Novel thermophilic beta-1, 4-endo-xylanase Xyn413

Abstract

The invention discloses thermophilic beta-1, 4-endo-xylanase Xyn413, the amino acid sequence of the thermophilic beta-1, 4-endo-xylanase Xyn413 is shown as SEQ ID No.1, and the thermophilic beta-1, 4-endo-xylanase Xyn413 is a novel beta-1, 4-endo-xylanase through amino acid sequence comparison and is derived from a metagenome of a hot spring of a hot pond in Yuanjiang County in Yunnan province. The thermophilic beta-1, 4-endo-xylanase Xyn413 provided by the invention can hydrolyze beech xylan, oat beta-glucan and lichenin, and has the activities of beta-1, 4-endo-xylanase and beta-1, 3-1, 4-glucanase, and the activity of the hydrolyzed xylan is the highest. The optimum reaction temperature of the beta-1, 4-endo-xylanase is 100 DEG C which is far higher than that of beta-1, 4-endo-xylanase reported at the present stage, after the beta-1, 4-endo-xylanase is placed at 80 DEG C for 24 hours, the residual enzyme activity is kept at 85.3%, and 5% ethanol can enhance the activity of the beta-1, 4-endo-xylanase, so that industrial application of lignocellulose biomass under a high-temperature condition is facilitated.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme genetic engineering and enzyme biochemical engineering technology, specifically relating to a novel thermophilic β-1,4-endoxylanase Xyn413. Background Technology

[0002] Xylan accounts for about one-third of all renewable organic carbon resources on Earth and is the second largest polysaccharide in nature after cellulose (Shi Xiaoyu et al. Isolation and identification of a xylan-degrading bacterium from the hindgut of termites. Bulletin of Microbiology, 2016, 43(3): 6). Xylan is one of the most important structural polysaccharides in plant cells. Together with lignin and cellulose, it constitutes an important component of plant cell walls and is also one of the main components of hemicellulose. Xylan is a complex polysaccharide whose sugar chain backbone is composed of xylose residues linked by β-1,4 glycosidic bonds. The xylan backbone is composed of xylan pyranose residues, and the side chains can be replaced by arabinose, methyl glucuronic acid, acetyl, etc. According to the type of side chain, xylan can be divided into four groups: homoxylan, arabinoxylan, glucuronic acid xylan, and arabinuronic acid xylan (Naidu et al. Carbohydrate Polymers, 2017, 179: 28-41).

[0003] Xylan is an important raw material in industrial production, such as energy, papermaking, and textiles. Due to its bioactivity, biocompatibility, biodegradability, and oxygen barrier properties, xylan has become an emerging biomaterial (Urtiga Set al. Eur J Pharm Biopharm, 2020, 151:199-208). The hydrolysate of xylan can produce byproducts such as ethanol, xylitol, and lactic acid after fermentation. Xylan can selectively stimulate the intestinal microbiota, which has a beneficial effect on the host and improves the host's health. It is a common prebiotic (Akpinar et al. Carbohydrate Research, 2009, 344(5): 660-6).

