A thermostable xylanase Lc-Xyn81 and its encoding gene and application

Abstract

The present invention discloses a thermostable xylanase Lc-Xyn81 and its encoding gene and application, and relates to the field of enzyme technology. The encoding gene sequence of the thermostable xylanase Lc-Xyn81 of the present invention is shown in SEQ ID NO.1. Lc-Xyn81 xylanase exhibits optimal activity under the conditions of 75°C and pH 6.6; at 65-75°C, it maintains more than 80% relative activity and has good stability at pH 4.0-8.0. In addition, Lc-Xyn81 can be Co 2+ 、Mn 2+ 、Cu 2+ The enzyme is activated by divalent metal ions such as β-glucan and β-glucan. The enzyme is active against beechwood, corncob, and bagasse xylans, with the resulting products primarily consisting of xylobiose and xylotetrose. These products also significantly promote the growth of Lactococcus lactis. Therefore, the enzyme has great potential for application in food, feed, papermaking, bioethanol, and particularly in prebiotic production.

Description

Technical Field

[0001] The present invention relates to the field of enzyme technology, and more particularly to a thermostable xylanase Lc-Xyn81 and its encoding gene and application. Background Art

[0002] Lignocellulose is the most abundant renewable biomass on Earth, primarily composed of cellulose, hemicellulose, and lignin. Most hemicelluloses are highly branched polymers composed of different sugars on the main and side chains. Xylan is the primary hemicellulose in plant cell walls. Xylan cannot be directly fermented and converted by microorganisms such as yeast. It must be degraded into xylose subunits and oligosaccharides by xylanase before it can be used to produce biofuels or other value-added products such as prebiotics.

[0003] Xylanases are the primary enzymes that hydrolyze xylan, cleaving the β-1,4 bonds between xylose residues on the hemicellulose backbone. According to the CAZy database (http: / / www.cazy.org), endo-1,4-β-xylanases are classified into several glycoside hydrolase (GH) families. The vast majority of xylanases belong to the GH10 and GH11 families and are widely distributed in prokaryotes and eukaryotes. Thermophilic and heat-resistant xylanases are often required in various industrial processes. For example, thermophilic and alkaliphilic xylanases are required in papermaking and pulp bleaching, while heat- and acid-resistant xylanases are preferred in the juice, bread, and feed industries. Currently, thermophilic xylanases suitable for industrial applications are relatively scarce.

[0004] Therefore, providing more thermostable xylanases is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a thermostable xylanase Lc-Xyn81 and its encoding gene and application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A thermostable xylanase Lc-Xyn81, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] Another object of the present invention is to provide an enzyme preparation comprising the above-mentioned thermostable xylanase Lc-Xyn81.

[0009] Another object of the present invention is to provide a gene encoding a thermostable xylanase Lc-Xyn81, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] Another object of the present invention is to provide an expression cassette containing the above encoding gene.

[0011] Another object of the present invention is to provide a recombinant vector containing the above-mentioned encoding gene, or a recombinant vector containing the above-mentioned expression cassette.

[0012] Another object of the present invention is to provide a recombinant microorganism containing the above-mentioned encoding gene, or a recombinant microorganism containing the above-mentioned expression cassette, or a recombinant microorganism containing the above-mentioned recombinant vector.

[0013] Another object of the present invention is to provide the use of the above-mentioned uricase, or the above-mentioned enzyme preparation, or the above-mentioned encoding gene, or the above-mentioned expression cassette, or the above-mentioned recombinant vector, or the above-mentioned recombinant microorganism in degrading xylan.

[0014] Another object of the present invention is to provide the use of the above-mentioned uricase, or the above-mentioned enzyme preparation, or the above-mentioned encoding gene, or the above-mentioned expression cassette, or the above-mentioned recombinant vector, or the above-mentioned recombinant microorganism in the preparation of prebiotics.

