High-temperature-resistant xylanase mutant, coding gene, preparation method and application of high-temperature-resistant xylanase mutant in weaned piglets

Abstract

The invention belongs to the technical field of bioengineering, and discloses a high-temperature-resistant xylanase mutant, a coding gene, a preparation method and application of the high-temperature-resistant xylanase mutant to weaned piglets. The invention discloses a method for improving the high temperature resistance of xylanase, which is characterized in that wild xylanase (the amino acid sequence is shown as SEQ ID NO: 1) is modified, S at the 76th site in the amino acid sequence is mutated into Y, K at the 169th site in the amino acid sequence is mutated into R, and N at the 198th site in the amino acid sequence is mutated into I. Through the mutation, the high temperature resistance of the wild xylanase can be obviously improved. After the obtained high-temperature-resistant xylanase is treated for 10 minutes at the temperature of 90 DEG C, the residual enzyme activity of the high-temperature-resistant xylanase can reach 21.85% or above under the condition of 37 DEG C, which is obviously higher than that of wild type xylanase which is almost completely inactivated under the same treatment condition, and the high-temperature-resistant xylanase can be used for preparing a feed additive or feed containing the xylanase so as to degrade xylan in the feed; the utilization rate of nutrient substances in the feed is improved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a heat-resistant xylanase mutant and its encoding gene, preparation method, and its application in weaned piglets. Background Technology

[0002] Xylan is a major type of hemicellulose found in the cell walls of grasses and the secondary cell walls of dicotyledonous plants. It is widely present in cereals such as corn, wheat, and barley, as well as byproducts such as bran, middlings, and rice bran. In monogastric animal feed (such as pigs and poultry), xylan in the feed ingredients has strong hydrophilicity, increasing the viscosity of digesta and thus hindering the digestion, absorption, and utilization of feed nutrients. It is an anti-nutritional factor that reduces feed digestibility and utilization and can cause intestinal health problems in livestock and poultry.

[0003] Xylanase is an enzyme that hydrolyzes the β-1,4 glycosidic bonds in the backbone of xylan polymer chains. It is mainly found in plant cell walls and is produced by various microorganisms, including fungi, bacteria, and yeast. Currently, a series of thermophilic microorganisms (optimal temperature range of 50-90℃) and hyperthermophilic microorganisms (optimal temperature exceeding 90℃) have been isolated, all of which are producers of xylanase.

[0004] Xylanase plays an important role in the feed industry, especially for raw materials rich in lignocellulose, such as wheat, barley, and soybeans. Treating feed with xylanase breaks down xylan in the raw materials and releases the nutrients encapsulated in the xylan, improving the digestibility and absorption rate of the feed in the animal's gastrointestinal tract and further enhancing its nutritional value.

[0005] However, in feed processing, feed ingredients typically undergo mixing, conditioning, preservation, conditioning, and pelleting. Furthermore, the conditioning-preservation-conditioning-pelleting process is a short-duration, non-uniform heating process. When the conditioner is set to 85℃, the local instantaneous temperature can reach 85-90℃, and the residence time of materials in different formulations ranges from 180-250 seconds. This can destroy the activity of enzyme preparations and limit their use. Therefore, xylanase applied in feed processing needs to possess good heat resistance. Simultaneously, it is also necessary to ensure that feed with added xylanase retains its effective function after animal ingestion at the digestive tract ambient temperature (approximately 37℃). Therefore, xylanase also needs to retain high enzyme activity after high-temperature treatment.

[0006] In conclusion, obtaining a xylanase that can simultaneously meet both of the above requirements is a pressing technical problem that needs to be solved. Summary of the Invention

[0007] The present invention aims to at least solve one of the technical problems existing in the prior art. It provides a thermoresistant xylanase that retains good enzymatic activity at the ambient temperature of the animal digestive tract (approximately 37°C) after being subjected to the high temperature of a granulation process (approximately 85°C).

[0008] The first objective of this invention is to provide a method for improving the high-temperature resistance of xylanase.

[0009] The second objective of this invention is to provide a heat-resistant xylanase.

[0010] The object of a third aspect of the present invention is to provide biomaterials related to the thermostable xylanase of the second aspect of the present invention.

[0011] The fourth aspect of this invention aims to provide the application of the heat-resistant xylanase of the second aspect of this invention or the biomaterial of the third aspect of this invention.

[0012] The fifth aspect of this invention aims to provide a product.

[0013] The sixth aspect of this invention aims to provide a feed additive or feed.

[0014] The seventh aspect of this invention aims to provide a method for degrading xylan.

[0015] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0016] In a first aspect, the present invention provides a method for improving the heat resistance of xylanase, comprising mutating S at position 76 to Y, K at position 169 to R, and N at position 198 to I in the amino acid sequence of xylanase; the amino acid sequence of said xylanase is shown in SEQ ID NO:1.

[0017] In a second aspect, the present invention provides a thermostable xylanase, the amino acid sequence of which is shown in SEQ ID NO:3.

[0018] A third aspect of the invention provides biomaterials related to the thermostable xylanase of the second aspect of the invention, said biomaterials being any one of a1)-a12):

[0019] a1) A nucleic acid molecule encoding the thermostable xylanase of the second aspect of the present invention;

[0020] a2) An expression cassette containing the nucleic acid molecule described in a1);

[0021] a3) A recombinant vector containing the nucleic acid molecules described in a1);

[0022] a4) A recombinant vector containing the expression cassette described in a2);

[0023] a5) Recombinant microorganisms containing the nucleic acid molecules described in a1);

[0024] a6) Recombinant microorganisms containing the expression cassette described in a2);

[0025] a7) Recombinant microorganisms containing the recombinant vector described in a3);

[0026] a8) Recombinant microorganisms containing the recombinant vector described in a4);

[0027] a9) Transgenic animal cell lines containing the nucleic acid molecules described in a1);

[0028] a10) Transgenic animal cell lines containing the expression cassette described in a2);

[0029] a11) Transgenic animal cell lines containing the recombinant vector described in a3);

[0030] a12) Transgenic animal cell lines containing the recombinant vector described in a4).

