CN107129976A - A kind of neutral high temperature xylanase and its coding gene and its application - Google Patents

Abstract

The present invention provides a neutral thermoxylanase CtXyn10A and its gene and application. The neutral thermoxylanase CtXyn10A provided by the present invention is a protein having an amino acid sequence shown in SEQ ID No. 3 and/or SEQ ID No. 5 in a sequence list, and/or a protein derived from a protein having an amino acid sequence shown in SEQ ID No. 3 and/or SEQ ID No. 5 in a sequence list, wherein one or more amino acid residues are substituted and/or deleted and/or added to the amino acid residue sequence shown in SEQ ID No. 3 and/or SEQ ID No. 5 in a sequence list and having xylanase activity. The xylanase CtXyn10A of the present invention has an optimum pH of 6.0-7.0 and an optimum temperature of 70°C, maintains more than 20% of enzyme activity at 90°C and pH 9.0, and has a specific activity of 461U/mg. The xylanase CtXyn10A of the present invention has the activities of xylanase, glucanase, and cellulase, is easy to be produced by industrial fermentation, and as a new type of broad-spectrum enzyme preparation, can be widely used in food, papermaking, energy industries, etc.

Description

A kind of neutral high temperature xylanase and its coding gene and its application

technical field

The invention belongs to the field of genetic engineering, and specifically relates to a neutral high-temperature xylanase, its encoding gene and its application.

Background technique

Xylan (xylan) is the main component of the second most abundant plant biomass in nature after cellulose (Lynd et al. Microbiology and Molecular Biology Review, 2002, 66:506–577), in terms of bioenergy have potential use value. Existing biotechnology can efficiently degrade the cellulose component in plants and convert it into fermentable glucose, which is produced by yeast to produce bioethanol. High-component xylan not only limits the combination reaction of cellulase and cellulose, inhibits the catalytic reaction, but also discharges into nature as a by-product, which can cause nutrient enrichment and damage the ecosystem (Polizeli et al.Applied Microbiology and Biotechnology, 2005, 67:577-591). Therefore, in the process of biomass bioconversion, xylanase is usually added to remove the physical barrier of xylan and promote the combination of cellulase and cellulose (Hu et al.Biotechnology for Biofuels, 2011, 4:14 ; Qing and Wyman Biotechnology for Biofuels, 2011,4:18), on the other hand the xylooligosaccharides generated can also be further degraded into fermentable xylose, providing 10-25% raw material for the production of bioethanol (Jin et al . Applied and Environmental Microbiology, 2004, 70:6816-6825).

Based on the amino acid sequence and structure of the catalytic domain, most xylanases (EC 3.2.1.8) are classified in families 10 and 11 of glycoside hydrolases. Compared with the 11th family xylanase that specifically degrades xylan, the 10th family xylanase has broad substrate specificity, in addition to acting on xylan, it can also catalyze cellulose, glucan Degradation of various substrates such as sugar, so it has a wider application prospect in the industrial field.

The existing bioethanol processing technology includes acid-base pretreatment of lignocellulose, long-term high-temperature enzyme reaction, which consumes a lot of energy and acid-base chemical reagents, resulting in a substantial increase in cost and serious environmental pollution. Therefore, the development of lignocellulose-degrading enzymes with neutral high temperature is of great significance in terms of energy saving, environmental protection, and ease of catalyzing reactions (Ji et al. Biotechnology for Biofuels, 2014, 7:130).

Contents of the invention

One object of the present invention is to provide a protein named Ctxyn10A, which is derived from Cladosporium tianshanense SL-14.

The protein of the present invention is the protein described in 1), 2), or 3) as follows:

1) A protein having the amino acid sequence shown in SEQ ID No. 3 in the sequence listing;

2) A protein having the amino acid sequence shown in SEQ ID No. 5 in the sequence listing;

3) The amino acid residue sequence shown in SEQ ID No. 3 and/or SEQ ID No. 5 in the sequence listing undergoes substitution and/or deletion and/or addition of one or several amino acid residues and has xylan Enzymatically active proteins derived from 1) and/or 2).

The amino acid sequence shown in SEQ ID No. 3 in the sequence listing consists of 352 amino acid residues; among them, the N-terminal 18 amino acids are the predicted signal peptide sequence, which has the amino acid sequence shown in SEQ ID No. 4 in the sequence listing.

The amino acid sequence shown in SEQ ID No. 5 in the sequence listing consists of 334 amino acid residues; that is, the mature protein of Ctxyn10A.

The Ctxyn10A protein in the above 1), 2) and 3) can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression. The coding gene of the Ctxyn10A protein in the above 1), 2) and 3) can be obtained by adding SEQID No. 1, SEQ ID No. 2, and/or SEQ ID No. 55-1059 in the sequence listing in the sequence listing The DNA sequence shown in the nucleotide sequence shown in the position is obtained by deleting one or several amino acid residue codons, and/or carrying out one or several base pairs of missense mutations.

The nucleic acid molecules encoding the Ctxyn10A protein also belong to the protection scope of the present invention.

The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, hnRNA or tRNA.

Another object of the present invention is to provide a coding gene, characterized in that the coding gene has one of the following nucleotide sequences:

1) a polynucleotide sequence encoding the protein of the present invention;

2) the nucleotide sequence shown in the 55th-1059th position of SEQ ID No.: 1, SEQ ID No.: 2, and/or SEQ ID No.: 2 in the sequence listing;

3) The polynucleotide sequence encoding the protein sequence of SEQ ID No.: 3 and/or SEQ ID No. 5 in the sequence listing;

4) a nucleotide sequence that can hybridize to any of the DNA sequences defined in 1), 2), and/or 3) under high stringency conditions;

5) A DNA sequence having more than 90% homology with any of the DNA sequences defined in 1), 2), 3), and/or 4) and encoding the same functional protein.