[0015] Another object of the present invention is to provide a method for degrading xylan, comprising treating xylan with the aforementioned thermostable xylanase Lc-Xyn81 or the aforementioned enzyme preparation; wherein the treatment temperature is 60-75°C and the pH is 4.0-8.0. Preferably, the treatment temperature is 75°C and the pH is 6.6.

[0016] Another object of the present invention is to provide a prebiotic that has a growth-promoting effect on lactic acid bacteria, which is obtained by degrading xylan using the above-mentioned thermostable xylanase Lc-Xyn81 or the above-mentioned enzyme preparation.

[0017] Beneficial effects: The present invention isolated a new xylanase gene (Lc-Xyn81) from the metagenomic data extracted from the hot spring samples collected from Niujie Hot Spring Street in Eryuan, Dali. Lc-Xyn81 xylanase exhibits optimal activity under the conditions of 75°C and pH 6.6. At 65-75°C, it maintains more than 80% relative activity. When Lc-Xyn81 is incubated at 70°C for 40-100 minutes, its activity is activated to more than 120%, and its half-life at 70°C is 180 minutes. The enzyme has good stability at pH 4.0-8.0, and its activity is activated to more than 140% after incubation at pH 5.0-7.0 for 12 hours. In addition, Lc-Xyn81 can be Co 2+ (128.55%), Mn 2+ (119.84%), Cu 2+Lc-Xyn81 is activated by divalent metal ions such as β-catenin (112.27%). Lc-Xyn81 is active against beechwood xylan (213.68 U / mg), corncob xylan (143.40 U / mg), and bagasse xylan (80.39 U / mg). The products of xylan degradation are primarily xylobiose and xylotetrose, and these products significantly promote the growth of Lactococcus lactis. Therefore, this enzyme has great potential for application in food, feed, papermaking, bioethanol, and particularly in prebiotic production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0019] Figure 1 is the phylogenetic tree of Lc-Xyn81 constructed based on amino acid sequence homology;

[0020] Figure 2 homology modeling for the Lc-Xyn81 protein;

[0021] Figure 3 The SDS-PAGE gel image and enzyme spectrum of Lc-Xyn81, where 1 is the protein marker; 2 is the bacteria; 3 is the purified protein; 4 is the enzyme spectrum;

[0022] Figure 4 are the enzymatic properties of Lc-Xyn81; A is the optimum temperature test result; B is the optimum pH test result; C is the thermal stability of Lc-Xyn81; D is the pH stability;

[0023] Figure 5 The hydrolysis products of beech xylan and corn cob xylan analyzed by TLC plate of Lc-Xyn81; wherein, 1 is the oligo-xylo-oligosaccharide Maker (X1 is xylose, X2 is xylobiose, X3 is xylotriose, and X4 is xylotetrose); 2 is the product of Lc-Xyn81 mixed with beech xylan; 3 is the product of inactivated enzyme mixed with beech xylan; 4 is the product of Lc-Xyn81 mixed with corn cob xylan; 5 is the product of inactivated enzyme mixed with corn cob xylan;

[0024] Figure 6The growth-promoting effect of xylan hydrolysate (prebiotic) of Lc-Xyn81 on intestinal flora; A is the growth curve of lactic acid bacteria, and B is the growth curve of Escherichia coli DH5α; * indicates that the growth-promoting effect after adding prebiotics is significantly different (P≤0.05), and ** indicates that the growth-promoting effect after adding prebiotics is significantly different (P≤0.01);

[0025] Figure 7 The coating effect of Lc-Xyn81's xylan hydrolysate (prebiotic) co-cultured with intestinal flora (simulated by Escherichia coli and Lactococcus lactis); columns A & C represent the control group, and columns B & D represent the experimental group supplemented with prebiotics. Blue circles represent E. coli, and red circles represent Lactobacillus. The lower left corner of columns A and B shows the ratio of E. coli to lactic acid bacteria (A:B = Lactococcus lactis NZ9000: E. coli). Columns C & D are pie charts showing the percentage ratio of the two strains. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0027] Example 1

