CN107129976B - Xylanase, coding gene thereof and application thereof - Google Patents

Abstract

The invention provides a neutral high temperature xylanase CtXyn10A and its gene and application. The neutral high temperature xylanase CtXyn10A provided by the present invention is a protein with the amino acid sequence shown in SEQ ID No. 3 and/or SEQ ID No. 5 in the sequence listing, and/or the SEQ ID No. .3 and/or the amino acid residue sequence shown in SEQ ID No. 5 has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has xylanase activity from the SEQ ID NO. A protein derived from the protein with the amino acid sequence shown in ID No. 3 and/or SEQ ID No. 5. The optimum pH of the xylanase CtXyn10A of the present invention is 6.0-7.0, the optimum temperature is 70° C., the enzyme activity is maintained at more than 20% at 90° C. and pH 9.0, and the specific activity is 461 U/mg; The enzyme CtXyn10A has the activities of xylanase, glucanase and cellulase, and is easy to be produced by industrial fermentation. As a new type of broad-spectrum enzyme preparation, it can be widely used in food, papermaking, energy industries, etc.

Description

A kind of xylanase, its encoding gene and its application

technical field

The invention belongs to the field of genetic engineering, in particular to a xylanase, its encoding gene and its application.

Background technique

Xylan is the second most abundant major component of plant biomass in nature after cellulose (Lynd et al. Microbiology and Molecular Biology Review, 2002, 66: 506-577), and in the field of bioenergy There is potential use value. Existing biotechnology can efficiently degrade cellulosic components in plants and convert them into fermentable glucose, which generates bioethanol under the action of yeast. High-component xylan not only limits the binding reaction of cellulase and cellulose, inhibits the catalytic reaction, but also can cause nutrient enrichment and damage the ecosystem when it is discharged into nature as a by-product (Polizeli et al. Applied Microbiology and Biotechnology, 2005, 67:577-591). Therefore, in the process of biomass biotransformation, 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 generated xylo-oligosaccharides can also be further degraded into fermentable xylose, providing 10-25% of the raw material for the production of bioethanol (Jin et al. . Applied and Environmental Microbiology, 2004, 70: 6816-6825).

Based on 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 family 11 xylanases that specifically degrade xylan, family 10 xylanases have broad substrate specificity. In addition to acting on xylan, they can also catalyze cellulose, glucan The degradation of various substrates such as sugars has wider application prospects in the industrial field.

The existing bioethanol processing technology includes acid-base pretreatment of lignocellulose and 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 neutral and high-temperature lignocellulose-degrading enzymes is of great significance in terms of energy saving, environmental protection, and easy catalytic reactions (Ji et al. Biotechnology for Biofuels, 2014, 7: 130).

SUMMARY 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 the following 1), 2), or 3):

1) a protein with the amino acid sequence shown in SEQ ID NO.3 in the sequence listing;

2) a protein with 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 is replaced and/or deleted and/or added by 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; wherein, the 18 amino acids at the N-terminal 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 Ctxyn10A mature protein.

The Ctxyn10A protein in the above 1), 2) and 3) can be artificially synthesized, or its encoding gene can be synthesized first, and then biologically expressed. The coding gene of the Ctxyn10A protein in the above 1), 2) and 3) can be obtained by adding the 55th position-1059 of SEQ ID NO.1, SEQ ID NO.2, and/or SEQ ID NO.2 in the sequence listing The DNA sequence shown in the nucleotide sequence shown at the position is obtained by deletion of codons of one or several amino acid residues, and/or missense mutation of one or several base pairs.

The nucleic acid molecule encoding the Ctxyn10A protein also belongs 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 position 55-1059 of SEQ ID NO.1, SEQ ID NO.2, and/or SEQ ID NO.2 in the sequence listing;

3) a 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 hybridizes to any of the defined DNA sequences 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 hybridized at 65°C with a solution of 6×SSC, 0.5% SDS, and then washed once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS each.

