Thermophilic xylanase LD3-Xyn6 and application thereof

Abstract

The invention discloses a thermophilic xylanase LD3-Xyn6 and an application of the thermophilic xylanase LD3-Xyn6. Belongs to the technical field of gene engineering. According to the invention, a brand new xylanase gene is identified and obtained from a metagenome of Yunnan Dali Jingu hot spring, and the recombinase is obtained through heterologous expression and purification. An enzymatic property characterization result shows that the optimum temperature of the enzyme is 85 DEG C, and the optimum pH value is 5.6; the half-life periods at 85 DEG C and 90 DEG C are 15 min and 10 min respectively, and excellent heat resistance and excellent temperature and pH stability are shown. In addition, K < + >, Ca < 2 + > and Mg < 2 + > have an activation effect on the enzyme activity. The xylanase can effectively degrade various natural xylans, and hydrolysates of the xylanase mainly comprise xylobiose and xylotetraose and are proved to be capable of remarkably promoting growth of lactococcus lactis. Therefore, the xylanase LD3-Xyn6 disclosed by the invention has a wide application prospect in the preparation of high-temperature-resistant feed additives and prebiotics.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to a thermophilic xylanase LD3-Xyn6 and its applications. Background Technology

[0002] Feed costs account for 60% to 80% of the total cost of livestock production, making them a core factor restricting the industry's economic efficiency. Therefore, improving feed utilization efficiency has always been a key objective of feed science research. In modern feed processing technology, pelleting, by compressing powdered feed into pellets and combining this with the addition of exogenous enzymes such as xylanase, can significantly improve feed palatability, increase nutrient digestibility, and promote animal growth performance. However, the high-temperature conditioning stage in this process (usually requiring temperatures between 70°C and 95°C), while optimizing feed quality, also presents significant challenges to the application of enzymes.

[0003] Currently, commercially available xylanases widely used in the feed industry are mainly derived from mesophilic microorganisms, with their optimal operating temperature generally between 50℃ and 65℃, exhibiting poor thermal stability. When subjected to high-temperature conditioning exceeding 75℃ during granulation, these enzyme molecules undergo irreversible denaturation and inactivation, resulting in extremely low enzyme activity residues in the final feed product, rendering them ineffective in the animal's digestive tract. This technological bottleneck severely restricts the application of enzyme preparations before granulation, forcing manufacturers to adopt costly and uneven post-coating processes, which not only increase production costs but also affect product quality stability.

[0004] Besides insufficient heat resistance, existing xylanases face other prominent problems in practical applications. First, their stability under complex feed conditions is poor. The animal digestive tract environment is complex, ranging from the acidic environment of the stomach to the neutral environment of the intestines, with a large pH range. Many existing xylanases experience a sharp decline in activity after deviating from their optimal pH, making it difficult to maintain their efficacy throughout the digestive tract. Second, various metal ions and chemicals in feed may inhibit enzyme activity, and existing enzyme preparations often lack protection against common ions (such as K+). + Ca 2+ Mg 2+ The tolerance or activation effect of ) leads to unstable performance in practical applications.

[0005] Furthermore, there is room for optimization in the catalytic product profiles of existing xylanases. Xylanase degradation products, specifically xylooligosaccharides (XOS), serve as high-quality prebiotics, selectively promoting the proliferation of beneficial gut bacteria. However, xylanases from different sources produce varying proportions of XOS components due to differences in their hydrolytic properties. Studies have shown that specific XOS components (such as xylobiose and xylotetraose) possess superior prebiotic activity, while xylanases capable of efficiently hydrolyzing xylan in various feed ingredients and primarily generating these high-value XOS components remain relatively scarce.

[0006] Therefore, the current feed industry urgently needs to develop a novel xylanase with excellent thermal stability, wide pH adaptability, good tolerance to common metal ions, and the ability to efficiently generate XOS with high prebiotic value. This is a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a thermophilic xylanase LD3-Xyn6 and its application. The enzyme has excellent heat resistance and stability, and its catalytic product has excellent prebiotic properties.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0009] The primary objective of this application is to provide a thermophilic xylanase LD3-Xyn6, the amino acid sequence of which is shown in SEQ ID NO.4.