[0031] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:4.

[0032] In some embodiments of the present invention, the cell carrier does not include reproductive material.

[0033] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0034] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

[0035] In some embodiments of the present invention, the non-viral vector includes at least one of plasmid vectors, cationic polymer vectors, chitosan, polyethyleneimine, nanoparticle vectors, and liposomes.

[0036] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a sclerotium, an F sclerotium, or an artificial chromosome.

[0037] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0038] In some embodiments of the present invention, the recombinant expression vector uses pSHY211 or pPET28a as the original expression vector.

[0039] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new plant or animal varieties.

[0040] In some embodiments of the present invention, the prokaryotic cells include bacteria well known in the art, such as Escherichia coli, Streptomyces, Bacillus subtilis, and Lactobacillus, which are capable of expressing the target protein.

[0041] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

[0042] In some embodiments of the present invention, the cells are typically, but not limited to, selected from at least one of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Candida albicans, Rhodotorula rubrum, Bacillus, Escherichia coli, Salmonella, Clostridium, Streptomyces, Staphylococcus aureus, Neisseria, and Shigella.

[0043] A fourth aspect of the invention provides the use of the thermostable xylanase of the second aspect of the invention or the biomaterial of the third aspect of the invention in any one of b1)-b4):

[0044] b1) Preparation of feed or feed additives;

[0045] b2) Hydrolysis of xylan;

[0046] b3) Prepare products for hydrolyzing xylan;

[0047] b4) Industry.

[0048] In some embodiments of the present invention, the xylan described in b2)-b3) includes at least one of beech xylan, corn cob xylan, bagasse xylan, and birch xylan.

[0049] In some preferred embodiments of the present invention, b4) refers to industrial applications such as food processing (e.g., to improve the mechanical processing properties of dough, improve the elasticity of bread, or delay bread staling), feed processing (e.g., to degrade xylan molecules in feed, reduce the viscosity of chyme in the digestive tract, thereby promoting the digestion and absorption of nutrients), textile processing (e.g., to reduce or replace chemical blending methods in the degumming process of cellulose), papermaking (e.g., to reduce the amount of alkali and subsequent chlorine used without reducing the strength of pulp, reduce the content of organochlorine in waste liquid, reduce the degree of pollution of the papermaking industry to the environment, waste paper deinking, pulp bleaching), biomass energy processing (e.g., for the production of biofuels from lignocellulosic biomass), agricultural waste recycling (e.g., to degrade lignocellulosic cellulose to obtain xylooligosaccharides), and industrial wastewater treatment.

[0050] A fifth aspect of the present invention provides a product comprising the heat-resistant xylanase of the second aspect of the present invention or the biomaterial of the third aspect of the present invention.

[0051] In some embodiments of the present invention, the product comprises one or more of a reagent, a food additive, and a food.

[0052] In some embodiments of the present invention, the reagents include detergents, bleach, etc.

[0053] In some embodiments of the present invention, the product further includes a food- or industrially acceptable carrier.

[0054] In some embodiments of the present invention, the product further contains additives that regulate enzyme activity.

[0055] In some embodiments of the present invention, the additive that regulates enzyme activity is an additive that enhances enzyme activity; preferably selected from: Urea, EDTA, Triton X-100, Mn 2+ K + or Co 2+ .

[0056] A sixth aspect of the present invention provides a feed additive or feed comprising the thermostable xylanase of the second aspect of the present invention.

[0057] In some embodiments of the present invention, the feed additive or feed further includes feed industry-acceptable carriers such as silica (including mesoporous silica), zeolite molecular sieves, porous glass, porous resins, activated carbon, β-cyclodextrin, maltodextrin, starch (including modified starch), gum arabic, alginate, chitosan, trehalose, gelatin, whey protein, soy protein isolate, hydrogenated vegetable oil, beeswax, polyvinyl alcohol, etc.

[0058] In some embodiments of the present invention, the feed further includes a basal diet.

[0059] In some embodiments of the present invention, the basal diet includes corn, wheat, broken rice, puffed soybeans, whey powder, soybean meal, soybean protein concentrate, wheat bran, soybean oil, limestone powder, dicalcium phosphate, amino acids, and a premix of multivitamins and minerals.

[0060] In some embodiments of the present invention, the amino acids include lysine (hydrochloride), methionine, threonine, and valine.

[0061] In some embodiments of the present invention, the compound vitamin and mineral premix includes at least one of vitamin A, vitamin D3, vitamin K3, vitamin B12, riboflavin, niacin, D-pantothenic acid, biotin, thiamine, pyridoxine, copper, iron, manganese, zinc, cobalt, iodine and selenium.

[0062] In some embodiments of the present invention, the nutritional levels of the basal diet are as follows: net energy 2400-2600 kcal / kg, crude protein content 170-180 g / kg, crude fiber content 24-28 g / kg, crude ash content 46-50 g / kg, calcium content 4-7 g / kg, total phosphorus content 0.4-0.6 g / kg, and digestible lysine content 10-13 g / kg.

[0063] In some embodiments of the present invention, the amount of the thermostable xylanase added to the basal diet is 0.01%-0.1%; further, 0.01%-0.06%; and even further, 0.02%-0.04%.

[0064] In some embodiments of the present invention, the feed can be prepared using conventional feed technology, such as crushing, mixing, conditioning, preserving and granulating corn, wheat, broken rice, puffed soybeans, whey powder, soybean meal, soybean protein concentrate, wheat bran, soybean oil, limestone powder, dicalcium phosphate, choline chloride, amino acids and compound vitamin and mineral premix.

[0065] In some embodiments of the present invention, the mixing time is 170-190 seconds.

[0066] In some embodiments of the present invention, the modulation adopts a "dual modulation, dual quality assurance" process, including dual modulators and dual quality assurance devices.

[0067] In some embodiments of the present invention, the modulation is timed to 170-190 seconds.

[0068] In some embodiments of the present invention, the granulation temperature is 80-90°C.

