E, respectively. The site-directed mutagenesis primers are shown in Table 1.

[0059] Table 1. Site-directed mutagenesis primer sequences

[0060]

[0061] Using the pET-22b(+) expression vector as a vector, a recombinant plasmid pET-22b-MtC1LPMO carrying the MtC1LPMO mature peptide encoding gene shown in SEQ ID No:1 was constructed. In the recombinant plasmid pET-22b-MtC1LPMO, the MtC1LPMO gene naturally encodes an N-terminal signal peptide (17 amino acids), and SEQ ID No:1 shows its mature peptide encoding sequence after removing the signal peptide; this sequence was cloned into the corresponding multiple cloning site of pET-22b(+) after double digestion with Nco I / Xho I, and fused in the same frame with the pelB leader sequence contained in the vector.

[0062] Using recombinant plasmid pET-22b-MtC1LPMO as a template, whole-plasmid PCR amplification was performed using high-fidelity DNA polymerase. The PCR product was treated with Dpn I restriction endonuclease at 37°C for 30 min to degrade the methylated template, followed by heat transformation into *E. coli* BL21(DE3) competent cells. The cells were then plated on LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C. Positive clones were picked the following day for colony PCR verification and sequencing confirmation. PCR gel verification results are shown below. Figure 2 .

[0063] The verified mutant strain was inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C and 200 rpm until OD500. 600 Approximately 0.6 to 0.8 mg of IPTG was added to a final concentration of 0.5 mM, and the mixture was induced at 37°C for 12 h. The cells were collected by centrifugation, sonicated, and the supernatant was used for Western blot verification and enzyme activity assay. Before the assay, the enzyme solution was fully incubated with copper ions to achieve copper saturation. The relative enzyme activity of each mutant was calculated using the wild-type MtC1LPMO enzyme activity as 100%.

[0064] Enzyme activity assay results ( Figure 3 The results showed that the relative enzyme activity of the A144E mutant reached 190% of that of the wild type, and that of the N114E mutant reached 151% of that of the wild type. Other sites, such as G29D / E, S65D / E, Y67D / E, Q147D / E, I66D / E, A150D / E, A144D, and N114D, did not show a significant increase in activity, indicating that the effectiveness of the negative charge introduction strategy is highly dependent on the local microenvironment of the site.

[0065] As can be seen, based on the 8 candidate sites determined in Example 1, this embodiment constructed 16 single-point mutants (G29D / E, S65D / E, Y67D / E, Q147D / E, I66D / E, A150D / E, A144D / E, N114D / E). After induction expression and enzyme activity verification, only the A144E and N114E mutants showed significant activity enhancement, reaching 190% and 151% of the wild type, respectively. The other mutants did not show significant activity enhancement. The nucleotide sequence of the N114E mutant is shown in SEQ ID No:3, and the amino acid sequence is shown in SEQ ID No:4; the nucleotide sequence of the A144E mutant is shown in SEQ ID No:5, and the amino acid sequence is shown in SEQ ID No:6.

[0066] SEQ ID No:3

[0067] CATTAATACATTACCAAGAGTTGGTACTGGTTCTGATTGGCAACATGTTAGAAGAGCTGATAATTGGCAAAATAATGGTTTTGTTGGTGACGTTAATTCTGAACAAATTAGATGTTTCCAGGCTACACCAGCTGGTGCTCAAGATGTTTATACTGTTCAAGCTGGTTCTACTGTTACATATCATGCTAATCCATCTATATATCACCCAGGTCCAATGCAATTTTATTTGGCTAGAGTTCCAGATGGTCAAGATGTTAAATCTTGGACAGGTGAAGGTGCTGTTTGGTTTAAAGTTTATGAAGAACAACCACAATTCGGTGCTCAATTAACATGGCCATCTGAGGGTAAATCTTCTTTTGAAGTTCCAATCCCATCTTGTATTAGAGCTGGTAATTATTTGTTGAGAGCTGAACATATTGCTTTGCATGTTGCTCAATCTCAAGGTGGTGCTCAATTTTATATTTCTTGTGCTCAATTGCAGGTTACTGGTGGTGGTTCTACTGAACCATCTCAAAAAGTTTCTTTTCCAGGTGCTTATAAATCTACTGACCCAGGTATTTTGATTAATATTAACTACCCAGTCCCAACTTCTTATCAAAATCCAGGTCCAGCTGTTTTTAGATGTCTCGAG

