Fungaltoxin degrading enzyme thermal stability enhancing method based on structural activity protection

Abstract

The invention discloses a method for enhancing the thermal stability of a mycotoxin degrading enzyme based on structural activity protection. The invention belongs to the technical field of biology, and relates to a mycotoxin degrading enzyme mutant, which is prepared by mutating wild mycotoxin degrading enzyme with an amino acid sequence of SEQ ID No: 1: mutating N at the 6th site of SEQ ID No: 1 into Y, and mutating N at the 6th site of SEQ ID No: 1 into Y at the 6th site of SEQ ID No: 1; and / or mutating the 15th site of SEQ ID No: 1 from W to I; and / or mutating the 92nd site of SEQ ID No: 1 from A to G; and / or the 114 site of SEQ ID No: 1 is mutated from D to L. Through a rational design strategy combining structure prediction and active site protection, damage to a catalytic core is effectively avoided, the mutation success rate and the experiment efficiency are greatly improved, dual guarantee of enzyme activity and temperature resistance is considered, and the method is more systematic and reliable compared with a conventional method.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for enhancing the thermal stability of fungal toxin-degrading enzymes based on structural activity protection. Background Technology

[0002] Existing mycotoxin-degrading enzymes are easily deactivated under high temperature, acidic conditions, and industrial processing conditions, which limits their application in silage fermentation, food processing, and other scenarios.

[0003] Traditional methods for improving temperature resistance mainly include random mutation + high-throughput screening, which suffers from low efficiency and the fact that most mutations destroy the catalytic core. While rational design based on stability prediction can improve stability, it lacks spatial protection for the catalytic active region, often leading to a decrease in activity. Summary of the Invention

[0004] The main problem this invention aims to solve is how to improve the stability of enzymes at high temperatures and broaden the application scenarios of enzymes at high temperatures.

[0005] To address the above problems, the present invention provides the following fungal toxin-degrading enzyme.

[0006] The fungal toxin degrading enzyme mutant provided by this invention comprises the following mutation of the wild-type fungal toxin degrading enzyme with the amino acid sequence SEQ ID No:1: Transform the 6th position of SEQ ID No:1 from N to Y; and / or Transform the 15th position of SEQ ID No:1 from W to I; and / or Change the 92nd position of SEQ ID No:1 from A to G; and / or The 114th position of SEQ ID No:1 was mutated from D to L.

[0007] Furthermore, the mutation types include: the 6th position of the mycotoxin degrading enzyme is mutated from N to Y; the 15th position of the mycotoxin degrading enzyme is mutated from W to I; the 92nd position of the mycotoxin degrading enzyme is mutated from A to G; and the 114th position of the mycotoxin degrading enzyme is mutated from D to L.

[0008] Furthermore, the amino acid sequence of the mutant is as follows: 1) The amino acid sequence is the protein of SEQ ID No:2 and SEQ ID No:3 in the sequence listing; 2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein in 1); 3) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of 1) or 2).

[0009] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0010] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0011] Furthermore, the coding sequence of the mutant is the DNA molecule described in 1), 2), or 3) below: 1) The nucleotide sequence is the DNA molecule of SEQ ID No:5 and SEQ ID No:6 in the sequence listing; 2) Has 75% or more identity with the nucleotide sequence defined in 1), and encodes a DNA molecule of the mutant described above; 3) Hybridizes to the nucleotide sequence defined in 1) or 2) under strict conditions and encodes the DNA molecule of the mutant described above.

[0012] Those skilled in the art can readily mutate the nucleotide sequence encoding the mutant of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that possess 75% or more of the same nucleotide sequence as the mutant isolated from this invention, as long as they encode the mutant and have mutant function, are derived from and equivalent to the nucleotide sequence of this invention.

[0013] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0014] In this article, identity refers to the similarity between amino acid sequences or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid or nucleotide sequences, then the identity value (%) can be obtained.

