Xylanase, optimal design method of xylanase, DNA molecule, recombinant plasmid and modified cell

Abstract

The invention relates to the field of enzyme engineering, in particular to xylanase, an optimal design method of the xylanase, a DNA (deoxyribonucleic acid) molecule, a recombinant plasmid and a modified cell. The invention relates to xylanase with an amino acid sequence of SEQ ID NO: 2. The xylanase has relatively high enzymatic activity and thermal stability. The sequence of the xylanase is obtained through optimization design. The method comprises the following steps: performing mechanism prediction on a matrix enzyme of multiple structural domains through an online protein structure prediction platform, simulating a motion trail of the matrix enzyme in a solution under an AMBER99SB force field by molecular dynamics simulation software, and analyzing and calculating to obtain a root mean square fluctuation value of different fragments in a connection segment of the matrix enzyme connected between adjacent functional structural domains; and deleting the segment with the maximum root mean square fluctuation value in the connection segment to obtain the amino acid sequence of the designed enzyme. The method is used for carrying out optimal design on the enzyme, so that the method has relatively high reliability and is beneficial to improving the enzyme activity and the thermal stability of the matrix enzyme.

Description

Technical Field

[0001] The present invention relates to the field of enzyme engineering, and in particular to a xylanase, a xylanase optimization design method, a DNA molecule, a recombinant plasmid and a modified cell. Background Art

[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 the advantage of precise control over reaction outcomes, biocatalytic transformation technology has gradually become an important means of production, not only effectively supplementing traditional chemical synthesis methods, but also showing the potential to gradually replace chemical synthesis technology. However, since synthesis-related reactions rarely have counterparts in nature, the challenge of biocatalysis lies in identifying enzyme catalysts suitable for the desired application and improving the catalytic activity of wild-type enzymes that can catalyze the target substrate.

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

[0004] The purpose of the present invention is to provide a method for optimizing and designing xylanase with higher reliability, and the optimized xylanase.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: A xylanase, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0006] In a second aspect, the present invention further provides a method for optimizing the design of xylanase, comprising: Using an online protein structure prediction platform, a structure prediction is performed based on the amino acid sequence of the matrix enzyme to obtain a predicted structure. The matrix enzyme is a multi-domain xylanase having a connecting segment connecting adjacent functional domains. According to the predicted structure, the motion trajectory of the matrix enzyme in the solution is simulated using molecular dynamics simulation software under the AMBER99SB force field to obtain a trajectory file; Analyzing the trajectory file using the molecular dynamics simulation software to remove periodic artifacts and calculate the root mean square fluctuation value; The segment with the highest root mean square fluctuation value in the amino acid sequence of the connecting segment is deleted to obtain the amino acid sequence of the designed enzyme.

[0007] Optionally, the method further includes: Using the protein structure prediction platform, structure prediction is performed based on the amino acid sequence of the designed enzyme to obtain a designed structure; According to the designed structure, simulating the motion trajectory of the designed enzyme in the solution using the molecular dynamics simulation software to obtain a simulation file; The simulation file is analyzed by the molecular dynamics simulation software to remove periodic artifacts, and the root mean square deviation and gyration radius are calculated. The root mean square deviation and gyration radius are obtained by analyzing the trajectory file, and the two are compared.

[0008] Optionally, the model construction method for simulating the motion trajectory includes: Under the AMBER99SB force field, the protein structure is placed in a rectangular 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 treat Coulomb interactions, periodic boundary conditions are applied, and the constructed system is energy minimized. A two-stage pretreatment operation is performed to ensure that the TIP3P water in the system reaches the preset temperature and pressure conditions. The system is simulated unconstrainedly, and a structure is output every time period to obtain the motion trajectory.

[0009] Optionally, in constructing the model 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.

