A beta-1,4-xylanase mutant and a method for constructing the same

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to a beta-1, 4-xylanase mutant and a construction method thereof. BACKGROUND

[0002] Xylan is the most abundant hemicellulose component in plant cell walls, a heteropolysaccharide, widely exists in various plants such as corn cob, sugarcane residue, straw and other agricultural waste, and is the most abundant polysaccharide in nature except cellulose. Beta-1, 4-xylanase is a glycoside hydrolase (O-glycoside hydrolase, EC 3.2.1.8) derived from Bacillus amyloliquefaciens, which can specifically hydrolyze the beta-1, 4-glycosidic bond in xylan to generate xylose and high-value products such as oligoxylan. In addition, as a food additive, it can play an important role in many fields such as fruit juice clarification, bread baking, beer processing and animal feed processing. Freshly squeezed fruit juice is rich in pectin, starch and xylan and other polysaccharides, which are prone to precipitate during processing. The use of xylanase to degrade xylan in fruit juice can reduce the viscosity and turbidity of the beverage; insoluble arabinoxylan produced during bread baking can affect the quality of dough and bread, and its high water holding capacity can compete with gluten for water, interfere with the formation of gluten network and protein cross-linking, and thus affect the rheological properties of the dough.

[0003] However, the natural properties of xylanase limit its wide application in industrial production. Its poor thermal stability and low catalytic activity make it difficult to efficiently function in complex industrial environments, and it cannot meet the needs of industrial production. Therefore, improving the enzymatic properties of xylanase, especially its thermal stability and catalytic activity, has become the key to promoting the industrial application of xylanase. Protein engineering technology combined with efficient screening mechanism provides an effective means for the modification of xylanase. Through fine modification of its molecular structure, the activity and stability of the enzyme can be significantly improved. This not only can improve the efficiency of food processing, reduce the amount of enzyme used, and reduce production costs, but also lays a theoretical foundation for the wide application of xylanase in industry, and promotes the sustainable development of the food industry. SUMMARY

[0004] An object of the present application is to provide a site-directed mutation strategy for beta-1, 4-xylanase, as well as the nucleotide sequence and amino acid sequence of wild-type beta-1, 4-xylanase and the optimal beta-1, 4-xylanase mutant. The present application also provides a method for efficient heterologous expression and protein purification of beta-1, 4-xylanase.

[0005] In order to achieve the above-mentioned and related purposes, the technical scheme provided by the present application is to modify the beta-1, 4 xylanase, first link the genes of beta-1, 4 xylanase, 6His-Tag, signal peptide Usp45 and P5 promoter together and introduce into competent L.lactis NZ9000 to construct a heterologous expression system of recombinant xylanase in L.lactis, then design multiple single and multiple mutants near Asn-8, Ser-22 and Ala-59 through site-directed mutagenesis, and construct, express and secrete the mutants, obtain the purified mutant enzyme by using metal affinity chromatography, and through screening, obtain the mutant Mut-1 (N8Y / S22P / A59K) with improved xylanase activity and thermal stability, which is helpful to improve the practical utilization value in high-temperature processing environment (such as feed manufacturing, biomass conversion, etc.)

[0006] In order to achieve the above-mentioned and related purposes, the technical scheme provided by the present application is: a beta-1, 4 xylanase mutant from Bacillus amyloliquefaciens, characterized in that the amino acid sequence of the beta-1, 4 xylanase mutant is shown in SEQ ID NO. 4.

[0007] The preferred technical scheme is that the amino acid sequence of the wild-type beta-1, 4 xylanase is shown in SEQ ID NO. 1, and the sequence modification on the amino acid level is performed on the wild-type beta-1, 4 xylanase, including replacing, deleting or adding one or more amino acids with equivalent functions.

[0008] The preferred technical scheme is that the 8th asparagine (Asn) is mutated into tyrosine (Tyr), the 22nd serine (Ser) is mutated into proline (Pro), and the 59th alanine (Ala) is mutated into lysine (Lys).

[0009] In order to achieve the above-mentioned and related purposes, the technical scheme provided by the present application is: a gene encoding the beta-1, 4 xylanase mutant from Bacillus amyloliquefaciens according to claim 1, characterized in that the nucleotide sequence is shown in SEQ ID NO. 3.

[0010] The preferred technical scheme is that the nucleotide sequence of the wild-type beta-1, 4 xylanase gene is shown in SEQ ID NO. 1, and the sequence modification on the nucleotide level is performed on the wild-type beta-1, 4 xylanase gene, including replacing, deleting or adding one or more codons with equivalent functions to obtain the target gene.

