Beta-1, 4-xylanase mutant and construction method thereof

Abstract

The construction method comprises the following steps: constructing a beta-1, 4-xylanase recombinant expression vector, namely introducing a recombinant vector pNZ8048-P5-XynA into competent lactococcus lactis by utilizing an electrotransformation technology to construct a wild type expression vector, and designing and constructing the beta-1, 4-xylanase mutant by utilizing site-specific mutagenesis on the basis of the wild type expression vector. The method comprises the following steps: introducing a multi-site mutant recombinant plasmid in which asparagine (Asn) at the eighth site of beta-1, 4-xylanase is mutated into tyrosine (Tyr), serine (Ser) at the 22nd site is mutated into proline (Pro) and alanine (Ala) at the 59th site is mutated into lysine (Lys) into competent lactococcus lactis to form a heterologous expression strain of the mutant beta-1, 4-xylanase (Mut-1); a mutant xylanase beta-1, 4-xylanase (Mut-1) with high activity is stably obtained through a metal affinity chromatography method (Ni < 2 + >). The specific enzyme activity of the mutant reaches 1929.30 + / -5.36 U / mg, and the optimal temperature is improved by 15 DEG C compared with that of a wild type.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a β-1,4-xylanase mutant and its construction method. Background Technology

[0002] Xylan is the most abundant hemicellulose component in plant cell walls. It is a heteropolysaccharide widely found in various plants, such as corn cobs, sugarcane bagasse, and straw—a type of agricultural waste. It is the most abundant polysaccharide in nature besides cellulose. β-1,4-xylanase is a glycosidase (O-glycoside hydrolase, EC 3.2.1.8) derived from *Bacillus amyloliquefaciens*. It specifically hydrolyzes the β-1,4-glycosidic bonds in xylan, producing high-value-added products such as xylose and xylooligosaccharides. Furthermore, as a food additive, it plays an important role in various fields, including juice clarification, bread baking, beer processing, and animal feed processing. Freshly squeezed juice is rich in polysaccharides such as pectin, starch, and xylan, which are prone to precipitation during processing. Using xylanase to degrade xylan in juice can reduce the viscosity and turbidity of the beverage. Insoluble arabinoxylan produced during bread baking can affect the quality of dough and bread. Its high water-holding capacity competes with gluten for water, interfering with gluten network formation and protein cross-linking, thus affecting the rheological properties of the dough. Adding xylanase can hydrolyze insoluble arabinoxylan, reducing its molecular weight, improving the physicochemical properties of the dough, and increasing the bread's extensibility and elasticity.

[0003] However, the inherent properties of xylanase limit its widespread application in industrial production. Its poor thermal stability and low catalytic activity make it difficult to function efficiently in complex industrial environments, failing to meet industrial production demands. Therefore, modifying the enzymatic properties of xylanase, especially improving its thermal stability and catalytic activity, has become crucial for promoting its industrial application. Protein engineering technology combined with efficient screening mechanisms provides an effective means for modifying xylanase. Through precise modification of its molecular structure, the enzyme's activity and stability can be significantly enhanced. This not only improves food processing efficiency, reduces enzyme usage, and lowers production costs, but also lays the theoretical foundation for the widespread industrial application of xylanase, promoting the sustainable development of the food industry. Summary of the Invention

[0004] One object of this invention is to provide a site-directed mutagenesis strategy for β-1,4 xylanase, as well as the nucleotide and amino acid sequences of wild-type β-1,4 xylanase and optimal β-1,4 xylanase mutants. This invention also provides an efficient method for heterologous expression and protein purification of β-1,4 xylanase.

[0005] To achieve the above and related objectives, the technical solution provided by this invention involves modifying β-1,4-xylanase. First, the genes for β-1,4-xylanase, 6His-Tag, signal peptide Usp45, and the P5 promoter are ligated into plasmid pNZ8048 and introduced into competent L. lactis NZ9000 cells to construct a heterologous expression system of recombinant xylanase in L. lactis. Then, through site-directed mutagenesis, multiple single and multiple mutants are designed near Asn-8, Ser-22, and Ala-59. These mutants are then constructed, expressed, and secreted. The purified mutant enzymes are obtained using metal affinity chromatography. Through screening, the mutant Mut-1 (N8Y / S22P / A59K) with improved xylanase activity and thermostability is obtained. This helps to enhance its practical utilization value in high-temperature processing environments (such as feed manufacturing and biomass conversion). To achieve the above and other related objectives, the technical solution provided by the present invention is: a β-1,4 xylanase mutant derived from Bacillus amyloliquefaciens, characterized in that: the amino acid sequence of the β-1,4 xylanase mutant is shown in SEQ ID NO.4.

[0006] The preferred technical solution is as follows: the amino acid sequence of wild-type β-1,4 xylanase is shown in SEQ ID NO.1, and the sequence of wild-type β-1,4 xylanase is modified at the amino acid level, including the substitution, deletion or addition of one or more amino acids with equivalent functions.