[0010] Another object of this application is to provide: a DNA molecule encoding the above-mentioned thermophilic xylanase LD3-Xyn6, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0011] Another object of this application is to provide: a biomaterial, said biomaterial being any of the following: 1) Expression cassettes containing the aforementioned DNA molecules; 2) A recombinant vector containing the above-mentioned DNA molecules, or a recombinant vector containing the expression cassette described in 1); 3) Recombinant bacteria containing the above-mentioned DNA molecules, or recombinant bacteria containing the expression cassette described in 1), or recombinant bacteria containing the recombinant vector described in 2); Another object of this application is to provide the use of the above-described DNA molecule or the above-described biological material in the preparation of thermophilic xylanase LD3-Xyn6.

[0012] Another object of this application is to provide the use of the above-mentioned thermophilic xylanase LD3-Xyn6 in the preparation of xylooligosaccharides.

[0013] Another object of this application is to provide the application of the above-mentioned thermophilic xylanase LD3-Xyn6 in the degradation of xylan, said xylan being derived from beech, corn cob and / or bagasse.

[0014] Another object of this application is to provide the application of the above-mentioned thermophilic xylanase LD3-Xyn6 in promoting the growth of Lactococcus lactis.

[0015] Another object of this application is to provide a method for degrading xylan, comprising contacting a xylan substrate with the thermophilic xylanase LD3-Xyn6 under reaction conditions of 85°C and pH 5.6.

[0016] As a preferred technical solution, the xylan substrate is corn cob xylan treated with high temperature and alkali or wheat bran xylan treated with high temperature and water.

[0017] Another object of this application is to provide a feed additive comprising the above-mentioned thermophilic xylanase LD3-Xyn6.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: The xylanase LD3-Xyn6 provided by this invention achieves the following significant technical effects compared to existing technologies: (1) Excellent thermal stability and process adaptability: The optimal reaction temperature of LD3-Xyn6 is as high as 85℃, and it has a half-life of 15 min and 10 min at 85℃ and 90℃, respectively. This characteristic enables it to withstand the high-temperature conditioning process of 70-95℃ in feed pelleting, which solves the technical bottleneck that conventional xylanase cannot be added before pelleting due to heat-sensitive inactivation, and lays the foundation for pretreatment addition and reducing production costs.

[0019] (2) Wide pH adaptability and storage stability: The enzyme exhibits the highest activity at pH 5.6 and excellent stability over a wide pH range of 4-11. This ensures that it can adapt to the complex digestive tract environment of animals, from the acidic environment of the stomach to the neutral environment of the intestine, maintain high efficiency, and facilitate the long-term storage of enzyme preparations.

[0020] (3) Beneficial ion activation effect and efficient catalytic ability: K + Ca 2+ and Mg 2+ Common metal ions significantly activate the enzyme activity of LD3-Xyn6, indicating that it exhibits superior catalytic performance in actual feed environments containing these ions. This enzyme can efficiently degrade various xylans derived from agricultural byproducts (such as beech, corn cob, and sugarcane bagasse xylan), demonstrating broad substrate adaptability.

[0021] (4) Targeted production of high-value prebiotics: The main products of LD3-Xyn6 hydrolyzed xylan are xylobiose and xylotetraose, which are high-quality prebiotics. Experiments have shown that its hydrolysis products can significantly promote the growth of beneficial bacteria such as Lactococcus lactis, thus giving the enzyme dual application value in improving animal intestinal health and producing functional feed additives. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 For example: A is: constructing a phylogenetic tree of LD3-Xyn6 based on amino acid sequence homology; B is: homology modeling of LD3-Xyn6.

[0024] Figure 2 SDS-PAGE gel image of thermophilic xylanase LD3-Xyn6; where M is the protein marker; 1 is the bacterial cell; 2 is the purified protein.

[0025] Figure 3 The following are the enzymatic properties of thermophilic xylanase LD3-Xyn6: A represents the optimal temperature of thermophilic xylanase LD3-Xyn6; B represents the optimal pH of thermophilic xylanase LD3-Xyn6; C represents the thermal stability of thermophilic xylanase LD3-Xyn6; and D represents the pH stability of thermophilic xylanase LD3-Xyn6.

[0026] Figure 4 For: Effect of metal ions on LD3-Xyn6 enzyme activity; Note: Activity is 100% without additives; Each value represents the mean ± SD; This indicates a significant difference in activity when additives are added (P≤0.05); This indicates a significant difference in activity when additives are added (P≤0.01).