[0028] 1. Sample collection and metagenomic sequencing:

[0029] Soil samples were collected from the hot spring street in Eryuan, Dali, Yunnan (latitude 25°98′72.38″N, longitude 99°92′37.66″E), cultured, and DNA isolated using a kit. Metagenomic sequencing was performed using a HiSeq 2500 instrument (Suzhou GENWIZ). De novo assembly was performed using the Velvet assembly program version 1.2.08 (Zerbino DR et al., 2008). Sequences were compiled using the IMG server (https: / / img.jgi.doe.gov / cgi-bin / mer / main.cgi). To further analyze the potential functions of individual genes and ORFs, we used the COG (Tatusov RL et al., 2001), KEGG (Nakaya A et al., 2012), and Pfam (Finn RD et al., 2007) databases.

[0030] 2. Sequence prediction and analysis:

[0031] Functional prediction analysis was performed using KEGG and COG, and functional xylanase genes were identified. Their domain structures were analyzed using the Pfam database. In this study, a new xylanase gene sequence, named lc-xyn81, was obtained from the metagenomic database. Its nucleotide sequence is shown in SEQ ID NO. 1, and its amino acid sequence is shown in SEQ ID NO. 2.

[0032]

[0033] MFRKVLPSLCLLLAVVTGSCTRAASGEEEAGRSAALAEDTLPALKEVFKSAFLIGAALNPEQFYERDARGAALIKTHFNSITPENVLKWESVHPEPGRYDFEAPDRFVEFGEKNGMFIVGHTLVWHSQTPAWVFQDRNGNPVSRDTLLQRMRDHIHTVVGRYRGRIHAWDVVNEALNEDGTLRQSPWLRIIGEDYIAKAFQFAHEADPSAELYYNDYSLENAPKRNGAVRLIRQLLDQGIPVAGVGLQGHNRMDWPTLAQQDSTIAAFAALGVKVMITELDIDVLPRAVRQQGAEVTLRAEAREELDPYRAGLPDSVQQALARRYADLFRVYLKHKDAIHRVTFWGVTDGDSWLNYWPVRGRTNYPLLFDREGQPKPAFHAVVQVAREQESASLP,SEQ ID NO.2。

[0034] The BLASTx and BLASTp programs (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) were used to compare the DNA and protein sequences of Lc-Xyn81, 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 Lc-Xyn81 protein sequence was compared using NCBI BLASTp. Sequences of xylanases from the same or neighboring genera with high similarity were selected as neighboring sequences, while sequences of xylanases from other genera with low similarity were selected as outgroups for constructing multiple alignments and phylogenetic trees. Multiple alignments with closely related Lc-Xyn81 protein sequences were performed using ClustalX (Thompson JD et al., 1997). Phylogenetic analysis was performed using the MEGA7 software package (Kumar S et al., 2016). A phylogenetic tree was constructed using the maximum likelihood (ML) method with a Poisson correction model. The Lc-Xyn81 sequence was compared with the protein database (http: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi) (Larkin MA et al., 2007). Homology modeling of the Lc-Xyn81 amino acid sequence was performed using Swiss model and Pymol software.

[0035] Results showed that the nucleotide sequence analysis of the lc-xyn81 full gene revealed a full length of 1107 bp; the ORF encodes 369 amino acid residues, of which amino acids 1-26 are a signal peptide sequence and amino acids 87-386 encode a sequence encoding the GH-10 family domain. The theoretical molecular size of the Lc-Xyn81 xylanase is 44.52 kDa, with a theoretical pI of 6. The amino acid sequence of Lc-Xyn81 shares 72.29%, 72.57%, and 72.29% homology with endo-1,4-β-xylanases from Blastocatellia bacterium (HST22061.1), Bryobacteraceae bacterium (HEY1242353.1), and Blastocatelliabacterium (HKS43363.1), respectively (see Appendix). Figure 1The protein structure model of Lc-Xyn81 was simulated. The protein model of Lc-Xyn81 has a typical TIM barrel structure of the GH10 family, which is a three-dimensional protein structure formed by alternating 8 β-pleated sheets and 8 α-helices (see Appendix). Figure 2 The active site of enzymes with a TIM barrel structure is usually located at the C-terminus of the β-strand. The β-bond is believed to be involved in heat resistance, which may be related to the formation of salt bridges in the β-strand (which contributes to the stability of the entire protein structure).

