Neutral xylanase and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses neutral xylanase and application thereof, and the neutral xylanase provided by the invention is salt-resistant and alkali-resistant xylanase and has the activity of internally cutting beta-1, 4-xylanase. In addition, the xylanase XynE102 can hydrolyze xylan into xylooligosaccharide, mainly xylobiose (X2) and xylotetraose (X4), and the xylanase XynE102 can hydrolyze xylan into xylooligosaccharide. Compared with the existing xylanase, the xylanase XynE102 provided by the invention shows better thermal stability and a wider pH range, the Km value of the xylanase XynE102 is lower and is 2.68 mg / mL, and the xylanase XynE102 has higher salt-tolerant activity (137.21 U / mg), which indicates that the xylanase XynE102 has higher catalytic efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a neutral xylanase and its application. Background Art

[0002] Xylanase has broad application prospects in the fields of food, feed, papermaking, bioenergy, etc. When applied, it often has to face extreme environments such as high salt and high temperature. Therefore, exploring xylanase in extreme environments has become a hot topic in current research. Xylan is a heteropolysaccharide and is the main hemicellulose component that makes up the plant cell wall. It is the second most abundant polysaccharide in nature after cellulose and is also the most abundant renewable hemicellulose resource on Earth. It is usually located in the secondary cell wall and primary cell wall of plants. In the cell wall structure, xylan, cellulose, and lignin interact through covalent and non-covalent bonds. Xylan is located at the interface between lignin and cellulose. Xylan exists in the form of O-acetyl-4-O-methylglucuronic acid in the hardwood of angiosperms and in the form of arabinose-4-O-methylglucuronic acid in the softwood of gymnosperms. The basic structural units of xylan are β1,4-disaccharide, β1,3-xylene, and β1,3 / 1,4-mixed linked xylan. However, the most common one is composed of a D-xylopyranose backbone linked by β-1,4, and a D-xylopyranose backbone linked by β-1,3 has also been reported in some algal organisms. Since xylan is a complex compound with multiphase properties and different sources can also lead to structural differences, its complete decomposition requires the action of multiple hydrolases with different modes of action and specificities.

[0003] Enzymes of the GH10 family are almost all endo-1,4-β-xylanases. Endo-β-1,4-xylanase is an O-glycoside hydrolase that catalyzes the hydrolysis of the main chain β-d-xylosidic bonds of the complex plant cell wall polysaccharide xylan. These enzymes are important components of the microbial xylan-degrading enzyme and plant cell wall-degrading enzyme systems. They are crucial for the degradation of xylan into fermentable sugars, contribute to the carbon cycle, and often play a role in the infection of plant cells by plant pathogens, where they can break down hemicellulose, arabinoxylan, and plant fibers. They are widely present in bacteria, archaea, fungi, and animals, etc. However, the protein subunits of fungal-derived xylanases are more complex than those of bacterial xylanases. The survival of xylanases is more vulnerable to challenges in high-temperature, high-pressure, strong acid-base environments. Microbial-derived xylanases in natural resources have reaction specificity and mild reaction conditions, a higher utilization rate of substrates, and can also promote the production of by-products. Among the hydrolase systems, β-1,4-endoxylanase is the most critical hydrolase, which mainly hydrolyzes the β-1,4-glycosidic bonds in the xylan molecule to hydrolyze xylan into small oligosaccharides and xylo-oligosaccharides such as xylobiose. In the CAZy database, β-1,4-xylanase was considered to be limited to only 2 GH families for quite a long time: glycoside hydrolase family 10 (GH10) and 11 (GH11). Xylanase is an important industrial enzyme that can hydrolyze xylan into xylo-oligosaccharides, which means that xylanase is currently mainly applied in fields such as food processing, animal feed, pulp bleaching, textile industry, and bioenergy. Adding xylanase in food processing can improve the quality of bread and make the dough soft and relaxed; when making feed, adding xylanase can reduce the flow resistance during the granule discharging process, improve production efficiency, and does not affect the physical quality of the granules; in papermaking, pulp is usually treated with xylanase before using chemical bleaching agents to hydrolyze and precipitate xylan, which is beneficial for pulp bleaching, increases the brightness of the pulp, and reduces the consumption of chemicals; in bioenergy, the combined action of xylanase and several other enzymes such as mannanase, ligninase, xylosidase, glucanase, and glucosidase can be used to produce biofuels from lignocellulosic biomass. In addition, the application of xylanase in decomposing xylan into xylo-oligosaccharides and xylose has received extensive attention in recent years. Now xylanase has been proven to be suitable for the production of various specific xylo-oligosaccharides. Xylanases in such extreme environments are often difficult to culture in standard laboratories. Summary of the Invention

[0004] An object of the first aspect of the present invention is to provide a xylanase.

[0005] An object of the second aspect of the present invention is to provide a nucleic acid molecule encoding the xylanase of the first aspect of the present invention.

[0006] An object of the third aspect of the present invention is to provide a biological material related to the nucleic acid molecule of the second aspect of the present invention.

[0007] The fourth aspect of the present invention aims to provide use of the xylanase of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention or the biomaterial of the third aspect of the present invention.

[0008] The fifth aspect of the present invention aims to provide a product.

[0009] The sixth aspect of the present invention aims to provide a method for preparing reducing sugar.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a xylanase, wherein the amino acid sequence of the xylanase is:

[0012] a) SEQ ID NO: 4; or

[0013] b) an amino acid sequence in which one or more amino acids in SEQ ID NO: 4 are replaced and / or deleted and / or added and which has the same function as the protein shown in SEQ ID NO: 4; or

[0014] c) an amino acid sequence that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% homology to SEQ ID NO:4 and has the same function as the protein shown in SEQ ID NO:4.