[0069] A seventh aspect of the present invention provides a method for degrading xylan, comprising treating a substrate with a thermostable xylanase of the second aspect of the present invention, a biomaterial of the third aspect of the present invention, or a product of the fifth aspect of the present invention, wherein the substrate is xylan and / or a substance containing xylan.

[0070] In some embodiments of the present invention, the xylan includes, but is not limited to, at least one of beech xylan, corn cob xylan, bagasse xylan, and birch xylan.

[0071] In some embodiments of the present invention, the xylan-containing substances include, but are not limited to, at least one of beech, corn cob, bagasse, birch, poplar, and oats.

[0072] The beneficial effects of this invention are:

[0073] This invention modifies wild xylanase (amino acid sequence as shown in SEQ ID NO:1) by mutating S at position 76 to Y, K at position 169 to R, and N at position 198 to I. Through these mutations, the high-temperature resistance of wild xylanase can be significantly improved.

[0074] This invention provides a thermoresistant xylanase. Experiments have shown that after treatment at 90℃ for 10 minutes, the residual enzyme activity of this thermoresistant xylanase, measured at 37℃, can reach over 21.85%, significantly higher than that of wild-type xylanase, which is almost completely inactivated under the same treatment conditions. Therefore, the thermoresistant xylanase provided by this invention is suitable for high-temperature pelleting processes in feed processing and can be used to prepare feed additives or feeds containing xylanase to degrade xylan (anti-nutritional factors) in feed, thereby improving the utilization rate of nutrients in feed. It has good application prospects in the feed industry and can be widely used in animal husbandry. Attached Figure Description

[0075] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0076] Figure 1 This is a gel electrophoresis image of the thermostable xylanase provided by the present invention.

[0077] Figure 2 The graph shows the Tm data for wild-type xylanase (XynA) and thermostable xylanase (XynA-1), with the vertical axis representing the instrument response and the horizontal axis representing temperature.

[0078] Figure 3 This is a comparison chart showing the percentage of residual activity of wild-type xylan crude enzyme (XynA) and thermostable xylan crude enzyme (XynA-1) after treatment at 90℃.

[0079] Figure 4 A comparison chart showing the percentage of residual activity of wild-type xylan purified enzyme (XynA) and thermostable xylan purified enzyme (XynA-1) after treatment at 90℃. Detailed Implementation

[0080] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0081] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0082] The term "recombinant gene" refers to DNA or RNA capable of expressing the xylanase of the present invention. Typically, the recombinant gene is initially synthesized in vitro via solid-phase phosphoramidite synthesis, TdT biosynthesis, or other suitable techniques known in the art. Once a template sequence is available, it can be amplified by PCR or other suitable techniques known in the art. With a recombinant bacterial strain, further large-scale amplification can be achieved by culturing the strain. In some embodiments, the recombinant gene may also include residual restriction enzyme sites, other accessory elements such as control elements (e.g., promoters), labeling substances (e.g., fluorescent labels), and other sequences that do not affect the expression of the target gene.

[0083] The term "expression" refers to the process by which DNA is transcribed into messenger RNA (mRNA) and then translated into protein.

[0084] The term "expression vector" refers to the ability to incorporate and express heterologous polynucleotide fragments into host cells. Many prokaryotic and eukaryotic expression vectors are commercially available. Choosing a suitable expression vector is within the knowledge of a technician.

[0085] The term "chassis cell" refers to a suitable host vector for expressing DNA containing the DNA of the present invention. The host can be any organism capable of containing and expressing the nucleic acids or genes disclosed herein, but is not limited thereto. Chassis cells can be prokaryotes or eukaryotes, single-celled or multicellular, including mammalian cells, plant cells, fungi, etc. According to existing technology, those skilled in the art can achieve heterologous expression of the recombinant DNA of the present invention in different disclosed chassis cells by adjusting parameters through a limited number of experiments. Chassis cells can be selected from at least one of *Escherichia coli*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Hansenula polymorpha*, *Candida*, *Rhodotorula*, *Bacillus*, *Escherichia coli*, *Salmonella*, *Clostridium*, *Streptomyces*, *Staphylococcus*, *Neisseria*, and *Shigella*. This invention merely lists types of chassis cells and does not constitute a limitation on the types of chassis cells. Chassis cells are preferably *Escherichia coli*, and suitable *E. coli* strains (including many others) include BL21(DE3), C600, DH5αF', 1113101, JM83, JM101, JM103, JM105, JM107, JM109, JM110, MC1061, MC4100, MM294, NM522, NM554, TGI, χ1776, XL1-Blue, and Y1089. + The above E. coli strains are all commercially available strains.

[0086] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0087] Example 1: Preparation of thermostable xylanase

[0088] Sequence design, modification, and high-throughput screening were performed on the wild-type xylanase (>endo-1,4-beta-xylanase [Pseudothermotoga hypogea DSM11164], amino acid sequence as shown in SEQ ID NO:1, nucleotide sequence encoding this wild-type xylanase as shown in SEQ ID NO:2), ultimately yielding a thermostable xylanase mutant (amino acid sequence as shown in SEQ ID NO:3, nucleotide sequence encoding this mutant as shown in SEQ ID NO:4). Specifically, compared to the wild-type xylanase, the amino acid sequence shows a mutation where S at position 76 is mutated to Y, K at position 169 is mutated to R, and N at position 198 is mutated to I.

[0089] MRKLFFAVVLVLVATCSASNGLEGESLRSLAEKLGIYIGYASINHFWTIPDSNRYMEMARREFNILTPENQMKWDSIHPEPDRYNFSYAERHVEFALENNMLVHGHTLVWHNQLPFWLNRQWTKEELLKVLEDHIKTVVGHFRGRVKIWDVVNEAVSDMGSYRETIWYKTIGPEY IEKAFVWARQADPEAILIYNDYNIETINPKSNFTYQLIKELKEKGVPIDGIGFQMHIDINGIDYDSFRNNLKRFADLGLKLYITEMDVRIPKSATQKDLDRQAEIYAKIFEICLENPAVQAIQFWGFTDKYSWVPGFFAGYDHALIFDKDYNPKPAYFAIKRVLEAKVSKGR(SEQ ID NO:1).