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

Still another object of the present invention is to provide a vector, expression cassette, transgenic cell line, recombinant bacteria, and/or microorganism, said vector, expression cassette, transgenic cell line, recombinant bacteria, and/or microorganism containing said 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, Saccharomyces cerevisiae, and/or S. polymorpha; more specifically, the starting bacteria of the recombinant bacteria are Pichia GS115; more specifically, the The recombinant bacteria is specifically GS115/Ctxyn10A; the microorganisms include Cladosporium tianshanense SL-14;

The recombinant expression vector can be constructed using existing expression vectors. The expression vector may also contain the 3' untranslated region of the exogenous gene, i.e., containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The poly(A) signal directs the addition of poly(A) to the 3' end of the mRNA precursor. 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, which can be used alone or with other promoters. Combined use; in addition, enhancers, including translation enhancers or transcription enhancers, can also be used when using the gene of the present invention to construct a recombinant expression vector. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, GFP gene, luciferase) that express enzymes that can produce color changes or luminescent compounds in microorganisms Gene, etc.), antibiotic markers with resistance (gentamycin marker, kanamycin marker, etc.) or anti-chemical reagent marker gene (such as herbicide resistance gene) and so on. Considering the safety of GMOs, transformants can be directly screened under stress without adding any selectable marker genes.

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

Still another object of the present invention is to provide a kind of preparation method of protein, described method comprises:

1) prepare the coding gene of the present invention;

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

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

Specifically, the method further 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 The microorganisms include Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus, and/or Trichoderma; more specifically, the microorganisms include Pichia, Saccharomyces cerevisiae, and/or S. polymorpha; more specifically, the The microorganism is Pichia pastoris GS115; specifically, the recombinant expression vector includes pPIC-Ctxyn10A.

The protein prepared by the protein preparation method also belongs to 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 the glycosylation structure.

Yet another object of the present invention is to provide the protein, encoding gene, recombinant vector, expression cassette, transgenic cell line, recombinant bacteria, and/or microorganism, primer pair, protein preparation method, and protein preparation method of the present invention. protein applications.

Specifically, the application includes the application in 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 xylo-oligosaccharide, xylobiose, xylo-monose, containing xylo-oligosaccharide, containing xylobiose, and/or containing xylo-monose related products;

5) as cellulase, glucanase and/or preparation of related products with cellulase, glucanase activity;

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

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

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

Specifically, the xylan includes beech xylan, soluble wheat arabinoxylan, birch xylan, and insoluble wheat arabinoxylan; the cellulose includes carboxymethyl cellulose, lichenin; the Glucans include barley glucans.

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 obtained by point mutation; the recombinant xylanase includes 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 the 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℃-90℃;

3) 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; 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 Ni2+, Co2+, Na+, Cr3+, Mg2+, Mn2+, K+, Cu2+, Ca2+, Pb2+, Zn2+, Fe3+, β-mercaptoethanol, EDTA, SDS; the concentrations of the metal ions and/or chemical reagents include 5mmol/L; the xylan includes Beech xylan, soluble wheat arabinoxylan, birch xylan, insoluble wheat arabinoxylan; the cellulose includes carboxymethyl cellulose, lichen polysaccharide; the glucan includes barley glucan.

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 present invention has the characteristics of high activity in the range of neutral (pH 5.0-8.0) and high temperature (50°C-75°C). In the present invention, the xylanase CtXyn10A produced by Cladosporium tianshanense SL-14 (ACCC 32710 of China Agricultural Culture Collection Center) is screened, and its optimum pH value is 6.0-7.0, in the range of pH5.0-8.0 It maintains more than 80% of the enzyme activity; the optimum temperature is 70 °C, and it has more than 70% enzyme activity at 60-75 °C; 10% enzyme activity at 0 °C; , dextran, cellulose have degradation. Neutral high temperature xylanases of this nature have not been reported.