[0010] Optionally, the two-stage preprocessing operations include: Use a V-rescale thermostat to set the temperature to a first temperature, maintain the number of particles, volume, and temperature in the model unchanged for a first time, and then use a Parrinello-Rahman barostat to adjust the pressure in the model to a first pressure, and maintain the number of particles, pressure, and temperature in the model unchanged for a second time.

[0011] 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.

[0012] In a third aspect, the present invention further provides a DNA molecule encoding the above-mentioned xylanase.

[0013] In a fourth aspect, the present invention further provides a recombinant plasmid carrying the above-mentioned DNA molecule.

[0014] In a fifth aspect, the present invention further provides a modified cell, wherein the modified cell comprises the above-mentioned DNA molecule.

[0015] According to the first aspect of the present invention, the activity and thermal stability of xylanase are improved by adjusting the sequence of xylanase.

[0016] According to a second aspect of the present invention, a multi-domain matrix enzyme was structurally analyzed using a protein structure prediction platform, and a model was constructed to simulate the enzyme's motion 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 exhibit greater flexibility and instability. In protein structures, excessive structural instability can lead to incomplete folding of the linker peptide or random conformational changes, thereby affecting the overall stability of the enzyme. Different functional domains in a matrix enzyme typically require coordinated operation to efficiently bind substrates and catalyze reactions. Excessive flexibility in the linker connecting adjacent functional domains can lead to significant shifts in the relative positions of the different functional domains, affecting their synergistic interaction and, consequently, catalytic activity. By removing segments with the largest RMSF values within the linker, excessive flexibility is reduced, improving the stability of the overall protein structure, while retaining sufficient flexibility to maintain relative motion between the different functional domains and thus the enzyme's catalytic efficiency.

[0017] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flow chart of the method for optimizing the design of xylanase shown in Example 1 of the present invention; Figure 2 This is a graph showing the root mean square fluctuation values of different segments within the connecting segment of the matrix enzyme shown in Example 1 of the present invention; Figure 3 A comparison diagram of the root mean square deviation and gyration radius of the base enzyme and the designed enzyme shown in Example 1 of the present invention; Figure 4 Statistical graph of the enzyme activity of the matrix enzyme and the designed enzyme shown in Example 1 of the present invention; Figure 5This is a diagram showing the changes in enzyme activity of the matrix enzyme and the designed enzyme after being heated as shown in Example 1 of the present invention. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0020] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present 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.

[0021] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0022] In addition, 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.

[0023] In the field of enzyme engineering, optimizing and modifying wild-type enzymes obtained from organisms is an important means of expanding enzyme applications. Enzyme modification can be achieved through methods such as complete replacement of functional domains, amino acid substitution at several sites, and deletion or insertion of fragments. However, the amino acid structure is relatively complex, so prior to modification, it is necessary to design the enzyme based on its characteristics to increase the probability of optimizing the performance of the modified enzyme.

[0024] Molecular dynamics (MD) simulation is a computational method used to study how matter changes over time at the atomic or molecular level. Based on Newtonian mechanics, it simulates 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 influence enzyme activity and stability and predict the effects of these mutations on enzyme performance, allowing them to quickly screen for potential modification targets without extensive experimentation.

[0025] However, due to unknown protein structures and cumbersome force field selection and pre-equilibration processes, the performance reliability of modified enzymes currently obtained through molecular dynamics simulations for xylanase protein design is 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 is a significant waste. To address these issues, the present invention optimized the design of the wild-type xylanase PuXyn, whose amino acid sequence is SEQ ID NO: 1. First, using a protein structure prediction platform, the accurate protein structure of the xylanase was determined. It was discovered that the enzyme possesses a long flexible loop region at its terminus, making it easier for molecular dynamics simulations to capture dynamic changes in this region. Secondly, the AMBER99SB force field was selected, which corrects the main chain dihedral angle parameters compared to the commonly used AMBER force field, improving the accuracy of simulation results. Finally, a position-constrained pre-equilibration strategy was employed to release degrees of freedom in the flexible region, avoiding irrationalities caused by initial structural deviations and ensuring consistent results through multiple equilibrium simulations. By using the above strategy to improve the accuracy of molecular dynamics simulation results, a modified xylanase with more advantageous performance was finally obtained, and its performance was verified.