Specifically, the homology is more than 95%; more specifically, more than 96%; more specifically, more than 97%; more specifically, more than 98%; more specifically, more than 99%.

The above-mentioned high stringency conditions can be 6×SSC, 0.5% SDS solution, hybridization at 65° C., and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS respectively.

Among them, the nucleotide sequence shown in SEQ ID №: 1 in the sequence listing has a total of 1202bp, and contains two intron sequences with lengths of 59bp and 84bp respectively, and the two intron sequences respectively have SEQ ID No. ID №: the 141st-199th nucleotide sequence and the 512th-595th nucleotide sequence shown in 1; the cDNA length after removing the two intron sequences is 1059bp, and has the SEQ ID in the sequence list No.: the nucleotide sequence of 2, encoding the amino acid sequence shown in SEQ ID No.: 3 in the sequence listing and a stop codon, that is, the Ctxyn10A protein of the present invention; having the 55th position of SEQ ID No.: 2 in the sequence listing - The nucleic acid fragment of the 1059th nucleotide sequence encodes the amino acid sequence shown in SEQ ID No. 5 in the sequence list, that is, the Ctxyn10A mature protein of the present invention.

Another object of the present invention is to provide a vector, an expression cassette, a transgenic cell line, a recombinant bacterium, and/or a microorganism, the vector, an expression cassette, a transgenic cell line, a recombinant bacterium, and/or a microorganism containing the encoding Gene.

Specifically, the vectors include recombinant cloning vectors and recombinant expression vectors; more specifically pPIC-Ctxyn10A; the starting bacteria of the recombinant bacteria include Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, and/or Trichoderma; More specifically, the starting bacteria of the recombinant bacteria include Pichia pastoris, Saccharomyces cerevisiae, and/or S. polymorpha; more specifically, the starting bacteria of the recombinant bacteria are Pichia GS115; more specifically, the The recombinant bacterium is specifically GS115/Ctxyn10A; the microorganism includes Cladosporium tianshanense SL-14;

The recombinant expression vector can be constructed with existing expression vectors. The expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyA signal directs the addition of polyA to the 3' end of the pre-mRNA. When using the gene to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, and they can be used alone or with other promoters Used in combination; in addition, when using the gene of the present invention to construct a recombinant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that express enzymes or luminescent compounds that can produce color changes in microorganisms (GUS gene, GFP gene, luciferase Genes, etc.), antibiotic resistance markers (gentamycin markers, kanamycin markers, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic organisms, the transformants can be directly screened by stress without adding any selectable marker gene.

Another object of the present invention is to provide a pair of primers that can amplify the full length of the coding gene or any fragment thereof.

Another object of the present invention is to provide a method for preparing protein, said method comprising:

1) Prepare the coding gene described in the present invention;

2) preparing a recombinant expression vector comprising the coding gene described in step 1);

3) Expressing the expression vector described in step 2) to obtain the target protein.

Specifically, the method also includes deglycosylation of the target protein; the specific step of expressing the expression vector in step 2) includes introducing the expression vector into a microorganism to express it; more specifically, the Microorganisms include Escherichia coli, yeast, bacillus, lactobacillus, aspergillus, and/or Trichoderma; again specifically, said microorganisms include Pichia pastoris, cerevisiae, and/or Saccharomyces polymorpha; again specifically, said The microorganism is Pichia pastoris GS115; specifically, the recombinant expression vector includes pPIC-Ctxyn10A.

The protein prepared by the protein preparation method also falls within the protection scope of the present invention; specifically, the protein includes the Ctxyn10A protein and the modified structure of the present invention; specifically, the modified structure includes a glycosylation structure.

Another object of the present invention is to provide the protein, coding gene, recombinant vector, expression cassette, transgenic cell line, recombinant bacterium, and/or microorganism, primer pair, protein preparation method, and preparation method of the present invention. protein application.

Specifically, the application includes at least one of the following 1)-8):

1) Application in the preparation of xylanase and/or related products containing xylanase;

2) Application in the preparation of xylanase mutants and/or related products containing xylanase mutants;

3) Application in the preparation of recombinant xylanase and/or related products containing recombinant xylanase;

4) Application in the preparation of xylooligosaccharides, xylobiose, xylomonose, xylooligosaccharide-containing, xylobiose-containing, and/or xylomonoose-containing related products;

5) as cellulase, dextranase and/or in the preparation of related products with cellulase and dextranase enzyme activity;

6) Application in degrading xylan, cellulose, and/or glucan;

7) Application in the preparation of products used in the fields of industry, agriculture, food, medicine, feed, energy, waste treatment and/or environmental protection;

8) Applications in the fields of industry, agriculture, food, medicine, feed, energy, waste treatment and/or environmental protection.

Specifically, the xylan includes beech wood xylan, soluble wheat arabinoxylan, birch wood xylan, insoluble wheat arabinoxylan; the cellulose includes carboxymethyl cellulose, lichenan; the Glucans include barley glucan.