The invention overcomes the deficiencies of the prior art, obtains the bacterial resources of high-temperature xylanase, and provides a new neutral high-temperature xylanase CtXyn10A with excellent properties and suitable for application in food, paper and energy industries. . The optimum pH of the xylanase CtXyn10A of the invention is 6.0-7.0, and it has high enzymatic activity at pH 5.0-8.0; pH stability is good; specific activity is 461 U/mg; and it has broad substrate specificity. Its neutral high temperature can reduce the energy cost of xylanase in industrial production. The pH has a wide range of adaptation, which can improve the digestible energy and metabolizable energy of aquatic feed, reduce the cost of formula, 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. Xylanase CtXyn10A can be used in brewing industry to reduce the viscosity of materials, which can help amylase to act on the starch layer, improve the utilization rate of starch and increase the yield of alcohol. It is also possible to convert xylan in paper industry wastes and agricultural wastes into xylo-oligosaccharides under normal temperature conditions, and xylo-oligosaccharides can be converted into valuable fuels by bacteria, yeast and fungi. Therefore, the application of the xylanase CtXyn10A in the energy industry also shows its great potential. Xylanase CtXyn10A hydrolysates (xylose and xylo-oligosaccharides) can be used in the food industry as thickeners, fat substitutes, prebiotic oligosaccharides and antifreeze food additives; in the pharmaceutical industry xylan and other substances Used in combination, it can delay the release of pharmaceutical ingredients. Xylanases and their hydrolyzates 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 the deglycosylated purified recombinant xylanase; lane 2 is the purified recombinant xylanase; lane 3 is the low molecular weight protein Marker.

Figure 2 is a graph showing the results of the optimal pH determination of the recombinant xylanase CtXyn10A.

Figure 3 is a graph showing the results of pH stability assay of recombinant xylanase CtXyn10A.

FIG. 4 is a graph showing the results of measuring the optimum temperature of recombinant xylanase CtXyn10A.

Figure 5 is a graph showing the results of thermostability assay of recombinant xylanase CtXyn10A.

Detailed ways

illustrate:

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

The molecular biology experimental methods that are not specified in the following examples are all carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or according to the kits and products. Instruction manual.

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

Test materials and reagents:

1, bacterial strain and carrier: 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, Chinese Academy of Agricultural Sciences Agricultural Culture Collection Center, 100081), its deposit number is: ACCC32710, the public can buy this strain from the center. Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen.

2. Enzymes and other biochemical reagents: Endonuclease was purchased from TaKaRa Company, and ligase was purchased from Invitrogen Company. Beech xylan was purchased from Sigma Company, and the others were all domestic reagents (all available from common biochemical reagent companies).

3. Culture medium:

(1) Cladosporium tianshanense SL-14 medium is potato juice medium: 1000 mL potato juice, 10 g glucose, 25 g agar, pH 5.0.

(2) E. coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).

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

(4) BMMY medium: divided by 0.5% methanol (V/V) instead of glycerol, other components are the same as BMGY, pH 4.0.

Example 1. Cloning of genomic DNA of neutral high temperature xylanase CtXyn10A

Extract the genomic DNA of Cladosporium tianshanense SL-14:

The mycelium that had been cultured in liquid for 3 days was filtered with sterile filter paper and put into a mortar, 2 mL of extract was added, and ground for 5 minutes, then the ground liquid was placed in a 50 mL centrifuge tube, and the solution was split in a 65°C water bath for 20 minutes, and the mixture was mixed every 10 minutes. 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 the supernatant was added with an equal volume of isopropanol, and after standing at room temperature for 5 min, centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried under vacuum, dissolved by adding an appropriate amount of TE, and placed at -20°C for later use.

Design and synthesize specific primers Ctxyn10A-F/Ctxyn10A-R according to the sequence analysis of Cladosporium tianshanense SL-14 genome frame diagram:

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

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

The total DNA of Cladosporium tianshanense SL-14 was used as a template, and the above-mentioned Ctxyn10A-F/Ctxyn10A-R were used as specific primers for PCR amplification.

The PCR reaction parameters 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 incubation at 72°C for 10 min after 30 cycles.

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

The 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 listing, with a total of 1202 bp. The nucleic acid fragment having the nucleotide sequence shown in SEQ ID NO. 1 in the sequence listing was named Ctxyn10A.

