Heat-resistant xylanase Lc-Xyn81 as well as coding gene and application thereof

Abstract

The invention discloses heat-resistant xylanase Lc-Xyn81 as well as a coding gene and application thereof, and relates to the technical field of enzymes. The sequence of the coding gene of the heat-resistant xylanase Lc-Xyn81 disclosed by the invention is as shown in SEQ ID NO. 1. The Lc-Xyn81 xylanase shows the optimal activity under the conditions that the temperature is 75 DEG C and the pH value is 6.6; at the temperature of 65-75 DEG C, the relative activity of 80% or above is maintained, and good stability is achieved at the pH of 4.0-8.0. In addition, Lc-Xyn81 can be activated by divalent metal ions such as Co < 2 + >, Mn < 2 + > and Cu < 2 + >. The enzyme has degradation activity on beech xylan, corncob xylan and bagasse xylan, the obtained product mainly comprises xylobiose and xylotetraose, and the product has a remarkable growth promoting effect on lactococcus lactis. Therefore, the enzyme has great application potential in food, feed, papermaking and bioethanol, especially in the production of prebiotics.

Description

Technical Field

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

[0002] Lignocellulose is the most abundant renewable polymer biomass on earth, and its main components include cellulose, hemicellulose and lignin. Most hemicelluloses are highly branched heteropolymers composed of different sugars on the main chain and side chains, among which xylan is the main hemicellulose in plant cell walls. Xylan cannot be directly fermented and converted by microorganisms such as yeast, but needs to be degraded into xylose subunits, oligoxylose, etc. under the action of xylanase before it can be used to produce biofuels or other value-added products such as prebiotics.

[0003] Xylanase is the main enzyme for hydrolyzing xylan, which can cut the β-1,4 bonds between the xylose residues on the hemicellulose backbone. According to the CAZy database (http: / / www.cazy.org), endo-1,4-β-xylanases are divided into multiple glycoside hydrolase (GH) families. Most xylanases belong to the GH10 and GH11 families and are widely distributed in prokaryotes and eukaryotes. In different industrial production processes, thermophilic and thermostable xylanases are often needed. For example, thermophilic and alkaliphilic xylanases are required in papermaking and pulp bleaching processes, while thermostable and acid-resistant xylanases are preferred in the juice, bread and feed industries. At present, thermophilic xylanases that can be used in industrial production 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 solution:

[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 encoding gene, or a recombinant vector containing the above expression cassette.

[0012] Another object of the present invention is to provide a recombinant microorganism containing the above encoding gene, or a recombinant microorganism containing the above expression cassette, or a recombinant microorganism containing the above 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, wherein the xylan is treated with the thermostable xylanase Lc-Xyn81 or the enzyme preparation; the treatment temperature is 60-75°C and the pH value is 4.0-8.0. Preferably, the treatment temperature is 75°C and the pH value is 6.6.

[0016] Another object of the present invention is to provide a prebiotic having 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 isolates 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, the relative activity is maintained at more than 80%. When Lc-Xyn81 is incubated at 70°C for 40-100 minutes, the activity is activated to more than 120%, and the half-life at 70°C is 180 minutes; the enzyme has good stability at pH 4.0-8.0, and the 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+(112.27%) and other divalent metal ions. Lc-Xyn81 is active against beechwood xylan (213.68U / mg), corncob xylan (143.40U / mg) and bagasse xylan (80.39U / mg). The products obtained by its degradation of xylan are mainly xylobiose and xylotetrose, and the products show a significant growth-promoting effect on Lactococcus lactis. Therefore, this enzyme has great application potential in food, feed, papermaking, bioethanol, and especially in the production of prebiotics. 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying 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 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 of Lc-Xyn81 were analyzed by TLC plate; wherein, 1 is a xylo-oligosaccharide Maker (X1 is xylose, X2 is xylobiose, X3 is xylotriose, and X4 is xylotetrose); 2 is a product of Lc-Xyn81 mixed with beech xylan; 3 is a product of inactivated enzyme mixed with beech xylan; 4 is a product of Lc-Xyn81 mixed with corn cob xylan; 5 is a 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 the xylan hydrolysate (prebiotic) of Lc-Xyn81 and the co-culture of intestinal flora (simulated by Escherichia coli and Lactococcus lactis); among them, columns A&C are the control group, and columns B&D are the experimental groups with added prebiotics. The blue circle represents Escherichia coli, and the red circle represents Lactobacillus. The lower left corner of columns A and B shows the ratio of Escherichia coli to lactic acid bacteria (A:B = Lactococcus lactis NZ9000: Escherichia coli). Columns C&D are pie charts of the percentage ratio of the two strains. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work 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), enriched and cultured, and DNA was isolated using a kit. 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 et al., 2008). The IMG server (https: / / img.jgi.doe.gov / cgi-bin / mer / main.cgi) was used to investigate the sequences. To further analyze the possible 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] KEGG and COG were used for function prediction analysis to screen out the functional genes of xylanase, and its structural domain was analyzed through the Pfam database. In this study, a new xylanase gene sequence was obtained from the metagenomic database, named lc-xyn81, whose 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] 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 structure of the amino acid sequence was deduced and analyzed by the EXPASY tool (http: / / web.expasy.org / protparam / ). The protein sequence of Lc-Xyn81 was compared in NCBI BLASTp, and the xylanase sequences of the same or neighboring genera with higher similarity were selected as neighboring sequences, and the xylanase protein sequences of other genera with lower similarity were selected as outgroups for constructing multiple comparisons and phylogenetic trees. Multiple comparisons were performed with closely related Lc-Xyn81 protein sequences using ClustalX (Thompson JD et al., 1997). Phylogenetic analysis was performed using the MEGA7 software package (Kumar S et al., 2016). The phylogenetic tree was constructed by the maximum likelihood (ML) method and the 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). The homology modeling of the amino acid sequence of Lc-Xyn81 was performed using Swiss model and Pymol software.