[0027] Figure 5 For: Effect of chemical reagents on LD3-Xyn6 enzyme activity; Note: Activity without additives is 100%; Each value represents the mean ± SD; This indicates a significant difference in activity when additives are added (P≤0.05); This indicates a significant difference in activity when additives are added (P≤0.01).

[0028] Figure 6 The following are the TLC plate analysis results for the beech xylan hydrolysis products of LD3-Xyn6: 1 is the xylooligosaccharide maker (X1 is xylose, X2 is xylobiose, X3 is xylotriose, and X4 is xylotetraose); 2 is the product of LD3-Xyn6 mixed with beech xylan; and 3 is the product of inactivated enzyme mixed with beech xylan.

[0029] Figure 7 The graph shows the trend of sugar production from substrates hydrolyzed by LD3-Xyn6.

[0030] Figure 8 The effect of xylan hydrolysates (prebiotics) of LD3-Xyn6 on the growth of gut microbiota is shown in Figure A, where A represents the growth curve of lactic acid bacteria and B represents the growth curve and fluorescence intensity graph of gut microbiota co-culture. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Statistical analysis of data from the embodiments of this application: Unless otherwise stated, all analyses were performed triplicate, and the mean was used for all analyses. Results were statistically analyzed using SPSS 20.0 and are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA, and Tukey's test was used for comparisons among multiple groups. In all comparisons, a p-value <0.05 was considered statistically significant.

[0033] Example 1 (1) Sample collection and metagenomic sequencing Soil samples were collected from Liandu Hot Spring in Dali, Yunnan (latitude 26°15′13.41″N, longitude 99°59′15.96″E), enriched, and DNA isolated using a kit. A HiSeq 2500 instrument (Suzhou GENWIZ) was used for metagenomic sequencing. De novo assembly was performed using the Velvet assembly program version 1.2.08 (Zerbino DR, & Birney E (2008) Velvet: Algorithms for de novo short readassembly using de Bruijn graphs. Genome Research, 18(5), 821–829). The IMG server (https: / / img.jgi.doe.gov / cgi-bin / mer / main.cgi) was used to investigate the sequences. To further analyze the potential functions of individual genes and orfs, we used the COG (Tatusov RL (2001) The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Research, 29(1), 22–28.), KEGG (Nakaya A, Katayama T, Itoh M, Hiranuka K, Kawashima S, Moriya Y, Goto S (2012) KEGG OC: a large-scale automatic construction of taxonomy-based ortholog clusters. Nucleic Acids Research, 41(D1), D353–D357) and Pfam (Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Bateman A (2007) The Pfam protein families database. Nucleic Acids Research, 36(Database), D281–D288) databases.

[0034] (2) Sequence prediction and sequence analysis of LD3-Xyn6 Functional prediction analyses using KEGG and COG were performed to screen for xylanase functional genes, and their domains were analyzed using the Pfam database. This invention obtained a novel xylanase gene sequence (named LD3-Xyn6) from a metagenomic database, synthesized the LD3-Xyn6 gene according to E. coli base preferences, and cloned it into the PSHY211 vector. The BLASTx and BLASTp programs (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) were used to compare the DNA and protein sequences of LD3-Xyn6, respectively. Signal peptide prediction was performed using signalp (http: / / www.cbs.dtu.dlk / services / signalp / ). The amino acid sequence structure was deduced and analyzed using the EXPASY tool (http: / / web.expasy.org / protparam / ). The protein sequence of LD3-Xyn6 was compared in NCBI BLASTp. Xylanase sequences of the same or neighboring genera with high similarity were selected as neighboring sequences, while xylanase protein sequences of other genera with low similarity were selected as outgroups for constructing multiple alignments and phylogenetic trees. Multiple alignments were performed using ClustalX with closely related LD3-Xyn6 protein sequences (Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876-4882). Phylogenetic analysis was performed using the MEGA7 software package (Kumar S, Stecher G, & Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution, 33(7), 1870–1874). Phylogenetic trees were constructed using the maximum likelihood (ML) method and a Poisson-corrected model. Clustal X was able to align LD3-Xyn6 and other xylanase-like sequences using UPGMA-guided tree codes and iterative alignment functionality.Multiple sequence alignments were performed between the LD3-Xyn6 sequence and the protein database (http: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi). Homology modeling of the amino acid sequence of LD3-Xyn6 was performed using Swiss model and Pymol software.

