A xylanase, DNA molecule, recombinant plasmid and modified cell

Abstract

The application relates to the field of enzyme engineering, in particular to a xylanase, a xylanase optimization design method, a DNA molecule, a recombinant plasmid and a modified cell. The xylanase with an amino acid sequence of SEQ ID NO: 2 has high enzyme activity and thermal stability. The sequence of the xylanase is obtained through optimization design. The mechanism of a multi-domain matrix enzyme is predicted through an online protein structure prediction platform, and the motion trajectory of the matrix enzyme in a solution is simulated under an AMBER99SB force field through molecular dynamics simulation software, the root mean square fluctuation values of different fragments in a connecting section between adjacent functional domains of the matrix enzyme are analyzed and calculated, the fragment with the maximum root mean square fluctuation value in the connecting section is deleted, and the amino acid sequence of the design enzyme is obtained. The optimization design of the enzyme through the method has high reliability and is helpful to improve the enzyme activity and thermal stability of the matrix enzyme.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, and particularly to a xylanase, DNA molecule, recombinant plasmid, and modified cell. Background Technology

[0002] In the field of synthetic chemistry, the precise synthesis of simple or complex functionalized molecules is often achieved through the innovative application of enzyme catalysis. With its advantage of precise control over reaction outcomes, biocatalytic conversion technology has gradually become an important means in production, not only effectively supplementing traditional chemical synthesis methods but also showing the potential to gradually replace them. However, since synthesis-related reactions rarely have natural counterparts, the challenge of biocatalysis lies in identifying suitable enzyme catalysts for the desired application and improving the catalytic activity of wild-type enzymes that can catalyze the target substrate.

[0003] PuXyn, a wild-type xylanase with the amino acid sequence SEQ ID NO: 1, is a known enzyme capable of degrading xylan substrates. However, this enzyme exhibits low activity in degrading xylan substrates and poor thermal stability, making it difficult to meet the degradation requirements in production. Therefore, it is necessary to engineer this enzyme to maintain its stability and activity under the required application conditions. Summary of the Invention

[0004] The purpose of this invention is to provide an optimized design method for xylanase with high reliability, and the optimized xylanase obtained therefrom.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A xylanase having the amino acid sequence SEQ ID NO: 2.

[0007] Secondly, the present invention also provides an optimized design method for xylanase, comprising:

[0008] The predicted structure is obtained by using an online protein structure prediction platform based on the amino acid sequence of the matrix enzyme. The matrix enzyme is a multi-domain xylanase and has a linker segment connecting adjacent functional domains.

[0009] Based on the predicted structure, the trajectory of the matrix enzyme in solution was simulated using molecular dynamics simulation software under the AMBER99SB force field to obtain a trajectory file.

[0010] The trajectory file was analyzed using the molecular dynamics simulation software to remove periodic artifacts and calculate the root mean square fluctuation value.

[0011] The segment with the highest root mean square fluctuation value in the amino acid sequence of the linker is deleted to obtain the amino acid sequence of the designed enzyme.

[0012] Optionally, the method further includes:

[0013] The designed structure is obtained by predicting the structure of the designed enzyme based on the amino acid sequence using the protein structure prediction platform.

[0014] Based on the design structure, the molecular dynamics simulation software is used to simulate the motion trajectory of the designed enzyme in solution to obtain a simulation file;

[0015] The simulation file is analyzed using the molecular dynamics simulation software to remove periodic artifacts and calculate its root mean square deviation and radius of gyration. The root mean square deviation and radius of gyration are obtained from the trajectory file and compared with each other.

[0016] Optionally, the method for constructing a model to simulate the motion trajectory includes:

[0017] Under the AMBER99SB force field, the protein structure is placed in a cuboid box filled with TIP3P water for solvation, and sodium or chloride ions are added to neutralize the charge. The distance between the protein and the box wall is set to a preset distance. The particle-mesh Ewald method is applied to handle Coulomb interactions, periodic boundary conditions are applied, and the energy of the constructed system is minimized. Two stages of pretreatment are used to ensure that the TIP3P water in the system reaches the preset temperature and pressure conditions. Unconstrained simulation of the system is performed, and one structure is output at each time period to obtain the motion trajectory.