[0036] 3. Lc-Xyn81 molecular cloning, heterologous expression, and purification

[0037] The following primers were used to amplify the full-length gene of lc-xyn81: lc-xyn81-F ( CATCATCATCATCATCAT GAA GAGGAGGAGGCCGGC CGGTC, SEQ ID NO.3) and lc-xyn81-R ( GTGCTCGAGTGCGGCCGCAAG GGGCAACGACGCGCTCTCCT G, SEQ ID NO. 4). The underlined sequence indicates a recombinant fragment homologous to the pSHY211 vector, which had been previously digested with EcoR I and Hind III. The PCR program consisted of 10 cycles at 95°C for 180 seconds, 98°C for 20 seconds, and 68°C for 150 seconds, followed by 30 cycles at 98°C for 20 seconds, 55°C for 30 seconds, and 72°C for 150 seconds, and a final extension at 72°C for 10 minutes. The PCR product was inserted into pSHY211 (Yin et al., Scientific Reports, 2023) using the pEASY-Uni Seamless Cloning and Assembly Kit (Quanjin Biotechnology, China) to generate the expression plasmid pSHY211-Lc-Xyn81. The lc-xyn81 gene was cloned and expressed using the DH5α method. Escherichia coli was grown in LB medium containing 50 μg / mL kanamycin. DNA isolation and purification kit was used for DNA isolation and purification (Sangon, China).

[0038] Escherichia coli DH5α cells harboring the recombinant plasmid pSHY211-Lc-Xyn81 were cultured and heterologously expressed. Transformants were cultured in 100 ml of LB broth containing 50 μg / mL kanamycin at 37°C with shaking at 180 rpm for 7 hours. The culture was then transferred to 25°C with shaking at 180 rpm for 12 hours. The culture was aliquoted into 50 ml centrifuge tubes, centrifuged at 4000 x g for 20 minutes, and the supernatant discarded before resuspending. The resuspended cells were ultrasonically disrupted and centrifuged at 12000 x g for 15 minutes at 4°C to collect the cell lysate. The cell lysate was purified using a Ni-chelating affinity column (Histrap, TransGen Biotech, China) according to the method previously reported by Yin et al. (Yin et al., 2023). Protein concentration was determined using a Bradford protein assay kit (Order No. C503031, Sangon Biotech, China) using bovine serum albumin as a standard.

[0039] The protein purified by Ni column was detected by 12% denaturing acrylamide gel (SDS-PAGE).

[0040] The results showed that the gene of lc-xyn81 was successfully cloned into pSHY211 as a His-tag fusion protein, which was further confirmed by sequencing. 2+ -NTA resin affinity chromatography purification. The purified protein showed a single band of 43KDa on 12% SDS-PAGE gel, which was similar to the theoretical molecular weight (see Appendix Figure 3 ).

[0041] 4. Enzymatic properties of Lc-Xyn81

[0042] (1) Lc-Xyn81 enzyme spectrum analysis

[0043] Prepare a 12% native acrylamide gel with a final beechwood xylan substrate concentration of 0.2%. After electrophoresis, soak the native acrylamide gel in 2.5% Trition-X100 in phosphate buffer (pH 7.6) at 4°C for 30 minutes. Remove the gel and soak it in phosphate buffer (pH 7.6) at 60°C for 2 hours. Stain and destain the gel with 0.2% Congo red and 1M NaCl, respectively. Destained bands with a red background indicate xylanase activity.