Specifically, the xylanase includes the protein of the present invention and/or the protein prepared by the protein preparation method of the present invention; the xylanase mutant includes the protein of the present invention and/or the protein of the present invention The protein prepared by the protein preparation method is a protein obtained by point mutation; the recombinant xylanase comprises homologous recombination of the protein of the present invention and/or the protein prepared by the protein preparation method of the present invention protein obtained later.

Specifically, the application includes application under the conditions described in the following 1), 2), 3), and/or 4):

1) pH includes 4.0-12.0;

2) The temperature includes 0°C-90°C;

3) An environment including metal ions, and/or chemical reagents;

4) Substrates include xylan, cellulose, and/or dextran.

Preferably, the pH includes 5.0-8.0; more preferably, the pH includes 6.0-7.0; most preferably, the pH is 6.5; preferably, the temperature includes 50°C-80°C; preferably, the The temperature includes 50°C-75°C; preferably, the temperature includes 60°C-75°C; most preferably, the temperature is 70°C; the metal ions and/or chemical reagents include Ni ²⁺ , Co ^{2 +} , Na ⁺ , Cr ³⁺ , Mg ²⁺ , Mn ²⁺ , K ⁺ , Cu ²⁺ , Ca ²⁺ , Pb ²⁺ , Zn ²⁺ , Fe ³⁺ , β-mercaptoethanol, EDTA, SDS; The concentration of said metal ion and/or chemical reagent comprises 5mmol/L; Said xylan comprises beech wood xylan, soluble wheat arabinoxylan, birch wood xylan, insoluble wheat arabinoxylan; Said fiber Vegetables include carboxymethyl cellulose, lichenan; the dextran includes barley dextran.

Another object of the present invention is to provide a preparation method of xylanase, the method comprising: extracting xylanase from Cladosporium tianshanense SL-14.

The xylanase CtXyn10A provided by the invention has high activity characteristics in both neutral (pH 5.0-8.0) and high temperature (50°C-75°C) ranges. The present invention screens out the xylanase CtXyn10A produced by Cladosporium tianshanense SL-14 (China Agricultural Culture Collection Center ACCC 32710), and its optimum pH value is 6.0-7.0, in the range of pH 5.0-8.0 Maintain more than 80% of the enzyme activity in the body; the optimum temperature is 70°C, and it has more than 70% of the enzyme activity between 60-75°C; there is still 10% of the enzyme activity at 0°C; and for a variety of xylans , dextran, and cellulose have degradative effects. A neutral high temperature xylanase of this nature has not been reported.

The present invention overcomes the deficiencies of the prior art, obtains fungal resources of high-temperature xylanase, and provides a new neutral high-temperature xylanase CtXyn10A with excellent properties and suitable for application in food, papermaking and energy industries . The optimal pH of the xylanase CtXyn10A of the present invention is 6.0-7.0, and has high enzyme activity at pH 5.0-8.0; the pH stability is good; the specific activity is 461U/mg; and it has broad substrate specificity. The characteristic of neutral high temperature can reduce the energy cost of xylanase in industrial production. It has a wide range of pH adaptability, which can improve the digestibility and metabolic energy of aquatic feed, reduce formula cost and reduce environmental pollution. Its broad substrate specificity can improve the nutritional value of grain processing by-products and improve the quality of feed products. The xylanase CtXyn10A can be used in the brewing industry to reduce the viscosity of the material, which can facilitate the action of the amylase on the starch layer, improve the utilization rate of the starch, and increase the yield of alcohol. It is also possible to convert xylan in paper industry waste and agricultural waste into xylooligosaccharides under normal temperature conditions, and xylooligosaccharides can be converted into valuable fuels by bacteria, yeast and fungi. Therefore, the application of xylanase CtXyn10A in the energy industry also shows its great potential. The hydrolyzate of xylanase CtXyn10A (xylose and xylooligosaccharides) can be used in the food industry as thickeners, fat substitutes, prebiotic oligosaccharides and antifreeze food additives; xylan and other substances in the pharmaceutical industry Used in combination, the release of the drug ingredients can be delayed. Xylanase and its hydrolysis products can be further converted into liquid fuels, solvents and low-calorie sweeteners.

Description of drawings

Fig. 1 is the SDS-PAGE analysis of the recombinant xylanase CtXyn10A expressed in the recombinant bacteria GS115/Ctxyn10A,

Among them, lane 1 is deglycosylated purified recombinant xylanase; lane 2 is purified recombinant xylanase; lane 3 is low molecular weight protein marker.

Fig. 2 is a diagram showing the optimum pH determination results of the recombinant xylanase CtXyn10A.

Fig. 3 is a graph showing the pH stability measurement results of the recombinant xylanase CtXyn10A.

Fig. 4 is a graph showing the optimum temperature determination results of the recombinant xylanase CtXyn10A.

Fig. 5 is a graph showing the thermal stability measurement results of the recombinant xylanase CtXyn10A.

detailed description

illustrate:

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The molecular biology experimental methods not specifically described in the following examples are carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or according to the kit and product Instructions are carried out.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Test materials and reagents:

1. Bacterial strains and carriers: Cladosporium tianshanense SL-14 used in the following examples is preserved in the Agricultural Culture Collection Center of the Chinese Academy of Agricultural Sciences (No. 12, Zhongguancun South Street, Haidian District, Beijing, Agricultural Culture Collection of the Chinese Academy of Agricultural Sciences Center, 100081), its preservation number is: ACCC32710, and the public can buy this bacterial strain from this center. Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.