[0026] The amino acid sequence of the xylanase protected in the present invention is SEQ ID NO: 2.

[0027] The wild xylanase with the amino acid sequence of SEQ ID NO: 1 was used as the matrix enzyme. After optimization and design by the above method, it was experimentally verified that higher activity and thermal stability were obtained, which has performance advantages over the matrix enzyme.

[0028] Second, see Figure 1 The present invention also applies to protect a method for optimizing the design of xylanase, comprising: S1. Using an online protein structure prediction platform, the structure of the matrix enzyme was predicted based on its amino acid sequence to obtain a predicted structure. The matrix enzyme is a multi-domain xylanase and has a connecting segment connecting adjacent functional domains.

[0029] S2. Based on the predicted structure, the motion trajectory of the matrix enzyme in the solution is simulated using molecular dynamics simulation software under the AMBER99SB force field to obtain a trajectory file.

[0030] S3. Analyze the trajectory file using molecular dynamics simulation software, remove periodic artifacts, and calculate the root mean square fluctuation value.

[0031] S4. Delete the segment with the highest root mean square fluctuation value in the amino acid sequence of the connecting segment to obtain the amino acid sequence of the designed enzyme.

[0032] A protein structure prediction platform was used to analyze the structure of a multi-domain enzyme and construct a model to simulate the enzyme's motion in solution. After removing duplicate data, the root mean square fluctuation (RMSF) of different regions of the enzyme was calculated. Regions with large RMSF values exhibit greater flexibility and instability. In protein structures, excessive structural instability can lead to incomplete folding of the linker or random conformational changes, thus affecting the overall stability of the enzyme. Different functional domains in enzymes often require coordinated operation for efficient substrate binding and catalysis. Excessive flexibility in the linker between adjacent functional domains can lead to significant shifts in the relative positions of the different domains, disrupting their synergistic interactions and, consequently, affecting catalytic activity. Removing segments with the highest RMSF values within the linker helps reduce excessive flexibility and improve overall protein stability, while retaining sufficient flexibility to maintain relative motion between the different functional domains and thus the enzyme's catalytic efficiency.

[0033] In some embodiments, the above method further comprises: 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.

[0034] S6. Based on the designed structure, the motion trajectory of the designed enzyme in the solution is simulated using molecular dynamics simulation software to obtain a simulation file.

[0035] S7. Analyze the simulation file using molecular dynamics simulation software to remove periodic artifacts, and calculate the root mean square deviation and gyration radius. Analyze the trajectory file to obtain the root mean square deviation and gyration radius, and compare the two.

[0036] The root mean square deviation (RMSD) measures the degree to which a molecule deviates from a reference structure during a simulation. A larger RMSD value means that the molecule has undergone a larger conformational change, generally indicating that the molecule has undergone a larger structural adjustment; a smaller RMSD value indicates that the molecule has maintained a stable structure and has not undergone significant conformational changes. The radius of gyration (Rg) represents the average distance between the atoms of a molecule and its center of mass. A smaller Rg value indicates that the molecule is more compact, generally meaning that the molecule remains in a more folded or compact state; whereas a larger Rg value indicates that the molecule is more unfolded or loose, generally meaning that the molecule may be in a relaxed or unfolded state. Therefore, proteins with smaller RMSD and Rg values have greater structural stability, which for enzymes generally means greater thermal stability. Comparing the RMSD and Rg values of the matrix enzyme and the designed enzyme helps ensure that the designed enzyme has lower RMSD and Rg values, thereby verifying that the designed enzyme has higher thermal stability than the matrix enzyme, which helps improve the reliability of the optimized design.