[0044] The results showed that the activity of the purified enzyme was confirmed by the enzyme spectrum, and the red background was decolorized at 38-45 kDa, confirming the enzyme activity in this band (see Appendix Figure 3 ).

[0045] (2) Lc-Xyn81 activity assay

[0046] The activity of the recombinant protein Lc-Xyn81 was measured using 1% xylan from different sources (beechwood, corncob, bagasse, and oat) as substrates. Reducing sugars produced by hydrolysis by recombinant protein Lc-Xyn81 were measured using the DNS (3,5-dinitrosalicylic acid) method (Miller et al., 1959) with a microplate reader at 540 nm. The enzyme activity unit (U) of Lc-Xyn81 is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute using 1% xylan as substrate under optimal reaction conditions (see Table 3).

[0047] (3) Characterization of enzymatic properties

[0048] The purified Lc-Xyn81 protein was assayed for enzyme activity at various temperatures (20-80°C) and pH 7 to determine its optimal temperature. The purified Lc-Xyn81 enzyme was then incubated in various buffers with a pH range of 3.0 to 12.0 (sodium citrate-disodium hydrogen phosphate buffer, pH 3.0-8.0; glycine-sodium hydroxide buffer, pH 8.0-12.0) at the optimal temperature and pH. To assess thermal and pH stability, the purified Lc-Xyn81 was incubated at various temperatures for various times (0, 20, 40, 60, 80, 100, and 120 minutes) and at pH 3.0 to 9.0 for various times (12 and 24 hours), and the residual enzyme activity was measured.

[0049] The results showed that the optimal reaction temperature for Lc-Xyn81 was 75°C, with over 60% relative activity maintained at 60-75°C. Thermal stability analysis showed that Lc-Xyn81 retained over 90% activity after heat treatment at 65°C for 60 minutes, with half-lives of 180 minutes at 70°C and 10 minutes at 75°C. Interestingly, after incubation at 70°C for 80 minutes, the enzyme became activated, with activity increasing to 149% before beginning to decline (see Appendix). Figure 4 ).

[0050] The optimal pH for Lc-Xyn81 is pH 6.6, and it maintains more than 50% relative activity between pH 6 and pH 9. pH stability analysis showed that the purified enzyme maintained more than 100% of its initial activity in the pH range of 4.0-8.0 after incubation at 4°C for 12 hours, and more than 60% of its activity after incubation for 24 hours (see Appendix). Figure 4 ).

[0051] (4) Effects of metal ions and chemical reagents on enzyme activity

[0052] The effects of metal ions and chemical reagents on the enzyme activity of Lc-Xyn81 were evaluated. 1mM and 10mM of various metal ions (K + Mg 2+ 、Fe 3+ , Ca 2+ 、Zn 2+ 、Co 2+ 、Cu 2+ 、Ag + 、Mn 2+ , Pb 2+ 、Ni 2+ ), 0.1%, 1%, and 10% chemical reagents, such as ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), polyoxyethylene stearate (Tween 80), methanol (MeOH), ethanol (EtOH), sodium dodecyl sulfate (SDS), urea (Urea), β-mercaptoethanol (β-ME), cetyltrimethylammonium bromide (CTAB), and dithiothreitol (DTT), were added to the reaction system. A control was tested using the same conditions as above, without any additives added to the reaction mixture.