2. Enzymes and other biochemical reagents: endonucleases were purchased from TaKaRa Company, and ligases were purchased from Invitrogen Company. Beech xylan was purchased from Sigma Company, and the others were domestic reagents (all of which can be purchased from common biochemical reagent companies).

3. Medium:

(1) Cladosporium tianshanense SL-14 medium is potato juice medium: 1000mL potato juice, 10g glucose, 25g agar, pH5.0.

(2) Escherichia coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH7.0).

(3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V).

(4) BMMY medium: replace glycerol with 0.5% methanol (V/V), the rest of the ingredients are the same as BMGY, pH 4.0.

Cloning of the genomic DNA of embodiment 1, neutral high-temperature xylanase CtXyn10A

Extract the genomic DNA of Cladosporium tianshanense SL-14:

Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL of extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, and mix every 10min. Homogenize once and centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol/chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

According to the sequence analysis of the genome framework of Cladosporium tianshanense SL-14, design and synthesize specific primers Ctxyn10A-F/Ctxyn10A-R:

Ctxyn10A-F: 5'-ATGCGTTTCACTGAGGTCTTCACTGCTC-3';

Ctxyn10A-R: 5'-TCACTTCTTGCCACCCTTGATGCCC-3';

PCR amplification was performed using the total DNA of Cladosporium tianshanense SL-14 as a template and the above-mentioned Ctxyn10A-F/Ctxyn10A-R as specific primers.

The parameters of the PCR reaction were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 60 sec, and after 30 cycles, incubation at 72°C for 10 min.

The above PCR reaction obtained a nucleic acid fragment of about 1200bp, which was recovered and connected with the pEASY-T3 vector and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

Sequencing results show that the sequence of the nucleic acid fragment amplified by the above PCR has the nucleotide sequence shown in SEQ ID No. 1 in the sequence table, with a total of 1202 bp. The nucleic acid fragment having the nucleotide sequence shown in SEQ ID No. 1 in the sequence listing is named Ctxyn10A.

Example 2, Acquisition of the gene encoding mature neutral high-temperature xylanase CtXyn10A

Extract the total RNA of Cladosporium tianshanense SL-14, use reverse transcriptase to obtain a strand of cDNA, and then use primers Ctxyn10A-F/Ctxyn10A-R to amplify the single-stranded cDNA, and the amplified product is recovered and sent to III Sequenced by Bo Biotechnology Co., Ltd.

Ctxyn10A-F: 5'-GGGGAATTCCACCCTTCGGCTCCCAAGGAC-3';

Ctxyn10A-R: 5'-GGGGCGGCCGCTCACTTCTTGCCACCCTTGATGCC-3';

After comparing the genome sequence of the nucleic acid fragment measured in Example 1 with the sequencing result of the above-mentioned PCR amplification product, it was found that the gene has 2 introns, the cDNA is 1059bp long, and has the nucleotide of SEQ ID No. 2 in the sequence table Sequence, the amino acid sequence shown in SEQ ID No. 3 in the coding sequence list and a stop codon. A protein with the amino acid sequence shown in SEQ ID №: 3 in the sequence listing, with a total of 352 amino acid residues, and the N-terminal 18 amino acids are the predicted signal peptide sequence, with the amino acid sequence shown in SEQ ID №: 4 in the sequence listing . The 141st-199th nucleotide sequence and the 512th-595th nucleotide sequence of SEQ ID No. 1 in the sequence listing are 59bp and 84bp intron sequences respectively

The sequencing results of the above PCR amplification products show that the nucleic acid fragments obtained by the above PCR amplification include EcoRI+NotI double restriction sites, and the middle of the above two restriction sites is the 55th position-1059 of SEQ ID №: 2 in the sequence table A nucleic acid fragment of a nucleotide sequence.

The nucleic acid fragment having the 55th-1059th nucleotide sequence of SEQ ID No.: 2 in the sequence listing is a nucleic acid fragment of the mature protein part encoded by the Ctxyn10A gene, encoding the amino acid sequence shown in SEQ ID No.: 5 in the sequence listing; The nucleic acid fragment encoding the mature protein of Ctxyn10A gene was homologously compared with the xylanase gene sequence on GenBank, the highest identity was 74%, and the highest amino acid sequence identity was 72%. The gene encoding xylanase isolated and cloned from is a new gene.

Embodiment 3, the preparation of neutral high-temperature xylanase CtXyn10A

The Pichia pastoris expression vector pPIC9 was subjected to double digestion (EcoRI+NotI), and the nucleic acid fragment encoding the mature protein prepared in Example 2 was double-digested (EcoRI+NotI) to cut out the gene fragment encoding the mature protein and The expression vector pPIC9 after double digestion was ligated.

Sequencing to verify the correctness of the sequence. The sequence of the foreign gene inserted in the resulting recombinant plasmid is SEQ ID No.: 2 nucleotides 55-1059, and the recombinant plasmid is named pPIC-Ctxyn10A.

The above-mentioned recombinant plasmid pPIC-Ctxyn10A was transformed into Pichia pastoris GS115; the correctness of the sequence was verified by extracting the plasmid from the positive recombinant bacteria, and the recombinant Pichia strain containing the recombinant plasmid pPIC-Ctxyn10A was named GS115/Ctxyn10A.

Take the recombinant GS115/Ctxyn10A strain, inoculate it in 300mL BMGY culture medium, shake it at 250rpm at 30°C for 48h, and collect the bacteria by centrifugation. Then resuspend in 100mL BMMY medium, shake culture at 250rpm at 30°C. After 72 hours of induction, the supernatant was collected by centrifugation, the protein was purified, and the activity of xylanase CtXyn10A was determined.