[0004] The depolymerization of xylan depends on β-1,4-xylanase (EC 3.2.1.8), which breaks the β-1,4 glycosidic bonds in the xylose backbone to produce xylooligosaccharides. The degradation of substrates by β-1,4-xylanase depends on the characteristics of the substrate molecule, such as chain length, degree of branching, and type of substituents. The initial hydrolysis product is β-D-xylanose oligomers, and later hydrolysis may produce small molecules such as monosaccharides, disaccharides, and trisaccharides of β-D-xylanose (Birsan et al. Biochem SocTrans, 1998, 26(2): 156-60; Polizeli et al. Appl Microbiol Biotechnol, 2005, 67(5): 577-91.). β-1,4-xylanase is widely distributed in microorganisms including bacteria, yeast, Aspergillus, and Trichoderma. Some fungi, plants, protozoa, and crustaceans can also produce xylanase (Carvalho et al. Food Research International, 2013, 51(1): 75-85). Xylanases from different microbial sources can be classified into several glycoside hydrolase families (GH5, 7, 8, 10, 11, 26, 30 and 43). Based on the similarity of the amino acid sequence of the catalytic domain of xylan hydrolases, xylan hydrolases mainly belong to GH10 and GH11 (Tony et al. Fems Microbiology Reviews, 2010, 29(1): 3-23; Cheng et al. J Biol Chem, 2014, 289(16): 11020-8; Hong et al. Appl Environ Microbiol, 2014, 80(7): 2084-93). In industrial applications, xylanases are required to withstand harsh conditions such as acidic and alkaline environments and high temperatures. For example, in the bleaching process of pulp, heat-resistant xylanases are needed to achieve catalytic action under hot alkaline conditions of high temperature and alkali pretreatment. In the bleaching and degumming process of flax and ramie fibers, the use of heat-resistant β-1,4-xylanases can reduce costs and pollution. In the liquefaction and saccharification process of malt, the use of thermophilic β-1,4-xylanases can reduce the viscosity of wort and increase the filtration speed.

[0005] Given the immense value and demand for thermophilic β-1,4-xylanase in industrial applications, the search and development of thermophilic β-1,4-xylanase relying on pure culture microorganisms has been severely limited. In contrast, hot spring metagenomic samples undoubtedly provide a rich resource for the development and application of thermophilic β-1,4-xylanase. Summary of the Invention

[0006] The first objective of this invention is to provide a thermophilic β-1,4-endoxylanase Xyn413, the amino acid sequence of which is shown in SEQ ID No.1. It originates from a hot spring metagenomic genome in Yuanjiang County, Yunnan Province. Amino acid sequence similarity analysis shows that this enzyme is a novel β-1,4-endoxylanase Xyn413.

[0007] A second object of the present invention is to provide an amino acid sequence encoding the β-1,4-endoxylanase Xyn413. This sequence comprises an amino acid sequence having at least 89% identity with the amino acid sequence of SEQ ID No. 1, or an amino acid sequence having at least 82% identity with the amino acid sequence of SEQ ID No. 2, or an amino acid sequence having at least 92% identity with the amino acid sequence of SEQ ID No. 3, or an amino acid sequence having at least 88% identity with the amino acid sequence of SEQ ID No. 4, or an amino acid sequence having at least 86% identity with the amino acid sequence of SEQ ID No. 5, or an amino acid sequence having at least 86% identity with the amino acid sequence of SEQ ID No. 6, or an amino acid sequence having at least 88% identity with the amino acid sequence of SEQ ID No. 7, or an amino acid sequence having at least 86% identity with the amino acid sequence of SEQ ID No. 8.

[0008] This invention claims protection for the amino acid sequence of the β-1,4-endoxylanase Xyn413.

[0009] This invention utilizes metagenomic sequencing of samples from the Yuanjiang hot spring in Yunnan Province. The β-1,4-endoxylanase Xyn413 gene, obtained through binning, assembly, and annotation, was codon-optimized and synthesized, then ligated with the enzyme-digested pET49b plasmid to obtain a recombinant plasmid containing the β-1,4-endoxylanase Xyn413 gene. The recombinant plasmid was then transformed into expression host bacteria. Escherichia coli (BL21 DE3) was cultured using IPTG, and the cells were collected by centrifugation. After the cells were broken up, they were purified by Ni-NTA affinity chromatography and detected by SDS-PAGE electrophoresis to obtain pure β-1,4-endoxylanase Xyn413.

[0010] Invention Effects The thermophilic β-1,4-endoxylanase Xyn413 provided by this invention exhibits hydrolytic activity against beech xylan, β-glucan, and lichen polysaccharides containing β-1,4-glycosidic bonds or β-1,3-1,4-glycosidic bonds, with the highest hydrolytic activity against xylan. The optimal reaction temperature of this enzyme is 100 °C, significantly higher than that of currently reported β-1,4-endoxylanases. After incubation at 80 °C for 24 h, the residual enzyme activity remains at 85.3%. The optimal reaction pH is 6.0, and 5% ethanol or 10% isopropanol can enhance the enzyme's activity. This is beneficial for the industrial application of lignocellulose biomass under high-temperature conditions, improving the enzymatic hydrolysis efficiency of xylan and possessing significant economic and social benefits. Attached Figure Description

[0011] Figure 1 This is a comparison diagram of the amino acid sequence of β-1,4-endoxylanase Xyn413 with the amino acid sequences of the six most similar xylanases from the GH10 family.