[0035] (3) Molecular cloning of LD3-Xyn6 After enrichment culture of soil samples collected from Liandu (LD3) hot spring, metagenomic DNA extracted using a soil DNA extraction kit was used as a template to amplify the full-length LD3-Xyn6 gene using the following primers: LD3-Xyn6-F: CATCATCATCATCATCATGAA GGGCTGGCCAAGACAAA AT, SEQ ID NO.1; LD3-Xyn6-R: GTGCTCGAGTGCGGCCGCAAG TGTCGGTGTTGGCGTCG GT, SEQ ID NO.2; The underlined sequence represents a recombinant fragment homologous to the pSHY211 vector (mentioned in the literature "Yin, YR, Li, XW, Long, CH,Li, L., Hang, YY, Rao, MD, ...&Yang, LQ (2023). Characterization of a GH10 extremely thermophilic xylanase from the metagenome of hot spring for prebiotic production. Scientific Reports, 13(1), 16053", which has been previously digested with EcoRI and HindIII).

[0036] The PCR program consisted of 10 cycles of 95℃ for 180s, 98℃ for 20s, and 68℃ for 150s, followed by 30 cycles of 98℃ for 20s, 55℃ for 30s, and 72℃ for 150s, and finally extension at 72℃ for 10 min.

[0037] The PCR product was inserted into pSHY211 using the pEASY-Uni Seamless Cloning and Assembly Kit (Quanjin Biotechnology, China) to obtain the expression plasmid pSHY211-LD3-Xyn6. The LD3-Xyn6 gene was cloned and expressed using the DH5α method. *E. coli* was grown on LB medium containing 50 µg / mL kanamycin. A DNA isolation and purification kit was used for DNA isolation and purification (Sangon, China).

[0038] Results analysis:

[0039] Xylanase LD3-Xyn6 amino acid sequence: MAAALLVFTVSVVTPKEADAGLAKTKFLGNVINSSIPSNFAVYWDQVTPENATKWGSVESSRDSMNWSTADMIYNYARSNGFPFKFHTLVWGSQEPGWISGLSAAEQQAEVIEWIQAAGQRYPDADFVDVVNEPLHAKPSYRNAIGGDGSTGWDWVIWSFEQARRAFPNSKL LINEYGVENDPNAASQYVQIINLLKSRGLIDGIGIQGHYFNLDTVSVSTLRTTLGMLAETGLPIYVSELDLSGDDATQLARYQEKFPILWEHPSVQGITLWGYIEGQTWRSGTHLITASGAERPALQWLRTYLAGSSSSPTPTPTPTPTRTPTPTPTPTPTPTPPT, SEQ ID NO.4.

[0040] Nucleotide sequence analysis of the LD3-Xyn6 xylanase genome showed a full length of 1023 bp; the ORF encodes 341 amino acid residues, of which amino acids 1-20 form the signal peptide sequence, and amino acids 53-305 encode the GH-10 family domain. The theoretically calculated molecular size is 35.33 kDa, and the theoretical pI is 5.05. The amino acid sequence of LD3-Xyn6 shares 76.62% similarity with xylanases from Cohnella (WP_019004722.1), Cohnella sp (REK64096.1), and uncultured bacterium (WVX55670.1). Figure 1 A). A protein structure model of LD3-Xyn6 was simulated ( Figure 1 B).

[0041] Example 2 Heterologous expression and purification of LD3-Xyn6 E. coli DH5α was cultured with pSHY211-LD3-Xyn6 to induce heterologous expression of the LD3-Xyn6 gene. Transformants were cultured in 100 ml LB medium containing 50 µg / mL kanamycin at 37°C with shaking at 180 rpm for 7 h. They were then transferred to 25°C and cultured with shaking at 180 rpm for 12 h. The culture medium was aliquoted into 50 ml centrifuge tubes, centrifuged at 4000×g for 20 min, and the supernatant was discarded and the cells were resuspended. The resuspended cells were sonicated and centrifuged at 12000×g for 15 min at 4°C to collect the cell lysate. The cell lysate was purified using a Ni chelate affinity column (Histrap, TransGen Biotech, China) to extract the free cell extract. Protein concentration was determined using a Bradford protein assay kit (Order NO. C503031, Sangon Biotech, China), with bovine serum albumin (BSA) as a standard. Proteins purified by Ni column were detected using 12% denaturing acrylamide gel electrophoresis (SDS-PAGE).