[0068] SEQ ID No:4

[0069] HYTLPRVGTGSDWQHVRRADNWQNNGFVGDVNSEQIRCFQATPAGAQDVYTVQAGSTVTYHANPSIYHPGPMQFYLARVPDGQDVKSWTGEGAVWFKVYEEQPQFGAQLTWPSEGKSSFEVPIPSCIRAGNYLLRAEHIALHVAQSQGGAQFYISCAQLQVTGGGSTEPSQKVSFPGAYKSTDPGILININYPVPTSYQNPGPAVFRCLE

[0070] SEQ ID No:5

[0071] CATTAATACATTACCAAGAGTTGGTACTGGTTCTGATTGGCAACATGTTAGAAGAGCTGATAATTGGCAAAATAATGGTTTTGTTGGTGACGTTAATTCTGAACAAATTAGATGTTTCCAGGCTACACCAGCTGGTGCTCAAGATGTTTATACTGTTCAAGCTGGTTCTACTGTTACATATCATGCTAATCCATCTATATATCACCCAGGTCCAATGCAATTTTATTTGGCTAGAGTTCCAGATGGTCAAGATGTTAAATCTTGGACAGGTGAAGGTGCTGTTTGGTTTAAAGTTTATGAAGAACAACCACAATTCGGTGCTCAATTAACATGGCCATCTAATGGTAAATCTTCTTTTGAAGTTCCAATCCCATCTTGTATTAGAGCTGGTAATTATTTGTTGAGAGCTGAACATATTGCTTTGCATGTTGAGCAATCTCAAGGTGGTGCTCAATTTTATATTTCTTGTGCTCAATTGCAGGTTACTGGTGGTGGTTCTACTGAACCATCTCAAAAAGTTTCTTTTCCAGGTGCTTATAAATCTACTGACCCAGGTATTTTGATTAATATTAACTACCCAGTCCCAACTTCTTATCAAAATCCAGGTCCAGCTGTTTTTAGATGTCTCGAG

[0072] SEQ ID No:6

[0073] HYTLPRVGTGSDWQHVRRADNWQNNGFVGDVNSEQIRCFQATPAGAQDVYTVQAGSTVTYHANPSIYHPGPMQFYLARVPDGQDVKSWTGEGAVWFKVYEEQPQFGAQLTWPSNGKSSFEVPIPSCIRAGNYLLRAEHIALHVEQSQGGAQFYISCAQLQVTGGGSTEPSQKVSFPGAYKSTDPGILININYPVPTSYQNPGPAVFRCLE

[0074] In summary, the negative charge introduction strategy is highly dependent on the local microenvironment at the substrate binding site, and not all neutral residues on the substrate binding surface are suitable for this strategy. Based on the above enzyme activity screening results, A144E and N114E, which showed significant enhancements in catalytic activity, were selected for subsequent molecular dynamics simulation mechanism analysis and the construction and application verification of immobilized enzyme preparations displayed on spore surfaces.

[0075] Example 3: Molecular Dynamics Simulation and Free Energy Analysis

[0076] To elucidate the molecular mechanism of the enhanced activity of mutants A144E and N114E obtained in Example 2, this example uses the Gromacs 2024.4 package to conduct 100 ns molecular dynamics simulations.

[0077] The system setup for the molecular dynamics simulation is as follows:

[0078] Protein: Amber 99 sb all-atomic force field;

[0079] Ligand cellohexasaccharide: AM1-BCC charge calculated using Acpype tool and matched to Gaff 2 force field;

[0080] Solvent: TIP4P-EW water model, with cubic periodic water box boundaries at least 1 nm from the composite surface; Na added. + and Cl - Neutralize the system charge.

[0081] Furthermore, the electrostatic and van der Waals cutoff distances were set to 1.4 nm, and the long-range electrostatics were simulated using the PME method with a time step of 2 fs. The simulation procedure included steepest descent energy minimization, NVT ensemble 300 K equilibrium 250 ps, ​​NPT ensemble 300 K and 1 Bar equilibrium 250 ps, ​​and 100 ns conventional molecular dynamics simulation. The simulation results are as follows: Figure 4-10 As shown.

[0082] As shown in the figure, in terms of overall stability, the RMSD of wild-type, A144E, and N114E all reached thermodynamic equilibrium after 20 ns, with the equilibrium RMSD values ​​stabilizing in the range of 0.15 to 0.20 nm. The RMSD curves of the three types highly overlapped, and no significant conformational transition events were observed, indicating that the mutation did not disrupt the overall LPMO framework stability. The RMSD fluctuations of A144E and N114E were slightly larger than those of the wild-type, suggesting that the mutation introduced a certain degree of conformational heterogeneity. Figure 4 ).