[0011] In order to achieve the above-mentioned and related purposes, the technical scheme provided by the present application is: a construction method of a beta-1, 4 xylanase mutant from Bacillus amyloliquefaciens, characterized by comprising the following steps:

[0012] Step 1: obtaining of wild type beta-1, 4 xylanase gene;

[0013] Step 2: construction of wild type beta-1, 4 xylanase gene expression vector;

[0014] Step 3: site-directed mutation amplification of wild type beta-1, 4 xylanase gene expression vector;

[0015] Step 4: construction of mutant beta-1, 4 xylanase gene expression vector;

[0016] Step 5: construction of expression mutant library and screening of high-enzyme-activity and high-thermal-stability mutants.

[0017] To achieve the above object and other related objects, the technical scheme provided by the present application is a protein purification method for Bacillus amyloliquefaciens-derived beta-1, 4 xylanase mutants, characterized in that His-Tag is added to the end of the ribonuclease, and then the beta-1, 4 xylanase mutants are eluted and purified by using metal affinity chromatography (Ni2+).

[0018] The present application has the following beneficial effects: (1) a construction method of Bacillus amyloliquefaciens-derived beta-1, 4 xylanase mutants and its encoding gene; (2) the expression of the beta-1, 4 xylanase mutants in Lactococcus lactis is established, and high-activity and high-thermal-stability mutant xylanase Mut-1 (N8Y / S22P / A59K) is stably obtained by heterologous expression and a metal affinity chromatography (Ni 2+ ) method. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : recombinant plasmid pNZ8048-P5-XynA.

[0020] Figure 2 : three-dimensional structure model of beta-1, 4 xylanase.

[0021] Figure 3 : electrophoresis of recombinant plasmid containing wild type beta-1, 4 xylanase.

[0022] Figure 4 : SDS-PAGE electrophoresis of beta-1, 4 xylanase mutants.

[0023] Figure 5 : determination of enzyme activity of wild type and mutant beta-1, 4 xylanase at different temperatures.

[0024] Figure 6: Structure analysis of β-1, 4 xylanase wild type and mutant N8Y (a: wild type, b: mutant N8Y).

[0025] Figure 7 : Structure analysis of β-1, 4 xylanase mutant N8Y and mutant Mut-1 (a: mutant N8Y, b: mutant Mut-1). DETAILED DESCRIPTION

[0026] The following specific examples illustrate the embodiments of the present application, and other advantages and effects of the present application can be easily understood by those skilled in the art from the content disclosed in the examples.

[0027] Reference should be made to Figures 1-7 It should be understood that the structures, proportions, sizes, etc. shown in the drawings attached to the present specification are only used to understand and read the content disclosed in the present specification by those skilled in the art, and do not have technical significance to limit the conditions under which the present application can be implemented, so any modification of the structure, change of the proportion relationship or adjustment of the size. The following examples are provided to better understand the present application, but not to limit the present application. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available from a conventional biochemical reagent store unless otherwise specified.

[0028] Some of the materials used in the present application are as follows:

[0029] (1) Strains and plasmids

[0030] Bacillus amyloliquefaciens BH072, from the laboratory of Hefei University of Technology.

[0031] Lactococcus lactis NZ9000, from the laboratory of Hefei University of Technology.

[0032] Plasmids: pNZ8048, from the laboratory of Hefei University of Technology; pNZ8048-P5-XynA, from the laboratory of Hefei University of Technology.

[0033] (2) Reagents

[0034] 1. Biochemical reagents: Xylose, oligoxylosyl (X2-X6) analysis standard, beechwood xylan purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.; glucose, agarose, D-xylose, chloramphenicol, anhydrous ethanol, acetic acid, acetonitrile (chromatographically pure), imidazole, glycerol, etc. Biochemical reagents were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; M17 broth was purchased from Haibo Biological Technology Co., Ltd. (Qingdao, China), DNS reagent, Coomassie R-250 was purchased from Beijing Solabio Co., Ltd.

[0035] 2. Kits and molecular biology reagents

[0036] The nucleic acid extraction and purification related kits such as EasyPure Genomic DNA Extraction Kit (containing RNase A), EasyPure HiPure Plasmid DNA Extraction Kit, EasyPure PCR Purification Kit and EasyPure Fast Gel Extraction Kit were purchased from Beijing Zisangjin Biotechnology Co., Ltd.; BCA protein concentration determination kit and SDS-PAGE gel preparation kit were purchased from Beijing Solabio Co., Ltd.; NcoI, KpnI, BglII and other restriction endonucleases, T4 ligase, Taq-Mix for PCR amplification, pfu high-fidelity enzyme, Ni-NTA 6FF (His-tag) protein agarose purification resin and DNA marker were purchased from Shanghai Sangon Biotech Co., Ltd.

[0037] (3) Main solutions and preparation

[0038] Table 1: Main solutions used in the application and preparation methods

[0039]

[0040] Example 1: Establishing a heterologous expression system of β-1, 4 xylanase using Lactococcus lactis as a host

[0041] 1. Extraction of genomic DNA

[0042] The extraction of genomic DNA was performed using Solarbio-Bacterial Genomic DNA Extraction Kit (product model: D1600-100):

[0043] (1) The laboratory-preserved B. amyloliquefaciens carrying β-1, 4 xylanase gene was inoculated on LB medium for streak plate culture for 16-24 h, and a single colony was picked and inoculated in LB liquid medium for culture for 16-24 h.