[0015] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0016] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0017] The present invention also provides biomaterials containing the nucleic acid molecules described above, wherein the biomaterials may be any of the following: B1) Expression cassettes containing the nucleic acid molecules described above; B2) A recombinant vector containing the nucleic acid molecules described above, or a recombinant vector containing the expression cassette described in B1); B3) Recombinant microorganisms containing the nucleic acid molecules described above, or recombinant microorganisms containing the expression cassette described in B1), or recombinant microorganisms containing the recombinant vector described in B2).

[0018] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0019] The vectors described herein are well known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the vector pET-28a(+).

[0020] B2) The recombinant vector may be the recombinant expression plasmids pET-28a(+)-M1 and pET-28a(+)-M2.

[0021] The structure of the pET-28a(+)-M1 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:5 between the NdeI and XhoI restriction sites of the starting vector pET-28a(+), while keeping the other sequences of the pET-28a(+) vector unchanged. The pET-28a(+)-M1 vector can express the mut2 mutant protein, whose amino acid sequence is SEQ ID No:2.

[0022] The structure of the pET-28a(+)-M2 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:6 between the NdeI and XhoI restriction sites of the starting vector pET-28a(+), while keeping the other sequences of the pET-28a(+) vector unchanged. The pET-28a(+)-M2 vector can express the mut4 mutant protein, whose amino acid sequence is SEQ ID No:3.

[0023] Furthermore, the recombinant microorganism may be Escherichia coli BL21(DE3).

[0024] The present invention also provides a method for preparing the mutant described above, wherein the method involves introducing a DNA molecule capable of expressing the mutant described above into a target microorganism to obtain a recombinant microorganism, culturing the recombinant microorganism, and obtaining the mutant.

[0025] The DNA molecules capable of expressing the mutants described above are the recombinant vectors pET-28a(+)-M1 and pET-28a(+)-M2.

[0026] This invention also provides a method for structurally designing thermostable mutant enzyme proteins, comprising the following steps: 1) Construction of the three-dimensional catalytic core protected area of ​​the target enzyme: The substrate binding site and catalytic residue set of the target enzyme were determined through molecular docking and structural analysis; a spherical spatial region with a radius of 10 Å was set as the "catalytic core protected area" with the geometric center of the catalytic residues as the center; the amino acid residues in this protected area were excluded from the mutation candidates in the subsequent design. 2) Precise mutation screening outside the three-dimensional catalytic core protected area of ​​the target enzyme: ΔΔG and / or ΔTm of each amino acid residue outside the protected area are calculated using a protein stability prediction model; only sites with ΔΔG < -1.0 kcal / mol or ΔTm > +5 ℃ are retained as candidate sites for enhanced thermostability; 3) Combinatorial Mutation Design and Construction: The candidate sites for enhancing thermostability obtained in step 2 are subjected to 1-4 position combination mutation design to obtain the target enzyme mutation sequence; 4) Product verification and adaptability optimization: The mutant sequence obtained in step 3 is codon optimized and expressed in a suitable host. The thermal stability and catalytic activity of the mutant enzyme under the target application conditions are measured, and the thermostable mutant enzyme protein is screened.

[0027] Furthermore, the target enzyme is a fungal toxin-degrading enzyme.

[0028] The specific application conditions can be as follows: the fungal toxin-degrading enzyme is incorporated into feed or grain to degrade zearalenone (ZEN) in a suitable reaction system (e.g., buffer solution at 20–60°C and pH 5.0–9.0), thereby reducing its residual level.

[0029] Furthermore, the target enzyme can be applied in feed processing, storage, and livestock production to improve feed safety and enhance livestock production performance and health.

[0030] The present invention also provides the application of the method described above in screening thermostable enzyme proteins or improving the stability of enzymes under high temperature conditions.

[0031] The application of the mutants described above in the high-temperature fermentation of silage also falls within the scope of protection claimed by this invention.