[0015] The present invention identifies a new xylanase gene (named xyne102) from the metagenome of Karamay saline-alkali land in Xinjiang. Its amino acid sequence homology with the xylanase protein from Cellvibrionaceae bacterium is 69.17%. The gene was cloned and expressed in Escherichia coli, and the xylanase XynE102 was obtained by Ni-NTA affinity chromatography purification, and the enzymatic properties were studied. The results showed that the xylanase XynE102 showed activity on beech xylan, but had no CMC-Na and microcrystalline cellulose activity. XynE102 showed the best activity at 50°C and pH 7, and was inactivated after incubation at 50°C for 60 minutes. After incubation at 50°C and pH 8.0-10.0 for 24 hours, the relative enzyme activity remained above 95%. XynE102 still retained 75% of its activity under 2.0M NaCl. It was inactivated by 1mM concentration of Mg 2+ , K + 、Co 2+ , Fe 2+ and Fe 3+ Plasma activated, Mn 2+ , Cu 2+ , Pb2+ Plasma inhibition; inhibited by Mg at a concentration of 10 mM 2+ , Al 3+ , K + , Ca 2+ and activated by plasma, inhibited by Mn 2+ , Fe 3+ , Co 2+ , Cu 2+ ions. Under the condition of 10 mM Ag + , XynE102 is completely inactivated. According to bioinformatics analysis, the xylanase XynE102 determined in the present invention is a salt-tolerant enzyme belonging to family 10 (GH10) and has potential application value in the fields of food (such as bread baking, seafood processing, prebiotic preparation), feed, papermaking, bioenergy, etc.

[0016] The second aspect of the present invention provides a nucleic acid molecule encoding the xylanase of the first aspect of the present invention.

[0017] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 3.

[0018] The third aspect of the present invention provides a biological material related to the nucleic acid molecule of the second aspect of the present invention, and the biological material includes at least one of b1) to b12):

[0019] b1) The nucleic acid molecule of the second aspect of the present invention;

[0020] b2) An expression cassette containing the nucleic acid molecule described in b1);

[0021] b3) A recombinant vector containing the nucleic acid molecule described in b1);

[0022] b4) A recombinant vector containing the expression cassette described in b2);

[0023] b5) A recombinant cell containing the nucleic acid molecule described in b1);

[0024] b6) A recombinant cell containing the expression cassette described in b2);

[0025] b7) A recombinant cell containing the recombinant vector described in b3);

[0026] b8) A recombinant cell containing the recombinant vector described in b4);

[0027] b9) A recombinant microorganism containing the nucleic acid molecule described in b1);

[0028] b10) A recombinant microorganism containing the expression cassette described in b2);

[0029] b11) A recombinant microorganism containing the recombinant vector described in b3);

[0030] b12) A recombinant microorganism containing the recombinant vector described in b4).

[0031] In some embodiments of the present invention, the cell vector does not include propagation materials.

[0032] In some embodiments of the present invention, the expression cassette includes a 5' transcriptional control region, an open reading frame encoding the fusion antibody of the first aspect of the present invention, translation control signals, a 3' untranslated region (3' UTR), and a transcription termination signal.

[0033] In some embodiments of the present invention, the 5' transcriptional control region contains a promoter (a general promoter can be used, such as a viral promoter (SV40 promoter) or a mammalian "housekeeping" promoter), a transcription start site, an enhancer, and / or a silencer element.

[0034] In some embodiments of the present invention, the 3' UTR can encode an AU-rich element. Through the 3'-5' exosome pathway, the AU-rich element (ARE) is a common regulator of mRNA stability and is usually located in the 3' UTR. The AU-rich element can contain one or more repeats of the sequence AUUUA. It may also contain one or more so-called US2B elements with the sequence AUAUAU.

[0035] In some embodiments of the present invention, the vector includes a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0036] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0037] In some embodiments of the present invention, the viral vector includes at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxviral vector, a Sendai viral vector, and a herpes simplex viral vector.

[0038] In some embodiments of the present invention, the non-viral vector includes at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0039] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, an F cosmid, or an artificial chromosome.

[0040] In some embodiments of the present invention, the plasmid vector can be an optional plasmid, and the viral vector can be an optional virus.

[0041] In some embodiments of the present invention, the recombinant expression vector uses pSHY211 or pPET28a as the original expression vector.

[0042] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new plant or animal varieties.

[0043] In some embodiments of the present invention, the prokaryotic cells include bacteria well-known in the art such as Escherichia coli, Streptomyces, Bacillus subtilis, Lactobacillus, etc., which can be used to express the target protein.

[0044] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

[0045] The fourth aspect of the present invention provides the application of the xylanase of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, or the biological material of the third aspect of the present invention in at least one of c1) to c5):

[0046] c1) Preparation of reducing sugars;

[0047] c2) Preparation of products for reducing sugar preparation;

[0048] c3) Hydrolysis of xylan;

[0049] c4) Preparation of products for xylan hydrolysis;

[0050] c5) Industry.

[0051] In some embodiments of the present invention, the reducing sugars in c1) to c2) include xylo-oligosaccharides or xylo-oligosaccharides.

[0052] In some embodiments of the present invention, the xylo-oligosaccharides include xylobiose and xylotetraose.

[0053] In some embodiments of the present invention, the xylan in c3) to c4) includes at least one of beechwood xylan, corncob xylan, bagasse xylan, and birchwood xylan.