[0044] (2) Take 1 ml of bacterial culture solution, centrifuge at 12000 rpm for 1 min, and try to aspirate the supernatant.

[0045] (3) Add 200ul solution A to the bacteria, shake or pipette to make the bacteria fully suspended (if it is gram-positive bacteria, add 20mg / ml lysozyme at this step), add 20ul RNase A (10mg / ml) to the suspension, mix well, and let stand at room temperature for 15-30min.

[0046] (4) Add 20ul proteinase K (10mg / ml) to the tube, mix well, and digest at 55℃ for 30-60min. The sample can be mixed several times by inverting the centrifuge tube during the digestion until the sample is completely digested, at which time the bacterial solution is clear and viscous.

[0047] (5) Add 200ul solution B to the tube, mix well, and if white precipitate appears, place at 75℃ for 15-30min, the precipitate will disappear, which does not affect the subsequent experiment. If the solution is not clear, it indicates that the sample is not completely digested, which can reduce the amount and purity of the extracted DNA and can also block the adsorption column.

[0048] (6) Add 200ul anhydrous ethanol to the tube, mix well, at which time flocculent precipitate can also appear, which does not affect the extraction of DNA, and the solution and flocculent precipitate can be added to the adsorption column and let stand for 2min.

[0049] (7) Centrifuge at 12000rpm for 2min, discard the waste liquid, and place the adsorption column in the collection tube.

[0050] (8) Add 600ul rinse solution (check whether anhydrous ethanol has been added before use) to the adsorption column. Centrifuge at 12000rpm for 1min, discard the waste liquid, and place the adsorption column in the collection tube.

[0051] (9) Add 600ul rinse solution to the adsorption column, centrifuge at 12000rpm for 1min, discard the waste liquid, and place the adsorption column in the collection tube.

[0052] (10) Centrifuge at 12000rpm for 2min, and place the adsorption column in a room temperature or 50℃ incubator for several minutes, which is to remove the residual rinse solution in the adsorption column, otherwise the ethanol in the rinse solution will affect the subsequent experiments such as enzyme digestion and PCR.

[0053] (11) Place the adsorption column in a clean centrifuge tube, and add 50-200ul elution solution preheated at 65℃ to the center of the adsorption membrane, let stand at room temperature for 5min, and centrifuge at 12000rpm for 1min.

[0054] (12) Centrifuge the eluate and add it to the adsorption column, let stand at room temperature for 2min, and centrifuge at 12000rpm for 2min to obtain high-quality bacterial genomic DNA.

[0055] 2. Primer design for recombinant plasmid

[0056] Before the recombinant construction of the target gene and plasmid pNZ8048, the target fragment is amplified by PCR. The primer needs to be designed to be complementary to the N and C terminal of the target fragment. First, the target gene sequence is compared with the shuttle plasmid gene sequence in clonemanager 8.0 software. The sequence complementary to the N and C terminal of the target fragment is designed as a primer. The region of the primer that is not complementary to the N and C terminal of the DNA is extended to add the corresponding restriction enzyme site. The N terminal restriction enzyme site is Nco I (CCATGG), and the C terminal is the restriction enzyme site Kpn I (GGTACC).

[0057] 3. Construction of recombinant plasmid

[0058] The recombinant expression vector of β-1,4 xylanase gene is constructed using the vector pNZ8048. The P5 constitutive promoter is contained in this vector pNZ8048, and its activity is not regulated by the intracellular and extracellular environmental signals (such as nutritional conditions, temperature, inducers, etc.). It can stably function in most stages of the growth of lactic acid bacteria. Usp45 is used as a signal peptide to secrete β-1,4 xylanase to the extracellular. When designing the primer, His-Tag is introduced at the end of β-1,4 xylanase to enable protein purification by metal affinity chromatography. The plasmid is amplified by Round PCR. When designing the primer, the selected heterologous signal peptide gene sequence is added. The amplification system and amplification program are as follows:

[0059] (1) Take out the commercial DNA polymerase, buffer, dNTPs, template DNA, primer, and sterile double distilled water from the -20℃ refrigerator, and place them on ice to thaw.

[0060] (2) In an ice bath, distribute each component into the PCR tube in the following order

[0061] PCR system:

[0062]

[0063] (3) Set the reaction program. Centrifuge the above mixture slightly, and immediately place it in the PCR instrument to start amplification.

[0064] 95℃ pre-denaturation for 5min, and then enter the cycle amplification stage: 94℃, 30s→57℃, 30s→72℃, 60s, cycle 30 times, and finally amplify at 72℃ for 10min.