[0018] Optionally, in the model construction of the motion trajectory, the simulation step size is set to any value between 1fs and 2fs, the van der Waals interaction cutoff value is set to any value between 10Å and 14Å, the preset distance is any value between 8Å and 12Å, the total time of the unconstrained simulation is any value between 15ns and 25ns, and the time period is any value between 70ps and 130ps.

[0019] Optionally, the preprocessing operations in the two stages include:

[0020] Use a V-rescale thermostat to set the temperature to the first temperature, keeping the number of particles, volume, and temperature in the model constant for the first time. Then use a Parrinello-Rahman pressure regulator to adjust the pressure in the model to the first pressure, keeping the number of particles, pressure, and temperature in the model constant for the second time.

[0021] Optionally, the first temperature is any value between 280K and 320K, the first time is any value between 80ps and 120ps, the first pressure is any value between 0.8 atmospheres and 1.2 atmospheres, and the second time is any value between 80ps and 120ps.

[0022] Thirdly, the present invention also provides a DNA molecule that encodes the aforementioned xylanase.

[0023] Fourthly, the present invention also provides a recombinant plasmid carrying the aforementioned DNA molecule.

[0024] Fifthly, the present invention also provides a modified cell, wherein the modified cell comprises the aforementioned DNA molecules.

[0025] According to a first aspect of the invention, the activity and thermal stability of xylanase are enhanced by adjusting its sequence.

[0026] According to a second aspect of the invention, a protein structure prediction platform is used to perform structural analysis on matrix enzymes with multiple structural domains, and a model is constructed to simulate the enzyme's movement trajectory in solution. After removing duplicate data, the root mean square fluctuation (RMSF) of different regions of the enzyme is calculated. Regions with larger RMSF values ​​exhibit greater flexibility and instability. In protein structures, excessive structural instability can lead to incomplete folding or random conformational changes of linker peptides, thereby affecting the overall stability of the enzyme. In matrix enzymes, different functional domains typically need to work in coordination to efficiently bind substrates and catalyze reactions. Overly flexible linker segments connecting adjacent functional domains can lead to excessive changes in the relative positions of different functional domains, affecting their synergistic effect and thus impacting catalytic activity. By removing the segments with the largest RMSF values ​​from the linker segments, excessive flexibility is reduced, improving the overall stability of the protein structure, while retaining sufficient flexibility to maintain the relative movement between different functional domains and maintain the enzyme's catalytic efficiency.

[0027] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0028] Figure 1 This is a flowchart of the optimized design method for xylanase as shown in Embodiment 1 of the present invention;

[0029] Figure 2 This is a graph showing the variation of root mean square fluctuation values ​​of different segments within the linker segment of the matrix enzyme shown in Embodiment 1 of the present invention.

[0030] Figure 3 This is a comparison chart of the root mean square deviation and radius of gyration of the matrix enzyme and the designed enzyme shown in Embodiment 1 of the present invention.

[0031] Figure 4 This is a statistical chart showing the specific enzyme activity of the matrix enzyme and the designed enzyme as shown in Example 1 of the present invention;

[0032] Figure 5 This is a graph showing the changes in enzyme activity of the matrix enzyme and the designed enzyme after heating, as shown in Example 1 of the present invention. Detailed Implementation

[0033] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0035] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0036] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] In the field of enzyme engineering, optimizing and modifying wild-type enzymes obtained from organisms is an important means of expanding the application areas of enzymes. Enzyme modification involves methods such as the complete replacement of functional domains, the substitution of amino acids at specific sites, and fragment deletion or insertion. However, amino acid structures are quite complex; therefore, before modification, it is necessary to design the modification based on the enzyme's characteristics to increase the probability of optimizing the modified enzyme's performance.