[0054] In some embodiments of the present invention, the industries described in c5) include the food industry (such as improving the machinability of dough, enhancing the elasticity of bread, or delaying the staling of bread), the feed industry (such as degrading xylan molecules in feed, reducing the chyme viscosity in the digestive tract, thereby promoting the digestion and absorption of nutrients), the textile industry (such as reducing or replacing chemical retting methods during the degumming process of cellulose), the paper industry (such as reducing the alkali usage and subsequent chlorine usage without reducing the pulp strength, reducing the content of organochlorine in the waste liquid, and reducing the environmental pollution degree of the paper industry), and the biomass energy industry (such as producing biofuels from lignocellulosic biomass).

[0055] The fifth aspect of the present invention provides a product comprising the xylanase of the first aspect of the present invention or the biological material of the third aspect of the present invention.

[0056] In some embodiments of the present invention, the product includes any one or more of detergents, reagents, food additives, feed additives, foods, and feeds.

[0057] In some embodiments of the present invention, the product further includes a food-grade or industrially acceptable carrier.

[0058] In some embodiments of the present invention, the product further contains an additive for regulating enzyme activity.

[0059] In some embodiments of the present invention, the additive for regulating enzyme activity is an additive for increasing enzyme activity; preferably selected from: PMSF, EDTA, Mn 2+ 、Fe 3+ or Co 2+ .

[0060] The sixth aspect of the present invention provides a method for preparing reducing sugars, including treating a substrate to be hydrolyzed with the xylanase of the first aspect of the present invention, the biological material of the third aspect of the present invention, or the product of the fourth aspect of the present invention, wherein the substrate includes xylan or a substance containing xylan.

[0061] In some embodiments of the present invention, the xylan includes at least one of beechwood xylan, corncob xylan, bagasse xylan, and birchwood xylan.

[0062] The beneficial effects of the present invention are:

[0063] In the present invention, a novel xylanase gene XynE102 was cloned from the genome of saline-alkali land in Karamay and expressed in Escherichia coli to obtain xylanase XynE102. Through detailed enzymatic property characterization of xylanase XynE102, it was found that xylanase XynE102 is a salt- and alkali-tolerant xylanase with the activity of endo-β-1,4-xylanase. In addition, xylanase XynE102 can hydrolyze xylan into xylo-oligosaccharides, mainly xylobiose (X2) and xylotetraose (X4). Compared with existing xylanases, the xylanase XynE102 provided by the present invention exhibits better thermal stability and a wider pH range, with a lower Km value of 2.68 mg / mL and higher salt tolerance activity (137.21 U / mg), indicating that xylanase XynE102 has higher catalytic efficiency. This xylanase XynE102 has potential application value in fields such as food (such as bread baking, seafood processing, prebiotic preparation), feed, papermaking, and bioenergy. Brief Description of the Drawings

[0064] Figure 1 For the domain and tertiary structure of XynE102 (A) and the phylogenetic tree diagram obtained by maximum likelihood analysis based on the amino acid sequence (B), the phylogenetic tree diagram shows the phylogenetic position of XynE102 relative to related xylanases, and bootstrap values (expressed as percentages of 1000 replicates) are given at the nodes.

[0065] Figure 2 For SDS-PAGE analysis of purified XynE102. In the figure, lanes: 1. Molecular weight protein standard, 2. Culture supernatant of the induced transformant containing DH5α-XynE102, 3. Purified XynE102, used after Ni-NTA affinity chromatography.

[0066] Figure 3 For the effects of temperature and pH on the activity and stability of recombinant enzyme XynE102; among them, A is the effect of temperature on the activity of XynE102, B is the effect of pH on the activity of XynE102, C is the effect of temperature on the stability of XynE102, and D is the effect of pH on the stability of XynE102.

[0067] Figure 4 For the effect of salt concentration on the activity of recombinant xylanase XynE102.

[0068] Figure 5 For the TLC analysis results of purified xylan in the hydrolysis products of xylan.

[0069] Figure 6 For the hydrolysis analysis results of pretreated lignocellulose. Detailed Description of the Invention

[0070] The content of the present invention will be further described in detail below through specific embodiments.

[0071] It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0072] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0073] Strains involved in the embodiment: Escherichia coli DH5α was used for the cloning and expression of the xylanase gene. Escherichia coli was cultured on LB medium containing 50 μg / mL kanamycin. The DNA isolation and purification kit was purchased from Sangon, China.

[0074] Statistical analysis: Unless otherwise stated, all experiments were performed three times, and all analyses were based on the mean values. The results were statistically analyzed using SPSS 20.0 and expressed as mean ± SD. One-way analysis of variance was used for statistical analysis, and Tukey's test was used for multiple group comparisons. In all comparisons, a p-value ≤ 0.05 was considered statistically significant.

[0075] The features and properties of the present invention will be further described in detail below in combination with the embodiments.

[0076] Example 1

[0077] A xylanase gene xyne102 of the GH10 family was isolated from the metagenomic data of the saline-alkali land in Karamay, Xinjiang Uygur Autonomous Region, China. The specific isolation process is as follows:

[0078] (1) Sample collection and metagenomic sequencing

[0079] Samples were collected from the saline-alkali land in Karamay, Xinjiang Uygur Autonomous Region, China (45.56104°N, 85.20898°E). The collected samples were quickly frozen on dry ice and used for the extraction of metagenomic DNA in the laboratory. These activities do not require specific permits. DNA isolation was performed using the power soil Kit (MOBIO dnasy PowerSoil Kit, USA) according to the operation manual. Metagenomic sequencing was carried out on the HiSeq 2500 instrument at GENWIZ in Suzhou. The de novo assembly was performed using the Velvet assembly program version 1.2.08 (Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008 May;18(5):821-9. doi:10.1101 / gr.074492.107. Epub 2008 Mar 18. PMID:18349386; PMCID:PMC2336801). The IMG server (https: / / img.jgi.doe.gov / cgi-bin / mer / main.cgi) was used to study the sequences. To further analyze the possible functions of individual genes and orfs, the COG, KEGG, and Pfam databases were used.