The results of SDS-PAGE are shown in Figure 1, and the results in Figure 1 show that the xylanase CtXyn10A was expressed in the Pichia pastoris recombinant strain GS115/Ctxyn10A. After the expressed xylanase CtXyn10A is purified, the content of the target protein reaches more than 90% of the total protein, which is pure by electrophoresis. After deglycosylation by ENDO-H, the molecular weight of the purified protein was 37.4kDa, consistent with the theoretical value.

Embodiment 4, the enzyme activity assay of neutral high-temperature xylanase CtXyn10A

The activity of the xylanase CtXyn10A prepared in the above-mentioned Example 3 was measured by the DNS method, and the specific method was as follows: at pH 6.5, at 70°C, 1 mL of the reaction system included 100 μL of an appropriate diluted enzyme solution, 900 μL of the substrate, and the reaction After 10 minutes, 1.5mL DNS was added to terminate the reaction, and boiled for 5 minutes. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions. It was determined that the enzyme activity of the neutral high-temperature xylanase CtXyn10A prepared in Example 3 was 160 U/mL.

Embodiment 5, the property determination of neutral high-temperature xylanase CtXyn10A

1. The optimum pH and pH stability assay methods of recombinant xylanase CtXyn10A are as follows:

The recombinant xylanase purified in Example 3 was subjected to enzymatic reactions at different pHs to determine its optimum pH. The substrate xylan was dissolved in 0.1 mol/L citric acid-disodium hydrogen phosphate buffer solution with different pH, and xylanase activity was measured at 30°C. The results are shown in Figure 2, the optimum pH of xylanase CtXyn10A is 6.0-7.0, and in the range of pH 5.0-8.0, the enzyme activity is maintained above 80% of the maximum enzyme activity. Xylanase was treated at 37°C for 60min in the above-mentioned buffers with different pH, and then the enzyme activity was measured at 70°C in the pH6.5 buffer system to study the pH tolerance of the enzyme. The results are shown in Figure 3, xylanase CtXyn10A is very stable between pH 4.0-12.0, and the remaining enzyme activity is above 80% after 60 minutes of treatment in this pH range, which shows that this enzyme has good pH stability .

2. The optimal temperature and thermostability determination methods of xylanase are as follows:

The determination of the optimal temperature of xylanase is carried out in the citric acid-disodium hydrogen phosphate buffer (pH6.5) buffer system and the enzymatic reaction at different temperatures. The temperature resistance was measured by treating xylanase at different temperatures for different times, and then measuring the enzyme activity at pH 6.5 and 70°C. The optimum temperature measurement results of the enzyme reaction are shown in Figure 4. The optimum temperature of xylanase CtXyn10A is 70°C, and the specific activity is 461U/mg; it has higher activity in the high temperature range of 50°C-75°C; There is more than 70% enzyme activity between -75°C; there is also 10% enzyme activity under 0°C; the thermostability test results of the enzyme are shown in Figure 5 (the results shown at 80°C in Figure 5 are substrate reaction time 10 minutes; 80 ℃ in Fig. 4, result and expression measure enzyme activity after no substrate 80 ℃ incubating for a certain period of time), xylanase CtXyn10A has very good stability at 60 ℃, is incubated at 70 ℃ for 60min, completely Loss of enzyme activity.

In addition, the enzyme activity of xylanase CtXyn10A at 90°C and pH9.0 was also measured, and the results showed that it maintained more than 20% of the enzyme activity.

The above experimental results show that xylanase CtXyn10A is a neutral high temperature enzyme with the characteristics of neutral high temperature.

3. The Km value determination method of xylanase is as follows:

Using different concentrations of beech wood xylan as the substrate, in the citric acid-disodium hydrogen phosphate buffer (pH6.5) buffer system, the enzyme activity was measured at 70°C, and its Km and Vmax at 70°C were calculated , kcat and kcat/Km values. It has been determined that the xylanase CtXyn10A has a km value of 0.64 mg/mL at 70°C using beech wood xylan as a substrate, a maximum reaction velocity Vmax of 450 μmol/min mg, a kcat value of 281/s and a kcat/ The Km value is 434ml/mg·s. The experimental results show that CtXyn10A has a strong affinity with the substrate, and has high catalytic efficiency under high temperature conditions, and its enzymatic properties are much better than fungal xylanases with similar sequences FoXyn10A (optimum pH 7.0, optimum temperature 45°C, Km 0.8mg/mL, Vmax 1.22μmol/min·mg; Christakopoulos et al.Carbohydrate Research, 1997, 302:191-195)

4. The influence of different metal ion chemical reagents on the enzyme activity of xylanase CtXyn10A was determined as follows:

Different concentrations of different metal ions and chemical reagents were added to the enzymatic reaction system to study the effects of various chemical reagents on the activity of xylanase CtXyn10A. The final concentration of various substances was 5mmol/L. Enzyme activity was measured at 70°C and pH 6.5. The results are shown in Table 1. Most of the ions and chemical reagents have no obvious effect on the activity of recombinant xylanase CtXyn10A at a concentration of 5 mmol, and SDS has a partial inhibitory effect. Mn ²⁺ can increase the recombinase activity to 1.32 times at 5mmol. The experimental results show that CtXyn10A has better chemical resistance, and adding a small amount of Mn ²⁺ properly can increase the enzyme activity, thereby reducing the application cost.