[0038] Molecular dynamics (MD) simulations are computational methods used to study how matter changes over time at the atomic or molecular level. Based on Newtonian mechanics, they simulate the motion and interactions of particles to predict the physical and chemical properties of matter. Using this technique to simulate the atomic-level motion of enzyme molecules can help researchers identify key amino acid residues that affect enzyme activity and stability, and predict the impact of these mutations on enzyme performance, thus enabling rapid screening of potential modification targets without extensive experimental work.

[0039] However, due to unknown protein structures and the cumbersome force field selection and pre-equilibrium processes, the performance reliability of modified enzymes obtained by molecular dynamics simulations for xylanase protein design is currently low. Verifying the performance of modified enzymes requires steps such as primer design, amplification, transfection, screening, and extraction and purification, which are costly and time-consuming. Therefore, experimental testing of modified enzymes with low reliability would result in significant waste. Based on these issues, this invention optimizes the design of the wild-type xylanase PuXyn (SEQ ID NO: 1). First, the accurate protein structure of the xylanase is obtained through a protein structure prediction platform, revealing a long, flexible loop region at the enzyme's end, which molecular dynamics simulations can more easily capture in dynamic changes. Second, the AMBER99SB force field is used, which corrects the dihedral angle parameters of the main chain compared to the commonly used AMBER force field, improving the accuracy of the simulation results. Finally, a position-restricted pre-equilibrium strategy is employed to release the degrees of freedom of the flexible region, avoiding irrationality caused by initial structural deviations, and multiple equilibrium simulations ensure consistent results. By improving the accuracy of molecular dynamics simulation results through the above strategies, a xylanase-modified enzyme with superior performance was finally obtained, and its performance was verified.

[0040] The amino acid sequence of the xylanase protected in this invention application is SEQ ID NO: 2.

[0041] Using wild xylanase with the amino acid sequence SEQ ID NO: 1 as the matrix enzyme, after optimization design in the above manner, it was experimentally verified that it has higher activity and thermal stability, and has performance advantages over the matrix enzyme.

[0042] Secondly, please see Figure 1This invention also applies for protection of an optimized design method for xylanase, comprising:

[0043] S1. Using an online protein structure prediction platform, the structure of the matrix enzyme is predicted based on its amino acid sequence. The predicted structure is obtained, indicating that the matrix enzyme is a multi-domain xylanase with a linker segment connecting adjacent functional domains.

[0044] S2. Based on the predicted structure, the trajectory of the matrix enzyme in solution is simulated using molecular dynamics simulation software under the force field of AMBER99SB, and the trajectory file is obtained.

[0045] S3. The trajectory file is analyzed using molecular dynamics simulation software to remove periodic artifacts and calculate the root mean square fluctuation value.

[0046] S4. Delete the segment with the highest root mean square fluctuation value in the amino acid sequence of the linker segment to obtain the amino acid sequence of the designed enzyme.

[0047] Structural analysis of multi-domain matrix enzymes was performed using a protein structure prediction platform, and models were constructed to simulate the enzyme's movement trajectory in solution. After removing duplicate data, the root mean square fluctuation (RMSF) of different regions of the enzyme was calculated. Regions with larger RMSF values ​​exhibited greater flexibility and instability. In protein structures, excessive structural instability can lead to incomplete folding or random conformational changes in linker peptides, thus affecting the overall stability of the enzyme. In matrix enzymes, different functional domains typically need to work in coordination to efficiently bind substrates and catalyze reactions. Overly flexible linker segments connecting adjacent functional domains can cause excessive changes in the relative positions of different functional domains, affecting their synergistic effect and thus impacting catalytic activity. Removing the segments with the largest RMSF values ​​from the linker segments helps reduce excessive flexibility, improves the overall stability of the protein structure, while retaining sufficient flexibility to maintain the relative movement between different functional domains and preserve the enzyme's catalytic efficiency.

[0048] In some embodiments, the above method further includes:

[0049] S5. Using a protein structure prediction platform, the structure of the designed enzyme is predicted based on its amino acid sequence to obtain the designed structure.