[0032] Compared to traditional blind mutation or random screening methods, this invention employs a rational design strategy that combines structural prediction with active site protection, effectively avoiding damage to the catalytic core and significantly improving mutation success rate and experimental efficiency. The proposed "10 Å exclusion region + combinatorial mutation + structural stability prediction" process provides dual protection for enzyme activity and temperature tolerance, making it more systematic and reliable than conventional methods. Attached Figure Description

[0033] Figure 1 shows the spatial distribution characteristics of the enzyme's substrate binding site and its structural protection zone. In Figure A, the entire protein is represented using a surface model, with the binding site region highlighted in orange. The nested diagram on the right is a magnified view of the local structure of the binding site, demonstrating the interactions between the substrate and surrounding key amino acid residues (S102, S103, E126, W184, H242, etc.), including hydrogen bonds and hydrophobic interactions. In Figure B, a structural protection zone with a radius of 10 Å is defined centered on the binding site residue and marked with a yellow translucent surface, showing the spatial location and embedding state of this region in the three-dimensional structure. The surrounding protein surface is represented in cyan. This structural protection zone covers the substrate binding site and its adjacent structural regions, representing the spatial environment that may participate in catalytic stability and substrate recognition, providing a basis for the structural microenvironment analysis of functional sites. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0036] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0037] The pET-28a(+) used in the following examples was purchased from Beijing Jishi Biotechnology Co., Ltd. (js-023).

[0038] Example 1: Construction of a three-dimensional catalytic core protected area for proteins and screening of thermostable mutation sites outside the protected area. 1. Obtaining the structure file Download the three-dimensional crystal structure (resolution ≤ 3.0 Å) of the target enzyme 8JLV from the Protein Database (PDB) (https: / / www.rcsb.org / ). If no crystal structure is available, AlphaFold3 prediction is used. Remove water molecules and non-structurally essential small molecules, and save as a PDB format file.

[0039] 2. Catalytic residue recognition AutoDock Vina was used to dock the substrate molecule to the protein structure. PyMOL was used to analyze the binding conformation and identify key residues directly involved in catalysis (such as G32, S102, S103, E126, P128, W184, H242, etc.). The key residues of this enzyme are: 32, 102, 103, 126, 128, 184, 242 (residue numbers should match the structure file). If the structure contains multiple chains, chain identifiers should be added to the command, for example: select active_site, chain A and resi 32+102+103+126+128+184+242.

[0040] 3. Definition of a protected area In PyMOL, a spherical spatial region is generated with the geometric center of the catalytic residue as the center and a radius of 10 Å (adjustable by ±2 Å) as the sphere. This region is defined as a 10 Å protected area. All amino acid residues within this region are labeled, and a list of residue numbers is saved as a subsequent mutation exclusion region.

[0041] Visualizing the protected area: The residues within the protected area are displayed as sticks and highlighted. Specifically, the target region (residues within 10 Å of the specified atom / ligand) is displayed as sticks. These residues within the 10 Å range are uniformly set to yellow to distinguish them from other areas and to highlight them.

[0042] ZEN forms a stable bond with enzyme active site residues (such as S102, S103, E126, W184, H242, etc.) through hydrogen bonds and hydrophobic interactions. Furthermore, defining a structural protection zone with a radius of 10 Å around the binding site clearly shows that this region covers the substrate binding pocket and its key supporting residues, constituting the microenvironment necessary for catalysis. Figure 1 (A and B in the middle).

[0043] Combined with the results of the mutation experiments, it was found that when the mutation design avoided the 10 Å protected zone, the enzyme not only maintained its original catalytic activity, but some mutants also exhibited significantly enhanced thermostability. Conversely, when the mutation fell into the protected zone, even under optimized expression and purification conditions, the obtained mutants generally lost their activity. This result verifies the rationality and necessity of the protected zone shown in Figure 1, indicating that the setting of this structural protected zone can effectively guide mutation design and achieve the goal of "improving thermostability while maintaining activity".

[0044] 4. Export the list of protected area residues Output the chain identifier, residue name, and residue number of each residue within the protected area (Table 1) for record-keeping or subsequent analysis: Table 1. List of Residues in Protected Areas

[0045] 5. Screening of temperature-resistant mutation sites outside protected areas 1) Filtering Scope Limitation Residues within the protected area were removed from the full sequence of wild-type fungal toxin-degrading enzymes, and the remaining residues were included in the candidate range.