[0065] The linearized plasmid fragment was connected using T4 DNA ligase at 16°C overnight, and the connection system was 4 μL 5×T4 DNA Ligase Buffer, 1 μL T4 DNA Ligase, 15 μL plasmid fragment, mixed and connected overnight to obtain a recombinant plasmid. The constructed recombinant plasmid is shown in Figure 1 The recombinant plasmid was electrotransformed into competent Lactococcus lactis NZ9000 to obtain L. lactis NZ900 (L. lactis-P5-XynA) capable of heterologously expressing recombinant β-1, 4 xylanase.

[0066] Example 2: Construction of β-1, 4 xylanase mutants using site-directed mutagenesis technology

[0067] 1. Selection of mutation site

[0068] The three-dimensional structure of the protein β-1, 4 xylanase was analyzed using pymol software, and then the substrate binding pocket and its surrounding amino acid residues were subjected to alanine scanning to obtain the binding free energy change value (ΔΔG) for analysis, thereby determining the amino acid to be mutated. The three-dimensional structure of the β-1, 4 xylanase is shown in Figure 2 The interaction of β-1, 4 xylanase with xylohexose is labeled, and finally Asn-8 is selected as the ideal mutation site. It is found that the thermal stability and catalytic activity of the mutant N8Y are greatly improved. In order to obtain a β-1, 4 xylanase with better performance, molecular docking simulation is then used to identify the flexible region that fluctuates greatly in the N8Y mutant enzyme, and finally obtain the multi-mutant N8Y / S22P / A59K (Mut-1), in which the 8th asparagine (Asn) of the β-1, 4 xylanase is mutated to tyrosine (Tyr), the 22nd serine (Ser) is mutated to proline (Pro), and the 59th alanine (Ala) is mutated to lysine (Lys).

[0069] Table 2: Primers used for mutation and verification

[0070]

[0071] 2. Preparation of competent cells of Lactococcus lactis

[0072] L. lactis NZ9000 was streaked on GM17 plate, and after incubation at 30°C for activation, a single colony was inoculated into a 5 mL test tube (liquid M17 competent medium) and incubated overnight. The bacterial solution in the test tube was again inoculated into 100 mL of liquid M17 competent medium, and incubated to the logarithmic growth phase (OD600 value of 0.5-0.6). The bacterial cells were collected by low-temperature centrifugation (7000 rpm, 20 min, 4°C), and washed three times with 50 mL of competent washing solution (pre-cooled to an ice-water mixture). Finally, the bacterial cells were resuspended with 1 mL of competent washing solution, divided using an EP tube, and stored at -80°C.

[0073] 3. Construction of mutant plasmid

[0074] 10 μL of L. lactis NZ9000 containing recombinant plasmid (L. lactis-P5-XynA) stored at -80°C in the laboratory was inoculated on M17 solid medium and incubated at 30°C for 24 h. A single colony was then inoculated into 5 mL of liquid medium and incubated for 16 h. The plasmid was extracted from the bacterial solution using a kit, and the extracted plasmid was used for electrophoresis to verify the correctness of the plasmid. The extraction of the plasmid was performed according to the instructions of the Solarbio-plasmid small amount extraction kit (catalog number: D1100):

[0075] (1) 1-5 mL of bacterial culture was centrifuged at 12000 rpm for 1 min, and the supernatant was carefully aspirated (when the bacterial solution is large, the bacterial cell precipitate can be collected in one centrifuge tube by multiple centrifugation);

[0076] (2) 250 μL of solution I (please check whether RNase A has been added) was added to the centrifuge tube containing the bacterial cell precipitate, and the bacterial cell precipitate was thoroughly suspended using a pipette or vortex shaker. Note: If the bacterial block is not thoroughly mixed, it will affect the lysis and result in low plasmid extraction and low purity;

[0077] (3) 250 μL of solution II was added to the centrifuge tube, and the bacterial cells were gently inverted 6-8 times to fully lyse the bacterial cells. Note: The mixing should be gentle to avoid contamination of bacterial genomic DNA. At this time, the bacterial solution should become clear and viscous, and the action time should not exceed 5 min to avoid damage to the plasmid;

[0078] (5) 350 μL of solution III was added to the centrifuge tube, and immediately gently inverted 6-8 times to thoroughly mix. At this time, a white flocculent precipitate will appear. The supernatant was carefully transferred to another clean centrifuge tube using a pipette, and the precipitate was carefully aspirated. Note: The solution III should be mixed immediately after addition to avoid local precipitation. If there is still a small amount of white precipitate in the supernatant, it can be removed by centrifugation again.