[0050] S6. Based on the designed structure, use molecular dynamics simulation software to simulate the motion trajectory of the enzyme in the solution and obtain the simulation file.

[0051] S7. Analyze the simulation file using molecular dynamics simulation software, remove periodic artifacts, and calculate its root mean square deviation and radius of gyration. Analyze the trajectory file to obtain its root mean square deviation and radius of gyration values, and compare the two.

[0052] Root mean square deviation (RMSD) measures the degree to which a molecule deviates from a reference structure during simulation. A larger RMSD value indicates a significant conformational change, typically representing a substantial structural adjustment; a smaller RMSD value indicates a stable structure without significant conformational change. Radius of gyration (Rg) represents the average distance between each atom of a molecule and its center of mass. A smaller Rg value indicates a more compact molecule, typically meaning it remains folded or compact; a larger Rg value indicates a more unfolded or loose molecule, typically meaning it may be in a relaxed or unfolded state. Therefore, proteins with smaller RMSD and Rg values ​​generally exhibit higher structural stability, while for enzymes, this usually signifies higher thermal stability. Comparing the RMSD and Rg values ​​of the base enzyme and the designed enzyme helps ensure that the designed enzyme has lower RMSD and Rg values, thus validating the higher thermal stability of the designed enzyme compared to the base enzyme and improving the reliability of this optimized design.

[0053] In some embodiments, the method for constructing a model simulating a motion trajectory includes:

[0054] Under the AMBER99SB force field, the protein structure was solvated in a cuboid box filled with TIP3P water, and sodium or chloride ions were added to neutralize the charge. A preset distance was set between the protein and the box wall. The particle-mesh Ewald method was applied to handle Coulomb interactions, periodic boundary conditions were applied, and energy minimization was performed on the constructed system. Two stages of pretreatment ensured that the TIP3P water in the system reached the preset temperature and pressure conditions. Unconstrained simulations were performed on the system, outputting one structure at a time interval to obtain the motion trajectory. A position-restricted pre-equilibrium strategy was employed to release the degrees of freedom in the flexible region, avoiding irrationality caused by initial structural deviations. Consistency of results was ensured through multiple equilibrium simulations.

[0055] In some embodiments, in the model construction of the motion trajectory, the simulation step size is set to any value from 1fs to 2fs, for example, any value from 1fs, 1.2fs, 1.4fs, 1.6fs, 1.8fs, and 2fs; the van der Waals interaction cutoff value is set to any value from 10Å to 14Å, for example, any value from 10Å, 11Å, 12Å, 13Å, and 14Å; the preset distance is any value from 8Å to 12Å, for example, any value from 8Å, 9Å, 10Å, 11Å, and 12Å; the total time of the unconstrained simulation is any value from 15ns to 25ns, for example, any value from 15ns, 17ns, 19ns, 21ns, 23ns, and 25ns; and the time period is any value from 70ps to 130ps, for example, any value from 70ps, 90ps, 110ps, and 130ps.

[0056] In some embodiments, the two-stage preprocessing operations include:

[0057] Use a V-rescale thermostat to set the temperature to the first temperature, keeping the number of particles, volume, and temperature in the model constant for the first time. Then use a Parrinello-Rahman pressure regulator to adjust the pressure in the model to the first pressure, keeping the number of particles, pressure, and temperature in the model constant for the second time.

[0058] In some embodiments, the first temperature is any value from 280K to 320K, for example, any value from 280K, 290K, 300K, 310K, and 320K; the first time is any value from 80ps to 120ps, for example, any value from 80ps, 90ps, 100ps, 110ps, and 120ps; the first pressure is any value from 0.8 atmospheres to 1.2 atmospheres, for example, any value from 0.8 atmospheres, 0.9 atmospheres, 1 atmosphere, 1.1 atmospheres, and 1.2 atmospheres; and the second time is any value from 80ps to 120ps, for example, any value from 80ps, 90ps, 100ps, 110ps, and 120ps.

[0059] Thirdly, the present invention also seeks to protect a DNA molecule that encodes the aforementioned xylanase.