Example 2. Acquisition of the CtXyn10A Gene Encoding Mature Neutral High Temperature Xylanase

Extract the total RNA of Cladosporium tianshanense SL-14, use reverse transcriptase to obtain a strand of cDNA, and then amplify the single-stranded cDNA with primers Ctxyn10A-F/Ctxyn10A-R, and the amplified product is recovered and sent to three Sequencing 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 and the sequencing result of the PCR amplification product, it was found that the gene has 2 introns, the cDNA is 1059 bp long, and has the nucleotide of SEQ ID NO. 2 in the sequence table. Sequence, encoding the amino acid sequence shown in SEQ ID NO. 3 in the Sequence Listing and a stop codon. A protein with the amino acid sequence shown in SEQ ID NO.3 in the sequence listing, a total of 352 amino acid residues, the 18 amino acids at the N-terminal are the predicted signal peptide sequence, with the amino acid sequence shown in SEQ ID NO.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 the intron sequences of 59bp and 84bp respectively

The sequencing result of the above-mentioned PCR amplification product shows that the nucleic acid fragment obtained by the above-mentioned PCR amplification includes the EcoRI+NotI double-enzyme cleavage site, and the middle of the above-mentioned two enzyme cleavage sites is the 55th position-1059 with SEQ ID NO.2 in the sequence table. A nucleic acid fragment of a nucleotide sequence.

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

Example 3. Preparation of neutral high temperature xylanase CtXyn10A

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

Sequencing verifies the correctness of the sequence. The sequence of the foreign gene inserted in the obtained recombinant plasmid is nucleotides 55-1059 of SEQ ID NO. 2, and the recombinant plasmid is named pPIC-Ctxyn10A.

The above recombinant plasmid pPIC-Ctxyn10A was transformed into Pichia GS115; the positive recombinant plasmid was extracted by sequencing to verify the correctness of the sequence, and the correctly sequenced recombinant Pichia strain containing the recombinant plasmid pPIC-Ctxyn10A was named GS115/Ctxyn10A.

The recombinant strain GS115/Ctxyn10A was taken, inoculated into 300 mL of BMGY medium, cultured with shaking at 250 rpm at 30°C for 48 hours, and then centrifuged to collect the bacterial cells. Then resuspended in 100 mL of BMMY medium, and cultured with shaking at 250 rpm 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. The results of Figure 1 show that the xylanase CtXyn10A is expressed in Pichia pastoris recombinant strain GS115/Ctxyn10A. After the expressed xylanase CtXyn10A is purified, the content of its target protein reaches more than 90% of the total protein, and reaches electrophoresis purity. After deglycosylation by ENDO-H, the molecular weight of the purified protein was 37.4 kDa, which was consistent with the theoretical value.

Example 4. Enzymatic activity assay of neutral high temperature xylanase CtXyn10A

The activity of the xylanase CtXyn10A prepared in the above Example 3 was determined by DNS method. The specific method is as follows: under the conditions of pH 6.5 and 70 °C, 1 mL of the reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, and the reaction After 10 min, 1.5 mL of DNS was added to stop the reaction, and boiled for 5 min. The OD value was measured at 540 nm after cooling. One unit of enzymatic activity (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.

Example 5. Determination of properties of neutral high temperature xylanase CtXyn10A

1. The optimal pH and pH stability determination methods of recombinant xylanase CtXyn10A are as follows:

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

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

The optimal temperature of xylanase was determined by carrying out the enzymatic reaction in a citric acid-disodium hydrogen phosphate buffer (pH6.5) buffer system and at different temperatures. The temperature resistance was measured by treating the xylanase at different temperatures for different times, and then measuring the enzyme activity at pH 6.5 and 70°C. The results of the optimal temperature determination of the enzyme reaction are shown in Figure 4. The optimal temperature of the xylanase CtXyn10A is 70 °C, and the specific activity is 461 U/mg; it has high activity in the high temperature range of 50 °C-75 °C; The enzyme activity is more than 70% at -75°C; there is still 10% enzyme activity at 0°C; the thermal stability test results of the enzyme are shown in Figure 5 (the result shown at 80°C in Figure 5 is the substrate reaction time 10 minutes; as shown in Figure 4, the enzyme activity was measured at 80°C, the results and expressions were incubated at 80°C without substrate for a certain period of time), the xylanase CtXyn10A was very stable at 60°C, and was incubated at 70°C for 60 minutes, completely Loss of enzymatic activity.