[0083] Regarding local flexibility, RMSF analysis revealed that the flexible region of the wild-type protein is mainly distributed in the surface loop region, while the core domain remains rigid. The A144E mutation significantly increased the RMSF value near residue 60, peaking at 0.27 nm. This position is adjacent to the mutation site (residue 144), indicating that the substitution of Ala144→Glu caused a dynamic rearrangement of the local structure. The N114E mutation mainly enhanced the flexibility of the regions around residues 100 and 210. Residue 100 is located near the substrate-binding surface of MtC1LPMO, and its enhanced flexibility may directly participate in the substrate recognition process. Both mutants showed higher fluctuations in the C-terminal region (approximately residues 180-220) compared to the wild type, suggesting that the mutation may regulate the functional dynamics of the protein through long-range allosteric effects. Figure 5 ).

[0084] Regarding structural compactness, radius of gyration (Rg) analysis showed that the Rg values ​​of the three systems fluctuated within the range of 1.63–1.67 nm, with an average difference of less than 0.01 nm. This indicates that although the mutation altered local flexibility, the overall folding compactness of LPMO was not significantly affected, and the protein maintained a stable globular conformation. Figure 6 ).

[0085] Regarding interaction networks, intramolecular hydrogen bonds ( Figure 7 Analysis revealed that the wild-type protein maintains approximately 8 intramolecular hydrogen bonds in equilibrium, while the mutants A144E and N114E have approximately 5 hydrogen bonds each, a reduction of nearly 40%. The disruption of the hydrogen bond network mainly occurs at the mutation site and its adjacent regions. In mutant A144E, the carboxyl side chain of Glu144 cannot form the original hydrophobic interaction of Ala144, and charge repulsion is introduced, leading to rearrangement of surrounding hydrogen bonds. The effect in N114E is similar, but the impact is wider, extending to the substrate binding surface. The association between reduced hydrogen bonds and increased RMSF indicates that the mutation sacrifices local rigidity for conformational freedom; this "rigidity-flexibility trade-off" provides a thermodynamic basis for dynamic sampling of functionally relevant regions. Solvent-accessible surface area (SASA) analysis further confirmed that there was no significant difference in surface exposure among the three systems, indicating that the mutation did not cause protein unfolding or large-scale conformational opening; its effect was mainly limited to the remodeling of the internal interaction network. Figure 8 ).

[0086] Regarding free energy morphology, the free energy landscape (FEL) constructed based on principal component analysis showed that the wild-type FEL exhibited a deep and concentrated energy basin, with the lowest free energy region located at RMSD≈0.15 nm and Rg≈1.645 nm. The basin edges were steep, indicating that the wild-type protein mainly sampled a single, stable conformational state with low conformational entropy. The FEL of mutant A144E showed a significantly wider and shallower energy basin, with the lowest energy point shifted towards higher RMSD (approximately 0.16–0.17 nm), and the free energy extremum increased. This indicates that the mutation broadened the conformational sampling space and stabilized the slightly open conformation deviating from the wild-type. The FEL characteristics of mutant N114E were similar to A144E, but the distribution was wider along the Rg direction. FEL analysis revealed that A144E and N114E reshaped the conformational ensemble distribution of LPMO by lowering the local energy barrier and increasing conformational entropy, which is beneficial for the induced fit process during substrate binding. Figure 9 ).

[0087] Regarding electrostatic potential remodeling, representative conformations were selected based on the MD equilibrium segment (last 50 ns), and surface electrostatic potential analysis was performed using molecular visualization software. The wild-type protein exhibited a relatively balanced surface electrostatic potential distribution, ranging from -41.58 to 41.58 kT / e. Its substrate binding surface was predominantly electrically neutral and weakly negative, without significant charge polarization. In contrast, due to the introduction of the carboxyl group in the Glu side chain, the extreme values ​​of the negative surface potential for A144E and N114E expanded to -41.89 kT / e and -43.62 kT / e, respectively, and a more significant negative potential aggregation region formed near the substrate binding surface. This spatial expansion and redistribution of the negative potential aggregation region is beneficial for enhancing the ion-dipole interaction and hydrogen bond network between the enzyme and substrate, promoting the stabilization of the polysaccharide chain on the protein surface. Figure 10 ).