[0080] (2) XynE102 sequence prediction and sequence analysis

[0081] Based on functional prediction, a xylanase gene sequence XynE102 was isolated from the metagenomic database. The DNA and protein sequences of XynE102 were aligned using the BLASTx and BLASTp programs (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), respectively. SignalP (http: / / www.cbs.dtu.dk / services / SignalP / ) was used to predict the signal peptide. The main structure of the amino acid sequence was deduced and analyzed using the EXPASY tool (http: / / web.expasy.org / protparam / ). Multiple alignments were performed using Clustal X with closely related protein sequences (retrieved from the NCBI database). Phylogenetic analysis was carried out using the MEGA7 software package. The phylogenetic tree was constructed using the maximum likelihood method and the Poisson correction model. The sequence of XynE102 was compared in the protein structure data of the Protein Data Bank (http: / / www.rcsb.org / ).

[0082] The similarity search was performed on the isolated xylanase sequence, and a new candidate xylanase gene sequence was obtained, named XynE102. The nucleotide sequence analysis of the XynE102 gene showed that the 1140bp ORF encoded the XynE102 protein with 379 amino acid residues. No obvious signal peptide sequence was found in XynE102, suggesting that the enzyme might be localized in the cytoplasm. The deduced signal peptide-free protein consisted of 379 amino acids, with a theoretically calculated molecular size of 43.56 kDa and a theoretical pI of 6.02. The amino acid sequence of XynE102 had a homology of 69.17% with the xylanase from Cellvibrionaceae bacterium (GenBank: HEY7885703.1); a homology of 61.74% with the xylanase from Marinimicrobium sp. ABcell2 (GenBank: WP_306731003.1), and a homology of 59.26% with the xylanase from Teredinibacter haidensis (GenBank: WP_075188369.1).

[0083] As can be seen from Figure 1 , XynE102 had a catalytic domain, which was similar to the GH10 family domain from Marinimicrobium sp. ABcell2. Like other GH10 enzymes, its tertiary structure showed a (β / α)8 or TIM-barrel fold( Figure 1 A in Figure 1 ). Phylogenetic analysis showed that XynE102 clustered with the xylanase from Marinimicrobium sp. ABcell2 (GenBank: WP_306731003.1)(

[0084] Example 2 Cloning, Expression and Purification of XynE102

[0085] The full-length xylanase gene was amplified by PCR, and the primers were: XynE102-F: CATCATCATCATCATCATGAA ATGCCGTTGTCCCGCCGCGATT (SEQ ID NO:1) and XynE102-R: GTGCTGAGTGCGGCCGCAAGCTAGGATTTCAGTTTTCGCAAAG (SEQ ID NO:2). The underlined sequence represents the homologous recombination fragment of the pSHY211 vector (Novagen, USA), which has been previously digested with EcoR I and Hind III enzymes. In this invention, all PCR processes were completed by TransStarFastPfu DNA polymerase (TransGen Biotech, China). The PCR program included: pre-denaturation at 95°C for 3 min, 10 cycles of denaturation at 98°C for 20 s and annealing at 68°C for 2.5 min; then 29 cycles of denaturation at 98°C for 20 s, annealing at 55°C for 30 s and extension at 72°C for 2.5 min, and finally extension at 72°C for 10 min. The PCR product was inserted into pSHY211 using the pEASY-Uni seamless cloning and assembly kit (TransGen Biotech, China) to obtain the expression plasmid pSHY211 (XynE102). Escherichia coli DH5α was used for the cloning and expression of the xylanase gene. Escherichia coli was grown on LB medium containing 50 μg / mL kanamycin. The DNA isolation and purification kit was purchased from (Sangon, China). The transformants were cultured in 200 mL of LB broth containing 50 μg / mL kanamycin at 37°C with a rotation speed of 200 rpm. To induce the expression of the recombinant xylanase XynE102, at the absorbance (OD 600When OD600 nm reached 0.6, 0.2 mL of 100 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to the cell suspension. Then, the suspension was cultured with shaking at 200 rpm at 20 °C for 8 h, centrifuged at 10,000×g for 15 min at 4 °C, and the induced cells were harvested. The above cells were disrupted by ultrasonic waves, and the lysate was centrifuged at 12,000×g for 30 min at 4 °C. After centrifugation, the cell-free extract was purified using a Ni-NTA affinity column (Histrap, TransGen Biotech, China) according to the method previously reported by Yin et al. (Yin YR, Hu QW, Xian WD, Feng Z, Zhou EM, Hong M, Min X, Zhi XY, and Li WJ (2017) Characterization of a neutral recombinant xylanase from Thermoactinospora rubra YIM 77501T, Antonie Van Leeuwenhoek, 110:429-436. https: / / doi.org / 10.1007 / s10482-016-0798-y). The purified XynE102 was loaded on a 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Protein bands were stained with Coomassie Brilliant Blue R-250. The protein concentration was determined using a Bradford protein assay kit (Sangon Biotech, China), with bovine serum albumin as the standard.

[0086] The xylanase gene without a signal sequence was successfully cloned into pSHY211-His and further confirmed by sequencing as a His-tag fusion protein. The recombinant enzyme XynE102 was purified by Ni-NTA affinity chromatography. The purified protein showed a single 40 kDa band against protein markers on 12% SDS-PAGE ( Figure 2 ).

[0087] The nucleotide sequence of the recombinant enzyme XynE102 is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4.