[0012] Figure 2 This is an SDS-PAGE electrophoresis result of the protein obtained from the expression of the β-1,4-endoxylanase Xyn413 gene in *E. coli* in Example X. M, Marker protein band; 1, E. coli BL21 (DE3) fermentation supernatant preheated sample; 2, sample containing pET49b-Xyn413 recombinant plasmid. E. coli BL21 (DE3) fermentation supernatant preheated sample; 3, pure β-1,4-endoxylanase Xyn413.

[0013] Figure 3 This is an example of the effect of pH on the activity of β-1,4-endoxylanase Xyn413 in Example 3.

[0014] Figure 4 The graph shows the results of pH stability measurements. The horizontal axis represents pH, and the vertical axis represents relative enzyme activity (in units).

[0015] Figure 5 The effect of temperature on the activity of β-1,4-endoxylanase Xyn413 in Example 3. The horizontal axis represents temperature (°C), and the vertical axis represents relative enzyme activity, in percent.

[0016] Figure 6 The results of temperature stability measurements are shown on the x-axis, which represents the incubation time, and on the y-axis, which represents the relative enzyme activity, in units.

[0017] Figure 7 The effect of metal ions on the activity of β-1,4-endoxylanase Xyn413 in Example 3.

[0018] Figure 8 shows the effect of organic reagents on the activity of β-1,4-endoxylanase Xyn413 in Example 3. (a) Effect of organic solvents on enzyme activity; (b) Effect of organic reagents on enzyme activity.

[0019] Figure 9 The amount of reducing sugar produced by the hydrolysis of sugarcane bagasse by β-1,4-endoxylanase Xyn413 in Example 4 is represented by the absorbance value at λ = 540 nm. CK represents the reducing sugar content in the supernatant of 1 ml of denatured β-1,4-endoxylanase Xyn413, and Test represents the reducing sugar content in the hydrolysate of 1 ml of β-1,4-endoxylanase Xyn413. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments to enable those skilled in the art to understand the invention. However, this description does not limit the invention in any way, and any modifications or improvements made based on the teachings of the present invention fall within the protection scope of the present invention. Unless otherwise specified, the methods in this embodiment are operated according to conventional methods.

[0021] This invention relates to a novel natural thermophilic β-1,4-endoxylanase, comprising an amino acid sequence having at least 89.3% amino acid identity with SEQ ID No. 1, or comprising an amino acid sequence having at least 82% amino acid identity with SEQ ID No. 2, or having an amino acid sequence having at least 92% amino acid identity with SEQ ID No. 3, or having an amino acid sequence having at least 88% amino acid identity with SEQ ID No. 4, or having an amino acid sequence having at least 86% amino acid identity with SEQ ID No. 5, or having an amino acid sequence having at least 86% amino acid identity with SEQ ID No. 6, or having an amino acid sequence having at least 88% amino acid identity with SEQ ID No. 7, or having an amino acid sequence having at least 86% amino acid identity with SEQ ID No. 8.

[0022] The theoretical molecular weight of the thermophilic β-1,4-endoxylanase is 67.91 kDa.

[0023] The optimal reaction temperature for the thermophilic β-1,4-endoxylanase is 100 °C, and the optimal pH is 6.0.