[0079] (6) Add the supernatant obtained in the previous step to the adsorption column (in the collection tube), and place it at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube;

[0080] (7) Add 600 μL of the washing solution I (check whether anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column into the collection tube;

[0081] (8) Add 700 μL of the washing solution II (check whether anhydrous ethanol has been added before use) to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column into the collection tube;

[0082] (9) Add 500 μL of the washing solution II to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column into the collection tube;

[0083] (10) Centrifuge at 12000 rpm for 2 min, and place the adsorption column in an open state at room temperature or in a 50°C incubator for several minutes, so as 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;

[0084] (11) Place the adsorption column into a clean centrifuge tube, and add 50-200 μL of the elution solution preheated in a 65°C water bath to the center of the adsorption membrane, place it at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min to obtain the purified plasmid.

[0085] The electrophoresis result is shown in Figure 4 Lane 1 is a large molecular weight DNA marker (100-2000 bp), and lanes 1, 2 and 3 are the target gene NcoI-usp45-xynA-KpnI, the size of which is about 1245 bp, and the bands shown in lanes 1, 2 and 3 are in the middle of the 1000-2000 bp marker, and the band size is consistent with the theory.

[0086] The amplification system is 10 μL of 5×fast pfu buffer, 4 μL of dNTPs, 1 μL of Pfu DNA polymerase, 1 μL of template, 1 μL of upstream primer, 1 μL of downstream primer, and 32 μL of ddH2O to make up the total reaction system to 50 μL.

[0087] The mutation primer described in Table 2 is used to take the recombinant plasmid pNZ8048-P5-XynA as a template, and the linearized mutant plasmid fragment is obtained by using the mutation primer to perform round PCR amplification. The detailed information of the mutation primer is shown in Table 2, and the amplification system and the amplification procedure are as follows:

[0088] (1) The commercialized DNA polymerase, buffer, dNTPs, template DNA, primer and sterile double distilled water are taken out from the freezer at -20℃, and are placed on ice to be thawed.

[0089] (2) In the ice bath, each component is added to the PCR tube in the following order.

[0090] PCR system:

[0091]

[0092] (3) The reaction procedure is set. The above mixture is centrifuged slightly, and is immediately placed in the PCR instrument to start amplification.

[0093] The system is used by the polymerase chain reaction (PCR) instrument 95℃ denaturation 30s, 57℃ annealing 60s, 72℃ extension 1min, 30 cycles, and finally 72℃ extension 7min.

[0094] After the PCR product is verified by agarose gel electrophoresis, the PCR product recovery kit is used to recover and store at 4℃ for use. The linearized plasmid fragment is connected by T4 DNA ligase at 16℃ overnight, and the connection system is 4 μL 5×T4 DNA ligase buffer, 1 μL T4 DNA ligase, 15 μL plasmid fragment. After mixing, it is connected overnight to obtain the mutant plasmid.

[0095] 4. Electrotransformation into competent cells

[0096] The mutant recombinant plasmid was purified to remove excess salt ions in the solution. Specifically, 20 ul of the recombinant plasmid was taken with a pipette and dropped into an ultrafiltration membrane, which was floating in ultrapure water. The smaller salt ions were allowed to pass through the filter membrane by osmosis, thereby avoiding the generation of a large current that would destroy the competent cells during electroporation. The desalination lasted for 30 min, after which the recombinant plasmid was collected. 5 ul of the recombinant plasmid, 50 ul of the competent L. lactis cells, and an electroporation cup were thawed and pre-cooled on ice, respectively. Then, the recombinant plasmid and the competent cells were mixed in the electroporation cup, which was placed on ice for three minutes. The electroporation parameters were adjusted to 200 Ohm, 2.5KV, and 25 μF. The electroporation cup was wiped dry and subjected to an electric shock. After the electric shock, the system in the electroporation cup was quickly transferred into 1 mL of M17 electroporation recovery liquid. After incubation at 30°C for 2 h, 100 ul, 200 ul, and 300 ul of the recovery liquid were taken and spread on resistance plates (GM17, CmR), respectively, and incubated overnight to form mature round white single colonies.

[0097] 5. Screening of mutant positive clones

[0098] The single colonies were picked for colony PCR verification to screen the recombinant strains of the mutant plasmid correctly introduced. Specifically, a sterilized toothpick was used to pick the single colonies in a sterile clean bench and dispersed in 10 ul of sterile water. The bacterial liquid was used as the template for colony PCR. The fragments at both ends of the target gene in the plasmid were used as sequencing primers. 1 ul of the upstream primer seq-pNZ8048-F, the downstream primer seq-pNZ8048-F, and the bacterial liquid were taken for colony PCR amplification to verify the correct existence of the target gene. If the bacterial liquid successfully passed the PCR verification, the remaining 9 ul of the bacterial liquid was re-cultured and the plasmid was extracted for sequencing.

[0099] The strains verified by sequencing were repeated to construct the mutant plasmid as described above. The primers used are shown in Table 2. Finally, a triple mutant site plasmid was constructed, which was introduced into competent cells by electroporation to construct a multi-mutant strain.