[0088] ATGCCGTTGTCCCGCCGCGATTTTTTATTGCGCACCGCTGTTGGTGCTGGATTCGCTCTAACCCTGAAGCACCAGGCTTTTGCGGCTGCTGCCAAAAAGACTGGTCTCGCCGACCTCTACAAAGGGGACTTTCTAATAGGCACCGCTATCAGTAACGCGACCATGGAGCGCCATGATCAAGCCATGCTCGACCTGATCAGCCGCGAATTCAACGCCATCACCGCAGAAAATGCGATGAAGTGGGGCGTGCTCAACCCGGAGCCGGGTATCTGGAACTGGGACACACCCGATAAGATGGTAGATTACGGCGTTGAGAACGATATGTTCATTATGGGACACACGCTGGTGTGGCATTCCCAGGCGCCCGAGTGGATTTTCAAGAACAAAAAGGGCGGTACCGCCAGTCGCGAGTTATTACTCAAGCGTATGGAAAGCTATATCGATACAGTCGCGGGCCGTTACAAAGGCAAGATCCAGGGCTGGGACGTCGTCAACGAAGCGGTTGATGAAGACAAAGGCTGGCGCAAGAGCCCCTGGTTCGAGATCATCGGTCCAGACTATATGGAGCATGCGTTCCGCTTGGCTCACGAGGCCGACCCCAAGGCCCACCTCATTTACAACGACTACAACATGCATTCGCCCGAGAAGCGCAAATTCCTGTTCAACATTATCAAGGATTACCAAAAGCGCAACGTGCCGATTCACGGTGTTGGTCTACAGGGCCACGTCGGCTTGTCCTACCCCGACCTGAACGAATTCGAGAACACTATTAAAGCCTGTCGCGATCACGGTCTGGCCGCTCACATCACGGAGCTCGATGTGGACGTACTACCCGTAGCCTGGGAGCACACCGGCGCGAATATTTCGGATATGCAGGAATACCGAGATGATCTCAACCCCTATACCGACGGGCTGCCCAAGAAAGTTCAACAAGAGCTGACTGACCGCTACGTACAGCTGTTTGAGCTGTTCCTGGAATATCGCGACACCATAGCCCGCGTCACTACCTGGGGCACCAGCGACCATGAAAGCTGGAAGAACGATTTCCCGGTGGTTGGCCGTACCAACTACCCTCTGTTGTTCGATCGCAATAATGAGCCAAAGCCTGCGTATCACGCTTTGCGAAAACTGAAATCCTAG(SEQ ID NO:3).

[0089] MPLSRRDFLLRTAVGAGFALTLKHQAFAAAAKKTGLADLYKGDFLIGTAISNATMERHDQAMLDLISREFNAITAENAMKWGVLNPEPGIWNWDTPDKMVDYGVENDMFIMGHTLVWHSQAPEWIFKNKKGGTASRELLLKRMESYIDTVAGRYKGKIQGWDVVNEAVDEDKGWRKSPWFEIIGPDYMEHAFRLAHEADPKAHLIYNDYNMHSPEKRKFLFNIIKDYQKRNVPIHGVGLQGHVGLSYPDLNEFENTIKACRDHGLAAHITELDVDVLPVAWEHTGANISDMQEYRDDLNPYTDGLPKKVQQELTDRYVQLFELFLEYRDTIARVTTWGTSDHESWKNDFPVVGRTNYPLLFDRNNEPKPAYHALRKLKS-(SEQ ID NO:4).

[0090] Example 3 Biochemical Characteristics of Recombinase XynE102 (Abbreviated as XynE102)

[0091] (1) Effects of Temperature and pH on the Activity of XynE102

[0092] The purified XynE102 was used at pH 4.0 - 10.0. The optimal temperature was determined by measuring the activity of XynE102 at different temperatures (10 - 75 °C) at the optimal pH. To evaluate the thermal stability and pH stability, the purified XynE102 was incubated at different temperatures (40, 45, 50, and 55 °C) for different times (0, 20, 40, 60, 80, 100, and 120 min) and at pH 4.0 to 10.0 for different times (12 h and 24 h), and the residual XynE102 activity was measured.

[0093] Determination of the activity of recombinant xylanase (i.e., XynE102): The activity of the recombinant enzyme XynE102 was determined by spectrophotometry at 540 nm using beechwood xylan (Sigma, USA) as the substrate. Reducing sugars were measured using xylose as the standard and the Miller DNS (3,5-dinitrosalicylic acid) method (for the specific measurement method, see "Miller GL (1959) Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry, 31(3), 426–428. https: / / doi.org / 10.1021 / ac60147a030"). One unit of XynE102 activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute. Unless otherwise stated, the values in the following text were determined by this method.

[0094] The experimental results are as Figure 3 shown. The optimal reaction temperature for XynE102 activity was 50 °C, and at 30–55 °C, the maximum activity exceeded 60% ( Figure 3 in A). The optimal pH for XynE102 activity was 7.0, and more than 65% of the maximum activity was maintained between pH 6.0 and 8.0 ( Figure 3 in B). Thermal stability analysis showed that XynE102 still retained 130% of its activity after heat treatment at 40 °C for 120 min, completely lost its activity after heat treatment at 45 °C for 80 min, completely lost its activity after heat treatment at 50 °C for 60 min, and completely lost its activity after heat treatment at 55 °C for 20 min. Its half-life at 50 °C was 7.62 min ( Figure 3 in C). pH stability analysis showed that after incubation at 50 °C for 12 h, more than 85% of the relative activity was retained at pH 8.0–10.0; after incubation for 24 h, more than 85% of the relative activity was retained at pH 7.0–10.0 ( Figure 3 in D). The primary activity was taken as 100%, and the values in the figure are the mean ± SD (n = 3).

