CN107129976B

CN107129976B - Xylanase, coding gene thereof and application thereof

Info

Publication number: CN107129976B
Application number: CN201710409349.4A
Authority: CN
Inventors: 姚斌; 马锐; 柏映国; 罗会颖; 黄火清; 苏小运; 王苑; 涂涛; 王亚茹; 孟昆
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Animal Science of CAAS
Priority date: 2017-06-02
Filing date: 2017-06-02
Publication date: 2020-07-14
Anticipated expiration: 2037-06-02
Also published as: CN107129976A

Abstract

The invention provides a neutral high-temperature xylanase CtXyn10A, a gene and application thereof. The neutral high-temperature xylanase CtXyn10A provided by the invention is a protein with an amino acid sequence shown in SEQ ID No.3 and/or SEQ ID No.5 in a sequence table, and/or a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residue sequence shown in SEQ ID No.3 and/or SEQ ID No.5 in the sequence table, has xylanase activity and is derived from the protein with the amino acid sequence shown in SEQ ID No.3 and/or SEQ ID No.5 in the sequence table. The xylanase CtXyn10A of the invention has the optimum pH value of 6.0-7.0, the optimum temperature of 70 ℃, the enzyme activity of more than 20 percent is kept at 90 ℃ and pH value of 9.0, and the specific activity is 461U/mg; the xylanase CtXyn10A has the activities of xylanase, glucanase and cellulase, is easy for industrial fermentation production, is used as a novel broad-spectrum enzyme preparation, and can be widely used in food, paper making, energy industry and the like.

Description

Xylanase, coding gene thereof and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to xylanase, a coding gene and application thereof.

Background

Xylan (xylan) is the second most abundant plant biomass in nature, second only to cellulose (L yd et al. Microbiology and Molecular Biology Review, 2002, 66: 506-577), with potential utility in terms of bioenergy, existing Biotechnology can efficiently degrade the cellulose component in plants and convert it into fermentable glucose, which produces bioethanol under the action of yeast. high-component xylans not only limit the binding reaction of cellulase to cellulose, inhibit catalytic reactions, but also as a by-product, discharge into nature can cause nutrient enrichment to destroy the ecosystem (Poleleli et al. applied Microbiology and Biotechnology, 2005, 67: 577-591.) thus in biomass bioconversion processes xylanase is usually added, on the one hand, the physical barrier of xylan is removed, facilitating the binding of cellulase to cellulose, on the other hand, xylose production is further promoted by xylanase et al. Biotechnology for xylose production, 2011: 14, and 14: 10-oligosaccharide production by fermentation, on the other hand, xylose production of biomass-produced oligosaccharide is also useful as biomass.

Based on the amino acid sequence and the structure of the catalytic domain, most xylanases (EC 3.2.1.8) are classified in

glycoside hydrolases families

10 and 11. Compared with xylanase of family 11 which specifically degrades xylan, xylanase of family 10 has wide substrate specificity, can act on xylan and can catalyze the degradation of various substrates such as cellulose, glucan and the like, thereby having wider application prospect in the industrial field.

The existing bioethanol processing technology comprises acid-base pretreatment of lignocellulose and long-time high-temperature enzyme reaction, thereby consuming a large amount of energy and acid-base chemical reagents, and causing great cost increase and serious environmental pollution. Therefore, the development of a neutral high-temperature lignocellulose degrading enzyme system has important significance in the aspects of energy conservation, environmental protection, easy catalytic reaction and the like (Ji et al. Biotechnology for Biofuels, 2014, 7: 130).

Disclosure of Invention

An object of the invention is to provide a protein, named Ctxyn10A, derived from Cladosporium tienshanense S L-14.

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

1) a protein having an amino acid sequence represented by SEQ ID NO.3 of the sequence Listing;

2) a protein having an amino acid sequence shown by SEQ ID NO.5 in the sequence Listing;

3) protein which is derived from 1) and/or 2) and has xylanase activity and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residue sequences shown in SEQ ID NO.3 and/or SEQ ID NO.5 in the sequence table.

The amino acid sequence shown by SEQ ID NO.3 in the sequence table consists of 352 amino acid residues; wherein, 18 amino acids at the N end are predicted signal peptide sequences and have amino acid sequences shown in SEQ ID NO.4 in a sequence table.

The amino acid sequence shown by SEQ ID NO.5 in the sequence table consists of 334 amino acid residues; namely Ctxyn10A mature protein.