[0007] The preferred technical solution is to mutate the 8th position asparagine (Asn) to tyrosine (Tyr), the 22nd position serine (Ser) to proline (Pro), and the 59th position alanine (Ala) to lysine (Lys).

[0008] To achieve the above and other related objectives, the technical solution provided by the present invention is: a gene encoding a β-1,4 xylanase mutant derived from Bacillus amyloliquefaciens as described in claim 1, characterized in that: the nucleotide sequence is as shown in SEQ ID NO.3.

[0009] The preferred technical solution is as follows: the nucleotide sequence of the wild-type β-1,4 xylanase gene is shown in SEQ ID NO.1. The wild-type β-1,4 xylanase gene is modified at the nucleotide level, including substitution, deletion or addition of one or more codons with equivalent functions, to obtain the target gene.

[0010] To achieve the above and other related objectives, the technical solution provided by this invention is: a method for constructing a β-1,4 xylanase mutant derived from Bacillus amyloliquefaciens, characterized by comprising the following steps: Step 1: Obtaining the wild-type β-1,4 xylanase gene; Step 2: Construction of the expression vector for the wild-type β-1,4 xylanase gene; Step 3: Site-directed mutagenesis amplification of the wild-type β-1,4 xylanase gene expression vector; Step 4: Construction of the mutant β-1,4 xylanase gene expression vector; Step 5: Construct expression mutant library and screen mutants with high enzyme activity and high thermal stability.

[0011] To achieve the above and other related objectives, the technical solution provided by the present invention is: a method for purifying the protein of a β-1,4 xylanase mutant derived from Bacillus amyloliquefaciens, characterized in that: His-Tag is added to the end of the ribonuclease, and then metal affinity chromatography (Ni2+) is used to elute and purify the β-1,4 xylanase mutant.

[0012] The beneficial effects of this application are that the present invention provides: (1) a method for constructing a β-1,4 xylanase mutant derived from Bacillus amyloliquefaciens and its encoding gene; (2) the expression of the β-1,4 xylanase mutant in Lactococcus lactis, and the expression of the mutant in Lactococcus lactis through heterologous expression and a metal affinity chromatography method (Ni 2+ We were able to stably obtain a mutant xylanase Mut-1 (N8Y / S22P / A59K) with high activity and high thermal stability. Attached Figure Description

[0013] Figure 1 : Recombinant plasmid pNZ8048-P5-XynA.

[0014] Figure 2 Three-dimensional structural model of β-1,4 xylanase.

[0015] Figure 3 Electrophoresis of recombinant plasmids containing wild-type β-1,4 xylanase.

[0016] Figure 4 SDS-PAGE electrophoresis of β-1,4 xylanase mutant.

[0017] Figure 5 β-1,4 xylanase wild-type and mutant enzyme activity was determined at different temperatures.

[0018] Figure 6 Structural analysis of wild-type and mutant N8Y β-1,4 xylanase (a: wild-type, b: mutant N8Y).

[0019] Figure 7Structural analysis of β-1,4 xylanase mutants N8Y and Mut-1 (a: mutant N8Y, b: mutant Mut-1). Detailed Implementation

[0020] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0021] Please see Figure 1-7 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes and to aid those skilled in the art in understanding and reading the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0022] The following are some of the materials used in this invention: (1) Strains and plasmids Bacillus amyloliquefaciens BH072 was obtained from the laboratory of Hefei University of Technology.

[0023] Lactococcus lactis NZ9000 was obtained from the laboratory of Hefei University of Technology.

[0024] Plasmids: pNZ8048, from the laboratory of Hefei University of Technology; pNZ8048-P5-XynA, from the laboratory of Hefei University of Technology.

[0025] (2) Reagents 1. Biochemical reagents: Xylose, xylooligosaccharide (X2-X6) analytical standards, and beech xylan were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; glucose, agarose, D-xylose, chloramphenicol, anhydrous ethanol, acetic acid, acetonitrile (chromatographic grade), imidazole, glycerol and other biochemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.; M17 broth was purchased from Haibo Biotechnology Co., Ltd. (Qingdao, China); DNS reagent and Coomassie R-250 were purchased from Beijing Solarbio Co., Ltd.

[0026] 2. Kits and Molecular Biology Reagents The EasyPure Genomic DNA Extraction Kit (containing RNase A), EasyPure HiPure Plasmid DNA Extraction Kit, EasyPure PCR Purification Kit, and EasyPure Rapid Gel Extraction Kit, among other nucleic acid extraction and purification kits, were purchased from Beijing TransGen Biotech Co., Ltd.; the BCA protein concentration assay kit and SDS-PAGE gel preparation kit were purchased from Beijing Solarbio Co., Ltd.; restriction endonucleases such as NcoI, KpnI, and BglII, T4 ligase, Taq-Mix for PCR amplification, PFU high-fidelity enzyme, Ni-NTA 6FF (His-tag) protein agarose purification resin, and DNA markers were all purchased from Shanghai Sangon Biotech Co., Ltd. (3) Main solutions and preparation Table 1: Main solutions and preparation methods used in this invention

[0027] Example 1: Establishment of a heterologous expression system for β-1,4-xylanase using Lactococcus lactis as the host. 1. Extraction of genomic DNA Genomic DNA was extracted using the Solarbio Bacterial Genomic DNA Extraction Kit (product model: D1600-100): (1) Take the Bacillus amyloliquefaciens carrying the β-1,4 xylanase gene stored in the laboratory and place it on LB medium for streak plate culture for 16-24h. Pick a single colony and inoculate it into LB liquid medium for 16-24h.