[0042] Results analysis: The LD3-Xyn6 gene was successfully cloned into pSHY211 as a His-tag fusion protein, which was further confirmed by sequencing. The recombinase LD3-Xyn6 was prepared using Ni... 2+ -NTA resin affinity chromatography purification. The purified protein showed a single band of 35.33 Kda on a 12% SDS-PAGE gel, similar to the theoretical molecular weight. Figure 2 ).

[0043] Example 3 Enzymatic property determination The activity of thermophilic xylanase LD3-Xyn6 was detected using the DNS (3,5-dinitrosalicylic acid) method. Xylanase activity is measured in units of enzyme activity released per minute equal to the amount of reducing sugar released.

[0044] (1) Determination of optimal temperature The activity of purified enzyme LD3-Xyn6 was determined by measuring its activity at different temperatures (20-100℃) and pH 7, and the optimal temperature was thus determined. Figure 3 A).

[0045] (2) Determination of optimal pH The purified enzyme LD3-Xyn6 was incubated in different buffers with a pH range of 3.0–10.0 (sodium citrate-disodium hydrogen phosphate buffer, pH 3.0–8.0; glycine-sodium hydroxide buffer, pH 8.0–10.0). After reacting at the optimal temperature, its optimal pH was determined. Figure 3 B).

[0046] (3) Determination of thermal stability and pH stability The purified LD3-Xyn6 was incubated at different temperatures (75℃, 80℃, 85℃, 90℃) for different times (0, 20, 40, 60, 80, 100, and 120 min) and at pH 3.0–11.0 for different times (12 and 24 h). The residual enzyme activity was then measured. Figure 3 C and Figure 3 D).

[0047] Results analysis: The optimal reaction temperature for LD3-Xyn6 is 85℃, and it retains more than 60% of its relative activity at 75-90℃. Figure 3 A). The optimal pH for LD3-Xyn6 is pH 5.6, and it maintains over 80% relative activity between pH 5 and pH 6.6. Figure 3 B). Thermal stability analysis showed that LD3-Xyn6 had a half-life of 15 min at 85℃ and 10 min at 90℃. After heat treatment at 80℃ for 20 min, its activity decreased by approximately 40%. Figure 3 C). pH stability analysis showed that the purified enzyme maintained more than 60% of its initial activity within the pH range of 4.0-11.0 after incubation at 4℃ for 12 h and 24 h; and the effect of incubation at different pH values ​​for 12 h or 24 h on enzyme activity was basically consistent. Figure 3 D).

[0048] (4) Effects of metal ions and chemical reagents on the activity of LD3-Xyn6 enzyme Various metal ions (K) at 1mM and 10mM + Mg 2+ Fe 3+ Ca 2+ Zn 2+ Co 2+ Cu 2+ Ag + Mn 2+ Pb 2+ Ni 2+ Ba 2+ Cd 2+ Al 3+ Fe 2+0.1% and 1% of chemical reagents, such as ethylenediaminetetraacetic acid (EDTA), benzyl sulfonyl fluoride (PMSF), polyoxyethylene stearate (Tween 80), methanol (MeOH), ethanol (EtOH), sodium dodecyl sulfate (SDS), urea (Urea), β-thioethanol (β-ME), hexadecyltrimethylammonium bromide (CTAB), dithiothreitol (DTT), polyethylene glycol octylphenyl ether (Triton X-100), isopropanol (IPA), and dimethyl sulfoxide (DMSO), were added to the reaction system. A control was tested under the same conditions as above, without any additives added to the reaction mixture. Figure 4 and Figure 5 ).

[0049] Results analysis: such as Figure 4 As shown, at ion concentrations of 1 mM or 10 mM, the activity of LD3-Xyn6 is affected by K. + Ca 2+ and Mg 2+ Activated, exhibiting varying degrees of activation (4%-21%); most ions slightly inhibited its activity in 10mM Zn 2+ Fe 2 + Under the influence of [unclear], its activity decreased to below 50%, of which 10 mM Fe [unclear] 3+ Al 3+ Cu 2+ Ag + It has a strong inhibitory effect on activity.