[0037] In some embodiments, a method for constructing a model for simulating a motion trajectory includes: Under the AMBER99SB force field, the protein structure was solvated in a rectangular box filled with TIP3P water, and sodium or chloride ions were added to neutralize the charge. The distance between the protein and the box wall was set to a preset distance. The particle-mesh Ewald method was applied to treat Coulomb interactions, periodic boundary conditions were applied, and the constructed system was energy minimized. A two-stage preconditioning operation ensured that the TIP3P water in the system reached the preset temperature and pressure conditions. The system was simulated unconstrained, and a structure was output every time period to obtain the motion trajectory. A position-restricted pre-equilibration strategy was used to release the degrees of freedom in the flexible region, avoiding irrationalities caused by initial structural deviations and ensuring the consistency of the results through multiple equilibrium simulations.

[0038] In some embodiments, in the model construction of the motion trajectory, the simulation step size is set to any value between 1fs and 2fs, for example, it can be any value between 1fs, 1.2fs, 1.4fs, 1.6fs, 1.8fs and 2fs, the van der Waals interaction cutoff value is set to any value between 10Å and 14Å, for example, it can be any value between 10Å, 11Å, 12Å, 13Å and 14Å, the preset distance is any value between 8Å and 12Å, for example, it can be any value between 8Å, 9Å, 10Å, 11Å and 12Å, the total time of the unconstrained simulation is any value between 15ns and 25ns, for example, it can be any value between 15ns, 17ns, 19ns, 21ns, 23ns and 25ns, and the time period is any value between 70ps and 130ps, for example, it can be any value between 70ps, 90ps, 110ps and 130ps.

[0039] In some embodiments, the two-stage pre-processing operation includes: Use a V-rescale thermostat to set the temperature to a first temperature, maintain the number of particles, volume, and temperature in the model unchanged for a first time, and then use a Parrinello-Rahman barostat to adjust the pressure in the model to a first pressure, and maintain the number of particles, pressure, and temperature in the model unchanged for a second time.

[0040] In some embodiments, the first temperature is any value between 280K and 320K, for example, it can be any value between 280K, 290K, 300K, 310K and 320K, the first time is any value between 80ps and 120ps, for example, it can be any value between 80ps, 90ps, 100ps, 110ps and 120ps, the first pressure is any value between 0.8 atmospheres and 1.2 atmospheres, for example, it can be any value between 0.8 atmospheres, 0.9 atmospheres, 1 atmosphere, 1.1 atmospheres and 1.2 atmospheres, and the second time is any value between 80ps and 120ps, for example, it can be any value between 80ps, 90ps, 100ps, 110ps and 120ps.

[0041] In a third aspect, the present invention also applies to protect a DNA molecule, which encodes the above-mentioned xylanase.

[0042] In a fourth aspect, the present invention also applies to protect a recombinant plasmid carrying the above-mentioned DNA molecule.

[0043] In a fifth aspect, the present invention also applies to protect a modified cell, which includes the above-mentioned DNA molecule.

[0044] Please refer to the following examples for details.

[0045] Example 1: See Figure 1 The optimization design method of xylanase shown in a preferred embodiment of the present application includes: S1. Using an online protein structure prediction platform, the structure of the matrix enzyme was predicted based on its amino acid sequence to obtain a predicted structure. The matrix enzyme is a multi-domain xylanase and has a connecting segment connecting adjacent functional domains.

[0046] S2. Based on the predicted structure, the motion trajectory of the matrix enzyme in the solution is simulated using molecular dynamics simulation software under the AMBER99SB force field to obtain a trajectory file.

[0047] S3. Analyze the trajectory file using molecular dynamics simulation software, remove periodic artifacts, and calculate the root mean square fluctuation value.

[0048] S4. Delete the segment with the highest root mean square fluctuation value in the amino acid sequence of the connecting segment to obtain the amino acid sequence of the designed enzyme.