[0028] (2) Take 1 ml of bacterial culture medium, centrifuge at 12000 rpm for 1 min, and remove the supernatant as much as possible.

[0029] (3) Add 200 μL of solution A to the bacterial cells, shake or pipette to fully suspend the bacterial cells (if it is a Gram-positive bacterium, lysozyme with a final concentration of 20 mg / ml can be added at this step), add 20 μL of RNase A (10 mg / ml) to the suspension, mix thoroughly by inverting, and let stand at room temperature for 15-30 min.

[0030] (4) Add 20 μL of proteinase K (10 mg / ml) to the tube, mix thoroughly, and digest at 55°C for 30-60 min. During digestion, the centrifuge tube can be inverted several times to mix until the sample is completely digested. At this time, the bacterial solution will be clear and viscous.

[0031] (5) Add 200 μL of solution B to the tube and mix thoroughly by inverting. If a white precipitate appears, place it at 75°C for 15-30 minutes. The precipitate will disappear and will not affect subsequent experiments. If the solution does not become clear, it indicates that the sample digestion is incomplete, which may lead to a decrease in the amount and purity of DNA extracted, and may also clog the adsorption column.

[0032] (6) Add 200 μL of anhydrous ethanol to the tube and mix thoroughly. At this time, flocculent precipitate may appear, which will not affect DNA extraction. The solution and flocculent precipitate can be added to the adsorption column and left to stand for 2 minutes.

[0033] (7) Centrifuge at 12000 rpm for 2 min. Discard the waste liquid and put the adsorption column into the collection tube.

[0034] (8) Add 600 μL of rinsing solution to the adsorption column (please check whether anhydrous ethanol has been added before use). Centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column into the collection tube.

[0035] (9) Add 600 μL of washing solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column into the collection tube.

[0036] (10) Centrifuge at 12000 rpm for 2 min, and place the adsorption column open at room temperature or in a 50℃ incubator for several minutes. The purpose is to remove the residual washing solution in the adsorption column. Otherwise, the ethanol in the washing solution will affect subsequent experiments such as enzyme digestion and PCR.

[0037] (11) Place the adsorption column into a clean centrifuge tube, add 50-200 μL of preheated elution solution to the center of the adsorption membrane, let it stand at room temperature for 5 min, and centrifuge at 12000 rpm for 1 min.

[0038] (12) Add the elution obtained by centrifugation back into the adsorption column, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to obtain high-quality bacterial genomic DNA.

[0039] 2. Design of recombinant plasmid primers Before constructing the recombination of the target gene and plasmid pNZ8048, PCR amplification of the target fragment was performed. Sequence amplification required primers complementary to the N and C ends of the target fragment. The target gene sequence was first compared with the shuttle plasmid gene sequence in CloneManager 8.0 software. A sequence complementary to both ends of the target fragment was designed as a primer. Regions on the primers not complementary to the N and C ends of the DNA were extended with corresponding restriction enzyme sites. The N-terminal restriction enzyme site was NcoⅠ (CCATGG), and the C-terminal restriction enzyme site was KpnⅠ (GGTACC).

[0040] 3. Construction of recombinant plasmids A recombinant expression vector for the β-1,4-xylanase gene was constructed using the pNZ8048 vector. This pNZ8048 vector contains a constitutive P5 promoter, whose activity is not regulated by intracellular or extracellular environmental signals (such as nutrient conditions, temperature, inducers, etc.), and it functions stably in most stages of lactic acid bacteria growth. Usp45 was used as the signal peptide to secrete β-1,4-xylanase extracellularly. In primer design, a His-Tag was introduced at the β-1,4-xylanase terminus to enable protein purification by metal affinity chromatography. The plasmid was amplified by round PCR, with the selected heterologous signal peptide gene sequence added during primer design. The amplification system and procedure are as follows: (1) Take out the commercial DNA polymerase, buffer, dNTPs, template DNA, primers and sterile double-distilled water from the -20℃ freezer and place them on ice to thaw.

[0041] (2) In an ice bath, add the components to the PCR tube in the following order. PCR system:

[0042] (3) Set the reaction program. Centrifuge the above mixture briefly and immediately place it in a PCR instrument to start amplification.

[0043] Pre-denaturation at 95℃ for 5 min, followed by cyclic amplification: 94℃, 30 s → 57℃, 30 s → 72℃, 60 s, 30 cycles, and finally amplification at 72℃ for 10 min.