[0060] Fourthly, this invention also seeks to protect a recombinant plasmid carrying the aforementioned DNA molecule.

[0061] Fifthly, the present invention also seeks to protect a modified cell containing the aforementioned DNA molecules.

[0062] Please refer to the following examples for details.

[0063] Example 1:

[0064] Please see Figure 1 The preferred embodiment of this application shows an optimized design method for xylanase, which includes:

[0065] S1. Using an online protein structure prediction platform, the structure of the matrix enzyme is predicted based on its amino acid sequence. The predicted structure is obtained, indicating that the matrix enzyme is a multi-domain xylanase with a linker segment connecting adjacent functional domains.

[0066] S2. Based on the predicted structure, the trajectory of the matrix enzyme in solution is simulated using molecular dynamics simulation software under the force field of AMBER99SB, and the trajectory file is obtained.

[0067] S3. The trajectory file is analyzed using molecular dynamics simulation software to remove periodic artifacts and calculate the root mean square fluctuation value.

[0068] S4. Delete the segment with the highest root mean square fluctuation value in the amino acid sequence of the linker segment to obtain the amino acid sequence of the designed enzyme.

[0069] In step S1, the protein structure prediction platform is the AlphaFold3 online server. Wild-type xylanase PuXyn from *Penicillium* species was used as the matrix enzyme. The NCBI accession number for the matrix enzyme is KAF7714546.1, and its amino acid sequence is SEQ ID NO: 1. The matrix enzyme includes a catalytic domain, a carbohydrate-binding domain, and a linker peptide connecting the two. The linker peptide is the linker segment.

[0070] In step S2, the molecular dynamics simulation software is the General-purpose Open-source Molecular-dynamics Algorithms and Systems (GROMACS) version 2022.6. The specific model construction method for simulating the movement trajectory of matrix enzymes in solution using this software includes:

[0071] The AMBER99SB force field was selected, and the water model was a three-point charged water model (TIP3P). The simulation step size was set to 2 fs. The particle-mesh Ewald (PME) method was applied to handle Coulomb interactions, and the van der Waals interaction cutoff was set to 12 Å. The protein structure was placed in a cuboid box filled with TIP3P water for solvation, and sodium or chloride ions were added to neutralize the charge. The distance between the protein and the box wall was set to 10 Å. Periodic boundary conditions were applied, and the energy of the constructed system was minimized. Two stages of pretreatment were performed to ensure that the TIP3P water in the system reached the preset temperature and pressure conditions. The system was subjected to a 20 ns unconstrained simulation, and one structure was output every 100 ps to obtain the motion trajectory.

[0072] The two-stage pretreatment process involved setting the temperature to 300K using a velocity rescale thermostat, maintaining constant particle count, volume, and temperature within the model, and holding the protein structure at equilibrium for 100 ps. Then, a Parrinello-Rahman barometer was used to adjust the pressure within the model to one atmosphere, maintaining constant particle count, pressure, and temperature within the model, and holding the protein structure at equilibrium for another 100 ps.

[0073] In step S3, GROMACS 2022.6 is used to further process and analyze the trajectory file obtained from the simulation. After removing periodicity from the trajectory, the root mean square fluctuation (RMSF), root mean square deviation (RMSD), and radius of gyration (Rg) are calculated. RMSF represents the offset of each atom or residue relative to its average position. Regions with larger RMSF values ​​typically represent areas with higher flexibility and greater conformational changes. Please refer to [link to relevant documentation]. Figure 2 The linker region of the matrix enzyme has the highest RMSF value, indicating that this region may exhibit strong flexibility and instability. Based on the above molecular dynamics simulation results, the RMSF values ​​at sites 350-373 in the linker region were deleted, and an enzyme with the amino acid sequence SEQ ID NO: 2 was constructed. This design retains sufficient flexibility to maintain the relative movement between the catalytic domain and the carbohydrate-binding domain, thus maintaining the enzyme's catalytic efficiency while reducing excessive flexibility and improving the overall stability of the protein structure.