[0088] Based on the above analysis, although the introduction of Glu114 and Glu144 disrupts the local intramolecular hydrogen bond network, it forms new intermolecular hydrogen bonds and ion-dipole interaction sites. This trade-off between "intramolecular hydrogen bond sacrifice and intermolecular polar interaction gain" is a microscopic manifestation of the negative charge introduction strategy for regulating the local electric field: by sacrificing local rigid constraints in exchange for conformational freedom, and simultaneously using negative charge implantation to enhance the enzyme-substrate polar interaction network, it promotes the stabilization of the substrate on the protein surface.

[0089] The above results demonstrate that this embodiment, through 100 ns molecular dynamics simulations and multidimensional thermodynamic analysis, systematically elucidates the molecular mechanism underlying the enhanced activity of A144E and N114E at the atomic scale. While maintaining overall protein folding stability, the mutants reshape the local electrostatic environment of the substrate binding surface through a "rigid-flexible trade-off" mechanism: the number of intramolecular hydrogen bonds decreases by approximately 40%, conformational entropy increases, the free energy landscape broadens and becomes shallower, and the extreme negative potential of the substrate binding surface significantly expands and forms an aggregation region. This synergistic effect promotes induced substrate binding and stabilization, providing a structure-kinetic theoretical explanation for the enhanced catalytic activity observed in Example 2 (A144E: 190%, N114E: 151%), and also providing a strategic framework for the rational design of LPMOs through "electrostatic microenvironment regulation."

[0090] Example 4: Construction of engineered bacteria for display on the surface of Bacillus subtilis

[0091] In this embodiment, either the A144E or N114E mutant was constructed into the Bacillus subtilis spore surface display system to prepare an immobilized enzyme preparation. The specific steps are as follows:

[0092] The A144E or N114E mutant gene was cloned into the Escherichia coli-Bacillus subtilis shuttle expression vector pHS-cotB, respectively, and the mutant gene was fused with the CotB spore shell anchoring domain to obtain CotB-MtC1LPMO-A144E and CotB-MtC1LPMO-N114E, respectively, ensuring that the protein is effectively anchored to the outer layer of the spore, thus obtaining the recombinant vector.

[0093] The recombinant vector was introduced into Bacillus subtilis WB800N competent cells via electroporation at 1.8 kV for 2.5 ms. After transformation, the cells were plated on LB agar plates containing 10 μg / mL each of chloramphenicol and kanamycin and incubated overnight at 37°C. Positive clones were selected for colony PCR verification and sequencing confirmation, yielding the engineered B. subtilis WB800N-pHS-cotB-MtC1LPMO-A144E and B. subtilis WB800N-pHS-cotB-MtC1LPMO-N114E displaying spore surface characteristics.

[0094] The verified engineered bacteria were inoculated into DSM liquid medium containing 10 μg / mL each of chloramphenicol and kanamycin, and cultured at 37°C and 180 rpm for 36 h. After culture, the bacterial cells were enriched by centrifugation at 8000 rpm for 10 min, and residual medium was removed. The collected precipitate was thoroughly resuspended in GTE buffer (50 mM glucose, 20 mM Tris-HCl pH 7.5, 10 mM EDTA, 2 mg / mL lysozyme) and incubated at 37°C for 30-60 min to lyse the vegetative cells. Spores were then collected by centrifugation, washed twice with PBS (pH 7.4) buffer at 4000 rpm for 10 min, and finally resuspended in PBS buffer. The OD of the spore suspension was then measured. 600 Adjusted to version 1.0 for use in subsequent experiments.

[0095] To verify the anchoring of CotB-MtC1LPMO-A144E and CotB-MtC1LPMO-N114E on the spore surface, 50 μL of the above spore suspension was resuspended in an equal volume of spore capsid protein extract (1.5% SDS, 50 mM DTT) and heated at 70 °C for 1 h to release the fusion proteins displayed on the surface from the capsid layer into the solution phase. Western blot analysis was performed using 6*his-tag mouse monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse IgG (Shanghai Sangon Biotech) as the secondary antibody. A corresponding empty vector control was also included, and the treatment was performed in parallel under identical conditions. Finally, chemiluminescence detection was performed using a kit from Yifeixue. The results showed a single specific band at the expected molecular weight, while the empty control (WB800N-pHS-cotB) showed no band, proving that CotB-MtC1LPMO-A144E and CotB-MtC1LPMO-N114E were successfully displayed on the surface of Bacillus subtilis spores. Figure 11 ), successfully obtained immobilized enzyme preparations for displaying on the surface of mutant CotB-MtC1LPMO-N114E spores and immobilized enzyme preparations for displaying on the surface of mutant CotB-MtC1LPMO-A144E spores.