[0044] The linearized plasmid fragment was ligated overnight at 16°C using T4 DNA ligase. The ligation system consisted of 4 μL 5×T4 DNALigase Buffer, 1 μL T4 DNA Ligase, and 15 μL plasmid fragment. After mixing, ligation was performed overnight to obtain the recombinant plasmid. The constructed recombinant plasmid is shown below. Figure 1As shown. Electroporation of the recombinant plasmid into competent Lactococcus lactis NZ9000 yields Lactococcus lactis NZ900 (L. lactis-P5-XynA) capable of heterologously expressing recombinant β-1,4 xylanase.

[0045] Example 2: Construction of β-1,4 xylanase mutant using site-directed mutagenesis 1. Selection of mutation sites The three-dimensional structure of the protein β-1,4 xylanase was analyzed using PyMOL software. Subsequently, an alanine scan was performed on its substrate binding pocket and surrounding amino acid residues to obtain the binding free energy change (ΔΔG), which was then analyzed to determine the amino acid to be mutated. The three-dimensional structure of β-1,4 xylanase is shown below. Figure 2 As shown, the interaction between β-1,4 xylanase and xylohexasaccharide was identified. Ultimately, Asn-8 was selected as the ideal mutation site, and the mutant N8Y showed significantly improved thermostability and catalytic activity. To obtain a β-1,4 xylanase with even better performance, molecular docking simulations were used to identify the more volatile flexible regions in the N8Y mutant enzyme. This resulted in the multi-mutant N8Y / S22P / A59K (Mut-1), where the asparagine (Asn) at position 8 of the β-1,4 xylanase was mutated to tyrosine (Tyr), the serine (Ser) at position 22 to proline (Pro), and the alanine (Ala) at position 59 to lysine (Lys).

[0046] Table 2: Primers used for mutation and validation

[0047] 2. Preparation of competent cells of Lactococcus lactis L. lactis NZ9000 was streaked onto a GM17 plate and activated by incubation at 30°C. Single colonies were picked and inoculated into 5 mL test tubes (liquid M17 competent medium) and incubated overnight. The bacterial culture in the test tubes was then transferred to 100 mL of liquid M17 competent medium and cultured until the logarithmic growth phase (OD600 value of 0.5-0.6). The cells were collected by low-temperature centrifugation (7000 rpm, 20 min, 4°C) and washed three times with 50 mL of competent washing buffer (pre-cooled to an ice-water mixture) (centrifugation, removal of supernatant, and resuspending). Finally, the cells were resuspended with 1 mL of competent washing buffer, aliquoted into EP tubes, and stored at -80°C.

[0048] 3. Construction of mutant plasmids 10 μL of L. lactis NZ9000 (L. lactis-P5-XynA) containing the recombinant plasmid, stored at -80℃ in the laboratory, was inoculated onto M17 solid medium and streaked. After incubation at 30℃ for 24 h, a single colony was picked and transferred to 5 mL of liquid medium and incubated for 16 h. The plasmid was extracted from the culture using a kit, and the extracted plasmid was subjected to electrophoresis to verify its correctness. Plasmid extraction was performed according to the Solarbio Plasmid Mini-Extraction Kit instructions (catalog number: D1100). (1) Take 1-5 mL of bacterial culture, centrifuge at 12000 rpm for 1 min, and remove the supernatant as much as possible (if there is a lot of bacterial culture, the bacterial precipitate can be collected into a centrifuge tube by multiple centrifugations). (2) Add 250 μL of Solution I (please check if RNase A has been added) to the centrifuge tube containing the bacterial precipitate, and thoroughly suspend the bacterial cell precipitate using a pipette or vortex mixer. Note: If the bacterial clump is not thoroughly mixed, it will affect lysis and result in lower plasmid extraction yield and purity; (3) Add 250 μL of Solution II to the centrifuge tube and gently invert it 6-8 times to fully lyse the bacteria. Note: Mixing must be gentle to avoid contaminating the bacterial genomic DNA. At this point, the bacterial solution should become clear and viscous. Do not leave it for more than 5 minutes to avoid damaging the plasmids. (5) Add 350 μL of solution III to the centrifuge tube, and immediately gently invert it 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12000 rpm for 10 min, and carefully transfer the supernatant to another clean centrifuge tube using a pipette, trying not to aspirate the precipitate. Note: Solution III should be mixed immediately after addition to avoid local precipitation. If there is still a small white precipitate in the supernatant, centrifuge again and take the supernatant. (6) Add the supernatant obtained in the previous step to the adsorption column (add the adsorption column to the collection tube), let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. (7) Add 600 μL of washing solution I to the adsorption column (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column into the collection tube; (8) Add 700 μL of washing solution II to the adsorption column (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column into the collection tube; (9) Add 500 μL of washing solution II to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column into the collection tube; (10) Centrifuge at 12000 rpm for 2 min, and place the adsorption column open at room temperature or in a 50℃ incubator for several minutes. The purpose is to remove the residual washing solution in the adsorption column. Otherwise, the ethanol in the washing solution will affect subsequent experiments such as enzyme digestion and PCR. (11) Place the adsorption column into a clean centrifuge tube, add 50-200 μL of elution solution preheated in a 65°C water bath to the center of the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min to obtain the purified plasmid.