[0024] Example 1 Expression of the natural β-1,4-endoxylanase gene Natural β-1,4-endoxylanase gene xyn413 Acquisition Soil samples were collected from the hot springs at Reshuitang in Yuanjiang County, Yunnan Province. Genomic DNA of the microbial community in the environment was extracted and subjected to next-generation sequencing of metagenomic DNA. The sequencing results underwent quality control, followed by binning, assembly, and annotation of the quality-controlled data to obtain amino acid sequences encoding known endoxylanases (e.g., sequences with over 20% sequence identity and an expected value (E-value) of 1e). 20 The open reading frame (ORF) of the amino acid sequence was used to design primers based on the base sequence information of the ORF. The candidate gene was cloned from metagenomic DNA of hot spring soil using PCR. The gene was then synthesized by Sangon Biotech using codon optimization in *E. coli*, resulting in the recombinant vector pET49b-Xyn413.

[0025] Amino acid sequence analysis of β-1,4-endoxylanase The structure and function of the β-1,4-endoxylanase amino acid sequence were predicted using the InterPro online tool (https: / / www.ebi.ac.uk / interpro / search / sequence / ) and the Foldseek Search online tool (https: / / search.foldseek.com / search?accession=2ZUM&source=PDB) web servers. Protein alignment was performed using ESPript 3.0.

[0026] The results are as follows Figure 1 As shown, β-1,4-endoxylanase Xyn413 has the highest similarity to glycoside hydrolases of the GH10 family, and the amino acid sequences most similar to those in the PDB database are derived from... C. bescii Cdan_2053 (PDBID: 6D5C), C. bescii CbXyn10C (PDB ID: 5OFJ), C. owensensis CoXynA (PDB ID:5Y3X), Thermobacillus composti TcXyn10A (PDB ID: 6WQW), Paenibacillus barcinonensis The Xyn10B (PDB ID: 3EMC) and Caldicellulosiruptor owensensi The similarity of CoXynA (PDB ID: 2Q8X) was 52.69%, 51.65%, 50.5%, 49.85%, 49.09% and 48.77%, respectively.

[0027] plasmid extraction The recombinant plasmid pET49b-Xyn413 for transformation was extracted using the SanPrep column-based plasmid DNA mini-extraction kit according to the manufacturer's instructions.

[0028] Transformation The recombinant plasmid powder (approximately 4 µg) pET49b-Xyn413, synthesized from the gene, was centrifuged at 12000 r / min for 2 min, and 40 µL of ddH2O was added, resulting in a final plasmid concentration of 100 ng / µL. This was then transferred into pre-prepared competent *E. coli* cells. While the competent cells were in an ice-water mixture, 20 µL of the recombinant plasmid was immediately added to BL21(DE3) competent cells and gently mixed with a pipette. The cells were then incubated on ice for 30 min. After the ice bath, the competent *E. coli* cells were placed in a 42 ℃ water bath for 90 s (to open the cell membrane and allow the plasmid to enter the cells), and immediately removed and placed back on ice for 5 min (to close the cell membrane). 500 µL of antibiotic-free LB broth was added to the centrifuge tubes, and the culture medium was incubated on a shaker at 37 ℃ and 220 r / min for 45 min. Mix well and take 200 µL of the above culture medium. Use a sterile spreader to spread it onto solid LB medium containing kanamycin sulfate (final concentration 50 µg / mL). After inoculation, place the culture medium upright in 37 ℃ medium for 30 min, then invert it for 12 h-16 h to obtain Escherichia coli BL21(DE3) strain containing recombinant plasmid.

[0029] Inducible expression of recombinant β-1,4-endoxylanase Single-clone small-volume (5 mL) culture: Pick a single clone of recombinant Escherichia coli BL21(DE3) into a 10 mL centrifuge tube, add 5 mL of LB resistant liquid medium (Kan 50 µg / mL), and culture overnight at 37 ℃ and 220 rpm in a constant temperature shaker for 12-16 h to obtain Escherichia coli BL21(DE3) seed liquid containing recombinant plasmid.

[0030] Expand training Take 3 mL of bacterial suspension (1% inoculum) and inoculate it into 300 mL of fresh LB liquid medium (Kan 50 µg / mL). Incubate at 37℃ and 220 rpm in a constant temperature shaker for 4-6 hours until OD. 600 =0.4-0.6, add 714 µL IPTG (final concentration 0.5 mM), incubate at 220 rpm for 20 h on a constant temperature shaker at 16 ℃, centrifuge at 8000 rpm / min for 5 min, discard the supernatant and collect the bacterial cells, ensuring that each centrifuge tube collects 4.0 g of bacterial cells, and store at -20 ℃ to obtain Escherichia coli BL21(DE3) bacterial cells that have completed the expression of natural β-1,4-endoxylanase.