[0046] 2) Stability prediction calculation Single-point mutation calculations were performed on the above candidate residues using a large language model (such as SPIRED-Fitness) to obtain the values ​​of ΔΔG (kcal / mol) and ΔTm (°C).

[0047] 3) Screening criteria Only sites with ΔΔG < -1.0 kcal / mol or ΔTm > +5℃ are retained as candidate sites for enhanced temperature resistance; the sites that meet the criteria are numbered and recorded for use in combinatorial mutation design.

[0048] The above screening process yielded N6Y, W15I, A92G, and D114L mutation sites.

[0049] Example 2: Combinatorial Mutation Design and Plasmid Construction 1. Select 1–4 sites from the candidate sites in Example 1 for combined mutation design, preferably sites that are not adjacent to each other and are located in different secondary structure segments to avoid local structural damage; ensure that mutations are not performed in disulfide bond forming regions and cofactor binding sites. Specifically, the selected mutation site combinations are “N6Y, W15I, A92G, D114L” and “N6Y, A92G”.

[0050] 2. Codon optimization Based on the amino acid sequence of the target protein after mutation (amino acid sequences are SEQ ID No:2 and SEQ ID No:3), codon optimization software (such as OptimumGene) was used. TM (Or CodonW) optimizes its nucleotide sequence to ensure that the frequency of codon usage matches Escherichia coli BL21The expression preference for (DE3) was optimized while avoiding rare codons, repetitive sequences, and unstable mRNA secondary structures. The coding sequences of the optimized mutant protein (nucleotide sequences SEQ ID No:5 and SEQ ID No:6) were synthesized by a commercial company and restriction endonuclease recognition sequences (NdeI and XhoI) matching the multiple cloning site (MCS) of the expression vector were introduced at both ends.

[0051] 3. Plasmid construction The optimized gene and expression vector pET-28a(+) were digested with NdeI and XhoI enzymes, respectively. The reaction was carried out at 16°C for 12 h using T4 DNA ligase to ligate the target gene to the multiple cloning site of the vector, forming recombinant expression plasmids pET-28a(+)-M1 and pET-28a(+)-M2. Wild-type protein was used as a control. The optimized nucleotide sequence of the wild-type protein is SEQ ID No:4.

[0052] The structure of the pET-28a(+)-M1 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:5 between the NdeI and XhoI restriction sites of the starting vector pET-28a(+), while keeping the other sequences of the pET-28a(+) vector unchanged. The pET-28a(+)-M1 vector can express the mut2 mutant protein, whose amino acid sequence is SEQ ID No:2.

[0053] The structure of the pET-28a(+)-M2 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:6 between the NdeI and XhoI restriction sites of the starting vector pET-28a(+), while keeping the other sequences of the pET-28a(+) vector unchanged. The pET-28a(+)-M2 vector can express the mut4 mutant protein, whose amino acid sequence is SEQ ID No:3.

[0054] After the pET-28a(+)-M1 and pET-28a(+)-M2 vectors were verified to be correct through sequencing, they were transformed into... E. coli BL21(DE3) chemocompetent cells were introduced using the heat shock method (42 ℃, 90 s), and then cultured on LB solid medium plates containing 50 μg / mL kanamycin at 37 ℃ for 12–16 h. Positive single clones were picked for subsequent protein expression.

[0055] 4. Expression and purification of mutant proteins 1) Expression The positive monoclonal strain obtained in step 3 was inoculated into 50 mL of LB medium (containing 50 μg / mL kanamycin) and pre-cultured at 37℃ and 220 rpm for 12 h; then transferred to 1 L of LB medium (with the same concentration of antibiotic) at a ratio of 1:100 and cultured until OD. 600 =0.6–0.8; add IPTG to 0.1 mM, cool to 16℃ and induce for more than 24 h.