[0090] ATGAGGAAGTTGTTCTTCGCGGTTGTCTTGGTGCTTGTAGCAACTTGCTCCGCA

[0091] TCGAATGGATTGGAGGGAGAATCCTTGAGGTCATTAGCCGAGAAACTCGGCATCTA

[0092] CATCGGTTATGCCTCTATCAACCATTTTTGGACTATTCCGGATTCCAACAGATATATG

[0093] GAGATGGCGAGGAGGGAATTCAACATACTCACGCCCGAGAACCAGATGAAGTGG

[0094] GATAGTATTCATCCGGAGCCTGACAGGTACAACTTCAGTTACGCAGAGCGTCATGT

[0095] CGAGTTCGCTTTGGAAAACAACATGCTCGTTCATGGTCACACCCTGGTTTGGCACA

[0096] ACCAGCTCCCGTTCTGGTTGAACAGACAATGGACCAAAGAAGAACTCCTGAAAGT

[0097] CCTTGAGGACCACATCAAAACAGTCGTTGGTCACTTCAGAGGAAGGGTGAAGATT

[0098] TGGGACGTGGTGAACGAAGCGGTCAGCGACATGGGCAGTTACAGAGAGACCATTT

[0099] GGTACAAGACTATAGGACCCGAGTACATCGAAAAGGCGTTCGTGTGGGCAAGACA

[0100] AGCCGATCCGGAAGCGATCCTTATATACAACGACTACAACATAGAAACGATCAATC

[0101] CCAAATCGAACTTCACCTACCAGCTAATCAAGGAACTGAAAGAAAAAGGTGTACC

[0102] GATAGACGGCATCGGTTTTCAAATGCACATAGACATCAACGGAATAGACTATGACA

[0103] GTTTCAGAAACAACTTGAAGAGGTTCGCTGATCTCGGTTTGAAGCTCTACATCACG

[0104] GAAATGGATGTGAGAATACCCAAGAGCGCAACTCAAAAAGACTTGGACAGGCAG

[0105] GCAGAAATCTACGCGAAGATCTTCGAAATCTGCTTGGAGAATCCTGCGGTCCAAG

[0106] CCATACAGTTCTGGGGTTTCACGGACAAGTATTCTTGGGTACCGGGCTTTTTCGCA

[0107] GGCTACGACCACGCTCTGATCTTTGATAAAGATTACAACCCCAAACCTGCGTATTTTGCGATAAAAAGGGTGCTCGAAGCCAAAGTGAGCAAAGGGCGCTGA(SEQ ID NO:2)。

[0108] MRKLFFAVVLVLVATCSASNGLEGESLRSLAEKLGIYIGYASINHFWTIPDSNRYM

[0109] EMARREFNILTPENQMKWDYIHPEPDRYNFSYAERHVEFALENNMLVHGHTLVWHN

[0110] QLPFWLNRQWTKEELLKVLEDHIKTVVGHFRGRVKIWDVVNEAVSDMGSYRETIWY

[0111] RTIGPEYIEKAFVWARQADPEAILIYNDYIIETINPKSNFTYQLIKELKEKGVPIDGIGFQ

[0112] MHIDINGIDYDSFRNNLKRFADLGLKLYITEMDVRIPKSATQKDLDRQAEIYAKIFEICL

[0113] ENPAVQAIQFWGFTDKYSWVPGFFAGYDHALIFDKDYNPKPAYFAIKRVLEAKVSKGR(SEQ ID NO:3)。

[0114] ATGAGAAAGCTTTTTTTCGCCGTCGTTCTTGTCTTAGTCGCTACGTGCAGTGCT

[0115] TCTAATGGCCTGGAAGGAGAGTCTTTGAGGTCACTAGCAGAGAAGTTGGGCATCT

[0116] ACATTGGTTATGCCTCTATTAATCACTTCTGGACTATACCAGACTCAAACAGATACA

[0117] TGGAAATGGCAAGACGAGAGTTCAACATTTTGACCCCAGAAAACCAGATGAAGTG

[0118] GGATTACATTCACCCTGAACCTGACAGATATAATTTTTCATATGCAGAAAGACACGT

[0119] GGAATTTGCACTGGAAAACAACATGTTAGTACATGGTCACACATTGGTATGGCATA

[0120] ACCAATTACCATTTTGGCTGAATAGGCAATGGACAAAGGAAGAGTTGTTAAAGGT

[0121] GCTGGAAGATCATATCAAAACCGTTGTTGGTCACTTCAGAGGTAGAGTGAAAATCT

[0122] GGGACGTCGTAAACGAAGCCGTTTCCGACATGGGAAGTTACAGAGAGACTATTTG

[0123] GTACCGTACCATTGGACCAGAGTACATTGAAAAAGCCTTTGTCTGGGCCAGACAA

[0124] GCAGATCCAGAAGCAATCTTGATCTACAACGACTACATCATCGAAACAATCAACCC

[0125] TAAGTCCAATTTTACCTACCAACTGATTAAGGAGTTGAAAGAAAAGGGAGTTCCAA

[0126] TTGATGGTATTGGCTTTCAAATGCATATTGATATTAACGGCATTGATTACGATTCTTT

[0127] TCGTAACAACTTGAAAAGATTCGCAGACTTGGGTCTTAAGCTGTATATTACCGAGA

[0128] TGGACGTTAGAATTCCTAAGTCAGCTACCCAGAAGGATCTGGATCGTCAAGCTGAA

[0129] ATCTACGCTAAAATTTTCGAGATCTGTTTAGAGAATCCAGCCGTTCAAGCCATTCAA

[0130] TTTTGGGGTTTTACTGATAAGTACTCTTGGGTCCCAGGTTTCTTTGCTGGTTATGAT

[0131] CACGCCCTGATATTTGATAAGGATTACAACCCAAAACCTGCTTATTTCGCCATAAAACGAGTTCTTGAAGCCAAAGTCTCTAAGGGCAGA (SEQ ID NO: 4).