[0074] In this embodiment, the optimized design method further includes:

[0075] S5. Through a protein structure prediction platform, perform structure prediction based on the amino acid sequence of the designed enzyme to obtain the designed structure.

[0076] S6. According to the designed structure, use molecular dynamics simulation software to simulate the movement trajectory of the designed enzyme in solution to obtain a simulation file.

[0077] S7. Analyze the simulation file through molecular dynamics simulation software, remove periodic artifacts, and calculate the values of its root mean square deviation and radius of gyration. Analyze the values of its root mean square deviation and radius of gyration according to the trajectory file, and compare the two.

[0078] The differences between steps S5, S6, and S7 and steps S1 to S3 are only that simulation and analysis are performed with the sequence of the designed enzyme, and in step S7, the value of RMSF does not need to be calculated, only the values of RMSD and Rg are calculated and compared with the values in step S3. Please refer to Figure 2 , it can be seen that after site deletion, the values of RMSD and Rg have both decreased significantly, further improving the reliability that the enzyme activity and thermal stability of the designed enzyme are superior to those of the matrix enzyme, and reducing the risk that the design result does not match the actual situation.

[0079] The amino acid sequence SEQ ID NO: 1 of the expressed xylanase was subjected to whole gene synthesis. The synthesized gene was optimized according to the codon preference of Pichia pastoris, and the synthesized nucleotide fragment SEQ ID NO: 3 was inserted between the EcoRI and NotI restriction sites of the pPIC9K plasmid, thereby obtaining the recombinant plasmid pPIC9KPuXyn expressing wild-type xylanase PuXyn. Using the recombinant plasmid pPIC9KPuXyn as a template, use KOD Fx Neo high-fidelity DNA polymerase (Toyobo Shanghai Biotechnology Co., Ltd., KFX-201) with the primer "Puc-1" with a base sequence of SEQ ID NO: 4 and the primer "349_ga2" with a base sequence of SEQ ID NO: 5 for PCR amplification. In this example, the direction of the base sequences of the primers is all 5'-3'. The gene fragment "PuXyn-up" corresponding to the 1-349 sites of the matrix enzyme and the designed enzyme was obtained through gel recovery and purification. The PCR system is shown in Table 1, where "primer_1" and "primer_2" respectively refer to the two primers used for amplification.

[0080]

[0081] Similarly, using the recombinant plasmid pPIC9KPuXyn as a template, PCR amplification and recovery purification were carried out in the same method using the primer "374_ga1" with the base sequence of SEQ ID NO: 6 and the primer "9K-ga2" with the base sequence of SEQ ID NO: 7 to obtain the gene fragment "PuXyn-down" corresponding to the 374-413 sites of the matrix enzyme.

[0082] The plasmid vector pPIC9KPuXyn was digested with the restriction enzymes EcoRI and NotI, and the plasmid vector gene fragment "pPIC9K-B" was obtained after recovery purification in the same method. The gene fragments "PuXyn-up", "PuXyn-down" and "pPIC9K-B" were ligated with plasmid fragments using a homologous recombination kit (TransGen Biotech Co., Ltd., CU101-01), and the ligation product was transformed into Escherichia coli TOP10 competent cells (Zhuangmeng International Biotechnology Co., Ltd., ZC104). Then it was spread on an LBK kanamycin-resistant plate for screening of positive transformants. Then the transformants identified correctly by colony PCR were inoculated into LB medium and the plasmids were extracted. After verification by sequencing of Sangon Biotech (Shanghai) Co., Ltd., the recombinant plasmid pPIC9K-PuXyn-Del 350-373 carrying the designed enzyme gene was obtained.

[0083] The BMGY medium and BMMY medium were prepared. Among them, the components of the BMGY medium included 1% yeast extract, 2% tryptone, 1.34% yeast nitrogen base without amino acids, 10% 1 mol / L potassium phosphate buffer (pH 6.0) and 1% glycerol, and after preparation, it was sterilized at 115 °C for 20 min by high-pressure steam. In the components of the BMMY medium, only 1% (v / v) glycerol was replaced with 1% (v / v) methanol, and the methanol was added after high-pressure sterilization. The above concentrations are all mass-volume percentages.