[0056] 2) Pyrolysis Collect bacterial cells by centrifugation at 8000 × g for 10 min at 4℃. Resuspend the precipitate in a lysis buffer matched to the initial purification conditions (e.g., 50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, pH 8.0; or an isotropic buffer system such as PBS or HEPES). The choice of buffer system can be adjusted according to the stability and activity requirements of the target protein, but the pH and the concentration of major salt ions must be kept relatively constant throughout the purification process. Sonicate the cells (300 W, 5 s on / 10 s off, cumulative 10 min), centrifuge at 12000 × g for 20 min at 4℃, and collect the supernatant.

[0057] 3) Purification Purification was performed using Ni-NTA affinity chromatography: Equilibration and loading: Pre-equilibrate the column with an equilibration buffer of the same system as the lysis buffer (such as Tris-HCl, PBS or HEPES system, with the pH kept consistent), and load the sample under the same conditions; Washing: Switch to washing buffer (keeping the same buffer system, increase the imidazole concentration to 50 mM, pH unchanged) to remove non-specifically bound proteins by gradually changing the imidazole concentration; Elution: Elution of the target protein was performed using elution buffer (the same buffer system with increased imidazole concentration); SDS-PAGE was used to determine purity and concentration.

[0058] 5. Temperature resistance and catalytic activity verification In a 25 mM Tris, 150 mM NaCl (pH 9.0) buffer solution, zearalenone (ZEN, final concentration 20 μg / mL) was used as a substrate and reacted at 40 °C for 10 min. The residual activity of wild-type and mutant strains after pretreatment at different temperatures (50 °C, 60 °C) was then detected.

[0059] Table 2. Determination of protein heat resistance at 50℃ and 60℃

[0060] In Table 2, the mutants (N6Y, A92G) were obtained under the condition of setting a 15 Å spherical spatial region as the "catalytic core protected area," thus proving that a 10 Å protection radius is sufficient to maintain protein activity. Further mutants (N6Y, W15I, A92G, D114L) obtained based on this condition exhibited stronger heat resistance, indicating that by reasonably limiting the radius of the protected area, not only can catalytic activity be maintained, but the heat resistance of the enzyme molecule can also be significantly improved.

[0061] The results showed that after incubation at 50℃ for 20 min, the wild-type enzyme was almost completely inactivated, while the mutants mut2 (mutant N6Y, A92G) and mut4 (mutant N6Y, W15I, A92G, D114L) maintained nearly 100% toxin degradation activity after 1 h of treatment, indicating significantly improved thermostability. After incubation at 60℃ for 2 min, the degradation rate of the wild-type enzyme was only 16%, while mut4 maintained 82.3% and mut2 maintained 43%. After incubation for 5 min, the degradation rate of the wild-type enzyme decreased to 5.68%, and that of mut4 decreased to 15.6%. These results indicate that the mutants significantly improved thermostability while maintaining high levels of activity.

[0062] To ensure the comparability of enzymatic parameters, an appropriate enzyme dosage was selected to maintain the ZEN degradation rate within a linear range of 10%–20%, and enzyme activity was calculated accordingly. The following formula was used: Activity (U / mg) = ((C0-C) / (C)) t )×V) / (t×m enzyme ).

[0063] Among them, C0 and C t The concentrations of ZEN before and after the reaction (mg / L) were obtained from the HPLC standard curve; V was the reaction volume (L); t was the reaction time (min); m enzyme The enzyme mass (mg) added during the reaction. The enzyme activity calculated when the degradation rate is maintained at 10%-20% can effectively reflect the true catalytic ability of different mutants.

[0064] The results showed that under these conditions, the enzyme activities of the three enzymes were on the same order of magnitude, indicating that the mutants had stronger temperature resistance while maintaining catalytic activity.

[0065] During the study, systematic experiments were conducted on ten different mutants. The results showed that their expression products were mainly deposited in inclusion bodies, making it difficult to obtain soluble protein. To improve expression levels, the induction conditions were optimized multiple times, including adjusting the IPTG concentration (0.1–0.4 mM), induction time (6–8 h), and induction temperature (20–25 °C). Although the expression of some mutants in the supernatant was enhanced after optimization, the overall amount of soluble protein remained low. Further purification yielded only about 0.2 mg / mL of enzyme solution, and functional assays revealed that these mutants ultimately lost all catalytic activity because they failed to avoid the 10 Å critical protected zone.