[0031] Crude enzyme acquisition Add 4.0 g of bacterial cells collected in a 50 mL centrifuge tube to 20 mL of non-denaturing lysis buffer, pipette to mix, centrifuge at 8000 rpm for 5 min, discard the supernatant, collect the bacterial cells, add 20 mL of non-denaturing lysis buffer and 20 µL of lysozyme, pipette to mix, vortex mix, and incubate on ice for 30 min. Use an ultrasonic homogenizer to homogenize the cells at 40 W, amplitude bar 6, program 1, ultrasonic operation for 2 s, pause for 8 s, homogenize on ice for 30 min, centrifuge at 8000 rpm for 15 min, collect the supernatant, preheat in a 70 ℃ constant temperature water bath for 2 h, centrifuge at 8000 rpm for 15 min, collect the supernatant to remove most of the impurities in the *E. coli*, obtaining a high-temperature purified crude enzyme solution of natural β-1,4-endoxylanase.

[0032] Example 2: Purification of β-1,4-endoxylanase Xyn413 First, the crude enzyme solution was concentrated, with 40 mL of crude enzyme solution concentrated to 10 mL. 4 mL of the concentrated enzyme solution was then purified using the GST protein purification kit, and the eluted sample was then spotted onto a Native-PAGE sample.

[0033] GST reagent kit purification steps: Take 1 mL of well-mixed 50% BeyoGold™ GST-tag Purification Resin and load it into an empty affinity chromatography column tube (3 mL) provided in this kit. Then, equilibrate with 0.5 mL of lysis buffer 2-3 times and add about 4 mL of bacterial lysis supernatant for binding. The column can be placed at 4℃ (16℃ in actual use) on a shaker at 80 rpm for 2 hours. Subsequently, the flow-through can be collected and the column can be loaded 3-5 times to ensure sufficient binding of the target protein.

[0034] Open the cap at the bottom of the purification column and allow the liquid inside the column to flow out under gravity. Collect about 1 mL of the flow-through liquid for subsequent analysis.

[0035] Wash the column 8 times, adding 1 mL of lysis buffer each time. Approximately 0.5 mL of the final wash solution can be collected for subsequent analysis and detection.

[0036] Elute the target protein 5 times, using 0.5 ml of elution buffer each time. (Preparation of elution buffer: Mix an appropriate amount of elution buffer (with added GSH) and 10×GSH solution in a 9:1 ratio, for example, mix 9 ml of elution buffer (with added GSH) and 1 ml of 10×GSH solution. The resulting solution is the elution buffer.) Collect each eluent into a separate centrifuge tube. The collected eluent is the purified GST-tagged protein sample.

[0037] After the Native-PAGE is completed, a transparent gel is obtained. First, use a clean scalpel to cut off the marker band and one of the channels. Place it on a horizontal shaker and shake it slowly for Coomassie Brilliant Blue staining for 30 minutes. Then, splice the band with the original unstained gel part. According to the stained band, cut off the unstained gel and put them into dialysis bags. Place them in a horizontal electrophoresis tank for horizontal electrophoresis for 40 minutes.

[0038] After dialysis electrophoresis, the solution in the dialysis bag is taken for corresponding enzyme activity detection. The bands with enzyme activity are collected and purified according to the required amount. After collection, the sample is concentrated by nitrogen blowing. The concentrated sample is then subjected to 10% SDS-PAGE electrophoresis and stained to confirm the purification of the target protein.

[0039] The results are as follows Figure 2 As shown, a band matching the target molecular weight was obtained, with a molecular weight of 100 kDa (containing GST and His tags).