The Ctxyn10A protein in the 1), 2) and 3) can be synthesized artificially, or can be obtained by synthesizing the coding gene and then carrying out biological expression. The coding gene of the Ctxyn10A protein in the 1), 2) and 3) can be obtained by deleting codons of one or more amino acid residues from DNA sequences shown by nucleotide sequences shown by the 55 th to 1059 th positions of SEQ ID NO.1, SEQ ID NO.2 and/or SEQ ID NO.2 in a sequence table and/or performing missense mutation of one or more base pairs.

The nucleic acid molecule for coding the Ctxyn10A protein also belongs to the protection scope of the invention.

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

It is still another object of the present invention to provide a coding gene, wherein the coding gene has one of the following nucleotide sequences:

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

2) a nucleotide sequence shown in the 55 th to 1059 th positions of SEQ ID NO.1, SEQ ID NO.2 and/or SEQ ID NO.2 in the sequence table;

3) a polynucleotide sequence encoding the protein sequence of SEQ ID NO.3 and/or SEQ ID NO.5 of the sequence list;

4) a nucleotide sequence that hybridizes to a DNA sequence defined in any one of 1), 2), and/or 3) under highly stringent conditions;

5) a DNA sequence which has more than 90% of homology with the DNA sequence defined in any one of 1), 2), 3) and/or 4) and encodes the same functional protein.

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

The high stringency conditions may be hybridization with 6 × SSC, 0.5% SDS solution at 65 ℃ followed by washing the membrane once with each of 2 × SSC, 0.1% SDS and 1 × SSC, 0.1% SDS.

Wherein, the nucleotide sequence shown in SEQ ID NO.1 in the sequence table is 1202bp in total, and comprises 2 intron sequences with the lengths of 59bp and 84bp respectively, and the 2 intron sequences respectively have a 141 th-199 th nucleotide sequence and a 512 th-595 th nucleotide sequence shown in the SEQ ID NO.1 in the sequence table; the cDNA with the 2 intron sequences removed has the length of 1059bp, has the nucleotide sequence of SEQ ID NO.2 in the sequence table, encodes the amino acid sequence shown by SEQ ID NO.3 in the sequence table and a stop codon, namely the Ctxyn10A protein; the nucleic acid fragment with the nucleotide sequence from 55 th site to 1059 th site of SEQ ID NO.2 in the sequence table encodes an amino acid sequence shown by SEQ ID NO.5 in the sequence table, namely the Ctxyn10A mature protein.

It is still another object of the present invention to provide a vector, an expression cassette, a transgenic cell line, a recombinant bacterium, and/or a microorganism, which contains the encoding gene.

The vector specifically comprises a recombinant cloning vector and a recombinant expression vector, and further specifically comprises pPIC-Ctxyn10A, wherein the starting bacteria of the recombinant bacteria comprise escherichia coli, yeast, bacillus, lactobacillus, aspergillus and/or trichoderma, and further specifically comprises pichia pastoris, beer yeast and/or polytypism yeast, and further specifically comprises pichia pastoris GS115, and further specifically comprises GS115/Ctxyn10A, and the microorganism comprises Cladosporium Cladoporium tianshanense S L-14;

the recombinant expression vector can be constructed using existing expression vectors. The expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added before the transcription initiation nucleotide, and can be used alone or combined with other promoters; in addition, when a recombinant expression vector is constructed using the gene of the present invention, an enhancer, including a translation enhancer or a transcription enhancer, may also be used. In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes expressing an enzyme or a luminescent compound which produces a color change in a microorganism (GUS gene, GFP gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin marker, kanamycin marker, etc.), or chemical-resistant marker genes (e.g., herbicide-resistant gene), etc. From the safety of transgenic organisms, the transformant can be screened directly in a stress environment without adding any selective marker gene.

It is still another object of the present invention to provide a primer pair which can amplify the full length of the encoding gene or any fragment thereof.

It is still another object of the present invention to provide a method for preparing a protein, the method comprising:

1) preparing the coding gene of the invention;

2) preparing a recombinant expression vector containing the coding gene of the step 1);

3) expressing the expression vector of step 2) to obtain the target protein.

Specifically, the method further comprises deglycosylation of the target protein; the specific step of expressing the expression vector in the step 2) comprises introducing the expression vector into a microorganism and expressing the expression vector; more specifically, the microorganism comprises escherichia coli, yeast, bacillus, lactobacillus, aspergillus, and/or trichoderma; more specifically, the microorganism comprises pichia pastoris, saccharomyces cerevisiae, and/or torula polymorpha; more specifically, the microorganism is pichia pastoris GS 115; specifically, the recombinant expression vector comprises pPIC-Ctxyn 10A.