[0095] (2) Effect of salt concentration on the activity of XynE102

[0096] NaCl at 0–1.0 M was added to the reaction system for xylanase activity determination (same as (1)) to evaluate the effect of salt concentration on the activity of XynE102.

[0097] The results are as Figure 4As shown, XynE102 exhibits a certain salt tolerance under salt environmental conditions. As the salt concentration increases, the activity of XynE102 continuously decreases. Under the condition of 0 - 2.0 M NaCl concentration, XynE102 still maintains an activity of over 70%. Under the condition of 4.0 M NaCl, there is still about 30% of the activity remaining.

[0098] (3) Effects of metal ions and chemical agents on XynE102

[0099] Evaluate the effects of metal ions and chemical reagents on the enzyme activity of XynE102. Use various metal ions (Mg 2+ , Al 3+ , K + , Ca 2+ , Mn 2+ , Fe 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Ag + , Cd 2+ , Ba 2+ , Pb 2+ ) at 1 mM and 10 mM; use chemical reagents such as 0.1%, 1% and 10% (SDS, Urea, CTAB, Tween80, Triton X - 100, DMSO, EtOH, MeOH, β - Me, IPA), EDTA (Ethylene Diamine Tetraacetic Acid), DTT (Deloitte ToucheTohmatsu), PMFS (Phenylmethylsufonyl Fluoride) and alcohols (methanol, ethanol and isopropanol) and add them into the reaction system for xylanase activity determination (same as (1)) respectively. Control conditions are tested using the same process as above, and no additives are added to the reaction mixture.

[0100] The effects of different metal ions on XynE102 are shown in Table 1. The activity of XynE102 is activated to 136.71 ± 2.46% and 129.17 ± 6.18% by Mn ions at 1 mM and 10 mM. At 10 mM concentration, XynE102 is inhibited by K 2+ (74.89 ± 5.40%), Ni + (70.63 ± 2.78%), Cu 2+ (21.78 ± 1.82%), Zn 2+ (74.89 ± 5.40%) and Pb 2+ (74.89 ± 5.40%) and Pb 2+(74.89 ± 5.40%) and other metal ions inhibit. At a concentration of 10 mM, Mn 2+ (129.17 ± 6.18%), Fe 3+ (113.25 ± 2.59%), Co 2+ (111.22 ± 1.98%) and other ions can significantly activate the activity of XynE102. It is worth noting that in the presence of Ag + , XynE102 will be completely inactivated.

[0101] The effects of chemical reagents on XynE102 are shown in Table 2. At a concentration of 0.1%, both PMSF (4.81 ± 0.97%) and CTAB (0.54 ± 0.29%) strongly inhibit the activity of XynE102, and SDS (93.58 ± 8.61%) and EtOH (91.16 ± 7.66%) have a slight inhibitory effect on the activity of XynE102; however, 1% SDS has no inhibitory effect on XynE102. EDTA has a promoting effect on the activity of XynE102. It is worth noting that XynE102 will be completely inactivated in the presence of 10% β-Me and IPA.

[0102] Table 1 Effects of metal ions on XynE102

[0103]

[0104] Table 2 Effects of chemical reagents on XynE102

[0105]

[0106]

[0107] Note: In Tables 1 - 2, * represents p ≤ 0.05, and ** represents p ≤ 0.01.

[0108] The activity of XynE102 can be slightly inhibited to 98.25 ± 0.21% and 96.50 ± 2.58% by 1 mM and 10 mM of Mg 2+ ions. This is because Mg 2+ has unique biological properties. Mg 2+ ions do not change the conformational properties of enzyme subunits at room temperature, but can have selective and differential effects on protein stability. In particular, the melting curve of the protein usually shifts to a lower temperature, indicating that Mg 2+ ions have an unstable effect on the β-sheet of the gyrase, resulting in a slight inhibition of enzyme activity. Ag + will completely inactivate XynE102. At a concentration of 10 mM, XynE102 is inhibited by Mn 2+ , Fe 3+, Co 2+ and Cu 2+ Metal ion inhibition, because Co 2+ , Mn 2+ and Cu 2+ and other metal ions have inhibitory effects on the activities of most xylanases, which is similar to the salt-tolerant β-1,3-xylanase Xyl88 reported in the prior art. In the presence of Ca 2+ , the activity of XynE102 is inhibited, probably because XynE102 does not have a Ca 2+ binding domain and cannot bind to Ca 2+ to enhance stability. At a concentration of 0.1%, both PMSF and CTAB can strongly inhibit the activity of XynE102, while 0.1% SDS has a slight inhibitory effect on the activity of XynE102. This is because SDS is a strong denaturing agent (anionic detergent) that can inhibit the activities of most enzymes at low concentrations, but 1% SDS has no inhibitory effect on XynE102. As a chelating agent, EDTA generally only chelates polyvalent metal ions such as Ca 2+ , Mg 2+ , etc., but XynE102 may be activated by Na + , which results in no effect of EDTA on the activity of XynE102. Therefore, when using XynE102, contact with Ag + , Mn 2+ , PMSF, CTAB, β-Me and IPA should be avoided.

[0109] (4) Substrate specificity

[0110] To study the substrate specificity of XynE102, the enzyme activities were determined using beechwood xylan, corncob xylan, sugarcane bagasse xylan, microcrystalline cellulose, CMC-Na, and cellobiose as substrates (1%, w / v). The kinetic constants of XynE102 were determined by reacting with different concentrations of substrates (0.1 - 20 mg / mL) at the optimal pH (7.0) and temperature (50 °C) for 5 min, and the maximum velocity Vmax and Km values of the reaction were calculated using the Lineweaver - Burk plot.