[0084] The recombinant plasmid pPIC9KPuXyn and the recombinant plasmid pPIC9K-PuXyn-Del350-373 were linearized with the restriction enzyme SalI respectively, and the two linearized vectors were purified and recovered and then electrotransformed into the Pichia pastoris GS115 competent cells purchased from Life Technologies Corporation. The positive transformants were screened on a histidine auxotrophic plate. The obtained positive transformants were respectively inoculated into the BMGY medium and cultured with shaking at 30 °C and 250 rpm for 20 h, then centrifuged at a low temperature at 6000 rpm for 5 min to collect the cell pellet and resuspended in the BMMY medium, and the concentration was adjusted to make the OD of the suspension 600The value was approximately 0.5. Fermentation was then carried out again at 30℃ and 250 rpm with shaking, and 1% methanol was added daily to reach a final concentration. After five days of fermentation, the mixture was centrifuged at 6000 rpm for 5 minutes at low temperature. The supernatant was collected to obtain enzyme solutions containing either the matrix enzyme or the designed enzyme. The enzyme solution containing the matrix enzyme was named WT, and the enzyme solution containing the designed enzyme was named Del 350-373.

[0085] The activities of the matrix enzyme and the designed enzyme were measured separately. 100 μL of 1% beech xylan substrate and 100 μL of enzyme solution of the same concentration were mixed, the pH was adjusted to approximately 5.0, and the mixture was reacted in a water bath at 50 °C for 30 min. After the reaction, 600 μL of DNS solution was added to terminate the reaction, and the mixture was then boiled in a water bath for 5 min. After cooling, the absorbance (A540) at 540 nm was measured, and the amount of reducing sugar released was read from the standard curve. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under given conditions. For calculating specific enzyme activity and plotting statistical graphs, please refer to [link to relevant documentation]. Figure 4 It can be seen that after deleting the highly unstable 350-373 sites, the specific activity of the designed enzyme was significantly increased, approximately 1.18 times that of the matrix enzyme.

[0086] The thermostability of the matrix enzyme and the designed enzyme was tested separately. The enzyme solution was incubated in a 70°C constant-temperature mixer for different times. After incubation, the precipitate was removed by centrifugation at 13300 rpm for 2 min, and then the activity was measured. The remaining enzyme activity was calculated using the activity of the enzyme solution that had not undergone heat incubation as a control. Please refer to [link to relevant documentation]. Figure 5 It can be seen that the matrix enzyme basically loses its catalytic activity after incubation at 70℃ for 2 hours, while the designed enzyme under the same conditions can still retain more than 80% of its catalytic activity. Even under the condition of incubation for 4 hours, the mutant still retains more than 50% of its catalytic activity.

[0087] Combination Figure 4 and Figure 5 The results show that the designed enzyme exhibits superior activity and thermostability compared to the matrix enzyme, thus demonstrating the effectiveness of enzyme protein modification design guided by molecular dynamics simulations. This novel designed enzyme helps expand the application range of matrix enzymes in lignocellulose degradation, food, and feed. This optimized design method for xylanases, by rapidly identifying amino acid sites or fragments that significantly influence enzyme activity and thermostability, guides mutant design, helping to substantially reduce the time and cost of traditional enzyme engineering and modification, while also demonstrating high reliability.

[0088] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0089] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A xylanase, characterized in that, The amino acid sequence thereof is SEQ ID NO:

2.

2. A DNA molecule, characterized in that, The DNA molecule encodes the xylanase as claimed in claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid carries the DNA molecule as claimed in claim 2.

4. A modified cell, wherein, The modified cell comprises the DNA molecule as claimed in claim 2. The amino acid sequence thereof is SEQ ID NO:

2. The DNA molecule encodes the xylanase as claimed in claim 1. The recombinant plasmid carries the DNA molecule as claimed in claim 2. The modified cell comprises the DNA molecule as claimed in claim 2.

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