5. Substrate specificity of xylanase CtXyn10A

Different substrates were added to the enzymatic reaction system to study the substrate specificity of xylanase CtXyn10A. Enzyme activity was measured at 70°C and pH 6.5. The results are shown in Table 2. The xylanase CtXyn10A has the activities of xylanase, cellulase and glucanase at the same time.

The results of this experiment show that xylanase CtXyn10A has broad substrate specificity. In addition to acting on xylan, it can also catalyze the degradation of various substrates such as cellulose and dextran, so it has more potential in the industrial field. Wide application prospects.

Table 1

Table 2

substrate Relative enzyme activity (%) Beech xylan (1%) 100.0 Soluble Wheat Arabinoxylan (1%) 130.6 Birch xylan (1%) 88.1 Insoluble Wheat Arabinoxylan (1%) 49.0 CMC (1%) 31.7 Lichenan (1%) 24.6 Barley Glucan (0.5%) 8.9

6. The analysis of xylanase CtXyn10A degrading beech xylan products is as follows:

100 μL of purified enzyme solution was added to 500 μL of 1% xylan, and incubated at the optimum temperature for 12 hours. The enzyme protein was precipitated with absolute ethanol, and the supernatant was analyzed with a 2500 chromatograph using high performance anion exchange chromatography-pulsed amperometric (HPAEC-PAD) detection method to analyze the sugar species in the product. The analysis results showed that the products of beech xylan degradation by xylanase CtXyn10A were mainly xylobiose and xylomonose. The content of xylobiose in the product is 68%, and the content of xylomonose is 24%. The results of this experiment show that xylanase CtXyn10A can be used to produce related products such as xylobiose and xylomonose.