[0053] The results are shown in Table 1. When the ion concentration was 1 mM or 10 mM, the activity of Lc-Xyn81 was inhibited by Co 2+ 、Cu 2+ 、Fe 3+ , Pb 2+ 、Mn 2+ 、Ni 2+ Mg 2+ Activated, and showed different degrees of activation effect; its activity was Zn 2+ , Ca 2+ , K + 、Ag + Inhibition, with Ag+ having the strongest inhibitory effect. In summary, most ions can activate the activity of Lc-Xyn81. As shown in Table 2, chemical reagents such as PMSF, SDS, EDTA, MeOH, EtOH, β-ME, DTT, Urea, and CTAB have an inhibitory effect on Lc-Xyn81, with high concentrations of SDS, DTT, CTAB, and β-ME having the strongest inhibitory effect on Lc-Xyn81. However, when the concentration of MeOH or EtOH is 10%, Lc-Xyn81 still retains over 80% of its activity, indicating that it may be tolerant to alcohols. Tween 80 can activate the activity of Lc-Xyn81. In the presence of 1% Tween 80, its maximum activity is activated to over 120%. In the presence of 1% Urea, PMSF, and EDTA, its activity remains above 70%.

[0054] Table 1 Effects of metal ions on Lc-Xyn81 enzyme activity

[0055]

[0056] Note: Activity without additives is 100%. Each value represents the mean ± SD. * indicates a significant difference in activity with the addition of additives (P ≤ 0.05). ** indicates a significant difference in activity with the addition of additives (P ≤ 0.01).

[0057] Table 2 Effects of chemical reagents on Lc-Xyn81 enzyme activity

[0058]

[0059]

[0060] Note: Activity without additives is 100%. Each value represents the mean ± SD. * indicates a significant difference in activity with the addition of additives (P ≤ 0.05). ** indicates a significant difference in activity with the addition of additives (P ≤ 0.01).

[0061] (5) Substrate specificity and kinetic analysis of Lc-Xyn81

[0062] The enzymatic activity of Lc-Xyn81 was measured using beechwood xylan, corncob xylan, oat xylan, bagasse xylan, carboxymethylcellulose sodium salt (CMC-Na), microcrystalline cellulose (Avicel), and cellobiose as substrates (1%, w / v). Kinetic constants for Lc-Xyn81 were determined at the optimal pH and temperature using various concentrations of the compounds, ranging from 0.1 to 20 mg / ml. The Km (Michaelis-Menten constant) and Vmax (maximum reaction velocity) of the reaction were calculated and determined from Lineweaver-Burk plots.

[0063] The results are shown in Table 3. The relative activity of beech xylan was 213.68±1.21 U / mg, while the relative activities of corncob xylan, bagasse xylan, and oat xylan were 143.40±8.32, 80.39±10.74, and 10.80±5.19 U / mg, respectively. However, Lc-Xyn81 had no activity against microcrystalline cellulose, sodium carboxymethyl cellulose, and cellobiose. Therefore, it was concluded that the optimal substrate for the recombinant enzyme Lc-Xyn81 was beech xylan, with its Km, Vmax, and Kcat values ​​of 4.62 mg / ml, 312.50 μmol / min / mg, and 242.49 s, respectively. -1 (See Table 4.) Lc-Xyn81 is active against different types of xylans and can degrade them.

[0064] Table 3 Activity of Lc-Xyn81 on different substrates

[0065]

[0066] Table 4 Characteristics of Lc-Xyn81

[0067]

[0068]

[0069] (6) TLC analysis of Lc-Xyn81

[0070] A reaction mixture consisting of 1% beechwood xylan and corncob xylan was incubated with 10 μg of purified Lc-Xyn81 xylanase at the optimal pH and temperature for 12 hours. The hydrolyzed products of beechwood xylan and corncob xylan were characterized by thin-layer chromatography (TLC) using silica gel 60 plates (Merck, Darmstadt, Germany). The solvent was n-butanol / acetic acid / water (2:1:1, v / v / v). Sugar content was determined by spraying freshly prepared 5% (v / v) H₂SO₄ in ethanol and treating at 120°C for 10 minutes. Xylose (X1), xylobiose (X2), xylotriose (X3), and xylotetrose (X4) were used as standards.

[0071] The results showed that the main hydrolysis products of Lc-Xyn81 xylanase were xylobiose (X2) and xylotetrose (X4), with a small amount of xylotriose (X3) (see Appendix Figure 5 ).