[0049] Electrophoresis results as follows Figure 4 As shown, lane 1 is a high molecular weight DNA marker (100-2000bp), and lanes 1, 2, and 3 are the target gene NcoI-usp45-xynA-KpnI, with a target gene size of approximately 1245bp. The bands shown in lanes 1, 2, and 3 are in the middle of the 1000-2000bp marker range, and the band size is consistent with the theory.

[0050] Using the extracted plasmid as a template, the amplification system consisted of 10 μL 5×fast pfu buffer, 4 μL dNTPs, 1 μL each of Pfu DNA polymerase, template, upstream primer, and downstream primer, and 32 μL ddH2O to bring the total reaction volume to 50 μL.

[0051] Using the mutant primers described in Table 2, and with the recombinant plasmid pNZ8048-P5-XynA as a template, round PCR amplification was performed to obtain the linearized mutant plasmid fragment. Detailed information on the mutant primers is listed in Table 2. The amplification system and procedure are as follows: (1) Take out the commercial DNA polymerase, buffer, dNTPs, template DNA, primers and sterile double-distilled water from the -20 ℃ freezer and place them on ice to thaw.

[0052] (2) In an ice bath, add the components to the PCR tube in the following order.

[0053] PCR system:

[0054] (3) Set the reaction program. Centrifuge the above mixture briefly and immediately place it in a PCR instrument to start amplification.

[0055] The system was denatured at 95°C for 30 seconds, annealed at 57°C for 60 seconds, and extended at 72°C for 1 minute using a polymerase chain reaction (PCR) instrument for 30 cycles, with a final extension at 72°C for 7 minutes.

[0056] After verification by agarose gel electrophoresis, the PCR products were recovered using a PCR product recovery kit and stored at 4°C for later use. The linearized plasmid fragment was ligated overnight at 16°C using T4 DNA ligase. The ligation system consisted of 4 μL 5×T4 DNALigase Buffer, 1 μL T4 DNA Ligase, and 15 μL plasmid fragment. After mixing, the mixture was ligated overnight to obtain the mutant plasmid.

[0057] 4. Electroporation to introduce competent cells The mutant recombinant plasmid was purified to remove excess salt ions from the solution. Specifically, 20 μL of the recombinant plasmid was pipetted into an ultrafiltration membrane floating in ultrapure water. This allowed smaller salt ions to permeate through the membrane via osmosis, preventing the generation of large currents during electroporation that could damage competent cells. After desalting for 30 minutes, the recombinant plasmid was collected. 5 μL of the recombinant plasmid, 50 μL of competent L. lactis cells, and an electroporation cuvette were thawed and pre-cooled on ice, respectively. The recombinant plasmid and competent cells were then mixed in the cuvette and placed on ice for three minutes. The electroporation parameters were adjusted to 200 Ohm, 2.5 kV, and 25 μF. The cuvette was dried and electroporation was performed. After electroporation, the system was quickly transferred to 1 mL of M17 electroporation recovery medium. After incubation at 30°C for 2 hours, 100 μL, 200 μL, and 300 μL of the recovery medium were plated onto antibiotic plates (GM17, CmR) and cultured overnight until mature, round, white single colonies formed.

[0058] 5. Screening of mutant positive clones Single colonies were picked for colony PCR verification to screen recombinant strains with correctly introduced mutant plasmids. The specific operation was as follows: a sterile toothpick was used to pick a single colony in a sterile laminar flow hood and disperse it in 10 μL of sterile water. This bacterial solution was used as a template for colony PCR. The fragments at both ends of the target gene in the plasmid were used as sequencing primers. 1 μL of the upstream primer seq-pNZ8048-F, the downstream primer seq-pNZ8048-F, and the bacterial solution were taken and colony PCR amplification was performed to verify the correct presence of the target gene. If the bacterial solution successfully passed the PCR verification, the remaining 9 μL of bacterial solution was recultured and the plasmid was extracted for sequencing.

[0059] The mutant plasmid was constructed by repeating the above procedure for the correctly sequenced strain. The primers used are shown in Table 2. Finally, a plasmid with three mutation sites was constructed, which was then electroporated into competent cells to construct a multi-mutant strain.

[0060] Example 3: Expression, purification, and investigation of enzyme properties of mutants 1. Expression of mutant enzymes Each *L. lactis* variant glycerol bacterium stored at -80 °C was activated on GM17 (CmR) plates. Smooth white colonies were then picked and reactivated in 5 mL of GM17 medium. At this point, the *L. lactis* strains exhibited strong growth activity. Subsequently, 1 mL of seed culture was added to 100 mL of fresh GM17 / CmR medium, and fermented at 30 °C. After 8 hours of incubation, the fermentation supernatant was collected by centrifugation and purified using nickel column affinity chromatography to obtain the mutant enzyme.