[0050] like Figure 5As shown, chemical reagents such as DMSO, IPA, EDTA, DTT, and CTAB inhibit LD3-Xyn6. High concentrations of SDS, PMSF, and β-ME strongly inhibit LD3-Xyn6, reducing its activity to below 10%. However, when the concentration of MeOH and EtOH is 1%, their effect on LD3-Xyn6 activity is weak; when the concentration is 10%, its activity is strongly inhibited, indicating that they may not be tolerant to high concentrations of alcohols. Tween 80, Urea, and Triton X-100 can activate the activity of LD3-Xyn6. In the paper industry, Tween 80, as a nonionic surfactant (emulsifier), can improve the uniformity of the coating to improve paper quality. Adding Urea during the pulping process can weaken the hydrogen bonding between fibers, improve fiber swelling and dispersion, thereby improving pulping efficiency and increasing pulp softness. Tween 80 and Urea can enhance the enzyme activity of LD3-Xyn6. Therefore, using pure LD3-Xyn6 xylanase in the paper industry will not affect its activity; on the contrary, LD3-Xyn6 xylanase can further promote the production of high-quality pulp. In traditional paper bleaching processes, the core function of low-concentration Triton X-100 is to improve paper bleaching performance by improving the contact efficiency between the enzyme and pulp, assisting in resin removal, and enhancing the enzymatic reaction effect. Currently, due to environmental protection and high-quality requirements, Triton X-100 has been gradually replaced by Tween 80 or some specialized pulp enzyme adjuvants.

[0051] (5) Substrate specificity and kinetic analysis of LD3-Xyn6 Enzyme activity was determined using beech xylan, corn cob xylan, sugarcane bagasse xylan, sodium carboxymethyl cellulose (CMC-Na), microcrystalline cellulose (Avicel), and cellobiose as substrates (1%, w / v).

[0052] At the optimal pH and temperature, the kinetic constants of LD3-Xyn6 were determined using compounds at concentrations ranging from 0.1 to 20 mg / ml. The Km (Michaelis-Menten constant) and Vmax (maximum reaction rate) of the reaction were calculated and determined using Lineweaver-Burk plots.

[0053] Table 1. Activity of LD3-Xyn6 on different substrates

[0054] Results Analysis: The results are shown in Table 1. The specific activity of beech xylan was 211 U / mg, while the specific activities of corn cob xylan, sugarcane bagasse xylan, and [other enzymes] were 191.65 and 169.57 U / mg, respectively. However, LD3-Xyn6 showed no activity against sodium carboxymethyl cellulose, microcrystalline cellulose, or cellobiose. Therefore, beech xylan is the optimal substrate for the recombinant enzyme LD3-Xyn6, with Km, Vmax, and Kcat values ​​of 1.87 mg / ml, 256.41 U / mg, and 150.98 s, respectively. -1 LD3-Xyn6 is active against different types of xylan and can degrade them.

[0055] Example 4 TLC analysis of xylanase LD3-Xyn6 (1) The reaction mixture consisting of 1% beech wood polysaccharide and 10 μg of purified enzyme was incubated at the optimal pH and optimal temperature for 12 h.

[0056] (2) Use a capillary tube to spot the enzymatically digested mixture and the standards (xylose X1, xylobiose X2, xylotriose X3, xylotetraose X4) onto the starting line of a silica gel 60 plate (Merck, Darmstadt, Germany). Place the spotted silica gel plate into a closed chromatography tank containing a mixed solvent of n-butanol / acetic acid / water (2:1:1, v / v / v), allowing the solvent to move from bottom to top by capillary action.

[0057] (3) Spray freshly prepared 5% (v / v) sulfuric acid-ethanol solution evenly onto the unfolded silica gel plate, then heat at 120°C for 10 minutes and detect the sugar content.

[0058] Results analysis: such as Figure 6 As shown, TLC analysis was performed on the xylan hydrolysis products of LD3-Xyn6. The results showed that the main hydrolysis products of LD3-Xyn6 were xylobiose (X2) and xylotetraose (X4), and a small amount of xylose.