[0066] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A mutant fungal toxin-degrading enzyme, characterized in that, This includes the following mutations to be made in the wild-type fungal toxin-degrading enzyme with the amino acid sequence SEQ ID No:1: Transform the 6th position of SEQ ID No:1 from N to Y; and / or Transform the 15th position of SEQ ID No:1 from W to I; and / or Change the 92nd position of SEQ ID No:1 from A to G; and / or The 114th position of SEQ ID No:1 was mutated from D to L.

2. The mutant according to claim 1, characterized in that, include: The 6th position of the mycotoxin degrading enzyme is mutated from N to Y; the 15th position of the mycotoxin degrading enzyme is mutated from W to I; the 92nd position of the mycotoxin degrading enzyme is mutated from A to G; and the 114th position of the mycotoxin degrading enzyme is mutated from D to L.

3. The mutant according to claim 1, characterized in that, The amino acid sequence of the mutant is as follows: 1) The amino acid sequence is the protein of SEQ ID No:2 and SEQ ID No:3 in the sequence listing; 2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein in 1); 3) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of 1) or 2).

4. The mutant according to claim 3, characterized in that, The coding sequence of the mutant is the DNA molecule described in 1) or 2) or 3) or 4) below: 1) The nucleotide sequence is the DNA molecule of SEQ ID No:5 and SEQ ID No:6 in the sequence listing; 2) Having 75% or more identity with the nucleotide sequence defined in 1), and encoding a DNA molecule of the mutant of claim 1; 3) Hybridizes under stringent conditions to the nucleotide sequence defined in 1) or 2) and encodes a DNA molecule of the mutant of claim 1.

5. A biomaterial containing the nucleic acid molecule of claim 4, characterized in that, The biomaterial is any one of the following: B1) An expression cassette containing the nucleic acid molecule described in claim 4; B2) A recombinant vector containing the nucleic acid molecule of claim 4, or a recombinant vector containing the expression cassette of claim B1; B3) A recombinant microorganism containing the nucleic acid molecule described in claim 4, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2).

6. The method for preparing the mutant according to any one of claims 1-4, characterized in that, The method involves introducing DNA molecules capable of expressing the mutants described in claims 1-4 into a target microorganism to obtain a recombinant microorganism, culturing the recombinant microorganism, and obtaining the mutant.

7. A method for structurally designing thermostable mutant enzyme proteins, characterized in that, Includes the following steps: 1) Construction of the three-dimensional catalytic core protected area of ​​the target enzyme: The substrate binding site and catalytic residue set of the target enzyme were determined through molecular docking and structural analysis; a spherical spatial region with a radius of 10 Å was set as the "catalytic core protected area" with the geometric center of the catalytic residue as the center; 2) Precise mutation screening outside the three-dimensional catalytic core protected area of ​​the target enzyme: ΔΔG and / or ΔTm of each amino acid residue outside the protected area are calculated using a protein stability prediction model; only sites with ΔΔG < -1.0 kcal / mol or ΔTm > +5℃ are retained as candidate sites for enhanced thermostability; 3) Combinatorial Mutation Design and Construction: The candidate sites for enhancing thermostability obtained in step 2 are subjected to 1-4 position combination mutation design to obtain the target enzyme mutation sequence; 4) Product verification and adaptability optimization: The mutant sequence obtained in step 3 is codon optimized and expressed in a suitable host. The thermal stability and catalytic activity of the mutant enzyme under the target application conditions are measured, and the thermostable mutant enzyme protein is screened.

8. The method according to claim 7, characterized in that, The target enzyme is a fungal toxin-degrading enzyme.

9. The application of the method of claim 7 or 8 in screening thermostable enzyme proteins or improving the stability of enzymes under high temperature conditions.

10. The application of the mutants described in claims 1-4 in high-temperature fermentation of silage.

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