[0132] 1. Preparation of xylanase

[0133] 1.1 Construction of Recombinant Cells

[0134] The coding genes for wild-type xylanase and xylanase mutants were used as target genes. The nucleotide sequences of the target genes were synthesized by Beijing Qingke Biotechnology Co., Ltd., and these nucleotide sequences were inserted into expression vectors respectively. The coding gene sequence for wild-type xylanase is shown in SEQ ID NO:2, and the coding gene sequence for the thermostable xylanase mutant is shown in SEQ ID NO:4.

[0135] Specifically, the plasmid is inserted into plasmid pET28a(+) to obtain the corresponding plasmid. The synthesized plasmid is then transformed into chassis cells (E. coli BL21(DE3)), thereby constructing E. coli strains containing different plasmids, i.e., obtaining recombinant bacteria. Many other plasmids and chassis cells are available in the prior art; this embodiment only provides one specific method.

[0136] 1.2 Expression of xylanase

[0137] The recombinant bacteria were inoculated into liquid LB medium (5g yeast extract, 10g peptone, 10g sodium chloride) and cultured at 37°C in a shaker (220 rpm) until OD reached. 600The pH was set to approximately 1.0, then IPTG was added to a concentration of 200 μM, and the temperature was lowered to 16°C for overnight incubation. After overnight incubation, the bacterial cells were collected by centrifugation at 4000g for 10 min and resuspended in 10% NTA buffer (50 mM Tri-HCl, 300 mM NaCl, pH 8.0). Next, the bacterial cells were sonicated and then centrifuged at 12000 rpm for 1 h. The supernatant was collected as the crude enzyme solution. Many other methods for inducing recombinant bacterial expression are available in the prior art; this embodiment only provides one specific method.

[0138] 1.3 Purification of xylanase

[0139] The crude enzyme solution was passed through Ni 2+ Resin affinity chromatography was performed because the target protein has a histidine tag at its C-terminus, which can bind to the resin. The resin was washed with NTA buffer and imidazole buffer, followed by elution with NTA buffer. The resulting solution was dialyzed to remove imidazole, with a dialysis bag cutoff volume of 1 kDa. The dialyzed solution was concentrated until the protein concentration was 0.2 mg / mL.

[0140] According to existing technology, those skilled in the art can purify xylanase by adjusting parameters through a limited number of experiments, which will not be described in detail here. There are many other available methods for purifying xylanase in the prior art; this embodiment only provides one specific solution.

[0141] Wild-type xylanase (denoted as XynA) and thermostable xylanase (denoted as XynA-1) were heterologously expressed and purified according to the above-described method for preparing xylanase. The prepared thermostable xylanase was verified by gel electrophoresis (SDS-PAGE), and the results are as follows: Figure 1 As shown, the size of the thermostable xylanase is consistent with expectations.

[0142] Example 2: Determination of the heat resistance and enzyme activity of xylanase

[0143] 1. Method for determining heat resistance (measuring Tm value)

[0144] The thermal stability (Tm) value characterizes the gradual breakdown of a protein's higher-order structure during heating, thus indicating its thermal stability. The Tm value was determined using a Bio-RAD CFX96 system, as follows:

[0145] First, dilute the dye. Take 2.5 μL of dye (5000×, named SYPRO Orange Protein GelStain) and add it to 22.5 μL of DMSO to form dye (500×). Then add 225 μL of sterile water to form SY (50×). The dye should be kept away from light as much as possible.

[0146] The reaction components consist of 2 μg of the target protein and 2 μL of SY (50×), with the final volume made up to 20 μL of sterile water. Prepare three replicates of 80 μL each, mixing the reaction components in an eight-tube PCR apparatus. Using a multi-channel pipette with a low-adsorption tip, transfer 20 μL of the reaction solution to a quantitative real-time PCR plate. The reaction program is as follows: preheat at 25°C for 10 min, with a melting temperature range of 25°C to 90°C, increasing by 0.5°C every 20 seconds.

[0147] The heat resistance of wild-type xylanase (XynA) and thermostable xylanase (XynA-1) was determined according to the above-described heat resistance test method. The test results are as follows: Figure 2 As shown. Figure 2 The largest fluctuation was observed in the protein's Tm value. It was found that the Tm value of the thermostable xylanase (XynA-1) of this invention is approximately 84°C, while the Tm value of the wild-type xylanase (XynA) is approximately 73.5°C, significantly lower than that of the mutant. This demonstrates that the structural stability and heat resistance of the thermostable xylanase of this invention are significantly improved.

[0148] 2. Methods for determining enzyme activity

[0149] The xylanase activity was determined in accordance with the national standard GB / T 23874-2009.

[0150] Considering that crude enzyme solutions may be used in industrial production, their activity was also determined.

[0151] Prepare the following solutions:

[0152] Phosphate-citric acid buffer: Stock solution A is 200mM disodium hydrogen phosphate solution, and stock solution B is 100mM citric acid solution. They are mixed in proportion to make the final concentration of the buffer solution 50mM.

[0153] Xylan solution: 0.5%, weigh 0.25g xylan, dissolve in phosphate-citric acid buffer, and bring to a final volume of 50mL.

[0154] DNS solution: Dissolve 18.2g of potassium sodium tartrate in 50mL of distilled water and heat at about 42℃. Add 0.63g of 3,5-dinitrosalicylic acid, 2.1g of NaOH, and 0.5g of phenol to the hot solution in sequence and stir until dissolved. After cooling, dilute to 100mL with distilled water and store in a brown bottle at room temperature. Use after 7 days.