sequence listing

<110> Institute of Feed, Chinese Academy of Agricultural Sciences

<120> A neutral high-temperature xylanase, its coding gene and its application

<160>5

<210>1

<211>1202

<212> DNA

<213> Cladosporium tianshanense SL-14

<400>1

atgcgtttca ctgaggtctt cactgctctt acgctggccg cttcggcggt tgcccaccct 60

tcggctccca aggacaagaa gggtttggcc actgctatga aggctcgtgg aagggagttt 120

atcggcacag cccttacact gtacgtggtt gattgatctc tcaaggattt cgactttttt 180

gctgacttca agattccagt cgcggcaacg agaccgaaga agccattgct cgcaacaacg 240

ccgacttcaa ctctttcacg ccagagaatg caatgaagtg ggaggccatt gagcctaacc 300

gcaacaactt caccttcagc gacgccgacc gctaccgcga ctgggccaag gccaataaga 360

aggaaatcca ctgccaacact cttgtctggc actctcagct ccctccttgg gtcgctgctg 420

gcaactacga caacaagacc ctgataggga tcatggaaaa ccacatcaag aacgtggctg 480

gccgctacaa ggacgtgtgc acccgctggg agtaagtgga cccgactttt ttttctcgac 540

tttcgacgaa cttttttccg gacttgcaac gaaccatcaa ctaacaccat aacagcgtag 600

tcaacgaggc gttggaggag gacggtacct accgctcctc acccttctac gacaccatcg 660

gcgaagcttt catcccaatc gccttcaaat tcgccaagaa gtacagcccc aagtccgagc 720

tcttctacaa cgactacaac ctcgagtaca atggcaacaa gaccctcggc gccaagcgca 780

tcgtcaagct ggtccagagc tacggcgtgc acatcgacgg cgtgggtctg caagcccact 840

tggcccagga agtcaccccg accgccggcg ccctgcccga ccaagccact ctcgagaccg 900

ttctcagagg cttcacttcc ctggacgtcg atgttgttta caccgagatt gatatccgca 960

tgaacaccccc cagcaccccg gccaagctca agacacaggc caaggctttc gagactgttg 1020

ctcgcagctg tcttgctgtc aagaggtgca ttggaatgac cgtttggggt atttcagacg 1080

ctttctcgtg gatccctggt gtattccctg gtgagggcgc tgcgcttctt tgggacgaga 1140

accttaagaa gaagccggct tacgatggct tctacaaggg catcaagggt ggcaagaagt 1200

ga 1202

<210>2

<211>1059

<212> DNA

<213> Cladosporium tianshanense SL-14

<400>2

atgcgtttca ctgaggtctt cactgctctt acgctggccg cttcggcggt tgcccaccct 60

tcggctccca aggacaagaa gggtttggcc actgctatga aggctcgtgg aagggagttt 120

atcggcacag cccttacact tcgcggcaac gagaccgaag aagccattgc tcgcaacaac 180

gccgacttca actctttcac gccagagaat gcaatgaagt gggaggccat tgagcctaac 240

cgcaacaact tcaccttcag cgacgccgac cgctaccgcg actgggccaa ggccaataag 300

aaggaaatcc actgccacac tcttgtctgg cactctcagc tccctccttg ggtcgctgct 360

ggcaactacg acaacaagac cctgataggg atcatggaaa accacatcaa gaacgtggct 420

ggccgctaca aggacgtgtg cacccgctgg gacgtagtca acgaggcgtt ggaggaggac 480

ggtacctacc gctcctcacc cttctacgac accatcggcg aagctttcat cccaatcgcc 540

ttcaaattcg ccaagaagta cagccccaag tccgagctct tctacaacga ctacaacctc 600

gagtacaatg gcaacaagac cctcggcgcc aagcgcatcg tcaagctggt ccagagctac 660

ggcgtgcaca tcgacggcgt gggtctgcaa gccccacttgg cccaggaagt caccccgacc 720

gccggcgccc tgcccgacca agccactctc gagaccgttc tcagaggctt cacttccctg 780

gacgtcgatg ttgtttacac cgagattgat atccgcatga acacccccag caccccggcc 840

aagctcaaga cacaggccaa ggctttcgag actgttgctc gcagctgtct tgctgtcaag 900

aggtgcattg gaatgaccgt ttggggtatt tcagacgctt tctcgtggat ccctggtgta 960

ttccctggtg agggcgctgc gcttctttgg gacgagaacc ttaagaagaa gccggcttac 1020

gatggcttct acaagggcat caagggtggc aagaagtga 1059

<210>3

<211>352

<212> PRT

<213> Cladosporium tianshanense SL-14

<400>3

Met Arg Phe Thr Glu Val Phe Thr Ala Leu Thr Leu Ala Ala Ser Ala

1 5 10 15

Val Ala His Pro Ser Ala Pro Lys Asp Lys Lys Gly Leu Ala Thr Ala

20 25 30

Met Lys Ala Arg Gly Arg Glu Phe Ile Gly Thr Ala Leu Thr Leu Arg

35 40 45

Gly Asn Glu Thr Glu Glu Ala Ile Ala Arg Asn Asn Ala Asp Phe Asn

50 55 60

Ser Phe Thr Pro Glu Asn Ala Met Lys Trp Glu Ala Ile Glu Pro Asn

65 70 75 80

Arg Asn Asn Phe Thr Phe Ser Asp Ala Asp Arg Tyr Arg Asp Trp Ala

85 90 95

Lys Ala Asn Lys Lys Glu Ile His Cys His Thr Leu Val Trp His Ser

100 105 110

Gln Leu Pro Pro Trp Val Ala Ala Gly Asn Tyr Asp Asn Lys Thr Leu

115 120 125

Ile Gly Ile Met Glu Asn His Ile Lys Asn Val Ala Gly Arg Tyr Lys

130 135 140

Asp Val Cys Thr Arg Trp Asp Val Val Asn Glu Ala Leu Glu Glu Asp

145 150 155 160

Gly Thr Tyr Arg Ser Ser Pro Phe Tyr Asp Thr Ile Gly Glu Ala Phe

165 170 175

Ile Pro Ile Ala Phe Lys Phe Ala Lys Lys Tyr Ser Pro Lys Ser Glu

180 185 190

Leu Phe Tyr Asn Asp Tyr Asn Leu Glu Tyr Asn Gly Asn Lys Thr Leu

195 200 205

Gly Ala Lys Arg Ile Val Lys Leu Val Gln Ser Tyr Gly Val His Ile

210 215 220

Asp Gly Val Gly Leu Gln Ala His Leu Ala Gln Glu Val Thr Pro Thr

225 230 235 240

Ala Gly Ala Leu Pro Asp Gln Ala Thr Leu Glu Thr Val Leu Arg Gly

245 250 255

Phe Thr Ser Leu Asp Val Asp Val Val Tyr Thr Glu Ile Asp Ile Arg

260 265 270

Met Asn Thr Pro Ser Thr Pro Ala Lys Leu Lys Thr Gln Ala Lys Ala

275 280 285

Phe Glu Thr Val Ala Arg Ser Cys Leu Ala Val Lys Arg Cys Ile Gly

290 295 300

Met Thr Val Trp Gly Ile Ser Asp Ala Phe Ser Trp Ile Pro Gly Val

305 310 315 320

Phe Pro Gly Glu Gly Ala Ala Leu Leu Trp Asp Glu Asn Leu Lys Lys

325 330 335

Lys Pro Ala Tyr Asp Gly Phe Tyr Lys Gly Ile Lys Gly Gly Lys Lys

340 345 350

<210>4

<211>18

<212> PRT

<213> Cladosporium tianshanense SL-14

<400>4

Met Arg Phe Thr Glu Val Phe Thr Ala Leu Thr Leu Ala Ala Ser Ala

1 5 10 15

Val Ala

<210>5

<211>334

<212> PRT

<213> Cladosporium tianshanense SL-14

<400>5

His Pro Ser Ala Pro Lys Asp Lys Lys Gly Leu Ala Thr Ala Met Lys

1 5 10 15

Ala Arg Gly Arg Glu Phe Ile Gly Thr Ala Leu Thr Leu Arg Gly Asn

20 25 30

Glu Thr Glu Glu Ala Ile Ala Arg Asn Asn Ala Asp Phe Asn Ser Phe

35 40 45

Thr Pro Glu Asn Ala Met Lys Trp Glu Ala Ile Glu Pro Asn Arg Asn

50 55 60

Asn Phe Thr Phe Ser Asp Ala Asp Arg Tyr Arg Asp Trp Ala Lys Ala

65 70 75 80

Asn Lys Lys Glu Ile His Cys His Thr Leu Val Trp His Ser Gln Leu

85 90 95