[0040] Example 3: Enzymatic properties and enzyme activity experiments of β-1,4-endoxylanase Xyn413 β-1,4-endoxylanase activity was determined using the dinitrosalicylic acid (DNS) method. The crude β-1,4-endoxylanase solution was pretreated in a 70 °C water bath for 2 h. After centrifugation at 8000 rpm / min for 15 min, the supernatant was diluted appropriately, and 50 µL was added to a 96-well PCR plate. In a 50 µL solution of 1% beech xylan (dissolved in 50 mM citrate-disodium hydrogen phosphate pH 6.0 buffer), the reaction was carried out at 90 °C, pH 6.0 for 20 min. Then, 100 µL of DNS was added, and the plate was incubated at 90 °C for 10 min. After cooling to room temperature on an ice box, 150 µL of deionized water was added to the 96-well microplate. 50 µL of the reaction solution was added to the microplate and mixed thoroughly. 540 nm The absorbance of the sample is measured using an ELISA reader. This method is used for the detection of the following enzymatic properties.

[0041] Enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze and produce 1 µmol of reducing sugar per minute under the test conditions.

[0042] Study on the optimal pH and pH tolerance of β-1,4-endoxylanase Xyn413 The optimal pH was determined at 90°C using different buffer systems, including disodium hydrogen phosphate-citrate buffer (200 mM, pH 3.0–7.0) and glycine-NaOH buffer (20 mM, pH 8.0–12.0). Enzyme activity at the optimal pH was defined as 100% activity.

[0043] The pH stability of xylanase was determined by pre-incubation at 80°C within a different pH range of 3.0–12.0 to estimate pH stability after 24 hours of pre-incubation. Residual activity was measured under standard assay conditions.

[0044] Based on the experimental results, such as Figure 3 The optimal reaction pH for β-1,4-endoxylanase Xyn413 is shown to be 6.0; Figure 4 As shown, after treatment in buffer solutions with pH 5.0–9.0 for 24 h, β-1,4-endoxylanase Xyn413 still retains more than 80% of its enzyme activity, demonstrating good pH tolerance.

[0045] Study on the optimal temperature and thermal stability of β-1,4-endoxylanase Xyn413 The optimal temperature was determined by measuring the enzyme's activity within the temperature range of 30–105°C, with the activity at the optimal temperature defined as 100%. The temperature stability of xylanase was determined by pre-incubating the enzyme without substrate at 70–90°C and sampling at different time intervals (1.0–48.0 h) to determine residual activity. Unincubated enzymes were used as controls.

[0046] like Figure 5 As shown, the optimal reaction temperature of the enzyme is 100°C, and it still retains 49.2% of its relative enzyme activity at 105°C; Figure 6 As shown, after 24 h at 80°C in a pH 6.0 buffer, 85.3% of the relative enzyme activity was still retained, while after 48 h at 90°C, 20% of the relative enzyme activity was still retained.

[0047] Effects of metal ions on enzyme activity To investigate the effects of different metal ions on the catalytic activity of β-1,4-endoxylanase Xyn413, the effects of different concentrations (0.1 mM, 1 mM, and 10 mM) of metal salts (NiCl2, CoCl2, FeSO4, CuSO4, Al2(SO4)3, KCl, NaCl, HgCl2, ZnSO4, MnSO4, and MgSO4) on enzyme activity were evaluated. Enzyme activity was measured under optimal reaction conditions of 90 °C and pH 6.0. Enzyme activity with metal ion deficiency was defined as 100%, and relative enzyme activity was defined as activity relative to the control.

[0048] like Figure 7 As shown, CuSO4, Al2(SO4)3 and HgCl2 can completely inactivate the enzyme at 10 mM, while NaCl and KCl can enhance the enzyme activity at all three experimental concentrations.

[0049] The effect of organic matter on enzyme activity To investigate the effects of different organic compounds on the catalytic activity of β-1,4-endoxylanase Xyn413, the effects of different concentrations (0.1%, 1%, and 10%) of SDS, Tween 20, 2-mercaptoethanol, urea, EDTA, methanol, ethanol, and isopropanol on enzyme activity were evaluated. The reactions were carried out at 90 °C and pH 6.0. The absence of organic compounds was defined as 100%, and relative enzyme activity was given relative to the control.