[0100] Example 3: Expression, purification, and enzyme property exploration of the mutant

[0101] 1. Expression of mutant enzyme

[0102] The L. lactis variant glycerol bacteria stored in a -80°C refrigerator were activated on a GM17 (CmR) plate. Then, smooth white colonies were picked and reactivated in 5 mL of GM17 medium. At this time, the growth activity of each L. lactis strain was strong. Subsequently, 1 mL of seed bacterial liquid was added to 100 mL of fresh GM17, CmR medium, and fermented in a 30°C constant temperature incubator. After 8 h of culture, the fermentation supernatant was obtained by centrifugation and subjected to nickel column affinity chromatography to obtain the purified mutant enzyme.

[0103] After fermentation, the bacterial liquid was centrifuged at 4°C, 2200xg in a high-speed refrigerated centrifuge for 15 min to make the bacterial cells fully sedimented, and the supernatant was taken for purification. The heterologous protein secreted into the fermentation broth and containing a histidine tag at the C-terminus was purified by fixed metal affinity chromatography. First, 2 mL of Ni-NTA filling mixture was taken in a centrifuge tube, and low-speed centrifugation was performed in a small refrigerated benchtop centrifuge with the parameter setting of 4°C, 200xg, and centrifugation for 10 min. After the nickel column filler was fully layered with agarose, the agarose layer was gently sucked off with a pipette. 10 mL of Lysis buffer was mixed with the nickel column, and the balance rotor was used for balancing for 10 min. After balancing, the supernatant was centrifuged and discarded, and the above operation was repeated three times. 45 mL of fermentation supernatant was mixed with the washed Ni-NTA column in a 50 mL centrifuge tube, and the balance rotor was used for balancing for 4 h to make the heterologous protein containing a 6His-tag at the C-terminus fully bind to the nickel column. Subsequently, 50 mL of Wash buffer was used to wash the mixture, and 1 column volume of Elution buffer containing imidazole was used for elution to dissociate the histidine tag from the nickel column. The target protein collected was ion exchanged in 0.2M, pH 7.2 PBS buffer through a dialysis membrane (<10 kDa) to remove the imidazole in the target protein solution, and the dialyzed sample could be stored at -80°C. 2+ In a 50 mL centrifuge tube, the mixture was balanced for 4 h on the balance rotor to make the heterologous protein containing a 6His-tag at the C-terminus fully bind to the nickel column. Subsequently, 50 mL of Wash buffer was used to wash the mixture, and 1 column volume of Elution buffer containing imidazole was used for elution to dissociate the histidine tag from the nickel column. The target protein collected was ion exchanged in 0.2M, pH 7.2 PBS buffer through a dialysis membrane (<10 kDa) to remove the imidazole in the target protein solution, and the dialyzed sample could be stored at -80°C.

[0104] The purified protein was subjected to SDS-PAGE electrophoresis, and the electrophoresis result is shown in FIG. 2. The molecular weight of the wild-type Barnase protein is 23.26 kDa, and a single amino acid mutation theoretically does not cause a large-scale change in the molecular weight of the protein, so the molecular weight of the mutant protein should also be 23.26 kDa. The electrophoresis result shows that the mutant protein band is located between 14-25 kDa, which is basically consistent with the theoretical molecular weight (23.26 kDa) of the mutant protein, verifying the correct expression of the target protein. Figure 5

[0105] 2. Measurement of enzyme content and activity

[0106] ​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 the method published by previous researchers. β-1, 4 xylanase can hydrolyze β-1, 4-glycosidic bonds to produce xylo-oligosaccharides and xylose with reducing properties. The amount of reducing sugar released is usually quantified using the 3, 5-dinitrosalicylic acid method (DNS). The enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute of substrate under optimal reaction conditions. The specific reaction system is as follows: 600 μL of 1% (w / v) xylan is added as a substrate to 200 μL of citrate buffer with a pH of 6.0, 200 μL of diluted β-1, 4 xylanase is added, and then immediately placed in a 50°C constant temperature water bath for 10 min. After the reaction is completed, 1.2 mL of DNS is added to terminate the reaction, and the color is developed in boiling water for 10 min. Finally, the reaction mixture is diluted 8 times, and the absorbance of each sample at 540 nm is measured on an enzyme marker. The enzyme activity of β-1, 4 xylanase is calculated. D-xylose is used as a standard reducing sugar, and it is prepared into a standard solution with a concentration of 0-1.0 mg / mL. 1 mL of D-xylose standard solution with different concentrations is mixed with 1.2 mL of DNS, and the color is developed. The absorbance of each standard sample at a wavelength of 540 nm is measured, and the standard curve is plotted with the concentration of D-xylose standard solution as the abscissa and the absorbance as the ordinate.

[0107] The kinetic parameters of wild-type Barnase and mutant enzymes were measured, respectively, and the results are shown in Table 3. The mutant enzyme showed 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.