[0155] Since the present invention will subsequently use xylanase to prepare feed, the feed conditioning-pelletizing process is a short-term, non-uniform heating process. Practice shows that when the conditioner is set to 85°C, the local instantaneous temperature can reach 85-90°C, and the residence time of materials of different formulation types is distributed in the range of 180-250s. The industry typically employs a more stringent 90℃, 10min test to ensure coverage of the worst-case conditions in actual feed processing, such as short-term overheating, equipment errors, and human error fluctuations (Kumar, V.; Dangi, AK; Shukla, P. Engineering Thermostable Microbial Xylanases Toward its Industrial Applications. Molecular biotechnology 2018, 60, 226-235, doi:10.1007 / s12033-018-0059-6.; Chen, Q.; Li, M.; Wang, X. Enzymology properties of two different xylanases and their impacts on growth performance and intestinal microflora of weaned piglets. Animal Nutrition 2016, 2, 18-23, doi:https: / / doi.org / 10.1016 / j.aninu.2016.02.003.). Therefore, to simulate the actual conditions of xylanase in feed processing, each sample was heat-treated before activity testing. The treatment method was as follows: the sample was diluted 1000 times (for crude enzyme solution, prepared by step 1.2 in Example 1) or 200 times (for pure enzyme, prepared by step 1.3 in Example 1) with phosphate-citrate buffer to obtain enzyme solution, and then incubated at 90°C for 10 min.

[0156] After cooling the enzyme solution to room temperature, add 10 μL of the enzyme solution to 100 μL of xylan solution and mix well. Incubate at 37°C for 10 min. Then, quickly add 100 μL of DNS reagent to terminate the reaction. After mixing, boil in a water bath for 5 min, cool rapidly, add 300 μL of deionized water and mix well. Take 150 μL of the reaction solution and measure A using a microplate reader. 540 value.

[0157] The standard curve was prepared as follows: Take 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μL of the prepared xylose standard solution (1 mg / mL) and place them in 1.5 mL centrifuge tubes. Add deionized water to a final volume of 100 μL, mix well, add 100 μL of DNS, mix thoroughly, boil in a water bath for 5 min, cool rapidly, add 300 μL of deionized water, mix well, and then measure A using a microplate reader with 150 μL of the reaction solution. 540 value.

[0158] Enzyme activity calculation method: The formula is X = Y / (M×T)×N, where the letters have the following meanings: X is the enzyme activity, in U / g or U / mL; Y is the amount of xylose calculated based on the absorbance measured and substituted into the standard curve, in μmol; T is the reaction time, in min; N is the dilution factor; and M is the amount of the sample to be tested, in g or mL. Perform three parallel determinations and take the average value; the relative deviation of the parallel determinations should be ≤5%.

[0159] Enzyme activity unit U is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute from a 0.5% xylan solution at 37°C and pH 5.5. Specific enzyme activity U / mL: The number of enzyme activity units contained in each mL of enzyme solution. Specific enzyme activity U / g: The number of enzyme activity units contained in each g of enzyme protein.

[0160] The enzyme activities of wild-type xylanase (XynA) and thermostable xylanase (XynA-1) were determined according to the above-described enzyme activity assay method. The specific data are shown in Tables 1 and 2.

[0161] As shown in Table 1, for the crude enzyme solution, after treatment at 90℃ for 10 min, the residual activity percentages (activity after heat treatment / activity without heat treatment) of XynA and XynA-1 were 8.21% and 46.75%, respectively. Therefore, the activity of the heat-resistant xylanase XynA-1 of this invention is significantly greater than that of the wild-type xylanase XynA under heat treatment at 90℃. The measurement results are as follows. Figure 3 As shown.

[0162] As shown in Table 2, for pure enzymes, after treatment at 90℃ for 10 min, the residual enzyme activity percentage of XynA was only 5.2%, while the residual activity percentage of XynA-1 was still 21.85%. The measurement results are as follows... Figure 4 As shown.

[0163] The above results indicate that, for both crude enzyme solution and pure enzyme, XynA-1 exhibits significantly better activity and residual activity percentage after heat treatment than XynA.

[0164] Table 1 Enzyme activity of crude enzyme solution at 37°C after high-temperature treatment.

[0165] XynA (U / mL) XynA-1 (U / mL) Heat treatment at 90℃ for 10 minutes 2252.5 8r89.2 No heat treatment 2815r.2 18800.6

[0166] Table 2 Enzyme activity of purified enzymes at 37°C after high-temperature treatment.

[0167] XynA(U / g) XynA-1 (U / g) Heat treatment at 90℃ for 10 minutes 2263.35 r695.35 No heat treatment 4526r.0 35219.9

[0168] Example 3

[0169] A weaned piglet diet comprises the following components by weight: 25 parts corn, 30 parts wheat, 10 parts broken rice, 8 parts extruded soybeans, 8 parts whey powder, 5 parts soybean meal, 5 parts soybean protein concentrate, 5 parts wheat bran, 1 part soybean oil, 0.6 parts limestone powder, 0.6 parts dicalcium phosphate, 0.5 parts L-lysine hydrochloride, 0.25 parts DL-methionine, 0.2 parts L-threonine, 0.1 parts L-valine, 0.1 parts zeolite powder, and 0.25 parts compound vitamin and mineral premix.

[0170] Each kilogram of the compound vitamin and mineral premix contains 150mg Fe (FeSO4), 100mg Zn (ZnSO4), 30mg Mn (MnSO4), 25mg Cu (CuSO4), 0.5mg I (KIO3), 0.3mg Co (CoSO4), 0.3mg Se (Na2SeO3), 2200IU vitamin A, 220IU vitamin D3, 0.5mg vitamin K3, 0.0175mg vitamin B12, 3.5mg riboflavin, 30mg niacin, 10mg d-pantothenic acid, 0.05mg biotin, 0.3mg thiamine, and 7mg pyridoxine.

[0171] The wheat mentioned above was produced in Anhui Province, with a bulk density greater than 780 g / L and a crude protein content greater than 12%; the corn was produced in Heilongjiang Province, with a bulk density greater than 710 g / L and a crude protein content greater than 7.2%.

[0172] The nutritional levels of the above-mentioned weaned piglet diet are as follows: net energy 2500 kcal / kg, crude protein 175 g / kg, crude fiber 26 g / kg, crude ash 48 g / kg, calcium 6 g / kg, total phosphorus 0.5 g / kg, and digestible lysine 11.5 g / kg.