[0111] The substrate specificity of XynE102 is shown in Table 3. XynE102 is active against beechwood-derived xylan (100 ± 2.1 U / mg), corncob-derived xylan (31 ± 2.7 U / mg), and sugarcane bagasse-derived xylan (30 ± 1.1 U / mg), but is inactive against CMC-Na, microcrystalline cellulose, and cellobiose. All substrates in this experiment were tested at 1% (w / v). The results are expressed as mean ± SD, and there are significant differences among different substrates at the P ≤ 0.5 significance level.

[0112] From Table 4, it can be seen that the optimal substrate of XynE102 is beechwood xylan. The optimal temperature and pH are 50 °C and 7 respectively, and the activity is 137.21 U / mg. The Km, Vmax and Kcat of the recombinant enzyme XynE102 for beechwood xylan are 2.68 mg / ml, 166.67 μmol / min / mg and 120.97 s -1 , respectively. The relative molecular mass and pI of XynE102 are 43.56 kDa and 6.02 respectively. The above indicates that XynE102 has good substrate affinity.

[0113] Table 3 Substrate specificity of XynE102

[0114]

[0115] Table 4 Kinetic constants of XynE102

[0116]

[0117] (5) TLC analysis of hydrolysis products

[0118] The hydrolysis products of xylan by XynE102 were analyzed by thin-layer chromatography. A reaction mixture consisting of 1% beechwood xylan and 10 μg of purified XynE102 was incubated at 90 °C for 1 h, and the hydrolysis products of beechwood xylan were characterized by thin-layer chromatography (TLC) using a silica gel 60 plate (Merck, Darmstadt, Germany). The coating solution was 1-butanol / acetic acid / water (2:1:1, v / v / v). The TLC plate was sprayed with freshly prepared 5% (v / v) H2SO4 ethanol and treated at 120 °C for 10 min to detect the sugar content. The sugar standards used in this study were X1 (xylose), X2 (xylobiose), X3 (xylotriose) and X4 (xylotetraose).

[0119] The results of TLC analysis of hydrolysis products are as Figure 5 shown. The hydrolysis products of xylan by the recombinant xylanase XynE102 were analyzed by TLC. The results showed that the main hydrolysis products of XynE102 were xylobiose and xylotetraose.

[0120] Example 4 Hydrolysis study of lignocellulose pretreated with recombinant enzyme XynE102 (abbreviated as XynE102)

[0121] The wheat bran was ground and passed through an 80-mesh sieve. Wheat bran and deionized water were added to a container at a material ratio of 1:20, and continuously heated in water at 90 °C for 2 h. The corn straw was filtered through a paper filter and dried at 80 °C for later use. For the alkali treatment of corn straw, the ground corn straw was passed through an 80-mesh sieve. Corn straw and 2% NaOH were added to a container at a material ratio of 1:20, and continuously heated in water at 80 °C for 2 h. The corn straw was filtered through a paper filter, rinsed with a large amount of deionized water until the filtrate was colorless, and then dried at 80 °C for later use. Then, 0.2 g of the pretreated lignocellulose was added to 5 mL of buffer (pH 7), and 0.05 mg of XynE102 was added to the reaction system. Then the mixture was incubated at 40 °C respectively. At 0, 2, 4, 6, 8, 10, 12, and 24 h after the start of the reaction, the reducing sugars produced were measured using DNS (same as Example 3). The control condition was a reaction solution without the enzyme.

[0122] The results are as Figure 6 shown. It can be seen that for both pretreated lignocelluloses, with the hydrolysis time, the concentration of reducing sugars produced is continuously increasing. After 24 h, the concentration of reducing sugars in the system of alkali-treated corn straw reaches 5.44 μmol / mL, while that in the system of water-pretreated wheat bran reaches 4.18 μmol / mL. It can be Figure 6 seen that the concentration of reducing sugars in both substrates reaches more than 80% of that after 24 h of reaction after 8 h of reaction. Incubating for 8 hours is the most economical and beneficial time.

[0123] In summary, a new xylanase gene XynE102 was identified from the metagenome of saline-alkali soil in Karamay, Xinjiang, China. Its enzymatic properties were determined and its biochemical characteristics were characterized to explore its main functions, which plays a crucial role in applying this enzyme to industry. Therefore, the present invention cloned the XynE102 gene, expressed it in Escherichia coli, purified XynE102 by Ni-NTA resin affinity chromatography, and then determined the enzymatic properties and predicted the functions of XynE102.

[0124] Salt-tolerant enzymes have advantages in the field of food processing. For example, the application of a novel salt-tolerant xylanase (rXynMF13A) in steamed bread (Wu J, Qiu C, Ren Y, Yan R, Ye X, Wang G. Novel Salt-Tolerant Xylanase from a Mangrove-Isolated Fungus Phoma sp. MF13 and Its Application in Chinese Steamed Bread. ACS Omega. 2018 Apr 30;3(4):3708-3716. doi:10.1021 / acsomega.8b00345. Epub 2018 Apr 2. PMID:30023876; PMCID:PMC6045339). Compared with rXynMF13A (Table 5), XynE102 showed better thermal stability and a wider pH range, indicating that XynE102 has greater application potential in the food processing process. Combining with the thermal stability of XynE102( Figure 3 in C), it can be found that XynE102 has good thermal stability at 40 °C, which may be related to the special charges or acidic residues on the surface of XynE102. These special substances strengthen the structure of the protein molecule, thus enhancing its thermal stability. The salt tolerance of salt-tolerant xylanase may be because salt ions form a membranous structure on the protein surface, fixing the protein of XynE102 into a certain spatial form, thus playing a stabilizing role in the structure of the protein. Although this structure part is loose, Na + can help the structure return to its original state.