[0035] The results showed that the nucleotide sequence analysis of the lc-xyn81 full gene showed that its full length was 1107bp; ORF encoded 369 amino acid residues, of which amino acids 1-26 were signal peptide sequences, and amino acid sequences 87-386 were sequences encoding the GH-10 family domain. The theoretical molecular size of Lc-Xyn81 xylanase was 44.52Kda and the theoretical pI was 6. The amino acid sequence of Lc-Xyn81 had 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 associated with 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. Molecular cloning, heterologous expression, isolation and purification of Lc-Xyn81

[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 included 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 finally incubation at 72°C for 10 minutes for extension. 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 obtain 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 containing the recombinant plasmid pSHY211-Lc-Xyn81 was cultured and heterologously expressed. The transformants were cultured in 100 ml LB culture medium containing 50 μg / mL kanamycin at 37°C and 180 rpm for 7 h. Then the culture was transferred to 25°C and 180 rpm for 12 h. The culture medium was divided into 50 ml centrifuge tubes, centrifuged at 4000xg for 20 min, and the supernatant was discarded and resuspended. After ultrasonic disruption, the resuspended cells were centrifuged at 12000xg for 15 min at 4°C, and the cell lysate was collected. 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). The protein concentration was detected using a Bradford protein assay kit (Order NO. C503031, Sangon Biotech, China), with bovine serum albumin as the 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 43 KDa 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% non-denaturing acrylamide gel with a final concentration of 0.2% beechwood xylan substrate. After electrophoresis, soak the non-denaturing acrylamide gel in 2.5% Trition-X100 phosphate buffer (pH 7.6) at 4°C for 30 min. Take out the gel and soak it in phosphate buffer (pH 7.6) at 60°C for 2 h. Stain and destain the gel with 0.2% Congo red and 1M NaCl, respectively. Decolorized bands with a red background indicate xylanase activity.

[0044] The results showed that the activity of the purified enzyme was confirmed by the zymogram, 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 determined using 1% xylan from different sources (beechwood xylan, corncob xylan, bagasse xylan and oat xylan) as substrates. The reducing sugars produced by the hydrolysis of the recombinant protein Lc-Xyn81 were determined by the DNS (3,5-dinitrosalicylic acid) method using a microplate reader at 540 nm (Miller et al., 1959). The enzyme activity unit (U) of Lc-Xyn81 is the amount of enzyme that releases 1 μmol of reducing sugar per minute with 1% xylan as substrate under the optimal reaction conditions (results are shown in Table 3).

[0047] (3) Characterization of enzyme properties

[0048] The purified Lc-Xyn81 protein was tested for enzyme activity at different temperatures (20-80°C) and pH 7 to determine the optimal temperature. The purified enzyme Lc-Xyn81 was then incubated in different buffers with a pH range of 3.0-12.0 (sodium citrate-sodium hydrogen phosphate buffer, pH 3.0-8.0; glycine-sodium hydroxide buffer, pH 8.0-12.0) and the optimal pH was determined after reaction at the optimal temperature. In order to evaluate the thermal and pH stability, the purified Lc-Xyn81 was incubated at different temperatures for different times (0, 20, 40, 60, 80, 100 and 120 min) and pH 3.0-9.0 for different times (12 and 24 h), and the residual enzyme activity was determined.

[0049] The results showed that the optimal reaction temperature of Lc-Xyn81 was 75°C, and it maintained more than 60% relative activity at 60-75°C. Thermal stability analysis showed that Lc-Xyn81 still maintained more than 90% activity after heat treatment at 65°C for 60 minutes, and its half-life at 70°C and 75°C was 180 minutes and 10 minutes, respectively. Interestingly, after incubation at 70°C for 80 minutes, the enzyme was activated, and the enzyme activity increased to 149% and then began to decline (see Appendix). Figure 4 ).