[0059] Example 5 (1) Preparation of xylan extracted by high temperature alkali and xylan extracted by high temperature water The collected wheat bran and corn cobs were first rinsed with tap water to remove dirt. They were then dried overnight at room temperature, ground, and passed through a 2-10 mm sieve. For xylan extraction, 50 g of each of the above materials were soaked in 1.5 L of 2% NaOH in an 80°C water bath for 90 min, cooled, and filtered through a cotton cloth filter. The pH of the filtrate was adjusted to 5 with glacial acetic acid. The alkali-soluble xylan was added to 1.5 L of 95% (final concentration 50% v / v) ethanol to precipitate, and allowed to stand overnight. The precipitate was centrifuged and washed several times with distilled water. It was then dried to constant weight in a 50°C hot air oven to obtain the high-temperature alkali-treated xylan substrate. High-temperature water-treated xylan substrates were prepared by replacing NaOH with pure water using the same method.

[0060] (2) High-temperature alkaline method and enzymatic hydrolysis of xylan extracted at high temperature 0.2 g of substrates treated with high temperature and alkali (corn cob, wheat bran) and substrates treated with high temperature and water (corn cob, wheat bran) were weighed and added to 80 µl of LD3-Xyn6 purified enzyme solution (protein concentration 991.25 µg / ml). The total volume was then adjusted to 1 mL with pH 5.6 buffer and incubated at 75 °C. OD540 nm was measured. Reducing sugar content was measured every 2 h until 12 h, and then every 12 h until 36 h. No enzyme solution was added to the control group.

[0061] Results analysis: The sugar production curves of LD3-Xyn6 on the four substrates showed significant differences overall. The sugar production of wheat bran treated with high-temperature water was significantly higher than that of the other three substrates, reaching a peak at 12 h and then stabilizing. Before 36 h, the sugar production of both alkali-treated corn cob and alkali-treated wheat bran continued to increase and was higher than that of the corn cob substrate treated with high-temperature water. Figure 7 The results showed that corn cobs treated with high temperature and alkali and wheat bran treated with high temperature and water were suitable direct hydrolysis substrates for LD3-Xyn6.

[0062] Example 6 The effect of prebiotics on the growth of lactic acid bacteria Prebiotic products were obtained by treating xylan substrates with xylanase LD3-Xyn6. The specific procedure is as follows: 60 mg of beech xylan was weighed and dissolved in 550 µL of pH 7 sodium citrate-disodium hydrogen phosphate buffer. 130 µg of purified LD3-Xyn6 xylanase was added, and the mixture was placed in a 70°C water bath and reacted overnight (16 h). The reaction solution was then filtered through a 0.45 µm sterile filter in a laminar flow hood to obtain the prebiotic products.

[0063] Add an equal volume of prebiotic product to 10 mL of LB liquid medium, then inoculate with 200 µL of *Lactococcus lactis* NZ9000. A control group without prebiotics was used, and each group was repeated in triplicate. The cultures were incubated at 37°C on a shaker, and bacterial count was measured at OD600. Bacterial count was measured every 2 hours until 12 hours, then every 12 hours until 60 hours. A growth curve of *Lactococcus lactis* NZ9000 was plotted.

[0064] To simulate the gut microbiota environment, *E. coli* and *Lactococcus lactis* NZ9000 were co-cultured in LB medium supplemented with prebiotics to investigate the effects of prebiotics on beneficial gut microbiota such as lactic acid bacteria. An EGFP fluorescent gene was introduced into *E. coli* and co-cultured with *Lactococcus lactis* NZ9000 as a negative control; a mixed medium of *E. coli* and *Lactococcus lactis* NZ9000 supplemented with prebiotics served as the experimental group. The amounts of *E. coli* and *Lactococcus lactis* NZ9000 were the same in both the control and experimental groups. Both groups were cultured in a shaker at 37°C. Bacterial cell count was measured at OD600 absorbance and fluorescence intensity at OD485 absorbance, with measurements taken every 2 hours to 12 hours, and then every 12 hours to 60 hours.