[0061] After fermentation, the bacterial culture was centrifuged at 4°C and 2200×g for 15 min in a high-speed refrigerated centrifuge to allow the cells to settle completely. The supernatant was then purified. Heterologous proteins secreted into the fermentation broth and containing histidine tags at the C-terminus were purified using fixed-metal affinity chromatography. First, 2 mL of the Ni-NTA packing mixture was placed in a centrifuge tube and centrifuged at low speed in a small refrigerated benchtop centrifuge at 4°C, 200×g for 10 min. Once the nickel column packing and agarose had separated into distinct layers, the agarose layer was gently removed using a pipette. 10 mL of Lysis buffer was thoroughly mixed with the nickel column, and the mixture was equilibrated using a balancing rotor for 10 min. After equilibration, the column was centrifuged, and the supernatant was discarded. This process was repeated three times. 45 mL of the fermentation supernatant was then mixed with washed Ni... 2+ The mixture was combined in a 50 mL centrifuge tube and equilibrated on a balanced rotor for 4 h to allow the heterologous protein containing the C-terminus 6His-tag to fully bind to the nickel column. The mixture was then washed with 50 mL of Wash buffer and eluted with one column volume of Elution buffer (containing imidazole) to dissociate the histidine tag from the nickel column. The collected target protein was ion-exchanged through a dialysis membrane (<10 kDa) in 0.2 M, pH 7.2 PBS buffer to remove imidazole from the target protein solution. The dialysis sample can be stored at -80°C.

[0062] The purified protein was subjected to SDS-PAGE electrophoresis, and the electrophoresis results are as follows: Figure 5 As shown, the molecular weight of wild-type Barnase protein is 23.26 kDa. Theoretically, a mutation of a single amino acid should not cause a large-scale change in the molecular weight of the protein. Therefore, the molecular weight of the mutant protein should also be 23.26 kDa. The electrophoresis results show that the mutant protein band is located between 14-25 kDa, which is basically consistent with its theoretical molecular weight (23.26 kDa), verifying the correct expression of the target protein.

[0063] 2. Measurement of enzyme content and activity The concentration of the purified mutant enzyme was determined using the BCA method, and the enzyme activity was measured according to the national standard (GB / T34222-2017) and previously published methods. β-1,4-xylanase hydrolyzes β-1,4-glycosidic bonds to produce reducing xylooligosaccharides and xylose. The amount of reducing sugar released is usually quantified using the 3,5-dinitrosalicylic acid method (DNS). Enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute from the substrate under optimal reaction conditions. The specific reaction system is as follows: 600 μL of 1% (w / v) xylan as substrate was added to 200 μL of citrate buffer (pH 6.0), followed by 200 μL of diluted β-1,4-xylanase. The mixture was then immediately placed in a 50℃ water bath for 10 min. After the reaction was complete, 1.2 mL of DNS was added to terminate the reaction, and the mixture was then incubated in boiling water for 10 min. Finally, the reaction mixture was diluted 8-fold, and the absorbance of each sample at 540 nm was measured using a microplate reader to calculate the enzyme activity of β-1,4-xylanase. D-xylose was used as the standard reducing sugar to prepare standard solutions with concentrations ranging from 0 to 1.0 mg / mL. 1 mL of different concentrations of D-xylose standard solution was mixed with 1.2 mL of DNS, and the mixture was subjected to color development. The absorbance of each standard sample at 540 nm was measured. A standard curve was plotted with the concentration of the D-xylan standard solution on the x-axis and the absorbance on the y-axis.

[0064] The kinetic parameters of wild-type Barnase and the mutant enzyme were measured separately, and the results are shown in Table 3. The mutant enzyme exhibited excellent catalytic activity, with a specific enzyme activity of 1929.30 ± 5.36 U / mg, which was 174.84% higher than that of the original enzyme. Table 3: Comparison of kinetic parameters between recombinant wild-type β-1,4-xylanase and mutant β-1,4-xylanase

[0065] Enzyme thermal stability assay The diluted β-1,4 xylanase was incubated with a 1% xylan mixture in a constant temperature water bath at 35 ℃–90 ℃ for 0–120 min. Immediately after incubation, the relative residual enzyme activity was measured, with the relative enzyme activity of BaXynA at 0 min incubation considered as 100%. Subsequently, a nonlinear fitting curve was generated based on the relative enzyme activity at different times. The nonlinear fitting is based on the exponential decay equation, usually calculated using formula (2-1). Here, y is the absorbance value changing with time, y0 is the initial absorbance value at 0 min incubation, t is the incubation time, and K is the decay rate constant, where Plateau is 0. The half-life is determined by K and calculated using formula (2-2), with a coefficient of determination of R², which can be used to determine the good agreement between the fitted curve and the experimental data.

[0066] y=(y0−Plateau)exp −Kt + Plateau (1) t1 / 2=ln2 / K (2) Table 4: Comparison of half-life of recombinant wild-type β-1,4-xylanase and mutant β-1,4-xylanase at various temperatures.