[0108] Table 3: Comparison of kinetic parameters of recombinant wild-type β-1, 4 xylanase and mutant β-1, 4 xylanase

[0109]

[0110] Enzyme thermal stability determination

[0111] 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.

[0112] y=(y0−Plateau)exp −Kt + Plateau (1)

[0113] t1 / 2=ln2 / K (2)

[0114] Table 4: Comparison of half-life of recombinant wild-type β-1,4-xylanase and mutant β-1,4-xylanase at various temperatures.

[0115]

[0116] 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℃.

[0117] 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 6). This non-covalent interaction can effectively fix the substrate in the substrate binding pocket, and also help to enhance the enzyme's ability to recognize the substrate, thereby improving the enzyme's catalytic activity and substrate specificity. In addition, the benzene ring of the Tyr side chain also forms a hydrophobic interaction with the side chain of Ala-18, and the benzene ring in the Tyr side chain is modified by the hydroxyl group, which not only reduces the freedom of the active pocket, but also increases the hydrophobic interaction. Therefore, after introducing Tyr into the active pocket, not only does it enhance the binding ability between the enzyme and the substrate, but also further stabilizes the local structure of the protein by restructuring the hydrogen bond network and introducing additional hydrophobic interactions, thereby improving the catalytic efficiency of the enzyme. Although the performance of the mutant enzyme is better than that of the wild type, the thermal stability is limited, so we look for other mutation sites to improve the dynamic correlation of the protein by using multi-site mutations, so that the amino acids can work together to significantly enhance the thermal stability of the protein.

[0118] We first predicted and located the non-active site flexible regions in the N8Y mutant enzyme through molecular dynamics simulation, and then combined Discovery Studio and various in silico tools to screen three double mutants N8Y / A59K, N8Y / S22P, N8Y / N159V that have significantly improved or little changed enzyme performance compared to N8Y using a multi-level prediction combination method. Therefore, we consider combining the mutation strategies of multiple flexible regions to further stabilize the structure of N8Y and improve the thermal stability and catalytic activity of the enzyme. Finally, we get the multi-mutant enzyme N8Y / S22P / A59K (Mut-1) with the most improved enzyme performance through verification. According to the analysis and comparison of the structures of mutant enzymes Mut-1 and N8Y.

[0119] The 22nd serine (Ser) is mutated to proline (Pro), which has a cyclic pyrrolidine structure that restricts the freedom of the main chain dihedral angle, thereby making the local region of the protein tend to form a relatively rigid conformation. This increase in rigidity can reduce the conformational fluctuations of the local region at high temperatures, thereby improving the thermal stability of Mut-1. In addition, Pro has stronger hydrophobicity than Ser, and its interaction with the solvent is relatively weak, which improves the stability of the protein in solution. When the 59th alanine (Ala) is mutated to lysine (Lys), Lys-59 forms additional hydrogen bonds with Val-57 and ASN-61 with a distance of 1.8 Å and 3.0 Å, respectively, enhancing the local conformational stability of the β-1,4-xylanase. In addition, compared to Ala, Lys is a positively charged basic amino acid with a long side chain, which can form more electrostatic interactions through its charge in addition to hydrogen bonds with surrounding amino acid residues ( Figure 7). Therefore, this mutation plays a dual role in stabilizing both local and global conformations throughout the protein structure, and this synergistic mechanism of multiple interactions effectively reduces the probability of conformational fluctuations and disintegration of Mut-1 under high temperature conditions. In summary, the above structural modification significantly improves the catalytic efficiency and thermal stability of the β-1, 4-xylanase by enhancing the hydrogen bond network and conformational rigidity.

[0120] Nucleotide sequence of wild-type β-1, 4-xylanase SEQ ID NO. 1:

[0121] ATGTTTAAGTTTAAAAAGAAATTCTTAGTTGGATTAACGGCAGCTTTCATAAGTATCAGCATGTTTTCGGCAACCGCCTCTGCAGCTGGCACAGATTACTGGCAAAATTGGACTGATGGGGGCGGAACAGTCAACGCAGTCAATGGATCTGGCGGGAATTACAGTGTTAATTGGTCTAATACCGGAAATTTCGTTGTTGGTAAAGGCTGGACTACAGGCTCGCCATTTAGAACAATAAACTATAATGCCGGAGTCTGGGCGCCGAATGGCAATGGATATTTGACTTTATATGGCTGGACGAGAGCACCTCTCATCGAATATTATGTAGTGGATTCATGGGGTACTTACAGACCTACCGGAACGTATAAAGGTACTGTAAAGAGTGATGGAGGTACATATGACATATATACAACGACACGTTATAACGCACCTTCCATTGATGGCGATAACACTACTTTTACGCAGTACTGGAGTGTTCGCCAGTCGAAGAGGCCGACCGGAAGCAACGCTGCAATCACTTTCAGCAATCATGTTAACGCATGGAAGAGTCATGGAATGAATCTGGGCAGTAATTGGGCTTACCAAGTCTTAGCGACAGAAGGATATAAAAGCAGCGGAAGTTCTAATGTAACAGTGTGGCATCACCATCACCATCACTAA