In addition, the enzymatic activity of xylanase CtXyn10A was also measured at 90°C and pH 9.0, and the results showed that it maintained more than 20% of the enzymatic activity.

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

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

Using different concentrations of beech xylan as the substrate, in the citric acid-disodium hydrogen phosphate buffer (pH 6.5) 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 was determined that the km value of this xylanase CtXyn10A using beech xylan as a substrate at 70 °C was 0.64 mg/mL, the maximum reaction rate Vmax was 450 μmol/min·mg, the kcat value was 281/s and kcat/ The Km value is 434ml/mg·s. The experimental results show that CtXyn10A has a strong affinity with the substrate, and has a high catalytic efficiency under high temperature conditions. Its enzymatic properties are much better than those of fungal xylanases with similar sequences. FoXyn10A (optimum pH 7.0, optimum temperature 45°C, Km 0.8 mg/mL, Vmax 1.22 μmol/min·mg; Christakopoulos et al. Carbohydrate Research, 1997, 302: 191-195)

4. The effect of different metal ion chemical reagents on the enzymatic 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 each substance was 5mmol/L. The enzyme activity was measured at 70°C and pH 6.5. The results are shown in Table 1. Most ions and chemical reagents had no significant effect on the activity of recombinant xylanase CtXyn10A at a concentration of 5 mmol, and SDS had a partial inhibitory effect. Mn2+ can increase the activity of recombinase to 1.32 times at 5mmol. The experimental results show that CtXyn10A has good chemical resistance, and appropriate addition of a small amount of Mn2+ can also 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 the xylanase CtXyn10A. The 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 experimental results show that the 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 glucan. Therefore, it has more advantages in the industrial field. Broad application prospects.

Table 1

Table 2 6. The analysis of xylanase CtXyn10A degradation products of beech wood xylan 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 h. The enzyme protein was precipitated with absolute ethanol, and the supernatant was analyzed by a 2500 chromatograph using a high performance anion exchange chromatography-pulsed amperometric (HPAEC-PAD) detection method. The analysis results showed that the main products of beech xylan degradation by xylanase CtXyn10A were xylobiose and xylomonose. The content of xylobiose in the product is 68%, and the content of xylomonose is 24%. The experimental results show that the xylanase CtXyn10A can be used to produce related products such as xylobiose and xyloose.

sequence listing

<110> Feed Institute, Chinese Academy of Agricultural Sciences

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

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<211>1202

<212> DNA

<213> Cladosporium tianshanense SL-14

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atcggcacag cccttacact gtacgtggtt gattgatctc tcaaggattt cgactttttt 180

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ccgacttcaa ctctttcacg ccagagaatg caatgaagtg ggaggccatt gagcctaacc 300

gcaacaactt caccttcagc gacgccgacc gctaccgcga ctgggccaag gccaataaga 360

aggaaatcca ctgccacact cttgtctggc actctcagct ccctccttgg gtcgctgctg 420

gcaactacga caacaagacc ctgataggga tcatggaaaa ccacatcaag aacgtggctg 480

gccgctacaa ggacgtgtgc acccgctggg agtaagtgga cccgactttt ttttctcgac 540

tttcgacgaa ctttttttccg 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

tgaacacccc 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 gcccacttgg 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 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 Pro

305 310 315 320

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

325 330

Claims (5)

1. A xylanase, wherein the amino acid sequence of the xylanase is shown in SEQ ID No: 3 or SEQ ID No: 5. 2 . A xylanase gene, characterized in that it encodes the xylanase of claim 1 . 3 . 3. A recombinant expression vector comprising the xylanase gene of claim 2. 4. a method for preparing xylanase, it is characterised in that the method comprises the following steps: (1) introducing the recombinant expression vector of claim 3 into a host cell; (2) Induce the expression of xylanase; (3) Separating and purifying the xylanase. 5. The application of the xylanase of claim 1 in hydrolyzing xylan.

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