[0173] The preparation method of the above-mentioned weaned piglet diet is as follows: Corn, wheat, broken rice, extruded soybeans, whey powder, soybean meal, soybean protein concentrate, wheat bran, soybean oil, limestone powder, dicalcium phosphate, L-lysine hydrochloride, DL-methionine, L-threonine, L-valine, and a premix of compound vitamins and minerals are pulverized, mixed, prepared, preserved, and granulated. Specifically, the pulverized corn particle size is 3.5 mm, and the pulverized wheat particle size is 2.5 mm; the mixing time is 180 s; the preparation adopts a "double preparation, double preservation" process, including double preparers and double preservers; the granulation temperature is 85℃, the preparation time is 180 s, the feeding speed is 35 Hz, the preserver frequency is 60 Hz, the ring die aperture is 3.0 mm, and the compression ratio is 1:8.

[0174] Example 4

[0175] A weaned piglet diet comprises the following components by weight: 25 parts corn, 30 parts wheat, 10 parts broken rice, 8 parts extruded soybeans, 8 parts whey powder, 5 parts soybean meal, 5 parts soybean protein concentrate, 5 parts wheat bran, 1 part soybean oil, 0.6 parts limestone powder (as a calcium source), 0.6 parts dicalcium phosphate, 0.5 parts L-lysine hydrochloride, 0.25 parts DL-methionine, 0.2 parts L-threonine, 0.1 parts L-valine, 0.02 parts wild-type xylanase XynA (the original enzyme activity of pure XynA is 45267 U / g, diluted to 5000 U / g using silica as a carrier), 0.08 parts zeolite powder (as a carrier), and 0.25 parts compound vitamin and mineral premix.

[0176] Each kilogram of the compound vitamin and mineral premix contains 150mg Fe (FeSO4), 100mg Zn (ZnSO4), 30mg Mn (MnSO4), 25mg Cu (CuSO4), 0.5mg I (KIO3), 0.3mg Co (CoSO4), 0.3mg Se (Na2SeO3), 2200IU vitamin A, 220IU vitamin D3, 0.5mg vitamin K3, 0.0175mg vitamin B12, 3.5mg riboflavin, 30mg niacin, 10mg d-pantothenic acid, 0.05mg biotin, 0.3mg thiamine, and 7mg pyridoxine.

[0177] The wheat mentioned above was produced in Anhui Province, with a bulk density greater than 780 g / L and a crude protein content greater than 12%; the corn was produced in Heilongjiang Province, with a bulk density greater than 710 g / L and a crude protein content greater than 7.2%.

[0178] The nutritional levels of the above-mentioned weaned piglet diet are as follows: net energy 2500 kcal / kg, crude protein 175 g / kg, crude fiber 26 g / kg, crude ash 48 g / kg, calcium 6 g / kg, total phosphorus 0.5 g / kg, and digestible lysine 11.5 g / kg.

[0179] The preparation method of the above-mentioned weaned piglet diet is the same as that in Example 3.

[0180] Example 5

[0181] A weaned piglet diet comprises the following components by weight: 25 parts corn, 30 parts wheat, 10 parts broken rice, 8 parts extruded soybeans, 8 parts whey powder, 5 parts soybean meal, 5 parts soybean protein concentrate, 5 parts wheat bran, 1 part soybean oil, 0.6 parts limestone powder, 0.6 parts dicalcium phosphate, 0.5 parts L-lysine hydrochloride, 0.25 parts DL-methionine, 0.2 parts L-threonine, 0.1 parts L-valine, 0.02 parts heat-resistant mutant xylanase XynA-1 (the original enzyme activity of pure XynA-1 is 35220 U / g, diluted to 5000 U / g using silica as a carrier), 0.08 parts zeolite powder, and 0.25 parts compound vitamin and mineral premix.

[0182] Each kilogram of the compound vitamin and mineral premix contains 150mg Fe (FeSO4), 100mg Zn (ZnSO4), 30mg Mn (MnSO4), 25mg Cu (CuSO4), 0.5mg I (KIO3), 0.3mg Co (CoSO4), 0.3mg Se (Na2SeO3), 2200IU vitamin A, 220IU vitamin D3, 0.5mg vitamin K3, 0.0175mg vitamin B12, 3.5mg riboflavin, 30mg niacin, 10mg d-pantothenic acid, 0.05mg biotin, 0.3mg thiamine, and 7mg pyridoxine.

[0183] The wheat mentioned above was produced in Anhui Province, with a bulk density greater than 780 g / L and a crude protein content greater than 12%; the corn was produced in Heilongjiang Province, with a bulk density greater than 710 g / L and a crude protein content greater than 7.2%.

[0184] The nutritional levels of the above-mentioned weaned piglet diet are as follows: net energy 2500 kcal / kg, crude protein 175 g / kg, crude fiber 26 g / kg, crude ash 48 g / kg, calcium 6 g / kg, total phosphorus 0.5 g / kg, and digestible lysine 11.5 g / kg.

[0185] The preparation method of the above-mentioned weaned piglet diet is the same as that in Example 3.

[0186] Example 6: Effects of xylanase on the growth performance of weaned piglets in wheat-based diets

[0187] This invention investigates the effect of xylanase on the growth performance of weaned piglets by feeding them with feed containing xylanase (such as the weaned piglet diets of Examples 4-5), as detailed below:

[0188] Three hundred and sixty Duroc × Landrace × Large White three-way crossbred weaned piglets with a uniform weight of approximately 7 kg were randomly selected. The experiment was divided into three treatments, with six replicates per treatment and 20 piglets per replicate. The control group was fed the weaned piglet diet of Example 3, the XynA group was fed the weaned piglet diet of Example 4, and the XynA-1 group was fed the weaned piglet diet of Example 5. The experiment lasted for 18 days, starting when the weaned piglets reached 7 kg. Piglets had free access to feed and water during the experiment, and were fed three times daily at 8:00, 12:00, and 16:00. Feed intake, diarrhea, and mortality were recorded daily, and piglets were weighed before the start of the experiment and again on day 18. Growth performance data were statistically analyzed.

[0189] Growth performance data include:

[0190] Record the average daily feed intake for days 1-18 during the trial period, weigh the animals on day 18, and calculate the average daily weight gain, FCR (feed ratio), and mortality rate.