[0096] In summary, this embodiment successfully constructed two engineered bacteria, *B. subtilis* WB800N-pHS-cotB-MtC1LPMO-A144E and WB800N-pHS-cotB-MtC1LPMO-N114E, for displaying spores. Western blot confirmed that the CotB-LPMO fusion protein is effectively anchored to the spore shell. This immobilized enzyme preparation can be directly collected and used without protein purification, providing a convenient enzyme preparation form for subsequent wheat straw degradation applications.

[0097] Example 5: Preparation of a mutant enzyme preparation of polysaccharide-lysing monooxygenase for wheat straw pretreatment

[0098] In this embodiment, the spore surface display engineered bacteria B. subtilis WB800N-pHS-cotB-MtC1LPMO-A144E and B. subtilis WB800N-pHS-cotB-MtC1LPMO-N114E prepared in Example 4 were used as enzyme preparations, along with a mixed enzyme, an electron donor, and Cu. 2+ The compound was used for the enzymatic pretreatment of wheat straw to investigate the ability of the polysaccharide monooxygenase mutant enzyme preparation to degrade lignocellulose.

[0099] The specific steps are as follows:

[0100] Wheat straw was crushed and passed through a 40-mesh sieve to obtain straw pellets. These pellets were then placed in a dry environment and divided into three groups: a control group, a wild-type group, and a mutant group. The reaction systems for each group were set up as follows:

[0101] (S1) Control group: straw pellets + mixed enzyme system + empty vector spore suspension (WB800N-pHS-cotB, i.e., blank spore suspension that does not show LPMO, spore suspension prepared by Bacillus subtilis WB800N carrying empty vector pHS-CotB under the same culture induction conditions).

[0102] (S2) Wild-type group: straw pellets + mixed enzyme system + wild-type CotB-MtC1LPMO enzyme preparation + ascorbic acid + Cu 2+ ;

[0103] (S3) N114E mutant group: straw pellets + mixed enzyme system + mutant CotB-MtC1LPMO-N114E spore surface display immobilized enzyme preparation + ascorbic acid + Cu 2+ .

[0104] (S4) A144E mutant group: straw pellets + mixed enzyme system + mutant CotB-MtC1LPMO-A144E spore surface display immobilized enzyme preparation + ascorbic acid + Cu 2+ .

[0105] In the control group and wild-type group, except for the different types of enzyme preparations, the dosage relationships of all other components were consistent with those in the mutant group.

[0106] Weigh 3g of straw pellets and, according to the group settings, mix the straw pellets with the reaction system, controlling the moisture content of the reaction system to 65%. After stirring evenly, place the mixture in a constant temperature and humidity incubator with a humidity of 70% and degrade at 37℃ for 72 h. After degradation, the cellulose content of the degradation products is determined using the filter bag method. The degradation rate is used as an indicator to evaluate the effect of immobilized enzyme preparation on wheat straw pretreatment.

[0107] In this reaction system, the amount of spore surface display enzyme preparation, based on the mass of straw pellets, is 20% (v / m, mL / g), meaning the ratio of spore surface display enzyme preparation to straw pellets is 20 mL: 100 g. The amount of mixed enzyme is 0.3% of the mass of straw pellets, and the mixed enzyme consists of mannanase, dextranase, xylanase, acid cellulase, and cellobiase mixed in a mass ratio of 1:1:1:1:1. The Cu in the reaction system... 2+ The concentration was 0.075 mM. The final concentration of the electron donor ascorbic acid was 4 mM.

[0108] The degradation results are shown in Table 2 and Figure 12 As shown.

[0109] Table 2. Effect of engineered bacteria on the surface of spores on catalytic degradation of wheat straw

[0110]

[0111] From Table 2 and Figure 12 The results show that the immobilized enzyme preparation, which exhibits a polysaccharide monooxygenase mutant on the spore surface, synergistically enhances the degradation rate of wheat straw cellulose with the mixed enzyme system (A144E group 37.38% vs. wild-type group 24.83% vs. control group 18.51%). This immobilized enzyme preparation can be directly used without complex purification, simplifying the industrial application process. The spores, as a natural carrier, possess good mechanical stability and environmental tolerance, providing potential advantages for enzyme immobilization, recovery, and reuse. This is expected to reduce enzyme preparation costs in large-scale applications and has application potential in the fields of second-generation bioenergy and biorefining.