[0065] Results analysis: The prebiotic products obtained by xylanase LD3-Xyn6 degrading xylan can promote the growth of Lactococcus lactis. The growth-promoting effect of prebiotics is as follows: Figure 8 As shown in Figure A, the growth of lactic acid bacteria was in the lag phase during the first 12 hours of culture, with little fluctuation in the growth rate. From 12 to 36 hours, the lactic acid bacteria in the group without prebiotics entered the logarithmic growth phase, and after 36 hours, the growth rate stabilized, entering the stationary phase. The lactic acid bacteria in the group with prebiotics showed an increasing growth rate from 12 to 24 hours, and the bacterial cell mass stabilized from 24 to 48 hours. Furthermore, the OD600 of the cells was significantly increased from 24 to 48 hours. Figure 8 A).

[0066] The effect of xylan hydrolysates (prebiotics) of LD3-Xyn6 on gut microbiota was simulated using a co-culture of *E. coli* and *Lactococcus lactis* NZ9000. In the control group, fluorescence intensity continued to increase over time, indicating a gradual increase in the number of *E. coli* and a gradual decrease in the number of lactic acid bacteria. However, in the group supplemented with prebiotics, the OD600 of lactic acid bacteria was significantly increased, and the fluorescence intensity was lower than in the unsupplemented group. This indicates a significant increase in the number of lactic acid bacteria compared to the control group, and that the number of lactic acid bacteria was significantly greater than that of *E. coli*. Figure 8 B). This indicates that the obtained prebiotic products have a certain promoting effect on the growth of lactic acid bacteria.

[0067] In summary, this invention identified a novel xylanase gene (LD3-Xyn6) from the Dali Liandu hot spring using enrichment culture and metagenomic techniques. The gene was obtained through specific PCR amplification, molecularly cloned and heterologously expressed in *E. coli*, and its enzymatic properties were investigated. Results showed that LD3-Xyn6 exhibited activity against beech xylan, corn cob xylan, and sugarcane bagasse xylan. LD3-Xyn6 showed optimal activity at 85℃ and pH 5.6. The half-life of LD3-Xyn6 was 15 min at 85℃ and 10 min at 90℃; after incubation at 4℃ for 12 h and 24 h, it maintained over 60% relative activity between pH 4 and pH 11. Furthermore, LD3-Xyn6 can be activated by K+. + Ca 2+ and Mg 2+ Activation. LD3-Xyn6 can degrade xylan from corn cobs treated with high temperature and alkali and xylan from wheat bran treated with high temperature and water. The hydrolysis products are mainly xylobiose and xylotetraose, and the products show a significant growth-promoting effect on Lactococcus lactis. In summary, the properties and characteristics of LD3-Xyn6 make it a strong candidate for feed additives and prebiotic production.

[0068] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0069] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A thermophilic xylanase LD3-Xyn6, characterized in that, The amino acid sequence of the thermophilic xylanase LD3-Xyn6 is shown in SEQ ID NO.

4.

2. A DNA molecule, characterized in that, The DNA molecule encodes the thermophilic xylanase LD3-Xyn6 of claim 1, and its nucleotide sequence is shown in SEQ ID NO.

3.

3. A biomaterial, characterized in that, The biomaterial is any one of the following: 1) An expression cassette containing the DNA molecule of claim 2; 2) A recombinant vector containing the DNA molecule of claim 2, or a recombinant vector containing the expression cassette of claim 1; 3) Recombinant bacteria containing the DNA molecule of claim 2, or recombinant bacteria containing the expression cassette of claim 1), or recombinant bacteria containing the recombinant vector of claim 2).

4. The use of the DNA molecule of claim 2 or the biomaterial of claim 3 in the preparation of thermophilic xylanase LD3-Xyn6.

5. The application of the thermophilic xylanase LD3-Xyn6 according to claim 1 in the preparation of xylooligosaccharides.

6. The application of the thermophilic xylanase LD3-Xyn6 according to claim 1 in the degradation of xylan, characterized in that, The xylan is derived from beech, corn cob, and / or bagasse.

7. The application of the thermophilic xylanase LD3-Xyn6 according to claim 1 in promoting the growth of Lactococcus lactis.

8. A method for degrading xylan, characterized in that, The reaction involves contacting the xylan substrate with the thermophilic xylanase LD3-Xyn6 described in claim 1 under reaction conditions of 85°C and pH 5.

6.

9. The method as described in claim 8, characterized in that, The xylan substrate is corn cob xylan treated with high temperature and alkali or wheat bran xylan treated with high temperature and water.

10. A feed additive, characterized in that, It contains the thermophilic xylanase LD3-Xyn6 as described in claim 1.

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