Pro Pro Trp Val Ala Ala Gly Asn Tyr Asp Asn Lys Thr Leu Ile Gly

100 105 110

Ile Met Glu Asn His Ile Lys Asn Val Ala Gly Arg Tyr Lys Asp Val

115 120 125

Cys Thr Arg Trp Asp Val Val Asn Glu Ala Leu Glu Glu Asp Gly Thr

130 135 140

Tyr Arg Ser Ser Pro Phe Tyr Asp Thr Ile Gly Glu Ala Phe Ile Pro

145 150 155 160

Ile Ala Phe Lys Phe Ala Lys Lys Tyr Ser Pro Lys Ser Glu Leu Phe

165 170 175

Tyr Asn Asp Tyr Asn Leu Glu Tyr Asn Gly Asn Lys Thr Leu Gly Ala

180 185 190

Lys Arg Ile Val Lys Leu Val Gln Ser Tyr Gly Val His Ile Asp Gly

195 200 205

Val Gly Leu Gln Ala His Leu Ala Gln Glu Val Thr Pro Thr Ala Gly

210 215 220

Ala Leu Pro Asp Gln Ala Thr Leu Glu Thr Val Leu Arg Gly Phe Thr

225 230 235 240

Ser Leu Asp Val Asp Val Val Tyr Thr Glu Ile Asp Ile Arg Met Asn

245 250 255

Thr Pro Ser Thr Pro Ala Lys Leu Lys Thr Gln Ala Lys Ala Phe Glu

260 265 270

Thr Val Ala Arg Ser Cys Leu Ala Val Lys Arg Cys Ile Gly Met Thr

275 280 285

Val Trp Gly Ile Ser Asp Ala Phe Ser Trp Ile Pro Gly Val Phe Pro

290 295 300

Gly Glu Gly Ala Ala Leu Leu Trp Asp Glu Asn Leu Lys Lys Lys Lys Pro

305 310 315 320

Ala Tyr Asp Gly Phe Tyr Lys Gly Ile Lys Gly Gly Lys Lys

325 330

Claims (10)

1. A protein, characterized in that, the protein is the protein described in 1), 2) or 3) as follows: 1) A protein having the amino acid sequence shown in SEQ ID No. 3 in the sequence listing; 2) A protein having the amino acid sequence shown in SEQ ID No. 5 in the sequence listing; 3) The amino acid residue sequence shown in SEQ ID No. 3 and/or SEQ ID No. 5 in the sequence listing undergoes substitution and/or deletion and/or addition of one or several amino acid residues and has xylan Enzymatically active proteins derived from 1) and/or 2). 2. A coding gene, characterized in that the coding gene has one of the following nucleotide sequences: 1) a polynucleotide sequence encoding the protein of claim 1; 2) the nucleotide sequence shown in the 55th-1059th position of SEQ ID No.: 1, SEQ ID No.: 2, and/or SEQ ID No.: 2 in the sequence listing; 3) The polynucleotide sequence encoding the protein sequence of SEQ ID No.: 3 and/or SEQ ID No. 5 in the sequence listing; 4) a nucleotide sequence that can hybridize to any of the DNA sequences defined in 1), 2), and/or 3) under high stringency conditions; 5) A DNA sequence having more than 90% homology with any of the DNA sequences defined in 1), 2), 3), and/or 4) and encoding the same functional protein. 3. A carrier, an expression cassette, a transgenic cell line, a recombinant bacterium, and/or a microorganism, characterized in that, said carrier, an expression cassette, a transgenic cell line, a recombinant bacterium, and/or a microorganism contain the coding genes. 4. A pair of primers, characterized in that the pair of primers can amplify the full length of the coding gene of claim 2 or any fragment thereof. 5. A method for preparing a protein and the protein prepared by said method, characterized in that said method comprises: 1) preparing the coding gene described in claim 2; 2) preparing a recombinant expression vector comprising the coding gene described in step 1); 3) Expressing the expression vector described in step 2) to obtain the target protein. 6. The protein of claim 1, the coding gene of claim 2, the recombinant vector, expression cassette, transgenic cell line, recombinant bacteria, and/or microorganism of claim 3, the primer pair of claim 4 , the preparation method of claim 5 and the application of the protein prepared by the preparation method. 7. The application according to claim 6, characterized in that the application includes at least one of the following 1)-8): 1) Application in the preparation of xylanase and/or related products containing xylanase; 2) Application in the preparation of xylanase mutants and/or related products containing xylanase mutants; 3) Application in the preparation of recombinant xylanase and/or related products containing recombinant xylanase; 4) Application in the preparation of xylooligosaccharides, xylobiose, xylomonose, xylooligosaccharide-containing, xylobiose-containing, and/or xylomonoose-containing related products; 5) as cellulase, dextranase and/or in the preparation of related products with cellulase and dextranase enzyme activity; 6) Application in degrading xylan, cellulose, and/or glucan; 7) Application in the preparation of products used in the fields of industry, agriculture, food, medicine, feed, energy, waste treatment and/or environmental protection; 8) Applications in the fields of industry, agriculture, food, medicine, feed, energy, waste treatment and/or environmental protection. 8. The application according to claim 7, wherein the xylanase comprises the protein of claim 1 or claim 5; the xylanase mutant comprises the protein of claim 1 or claim 5 The protein obtained by point mutation of the protein; the recombinant xylanase includes the protein obtained by homologous recombination of the protein described in claim 1 or claim 5 . 9. The application according to any one of claims 6, 7, and/or 8, characterized in that, the application includes applications under the conditions described in the following 1), 2), 3), and/or 4) : 1) pH includes 4.0-12.0; 2) The temperature includes 0°C-90°C; 3) An environment including metal ions, and/or chemical reagents; 4) Substrates include xylan, cellulose, and/or dextran. 10. A method for preparing xylanase, characterized in that the method comprises: extracting xylanase from Cladosporiumtianshanense SL-14.