[0049] In step S1, the protein structure prediction platform was the AlphaFold3 online server. The wild-type xylanase PuXyn from Penicillium species was used as the base enzyme. The NCBI accession number for the base enzyme is KAF7714546.1, and the amino acid sequence is SEQ ID NO: 1. The base enzyme consists of a catalytic domain, a carbohydrate-binding domain, and a linker peptide connecting the two. The linker peptide is the linker segment.

[0050] In step S2, the molecular dynamics simulation software is the General-purpose Open-source Molecular-dynamics Algorithms and Systems (GROMACS) software version 2022.6. The motion trajectory of the substrate enzyme in solution is simulated using this software. The specific model construction method includes: The AMBER99SB force field was used, and the three-point charge water model (TIP3P) was used as the water model. The simulation step size was set to 2 fs. The particle-mesh Ewald (PME) method was applied to account for Coulomb interactions, with a van der Waals interaction cutoff of 12 Å. The protein structure was solvated in a rectangular box filled with TIP3P water, 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 constructed system was energy minimized. A two-stage preconditioning operation was performed to ensure that the TIP3P water in the system reached the preset temperature and pressure conditions. The system was simulated unconstrained for 20 ns, with a structure output every 100 ps to obtain the motion trajectory.

[0051] The two-stage preprocessing involved setting the temperature to 300 K using a velocity rescaling (V-rescale) thermostat, maintaining the model's particle number, volume, and temperature constant for 100 ps to maintain protein structural equilibrium. The model's pressure was then adjusted to 1 atmosphere using a Parrinello-Raman barostat, maintaining the model's particle number, pressure, and temperature constant for 100 ps to maintain protein structural equilibrium.

[0052] In step S3, the trajectory files obtained from the simulation were further processed and analyzed using GROMACS 2022.6. After removing periodicity from the trajectory, the root mean square fluctuation (RMSF), root mean square deviation (RMSD), and radius of gyration (Rg) of the trajectory were calculated. RMSF represents the offset of each atom or residue relative to its average position. Regions with large RMSF values generally indicate greater flexibility and conformational changes in these regions. See [ 15 ] for more information. Figure 2 The RMSF value corresponding to the linker region of the matrix enzyme was the largest, indicating that this region may exhibit strong flexibility and instability. Based on the above molecular dynamics simulation results, positions 350-373 with the largest RMSF value in the linker region were deleted to construct a designed enzyme with the amino acid sequence of SEQ ID NO: 2. This design retains sufficient flexibility to maintain relative motion between the catalytic domain and the carbohydrate binding domain, maintaining the enzyme's catalytic efficiency while reducing excessive flexibility and improving the stability of the overall protein structure.

[0053] In this embodiment, the optimization design method further includes: 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.

[0054] S6. Based on the designed structure, the motion trajectory of the designed enzyme in the solution is simulated using molecular dynamics simulation software to obtain a simulation file.

[0055] S7. Analyze the simulation file using molecular dynamics simulation software to remove periodic artifacts, and calculate the root mean square deviation and gyration radius. Analyze the trajectory file to obtain the root mean square deviation and gyration radius, and compare the two.

[0056] The only difference between steps S5, S6, and S7 and steps S1 to S3 is that the designed enzyme sequence is used for simulation and analysis, and in step S7, the RMSF value does not need to be calculated, only the RMSD and Rg values are calculated and compared with the values in step S3. Figure 2 It can be seen that after the site deletion, the RMSD and Rg values have decreased significantly, further improving the reliability of the enzymatic activity and thermal stability of the designed enzyme being better than the matrix enzyme, and reducing the risk of the design results being inconsistent with the actual results.

[0057] The amino acid sequence of SEQ ID NO:1 expressing the xylanase was fully synthesized. 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 generating the recombinant plasmid pPIC9K-PuXyn expressing the wild-type xylanase PuXyn. PCR amplification was performed using KOD Fx Neo high-fidelity DNA polymerase (Toyobo Shanghai Biotechnology Co., Ltd., KFX-201) with primers "Puc-1" (SEQ ID NO:4) and "349_ga2" (SEQ ID NO:5). In this example, the primer sequences were oriented 5'-3'. The gene fragments corresponding to positions 1-349 of the base enzyme and the designed enzyme, "PuXyn-up," were obtained by gel extraction and purification. The PCR system is shown in Table 1, where "Primer_1" and "Primer_2" refer to the two primers used for amplification, respectively.