[0191] Average daily weight gain (ADG) = (final weight - initial weight) (kg) / number of experimental days (days);

[0192] FCR = Cumulative feed consumption (kg) / (Final weight - Initial weight) (kg);

[0193] Mortality rate (%) = (Number of deaths during the period + Number of culled heads during the period) / Number of heads in stock at the beginning of the period × 100%;

[0194] Diarrhea scores were recorded for each pen of piglets daily, the number of piglets with diarrhea was counted, and the average diarrhea rate over 18 days was calculated. Diarrhea rate (%) = number of piglets with diarrhea during the experimental period / (number of piglets in the experiment × number of days in the experiment) × 100%.

[0195] Determination of apparent total intestinal digestibility of nutrients: During the last week of the growth trial, 0.3% chromium trioxide was mixed into the diet of weaned piglets. Feces were collected during the first 4 days of acclimatization and the last 3 days. Feces were collected from each pen, fixed with hydrochloric acid, and frozen at -20℃. The samples were then sent for analysis to determine the apparent total intestinal digestibility of nutrients (including dry matter, crude protein, and starch) in the feed and feces. The apparent total intestinal digestibility of nutrients was determined using the indicator method. The apparent digestibility (%) of a nutrient is calculated as follows: 100 - A1F2 / A2F1 × 100, where: F1 is the nutrient content (%) in the feed; F2 is the nutrient content (%) in the feces; A1 is the Cr2O3 content (%) in the weaned piglet diet; and A2 is the Cr2O3 content (%) in the feces.

[0196] The effects of xylanase on the growth performance and nutrient digestibility of weaned piglets were compared through experiments. The experimental results are shown in Tables 3 and 4 below.

[0197] Table 3 shows that, compared with the control group, the XynA-1 group significantly increased the daily weight gain of weaned piglets by 13.3% (P<0.05); the final weight and daily weight gain of the XynA group were between the two groups, but the difference with the control group was not statistically significant. None of the treatments had a significant effect on daily feed intake, feed conversion ratio, or diarrhea rate (P>0.05).

[0198] Table 4 shows that, compared with the control group, the XynA-1 group significantly increased the apparent digestibility of dry matter, crude protein, insoluble dietary fiber, and total dietary fiber in weaned piglets by 4.2%, 6.8%, 14.7%, and 8.66%, respectively (P<0.05). The above indicators in the XynA group were all between those of the control and XynA-1 groups, but did not reach a statistically significant level. None of the treatments had a significant effect on the digestibility of starch and soluble dietary fiber (P>0.05).

[0199] In terms of average daily weight gain, the XynA-1 group showed a significant difference of 30 grams per day compared to the XynA group. Furthermore, the feed conversion ratio showed a trend of decreasing, improving by 0.03 compared to the wild-type. These small improvements can bring significant economic value in large-scale livestock production. Regarding nutrient digestibility, both showed a trend of improvement, with XynA-1 showing a greater numerical improvement compared to XynA. These results indicate that both xylanases XynA and XynA-1 have certain improvement effects compared to the control group. However, under the same enzyme activity, the heat-resistant mutant xylanase XynA-1 showed a greater improvement in daily weight gain and apparent nutrient digestibility in piglets, indicating that it is more suitable for complex feed pelleting conditions and can retain more enzyme activity than the wild-type enzyme preparation. In actual feed production, while ensuring enzyme activity effectiveness, it is advisable to appropriately reduce the amount added to lower the formulation cost.

[0200] Table 3. Effects of xylanase addition on growth performance of weaned piglets

[0201]

[0202] Note: Data in the same row are labeled a and b to indicate significant differences (P<0.05). The control group was fed a basal diet; the XynA group and the XynA-1 group were fed a basal diet supplemented with 0.02% wild-type xylanase and thermostable mutant xylanase, respectively.

[0203] Table 4. Effects of xylanase addition on apparent total digestive tract nutrient digestibility in weaned piglets.

[0204]

[0205] Note: Data in the same row are labeled a and b, indicating significant differences (P<0.05).

[0206] In conclusion, the addition of 0.02% of the heat-resistant mutant xylanase XynA-1 (i.e., the weaned piglet diet of Example 5) can significantly improve the utilization efficiency of nutrients such as dry matter, crude protein and dietary fiber in the diet of weaned piglets, thereby improving the growth performance of weaned piglets. This indicates that the xylanase XynA-1 has potential application value in promoting the growth performance of weaned piglets.

[0207] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for improving the high-temperature resistance of xylanase, comprising mutating S at position 76 to Y, K at position 169 to R, and N at position 198 to I in the amino acid sequence of xylanase; wherein the amino acid sequence of xylanase is shown in SEQ ID NO:

1.

2. A thermostable xylanase, the amino acid sequence of which is shown in SEQ ID NO:

3.

3. A biomaterial related to the thermostable xylanase of claim 2, wherein the biomaterial is any one of a1)-a12): a1) The nucleic acid molecule encoding the thermostable xylanase of claim 2; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic animal cell lines containing the nucleic acid molecules described in a1); a10) Transgenic animal cell lines containing the expression cassette described in a2); a11) Transgenic animal cell lines containing the recombinant vector described in a3); a12) Transgenic animal cell lines containing the recombinant vector described in a4).

4. The biomaterial according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

4.

5. The use of the thermostable xylanase of claim 2 or the biomaterial of claim 3 or 4 in any one of b1)-b4): b1) Preparation of feed or feed additives; b2) Hydrolysis of xylan; b3) Prepare products for hydrolyzing xylan; b4) Industry.

6. A product comprising the heat-resistant xylanase of claim 2 or the biomaterial of claim 3 or 4.

7. The product according to claim 6, characterized in that, The product comprises one or more of the following: reagents, food additives, and food.

8. A feed additive or feed comprising the thermostable xylanase of claim 2.

9. The feed additive or feed according to claim 8, characterized in that, The feed also includes a basal diet.

10. A method for degrading xylan, comprising treating a substrate with a thermostable xylanase as described in claim 2, a biomaterial as described in claim 3 or 4, or a product as described in claim 6 or 7, wherein the substrate is xylan and / or a substance containing xylan.

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