[0112] In summary, this invention proposes a negative charge introduction design strategy by using homology modeling and molecular docking to identify key neutral residues within the active site range of the substrate binding surface of MtC1LPMO. This strategy involves directionally mutating neutral residues near the active site into negatively charged glutamic acid and aspartic acid. By introducing negative charges, the local electrostatic potential of the active site is altered, thereby regulating substrate binding affinity and catalytic reaction energy barriers. Through virtual screening, site-directed mutagenesis, enzyme activity verification, and molecular dynamics simulation analysis, a cleavage polysaccharide monooxygenase mutant with significantly enhanced catalytic activity was obtained. The amino acid sequence of the cleavage polysaccharide monooxygenase mutant is shown in SEQ ID No:4 or SEQ ID No:6. The catalytic activities of the mutant are 151% and 190% of the wild type, respectively, demonstrating excellent application potential in the degradation of lignocellulose. This invention also constructs an immobilized enzyme preparation displaying the cleavage polysaccharide monooxygenase mutant on the spore surface. This preparation can be directly used without complex purification, and the spores exhibit strong stress resistance and reusability, significantly reducing industrial application costs.

[0113] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A polysaccharide-lysing monooxygenase mutant, characterized in that, The polysaccharide monooxygenase mutant is based on SEQ ID No:2 and undergoes any one or more of the following mutations: (a) Mutate the alanine residue at position 144 to glutamic acid; (b) Mutate the asparagine residue at position 114 to glutamic acid.

2. The polysaccharide-lysing monooxygenase mutant according to claim 1, characterized in that, The amino acid sequence of the polysaccharide monooxygenase mutant is shown in SEQ ID No:4 or SEQ ID No:

6.

3. A biomaterial, characterized in that, The biomaterials include any one of polynucleotides, expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria; Wherein, the polynucleotide encodes the polysaccharide monooxygenase mutant of claim 1 or 2; The expression cassette contains the polynucleotide; The recombinant vector carries the gene or carries the expression cassette; The recombinant cells or bacteria carry the gene or expression cassette, or contain the recombinant vector, or are capable of expressing the polysaccharide monooxygenase mutant of claim 1 or 2.

4. The biomaterial according to claim 3, characterized in that, The nucleotide sequence of the polynucleotide includes SEQ ID No:3, SEQ ID No:5, or their degenerate sequences; The recombinant vector comprises the Escherichia coli-Bacillus subtilis shuttle expression vector pHS-cotB and a polynucleotide encoding the cleavage polysaccharide monooxygenase mutant of claim 1; the polynucleotide is expressed by fusion with the CotB spore coat anchoring domain. The recombinant cells or recombinant bacteria include engineered bacteria displayed on the surface of spores.

5. An enzyme preparation, characterized in that, The enzyme preparation comprises the polysaccharide cleavage monooxygenase mutant of claim 1 or 2.

6. The enzyme preparation according to claim 5, characterized in that, The enzyme preparation includes an immobilized enzyme preparation displaying a polysaccharide monooxygenase mutant on the spore surface.

7. The use of the polysaccharide monooxygenase mutant of claim 1 or 2, or the biomaterial of any one of claims 3-4, or the enzyme preparation of any one of claims 5-6 in the degradation of lignocellulose.

8. A method for degrading lignocellulose, characterized in that, The method includes adding the polysaccharide monooxygenase mutant of claim 1 or 2, or the biological material of any one of claims 3-4, or the enzyme preparation of any one of claims 5-6 to lignocellulose.

9. The method according to claim 8, characterized in that, The method includes: The substrate particles containing lignocellulose, immobilized enzyme preparations, mixed enzyme systems, electron donor ascorbic acid, and Cu were used. 2+ The mixture was used to form a reaction system, and the water content of the reaction system was controlled. The degradation was carried out at 37℃ for 72 h. The mixed enzyme system comprises a mixture of mannanase, glucanase, acid cellulase, cellobiase, and xylanase in a mass ratio of 1:1:1:1:

1.

10. The method according to claim 9, characterized in that, The water content of the reaction system was controlled at 65%, and the degradation was carried out at 37℃ for 72 hours. Based on the mass of substrate particles containing lignocellulose, the immobilized enzyme preparation was added at 20% (mL / g), the mixed enzyme system was added at 0.3% wt, the final ascorbic acid concentration was 4 mM, and Cu... 2+ The final concentration was 0.075 mM.

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