[0058]

[0059] Using the recombinant plasmid pPIC9K-PuXyn as a template, PCR amplification and recovery purification were performed in the same way 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-lower" corresponding to the 374-413 sites of the matrix enzyme.

[0060] The plasmid vector pPIC9K-PuXyn was digested with the restriction enzymes EcoRI and NotI and purified using the same method to obtain the plasmid vector gene fragment "pPIC9K-B." The gene fragments "PuXyn-Upper" and "PuXyn-Lower" were ligated with "pPIC9K-B" using a homologous recombination kit (Beijing Quanshijin Biotechnology Co., Ltd., CU101-01). The ligation products were transformed into competent Escherichia coli TOP10 cells (Beijing Zhuangmeng International Biogene Technology Co., Ltd., ZC104) and plated on LBK kanamycin-resistant plates for selection of positive transformants. Correct transformants, identified by colony PCR, were then inoculated into LB medium, and the plasmid was extracted. Sequencing verification was performed by Shanghai Sangon Biotechnology Co., Ltd. to obtain the recombinant plasmid pPIC9K-PuXyn-Del 350-373 carrying the designed enzyme gene.

[0061] Prepare BMGY and BMMY medium. BMGY medium consists of 1% yeast extract, 2% tryptone, 1.34% amino-free yeast nitrogen base, 10% 1 mol / L potassium phosphate buffer (pH 6.0), and 1% glycerol. After preparation, autoclave sterilize at 115°C for 20 minutes. In BMMY medium, only the 1% (v / v) glycerol is replaced with 1% (v / v) methanol, which is added after autoclaving. All concentrations are expressed as mass-volume percentages.

[0062] The recombinant plasmids pPIC9K-PuXyn and pPIC9K-PuXyn-Del 350-373 were linearized with the restriction endonuclease SalI, and the two linearized vectors were purified and recovered and electroporated into Pichia pastoris GS115 competent cells purchased from Life Technologies Corporation, USA. Positive transformants were screened on histidine auxotrophic plates, and the obtained positive transformants were inoculated into BMGY medium. After shaking culture at 30°C and 250 rpm for 20 h, the cells were centrifuged at 6000 rpm for 5 min, the bacterial pellets were collected and resuspended in BMMY medium, and the concentration of the suspension was adjusted to make the OD of the suspension 600 The value of α was approximately 0.5, and fermentation was continued at 30°C, 250 rpm, with shaking. Methanol was added daily to a final concentration of 1%. After five days of fermentation, the supernatant was centrifuged at 6000 rpm for 5 minutes. The supernatant was recovered to obtain an enzyme solution containing the matrix enzyme or the designed enzyme. The enzyme solution containing the matrix enzyme was designated WT, and the enzyme solution containing the designed enzyme was designated Del 350-373.

[0063] Detect the activity of the matrix enzyme and the designed enzyme separately. Mix 100 μL of 1% beechwood xylan substrate and 100 μL of enzyme solution of the same concentration, adjust the pH to about 5.0, and react in a water bath at 50°C for 30 minutes. After the reaction is completed, add 600 μL of DNS solution to terminate the reaction and boil it in a water bath for 5 minutes. After cooling, measure the absorbance value A540 at a wavelength of 540 nm and read the content of released reducing sugars against the standard curve. 1 enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under given conditions. For calculation of specific enzyme activity and drawing statistical graphs, please refer to Figure 4 It can be seen that after deleting the highly unstable 350-373 site, the specific enzymatic activity of the designed enzyme was significantly improved, which was about 1.18 times that of the base enzyme.