[0067] Table 4 shows that at medium to high temperatures, the half-life of the mutant enzyme was significantly longer than that of the wild type, indicating a significant improvement in the thermostability of the mutant enzyme. Figure 6 The mutant β-1,4 xylanase showed significantly improved thermal stability at high temperatures compared to the wild type. The wild type β-1,4 xylanase had an enzyme activity of less than 40% at 60℃, while the mutant enzyme activity remained above 80% at 60℃. Furthermore, it maintained high activity at high temperatures of 70-90℃, while the wild type rapidly became inactive at 70-90℃.

[0068] We simulated the substrate-binding pocket of β-1,4-xylanase using homology modeling and molecular docking techniques. Alanine scanning identified key amino acid residues in β-1,4-xylanase, and we then site-specifically modified amino acid residues near the substrate-binding pocket. We first identified the N-terminal Asn-8 residue as an ideal single mutation site, obtaining the mutant N8Y (where the 8th asparagine residue is mutated to tyrosine). This mutant exhibited significantly improved thermal stability and catalytic activity compared to the wild type. By comparing the structures of the wild type and N8Y, we found that when Asn is mutated to Tyr, two additional hydrogen bonds are formed between Tyr and the 1-xylan and 2-xylan units at the reducing end of the xylan substrate. Figure 6This non-covalent interaction effectively immobilizes the substrate in the substrate-binding pocket and enhances the enzyme's ability to recognize the substrate, thereby improving the enzyme's catalytic activity and substrate specificity. Furthermore, a hydrophobic interaction is formed between the benzene ring of the Tyr side chain and the side chain of Ala-18, and the hydroxyl modification of the benzene ring in the Tyr side chain not only reduces the degrees of freedom of the active pocket but also increases hydrophobic interactions. Therefore, the introduction of Tyr into the active pocket not only enhances the binding ability between the enzyme and the substrate but also further stabilizes the local structure of the protein and improves the enzyme's catalytic efficiency by reconstructing the hydrogen bond network and introducing additional hydrophobic interactions. Although the mutant enzyme's performance is improved compared to the wild type, the improvement in thermal stability is limited. Therefore, we searched for other mutation sites and used multi-site mutations to improve the dynamic correlation of the protein, enabling synergistic effects between amino acids, thereby significantly enhancing the protein's thermal stability.

[0069] We first predicted and located the inactive flexible regions in the N8Y mutant enzyme using molecular dynamics simulations. Then, combining Discovery Studio and various in silico tools, we employed a multi-level prediction combination method to screen three double mutants—N8Y / A59K, N8Y / S22P, and N8Y / N159V—with significantly improved or minimally changed enzyme performance compared to N8Y. Therefore, we considered combining mutation strategies for multiple flexible regions to further stabilize the structure of N8Y and improve the enzyme's thermostability and catalytic activity. Finally, after verification, we obtained the multi-mutant enzyme N8Y / S22P / A59K (Mut-1) with the greatest performance improvement. This was based on the analysis and comparison of the structures of mutant enzyme Mut-1 and N8Y.

[0070] The mutation of serine (Ser) at position 22 to proline (Pro) restricts the degree of freedom of the dihedral angles of the main chain due to the cyclic pyrrolidine structure of Pro, thus causing local regions of the protein to tend to form a more rigid conformation. This enhanced rigidity reduces conformational fluctuations in local regions at high temperatures, thereby improving the thermal stability of Mut-1. Furthermore, Pro is more hydrophobic than Ser, with relatively weaker interactions with solvents, improving the stability of the protein in solution. When alanine (Ala) at position 59 is mutated to lysine (Lys), Lys-59 forms additional hydrogen bonds with Val-57 and ASN-61 at distances of 1.8 Å and 3.0 Å, respectively, enhancing the local conformational stability of β-1,4-xylanase. Moreover, compared to Ala, Lys is a positively charged basic amino acid with a longer side chain, which, in addition to forming hydrogen bonds with surrounding amino acid residues, may also participate in more electrostatic interactions through its charge. Figure 7Therefore, this mutation plays a dual role in stabilizing the local and global conformation of the protein. This multi-interaction synergistic mechanism effectively reduces the probability of conformational fluctuations and disintegration of Mut-1 under high-temperature conditions. In summary, the above structural modifications significantly improve the catalytic efficiency and thermal stability of β-1,4-xylanase by enhancing the hydrogen bond network and conformational rigidity.