[0125] The hydrolysis products of the recombinant enzyme XynE102 are xylobiose and xylotetraose. Many studies have shown that xylooligosaccharides, as a new type of prebiotic, can play an important role in promoting the growth of probiotics and balancing the stability of the human intestinal flora. This indicates that XynE102 has potential application value in the production of prebiotics. At present, a large number of studies have reported salt-tolerant xylanases. The kinetic parameters of the recombinant enzyme XynE102 discovered in this invention were compared with other enzymes reported in the literature, and it was found that the Km value of XynE102 was lower, at 2.68 mg / mL (Table 5). Compared with other salt-tolerant enzymes, XynE102 has high activity (137.21 U / mg), which means that XynE102 has higher catalytic efficiency.

[0126] Table 5 Comparison of XynE102 with other salt-tolerant enzymes

[0127]

[0128]

[0129] Note: The reference for ① is Xu B, Dai L, Li J, Deng M, Miao H, Zhou J, Mu Y, Wu Q, Tang X, Yang Y, Ding J, Han N, Huang Z. Molecular and Biochemical Characterization of a Novel Xylanase from Massilia sp. RBM26 Isolated from the Feces of Rhinopithecus bieti. J Microbiol Biotechnol. 2016 Jan;26(1):9 - 19. doi:10.4014 / jmb.1504.04021. PMID:26387816;

[0130] The reference for ② is Teo SC, Liew KJ, Shamsir MS, Chong CS, Bruce NC, Chan KG, Goh KM. Characterizing a Halo-Tolerant GH10 Xylanase from Roseithermus sacchariphilus Strain RA and Its CBM-Truncated Variant. Int J Mol Sci. 2019 May 9;20(9):2284. doi:10.3390 / ijms20092284. PMID:31075847; PMCID:PMC6539836.v;

[0131] The reference for ③ is Huang X, Lin J, Ye X, Wang G. Molecular Characterization of a Thermophilic and Salt-and Alkaline-Tolerant Xylanase from Planococcus sp. SL4, a Strain Isolated from the Sediment of a Soda Lake. J Microbiol Biotechnol. 2015 May;25(5):662 - 71. doi:10.4014 / jmb.1408.08062. PMID:25381738;

[0132] Reference for ④ is Wu J, Qiu C, Ren Y, Yan R, Ye X, Wang G. Novel Salt-Tolerant Xylanase from a Mangrove-Isolated Fungus Phoma sp. MF13 and Its Application in Chinese Steamed Bread. ACS Omega. 2018 Apr 30; 3(4): 3708-3716. doi: 10.1021 / acsomega.8b00345. Epub 2018 Apr 2. PMID: 30023876; PMCID: PMC6045339;

[0133] Reference for ⑤ is Yi Z, Cai Z, Zeng B, Zeng R, Zhang G. Identification and Characterization of a Novel Thermostable and Salt-Tolerant β-1,3 Xylanase from Flammeovirga pacifica Strain WPAGA1. Biomolecules. 2020 Sep 7; 10(9): 1287. doi: 10.3390 / biom10091287. PMID: 32906756; PMCID: PMC7563424.

[0134] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the gist of the present invention. In addition, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Claims

1. A xylanase, characterized in that, The amino acid sequence of the xylanase is as follows: a) SEQ ID NO: 4; or b) an amino acid sequence obtained by substituting and / or deleting and / or adding one or several amino acids to SEQ ID NO: 4 and having the same function as the protein shown in SEQ ID NO: 4; or c) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% homology with SEQ ID NO: 4 and having the same function as the protein shown in SEQ ID NO:

4.

2. A nucleic acid molecule encoding the xylanase according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein The nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO:

3.

4. A biological material related to claim 2 or 3, characterized in that, The biological material comprises at least one of b1) to b12): b1) the nucleic acid molecule according to claim 2 or 3; b2) an expression cassette containing the nucleic acid molecule described in b1); b3) a recombinant vector containing the nucleic acid molecule described in b1); b4) a recombinant vector containing the expression cassette described in b2); b5) a recombinant cell containing the nucleic acid molecule described in b1); b6) a recombinant cell containing the expression cassette described in b2); b7) a recombinant cell containing the recombinant vector described in b3); b8) a recombinant cell containing the recombinant vector described in b4); b9) a recombinant microorganism containing the nucleic acid molecule described in b1); b10) a recombinant microorganism containing the expression cassette described in b2); b11) a recombinant microorganism containing the recombinant vector described in b3); b12) a recombinant microorganism containing the recombinant vector described in b4).

5. Use of the xylanase according to claim 1, the nucleic acid molecule according to claim 2 or 3, or the biological material according to claim 4 in at least one of c1) to c5): c1) Preparation of reducing sugars; c2) Preparation of products for reducing sugar preparation; c3) Hydrolysis of xylan; c4) Preparation of products for xylan hydrolysis; c5) Industry.

6. The application according to claim 5, wherein The reducing sugars described in c1) to c2) include xylooligosaccharides or xylo-oligosaccharides; and / or, the xylan described in c3) to c4) includes at least one of beechwood xylan, corncob xylan, bagasse xylan, and birchwood xylan.

7. The application according to claim 5, characterized in that, The industry described in c5) is selected from the food industry, feed industry, textile industry, paper industry, and biomass energy industry.

8. A product comprising the xylanase according to claim 1 or the biological material according to claim 4.

9. The product according to claim 8, characterized in that, The product comprises one or more of a detergent, a reagent, a food additive, a feed additive, food, and feed.

10. A method for preparing reducing sugars, comprising treating a substrate to be hydrolyzed with the xylanase according to claim 1, the biological material according to claim 4, or the product according to claim 8 or 9, wherein the substrate comprises xylan or a substance containing xylan.

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