[0050] As shown in Figure 8, Tween 20 can enhance enzyme activity at three concentrations: 0.1%, 1%, and 10%. After treatment with 5% and 10% methanol, the enzyme activity was retained at 87.7% and 88.2%, respectively. 5% ethanol can enhance enzyme activity, but 50% ethanol completely destroys the enzyme activity. 5% and 10% isopropanol can enhance enzyme activity.

[0051] Example 4: Hydrolysis of sugarcane bagasse by β-1,4-endoxylanase Xyn413 Sugarcane bagasse was dried in a 60°C oven for 24 hours, then pulverized and passed through a 42-mesh sieve. 1.25 g of the sample was placed in a 500 ml Erlenmeyer flask, and 34 ml of pH 6.0 citrate-disodium hydrogen phosphate buffer was added, followed by 1 ml of crude β-1,4-endoxylanase Xyn413 enzyme solution for hydrolysis. The reaction system was stirred at 120 rpm using a 90°C magnetic stirrer. A control group was prepared by adding 1 ml of denatured and inactivated β-1,4-endoxylanase. After 4 hours of reaction, the reducing sugar content in the solution was determined using the DNS method.

[0052] The results are as follows Figure 9 As shown, after 4 hours of reaction, the addition of 1 ml of denatured β-1,4-endoxylanase resulted in an absorbance value of only 0.04 at λ=540 in the supernatant corresponding to the reducing sugar content; while the addition of 1 ml of non-denatured β-1,4-endoxylanase resulted in an absorbance value of 0.43 at λ=540 in the supernatant corresponding to the reducing sugar content. The reducing sugar content in the experimental and control reaction systems showed a highly significant difference.

Claims

1. A novel thermophilic β-1,4-endoxylanase Xyn413 / β-1,3-1,4-glucanase Xyn413, the amino acid sequence of which is shown in SEQ ID No.

1.

2. A novel thermophilic β-1,4-endoxylanase Xyn413 / β-1,3-1,4-glucanase Xyn413, the amino acid sequence of which is shown in SEQ ID No.

1.

3. The enzyme or its functional analogue according to claim 1, wherein the amino acid sequence has at least 82% identity with the polypeptide sequence shown in SEQ ID No. 2, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.

4. The enzyme or its functional analogue according to claim 1, wherein the amino acid sequence has at least 92% identity with the polypeptide sequence shown in SEQ ID No. 3, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.

5. The enzyme or its functional analogue according to claim 1, wherein the amino acid sequence has at least 88% identity with the polypeptide sequence shown in SEQ ID No. 4, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, and most preferably at least 99% identity.

6. The enzyme or its functional analogue according to claim 1, wherein the amino acid sequence has at least 86% identity with the polypeptide sequence shown in SEQ ID No. 5, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.

7. The enzyme or its functional analogue according to claim 1, wherein the amino acid sequence has at least 86% identity with the polypeptide sequence shown in SEQ ID No. 6, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.

8. The enzyme or its functional analogue according to claim 1, wherein the amino acid sequence has at least 88% identity with the polypeptide sequence shown in SEQ ID No. 7, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.

9. The enzyme or its functional analogue according to claim 1, wherein the amino acid sequence has at least 86% identity with the polypeptide sequence shown in SEQ ID No. 8, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.

10. A DNA sequence encoding the β-1,4-endoxylanase or functional analogue as described in any one of claims 1-9.

11. A recombinant expression vector containing the DNA sequence of claim 10.

12. A host cell obtained by transformation, transduction, or transfection of the recombinant expression vector of claim 11.

13. The use of the DNA sequence according to any one of claims 10-12 in the preparation of β-1,4-endoxylanase and β-1,3-1,4-glucanase.

14. The application of the enzymes or functional analogs according to claims 1-9 and the enzymes prepared according to claim 13 in the fields of textiles, pulp production, papermaking and lignocellulose hydrolysis.

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