[0122] Amino acid sequence of wild-type β-1, 4-xylanase SEQ ID NO. 2:

[0123] MFKFKKKFLVGLTAAFISISMFSATASAAGTDYWQNWTDGGGTVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLYGWTRAPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDNTTFTQYWSVRQSKRPTGSNAAITFSNHVNAWKSHGMNLGSNWAYQVLATEGYKSSGSSNVTVWHHHHHH

[0124] Nucleotide sequence of mutant beta-1, 4 xylanase (Mut-1 ) SEQ ID NO. 3:

[0125] ATGTTTAAGTTTAAAAAGAAATTCTTAGTTGGATTAACGGCAGCTTTCATAAGTATCAGCATGTTTTCGGCAACCGCCTCTGCAGCTGGCACAGATTACTGGCAATATTGGACTGATGGGGGCGGAACAGTCAACGCAGTCAATGGACCTGGCGGGAATTACAGTGTTAATTGGTCTAATACCGGAAATTTCGTTGTTGGTAAAGGCTGGACTACAGGCTCGCCATTTAGAACAATAAACTATAATGCCGGAGTCTGGAAACCGAATGGCAATGGATATTTGACTTTATATGGCTGGACGAGAGCACCTCTCATCGAATATTATGTAGTGGATTCATGGGGTACTTACAGACCTACCGGAACGTATAAAGGTACTGTAAAGAGTGATGGAGGTACATATGACATATATACAACGACACGTTATAACGCACCTTCCATTGATGGCGATAACACTACTTTTACGCAGTACTGGAGTGTTCGCCAGTCGAAGAGGCCGACCGGAAGCAACGCTGCAATCACTTTCAGCAATCATGTTAACGCATGGAAGAGTCATGGAATGAATCTGGGCAGTAATTGGGCTTACCAAGTCTTAGCGACAGAAGGATATAAAAGCAGCGGAAGTTCTAATGTAACAGTGTGGCATCACCATCACCATCACTAA

[0126] The amino acid sequence of the mutant beta-1, 4 xylanase (Mut-1) is SEQ ID NO. 4:

[0127] MFKFKKKFLVGLTAAFISISMFSATASAAGTDYWQYWTDGGGTVNAVNGPGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWKPNGNGYLTLYGWTRAPLIEYYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDNTTFTQYWSVRQSKRPTGSNAAITFSNHVNAWKSHGMNLGSNWAYQVLATEGYKSSGSSNVTVWHHHHHH

[0128] The above merely provides preferred embodiments of the present application but not for limiting the present application. Although the present application has been described in detail with reference to the foregoing embodiments, the technical solutions recorded in the foregoing embodiments can still be modified by those skilled in the art, or some technical features thereof can be replaced equivalently. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

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

4.

2. The beta-1,4-xylanase mutant according to claim 1, characterized in that: The beta-1,4-xylanase mutant is obtained by sequence modification at the amino acid level on a wild-type beta-1,4-xylanase, wherein the amino acid sequence of the wild-type beta-1,4-xylanase is shown as SEQ ID NO.

2.

3. The beta-1,4-xylanase mutant according to claim 2, characterized in that: The modification includes replacing the asparagine at position 8 with tyrosine, replacing the serine at position 22 with proline, and replacing the alanine at position 59 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 as SEQ ID NO.

3.

5. The gene of claim 4, wherein: The gene is obtained by nucleotide modification on a wild-type beta-1,4-xylanase gene, wherein the nucleotide sequence of the wild-type beta-1,4-xylanase gene is shown as SEQ ID NO.

1.

6. A vector having the gene according to claim 4 or 5.

7. A host cell, characterized in that, A vector comprising the vector according to claim 6.

8. A method of producing the β-1,4-xylanase mutant of claim 1, characterized by, The method comprises the following steps: a) obtaining a wild-type beta-1,4-xylanase gene; b) constructing a wild-type beta-1,4-xylanase gene expression vector; c) performing site-directed mutation amplification on the wild-type beta-1,4-xylanase gene; d) constructing a beta-1,4-xylanase mutant gene expression vector; e) constructing a mutant library and screening mutants with high enzyme activity and high thermal stability.

9. A method of purifying the β-1,4-xylanase mutant of claim 1, characterized by: The beta-1,4-xylanase mutant is obtained by adding His-Tag at the C-terminal of the beta-1,4-xylanase mutant and using metal affinity chromatography to elute and purify the mutant.

10. Use of a β-1,4-xylanase mutant according to claim 1 in food processing, feed manufacture or biomass conversion, characterized in that: The beta-1,4-xylanase mutant is used for degrading xylan in high-temperature processing environments.

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