[0050] The optimum pH of 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 h, and more than 60% of its activity after incubation for 24 h (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% of chemical reagents, such as ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), polyoxyethylene stearate (Tween80), methanol (MeOH), ethanol (EtOH), sodium dodecyl sulfate (SDS), urea (Urea), β-mercaptoethanol (β-ME), hexadecyltrimethylammonium bromide (CTAB), dithiothreitol (DTT), were added to the reaction system. The control was tested under the same conditions as above, and no additives were 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, among which Ag+ has the strongest inhibitory effect. In short, 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 inhibitory effects on Lc-Xyn81, among which high concentrations of SDS, DTT, CTAB, and β-ME have strong inhibitory effects on Lc-Xyn81. However, when the concentration of MeOH and EtOH is 10%, the activity of Lc-Xyn81 can still retain more than 80%, indicating that it may be tolerant to alcohols. Tween 80 can activate the activity of Lc-Xyn81. In the presence of 1% Tween80, its maximum activity is activated to more than 120%; in the presence of 1% Urea, PMSF, and EDTA, its activity is still maintained at more than 70%.

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

[0055]

[0056] Note: The activity without additives is 100%. Each value represents the mean ± SD. * indicates that the activity is significantly different when the additive is added (P ≤ 0.05). ** indicates that the activity is significantly different when the additive is added (P ≤ 0.01).

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

[0058]

[0059]

[0060] Note: The activity without additives is 100%. Each value represents the mean ± SD. * indicates that the activity is significantly different when the additive is added (P ≤ 0.05). ** indicates that the activity is significantly different when the additive is added (P ≤ 0.01).

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

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

[0063] The results are shown in Table 3. The relative activity of beech xylan is 213.68±1.21U / mg, while the relative activities of corncob xylan, bagasse xylan and oat xylan are 143.40±8.32, 80.39±10.74 and 10.80±5.19U / mg respectively. However, Lc-Xyn81 has no activity on microcrystalline cellulose, sodium carboxymethyl cellulose and cellobiose. It can be concluded that the best substrate for the recombinant enzyme Lc-Xyn81 is beech xylan, and its Km, Vmax and Kcat for beech xylan are 4.62mg / ml, 312.50μmol / min / mg and 242.49s / min 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] The reaction mixture consisting of 1% beechwood xylan and corncob xylan and 10 μg purified Lc-Xyn81 xylanase was incubated at the optimal pH and temperature for 12 h. The hydrolyzates 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). Freshly prepared 5% (v / v) H2SO4 was sprayed in ethanol and treated at 120°C for 10 min to detect the sugar content. 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 at pH 7.0, and 115 μg of purified Lc-Xyn81 xylanase was added and placed in a 60°C water bath for overnight reaction (16 h). The reaction solution was filtered with a 0.45 μm sterile filter in a clean bench to prepare a prebiotic product.

[0076] (2) Escherichia coli and lactic acid bacteria seed solution 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 an equal amount 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 Detect the amount of bacteria at the absorbance value. Detect the amount of bacteria every 2 hours until 14 hours, and then detect it at intervals of 6 hours and 12 hours until 48 hours. Draw the growth curves of lactic acid bacteria and Escherichia 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 then 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 group with added prebiotics and the group without added prebiotics, but the amount of lactic acid bacteria in the group with added prebiotics was relatively high. After 14 hours, the growth rate of lactic acid bacteria in the group without added prebiotics tended to stabilize and entered the stable period, while the growth rate of lactic acid bacteria in the group with added prebiotics showed a continuous upward trend, and the OD value of the bacteria was 0.04477 at 24-48 hours. 600 Significantly increased, 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 the colonies of microorganisms such as lactic acid bacteria that are beneficial to intestinal health were studied with and without the addition of 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 amounts of Escherichia coli and lactic acid bacteria in the control and experimental groups were 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 with the increase of 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 with the control group, and at 36h the number of lactic acid bacteria was significantly more than that of E. coli (see Appendix Figure 7 ). This indicates that the obtained prebiotic product has a promoting effect on the growth of lactic acid bacteria.

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

[0081] The present invention relates to references:

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[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 enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather 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 It comprises the thermoresistant xylanase Lc-Xyn81 described in 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 containing the coding gene according to claim 3, or a recombinant vector containing the expression cassette according to claim 4.

6. A recombinant microorganism comprising the coding 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 urate oxidase 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 urate oxidase 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 thermostable 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.

10. A prebiotic having a growth-promoting effect on lactic acid bacteria, characterized in that: The product is obtained by degrading xylan using the thermostable xylanase Lc-Xyn81 described in claim 1 or the enzyme preparation described in claim 2.

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