[0071] The nucleotide sequence of wild-type β-1,4-xylanase is SEQ ID NO.1: ATGTTTAAGTTTAAAAAGAAATTCTTAGTTGGATTAACGGCAGCTTTCATAAGTATCAGCATGTTTTCGGCAACCGCCTCTGCAGCTGGCACAGATTACTGGCAAAATTGGACTGATGGGGGCGGAACAGTCAACGCAGTCAATGGATCTGGCGGGAATTACAGT GTTAATTGGTCTAATACCGGAAATTTCGTTGTTGGTAAAGGCTGGACTACAGGCTCGCCATTTAGAACAATAAACTATAATGCCGGAGTCTGGGCGCCGAATGGCAATGGATATTTGACTTTATATGGCTGGACGAGAGCACCTCTCATCGAATATTATGTAGTG GATTCATGGGGTACTTACAGACCTACCGGAACGTATAAAGGTACTGTAAAGAGTGATGGAGGTACATATGACATATATACAACGACACGTTATAACGCACCTTCCATTGATGGCGATAACACTACTTTTACGCAGTACTGGAGTGTTCGCCAGTCGAAGAGGCCG ACCGGAAGCAACGCTGCAATCACTTTCAGCAATCATGTTAACGCATGGAAGAGTCATGGAATGAATCTGGGCAGTAATTGGGCTTACCAAGTCTTAGCGACAGAAGGATATAAAAGCAGCGGAAGTTCTAATGTAACAGTGTGGCATCACCATCACCATCACTAA The amino acid sequence of wild-type β-1,4-xylanase, SEQ ID NO.2: MFKFKKKFLVGLTAAFISISMFSATASAAGTDYWQNWTDGGGTVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLYGWTRAPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDNTTFTQYWSVRQSKRPTGSNAAITFSNHVNAWKSHGMNLGSNWAYQVLATEGYKSSGSSNVTVWHHHHHH Nucleotide sequence of mutant β-1,4-xylanase (Mut-1), SEQ ID NO.3: ATGTTTAAGTTTAAAAAGAAATTCTTAGTTGGATTAACGGCAGCTTTCATAAGTATCAGCATGTTTTCGGCAACCGCCTCTGCAGCTGGCACAGATTACTGGCAATATTGGACTGATGGGGGCGGAACAGTCAACGCAGTCAATGGACCTGGCGGGAATTACAGTGTTAATTGGTCTAATACCGGAAATTTCGTTGTTGGTAAAGGCTGGACTACAGGCTCGCCATTTAGAACAATAAACTATAATGCCGGAGTCTGGAAACCGAATGGCAATGGATATTTGACTTTATATGGCTGGACGAGAGCACCTCTCATCGAATATTATGTAGTGGATTCATGGGGTACTTACAGACCTACCGGAACGTATAAAGGTACTGTAAAGAGTGATGGAGGTACATATGACATATATACAACGACACGTTATAACGCACCTTCCATTGATGGCGATAACACTACTTTTACGCAGTACTGGAGTGTTCGCCAGTCGAAGAGGCCGACCGGAAGCAACGCTGCAATCACTTTCAGCAATCATGTTAACGCATGGAAGAGTCATGGAATGAATCTGGGCAGTAATTGGGCTTACCAAGTCTTAGCGACAGAAGGATATAAAAGCAGCGGAAGTTCTAATGTAACAGTGTGGCATCACCATCACCATCACTAA Amino acid sequence of mutant β-1,4-xylanase (Mut-1), SEQ ID NO.4: MFKFKKKFLVGLTAAFISMFSATASAAGTDYWQYWTDGGGTVNAVNGPGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWKPNGNGYLTLYGWTRAPLIEYYV VDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDNTTFTQYWSVRQSKRPTGSNAAITFSNHVNAWKSHGMNLGSNWAYQVLATEGYKSSGSSNVTVWHHHHHH The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A β-1,4-xylanase mutant, characterized in that, The amino acid sequence of the β-1,4-xylanase mutant is shown in SEQ ID NO.

4.

2. The β-1,4-xylanase xylanase mutant according to claim 1, characterized in that: A mutant β-1,4-xylanase was obtained by modifying the amino acid sequence of the wild-type β-1,4-xylanase, wherein the amino acid sequence of the wild-type β-1,4-xylanase is shown in SEQ ID NO.

2.

3. The β-1,4-xylanase mutant according to claim 2, characterized in that: The modification includes replacing the 8th position asparagine with tyrosine, the 22nd position serine with proline, and the 59th position alanine with lysine.

4. A gene encoding the β-1,4-xylanase mutant of claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

5. The gene according to claim 4, characterized in that: The gene was obtained by modifying the wild-type β-1,4-xylanase gene with nucleotides, wherein the nucleotide sequence of the wild-type β-1,4-xylanase gene is shown in SEQ ID NO.

1.

6. A vector having the DNA molecule as described in claim 4 or 5.

7. A host cell, characterized in that, It includes the carrier as described in claim 6.

8. A method for preparing the β-1,4-xylanase mutant according to claim 1, characterized in that, The steps include: a) Obtain the wild-type β-1,4-xylanase gene; b) Construct a wild-type β-1,4-xylanase gene expression vector; c) Site-directed mutagenesis amplification of the wild-type β-1,4-xylanase gene; d) Construct a gene expression vector for the β-1,4-xylanase mutant; e) Construct expression mutant libraries and screen for mutants with high enzyme activity and high thermal stability.

9. A method for purifying the β-1,4-xylanase mutant of claim 1, characterized in that: His-Tag was added to the C-terminus of the β-1,4-xylanase mutant, and the mutant was eluted and purified using metal affinity chromatography.

10. The application of the β-1,4-xylanase mutant of claim 1 in food processing, feed manufacturing, or biomass conversion, characterized in that: The β-1,4-xylanase mutant is used to degrade xylan in high-temperature processing environments.

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