[0072] Example 2

[0073] Lc-Xyn81 degrades xylan to produce prebiotics

[0074] (1) Use xylanase Lc-Xyn81 to treat xylan substrate to obtain prebiotic products.

[0075] 120 mg of beechwood xylan was weighed and dissolved in 1200 μL of sodium citrate-sodium hydrogen phosphate buffer (pH 7.0). 115 μg of purified Lc-Xyn81 xylanase was added and the mixture was reacted overnight (16 h) in a 60°C water bath. The reaction solution was filtered through a 0.45 μm sterile filter in a clean bench to produce the prebiotic product.

[0076] (2) Escherichia coli and lactic acid bacteria seed liquid were cultured in 10 mL of LB liquid medium (yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L) (37°C, 48 h) and diluted to OD 600The value was 0.31, and the dilution was used as the subsequent inoculum. After adding equal amounts of prebiotic products to 10 mL of LB liquid culture medium, 200 μL of lactic acid bacteria inoculum and Escherichia coli inoculum were inoculated respectively. The group without prebiotics was used as the control, and three replicates were performed for each group. Incubate at 37 ° C in a shaking incubator, OD 600 Measure bacterial counts at absorbance values. Measure bacterial counts every 2 hours until 14 hours, and then at 6-hour and 12-hour intervals until 48 hours. Plot growth curves for lactic acid bacteria and E. coli.

[0077] The results showed that the growth of lactic acid bacteria was in the lag phase in the first 10 hours of culture, and after 10 hours, the lactic acid bacteria began to enter the logarithmic growth phase. There was no significant difference in the growth rate of lactic acid bacteria in the first 14 hours between the prebiotics-added group and the non-prebiotics-added group, but the amount of lactic acid bacteria in the prebiotics-added group was relatively higher. After 14 hours, the growth rate of lactic acid bacteria in the non-prebiotics-added group tended to stabilize and entered the stable period, while the growth rate of lactic acid bacteria in the prebiotics-added group showed a continuous upward trend, and the OD value of the bacteria was significantly higher at 24-48 hours. 600 Significantly improved, 3-5 times that of the group without addition (see Appendix Figure 6 However, for E. coli DH5α, there was no significant difference in the growth of E. coli with or without the addition of prebiotics (see Appendix Figure 6 ). This indicates that the xylan hydrolysate of Lc-Xyn81 has a significant growth-promoting effect on lactic acid bacteria as a prebiotic, but has no growth-promoting effect on Escherichia coli.

[0078] (3) Escherichia coli and lactic acid bacteria were co-cultured in LB medium (pH 7.0) to simulate the intestinal flora environment (same method as above), and prebiotics were added to a final concentration of 4 mg / mL. The effects of prebiotics on microbial colonies such as lactic acid bacteria that are beneficial to intestinal health were studied with and without prebiotics. The EGFP fluorescent gene was introduced into Escherichia coli and co-cultured with lactic acid bacteria as a negative control group, in which no prebiotics were added. The amount of Escherichia coli and lactic acid bacteria in the control and experimental groups was the same. The control and experimental groups were cultured in a shaker at 37°C, mixed at 24h, 36h, and 48h, and 1mL of the mixed bacterial solution was pipetted into a sterile centrifuge tube and diluted 10 -7 Then, 100 μL of the mixed bacterial solution was aspirated for coating.

[0079] The results showed that the relative content of lactic acid bacteria at 24h (45% and 46%), 36h (4% and 69%), and 48h (2% and 34%) were not added and added prebiotics, respectively. In the control group, the number of E. coli gradually increased over time, while the number of lactic acid bacteria was extremely rare; in the experimental group with added prebiotics, both E. coli and lactic acid bacteria grew, and the number of lactic acid bacteria increased significantly compared to the control group. At 36h, the number of lactic acid bacteria was significantly greater than that of E. coli (see Appendix). Figure 7 ). This shows that the obtained prebiotic product has a promoting effect on the growth of lactic acid bacteria.