[0064] The thermal stability of the matrix enzyme and the designed enzyme was tested separately. The enzyme solution was incubated in a thermomixer at 70°C for different time periods. After incubation, the solution was centrifuged at 13,300 rpm for 2 minutes to remove the precipitated inactivated enzyme. The activity was then tested again. The residual enzyme activity was calculated using the activity of the enzyme solution that had not been heat-incubated as a control. Figure 5 It can be seen that the base enzyme basically lost its catalytic activity after incubation at 70℃ for 2h, while the designed enzyme can still retain more than 80% of its catalytic activity under the same conditions. Even after incubation for 4h, the mutant still retains more than 50% of its catalytic activity.

[0065] Combine Figure 4 and Figure 5 The results show that the designed enzyme has superior activity and thermostability compared to the matrix enzyme, demonstrating the effectiveness of molecular dynamics simulation-guided enzyme protein engineering. This new type of designed enzyme will help expand the application of matrix enzymes in lignocellulose degradation, food, and feed. This xylanase optimization design method guides mutant design by rapidly identifying amino acid sites or fragments that have a significant impact on enzyme activity and thermostability, significantly reducing the time and cost of traditional enzyme engineering and providing high reliability.

[0066] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0067] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

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

2.

2. A method for optimizing the design of xylanase, characterized in that: include: Using an online protein structure prediction platform, a structure prediction is performed based on the amino acid sequence of the matrix enzyme to obtain a predicted structure. The matrix enzyme is a multi-domain xylanase having a connecting segment connecting adjacent functional domains. According to the predicted structure, the motion trajectory of the matrix enzyme in the solution is simulated using molecular dynamics simulation software under the AMBER99SB force field to obtain a trajectory file; Analyzing the trajectory file using the molecular dynamics simulation software to remove periodic artifacts and calculate the root mean square fluctuation value; The segment with the highest root mean square fluctuation value in the amino acid sequence of the connecting segment is deleted to obtain the amino acid sequence of the designed enzyme.

3. The method for optimizing the design of xylanase according to claim 2, wherein: Also includes: Using the protein structure prediction platform, structure prediction is performed based on the amino acid sequence of the designed enzyme to obtain a designed structure; According to the designed structure, simulating the motion trajectory of the designed enzyme in the solution using the molecular dynamics simulation software to obtain a simulation file; The simulation file is analyzed by the molecular dynamics simulation software to remove periodic artifacts, and the root mean square deviation and gyration radius are calculated. The root mean square deviation and gyration radius are obtained by analyzing the trajectory file, and the two are compared.

4. The method for optimizing the design of xylanase according to claim 2, wherein: The model construction method for simulating the motion trajectory includes: Under the AMBER99SB force field, the protein structure is placed in a rectangular box filled with TIP3P water for solvation and sodium ions 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 treat Coulomb interactions, periodic boundary conditions are applied, and the constructed system is energy minimized. A two-stage pretreatment operation is performed to ensure that the TIP3P water in the system reaches the preset temperature and pressure conditions. The system is simulated unconstrainedly, and a structure is output every time period to obtain the motion trajectory.

5. The method for optimizing the design of xylanase according to claim 4, wherein: 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.

6. The method for optimizing the design of xylanase according to claim 4, wherein: The two-stage preprocessing operations include: Use a V-rescale thermostat to set the temperature to a first temperature, maintain the number of particles, volume, and temperature in the model unchanged for a first time, and then use a Parrinello-Rahman barostat to adjust the pressure in the model to a first pressure, and maintain the number of particles, pressure, and temperature in the model unchanged for a second time.

7. The method for optimizing the design of xylanase according to claim 6, wherein: 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.

8. A DNA molecule, characterized in that The DNA molecule encodes the xylanase according to claim 1.

9. A recombinant plasmid, characterized in that The recombinant plasmid carries the DNA molecule according to claim 8.

10. A modified cell, characterized in that The modified cell comprises the DNA molecule according to claim 8.

Images