[0080] Unless otherwise stated, all analyses were performed three times, and the mean values ​​were used for all analyses. Statistical analysis was performed using SPSS 20.0, and the results are presented as mean ± SEM. One-way analysis of variance was used for statistical analysis, and multiple group comparisons were performed using the Tukey test. In all comparisons, a p value < 0.05 was considered statistically significant.

[0081] The present invention relates to references:

[0082] Zerbino DR,&Birney E(2008)Velvet:Algorithms for de novo short readassemblyusing de Bruijn graphs.Genome Research,18(5),821-829.

[0083] Tatusov RL(2001)The COG database: new developments in phylogenetic classification ofproteins from complete genomes. Nucleic Acids Research, 29(1),22–28.

[0084] 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 orthologclusters. Nucleic Acids Research, 41(D1),D353–D357.

[0085] 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.

[0086] Thompson JD,Gibson TJ,Plewniak F,Jeanmougin F,Higgins DG(1997)TheCLUSTAL_X windows interface:flexible strategies for multiple sequencealignment aidedby quality analysis tools.Nucleic Acids Res 25:4876-4882.

[0087] Kumar S,Stecher G,&Tamura K(2016)MEGA7:Molecular EvolutionaryGenetics Analysis Version 7.0for Bigger Datasets.Molecular Biology andEvolution,33(7),1870–1874.

[0088] Larkin MA,Blackshields G,Brown NP,Chenna R,McGettigan PA,McWilliam H,Valentin F,Wallace IM,Wilm A,Lopez R,Thompson JD,Gibson TJ,Higgins DG(2007)Clustal W and Clustal X version 2.0.Bioinformatics,23(21),2947-2948.

[0089] Yin Y R,Meng Z H,Hu Q W,et al.The hybrid strategy ofThermoactinosporarubra YIM 77501T for utilizing cellulose as a carbon source at differenttemperatures[J].Frontiers inMicrobiology,2017,8:942.

[0090] Yin, Yi-Rui, et al. "Characterization of a GH10 extremely thermophilicxylanase from the metagenome ofhot spring for prebiotic production." Scientific Reports 13.1(2023):16053.

[0091] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0092] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A thermostable xylanase Lc-Xyn81, characterized in that The amino acid sequence of Lc-Xyn81 is shown in SEQ ID NO.

2.

2. An enzyme preparation, characterized in that The invention comprises the thermostable xylanase Lc-Xyn81 according to claim 1.

3. A gene encoding a thermostable xylanase Lc-Xyn81, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

4. An expression cassette containing the coding gene according to claim 3.

5. A recombinant vector comprising the coding gene according to claim 3, or a recombinant vector comprising the expression cassette according to claim 4.

6. A recombinant microorganism comprising the encoding gene according to claim 3, or a recombinant microorganism comprising the expression cassette according to claim 4, or a recombinant microorganism comprising the recombinant vector according to claim 5.

7. Use of the thermostable xylanase Lc-Xyn81 according to claim 1, or the enzyme preparation according to claim 2, or the encoding gene according to claim 3, or the expression cassette according to claim 4, or the recombinant vector according to claim 5, or the recombinant microorganism according to claim 6 in degrading xylan.

8. Use of the thermostable xylanase Lc-Xyn81 according to claim 1, or the enzyme preparation according to claim 2, or the encoding gene according to claim 3, or the expression cassette according to claim 4, or the recombinant vector according to claim 5, or the recombinant microorganism according to claim 6 in the preparation of prebiotics.

9. A method for degrading xylan, characterized in that: Xylan is treated with the heat-resistant xylanase Lc-Xyn81 according to claim 1 or the enzyme preparation according to claim 2; the treatment temperature is 60-75° C. and the pH value is 4.0-8.0.

Images