The protein prepared by the protein preparation method also belongs to the protection scope of the invention; specifically, the protein comprises the Ctxyn10A protein and a modified structure; in particular, the modified structure comprises a glycosylation structure.

The invention further aims to provide the protein, the coding gene, the recombinant vector, the expression cassette, the transgenic cell line, the recombinant bacterium and/or the microorganism, the primer pair, the protein preparation method and the application of the protein prepared by the preparation method.

Specifically, the application includes an application in at least one of the following 1) to 8):

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

2) the application in preparing xylanase mutants and/or related products containing the xylanase mutants;

3) the application in preparing recombinant xylanase and/or related products containing the recombinant xylanase;

4) the application in the preparation of xylo-oligosaccharide, xylobiose, xylo-oligosaccharide-containing, xylobiose-containing and/or xylo-saccharide-containing related products;

5) the application of the cellulase and the glucanase and/or the preparation of products related to the enzymatic activities of the cellulase and the glucanase;

6) use in the degradation of xylan, cellulose, and/or glucan;

7) use in the preparation of a product for application in the industrial, agricultural, food, pharmaceutical, feed, energy, waste treatment and/or environmental protection fields;

8) in the industrial, agricultural, food, pharmaceutical, feed, energy, waste treatment and/or environmental protection fields.

Specifically, the xylan comprises beechwood xylan, soluble wheat arabinoxylan, birch xylan, insoluble wheat arabinoxylan; the cellulose comprises carboxymethyl cellulose and lichenin; the glucan comprises barley glucan.

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

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

1) the pH is comprised between 4.0 and 12.0;

2) the temperature is 0-90 ℃;

3) an environment comprising metal ions, and/or chemical agents;

4) the substrate comprises xylan, cellulose, and/or glucan.

Preferably, the pH comprises 5.0-8.0, more preferably, the pH comprises 6.0-7.0, most preferably, the pH is 6.5, preferably, the temperature comprises 50-80 ℃, preferably, the temperature comprises 50-75 ℃, preferably, the temperature comprises 60-75 ℃, most preferably, the temperature is 70 ℃, the metal ions, and/or chemical agents comprise Ni2+, Co2+, Na +, Cr3+, Mg2+, Mn2+, K +, Cu2+, Ca2+, Pb2+, Zn2+, Fe3+, β -mercaptoethanol, EDTA, SDS, the concentration of the metal ions, and/or chemical agents comprises 5 mmol/L, the xylans comprise beechwood, soluble wheat arabinoxylans, birchwood xylans, insoluble wheat arabinoxylans, the celluloses comprise carboxymethyl cellulose, lichen polysaccharides, and the glucans comprise barley glucans.

It is still another object of the present invention to provide a method for preparing xylanase, which comprises extracting xylanase from Cladosporium tianshanense S L-14.

The xylanase CtXyn10A provided by the invention has the characteristic of high activity in the neutral (pH 5.0-8.0) and high temperature (50-75 ℃), the xylanase CtXyn10A produced by Cladosporium tienshanense S L-14 (China agricultural culture Collection ACCC 32710) is screened, the optimum pH value is 6.0-7.0, more than 80% of enzyme activity is maintained in the pH range of 5.0-8.0, the optimum temperature is 70 ℃, more than 70% of enzyme activity is maintained at 60-75 ℃, 10% of enzyme activity is also obtained at 0 ℃, and the xylanase CtXyn10A has degradation effect on various xylans, glucans and celluloses.

The invention overcomes the defects of the prior art, obtains bacterial resources of the high-temperature xylanase, and provides a new neutral high-temperature xylanase CtXyn10A with excellent properties, which is suitable for application in food, paper and energy industries. The optimum pH value of the xylanase CtXyn10A is 6.0-7.0, and the xylanase has high enzyme activity at the pH value of 5.0-8.0; the pH stability is good; the specific activity is 461U/mg; has wide substrate specificity. The characteristic of neutral high temperature can reduce the energy cost of xylanase in industrial production. The pH application range is wide, the digestion energy and the metabolism energy of the aquatic feed can be improved, the formula cost is reduced, and the environmental pollution is reduced. The wide substrate specificity can improve the nutritive value of grain processing byproducts and improve the quality of feed products. The xylanase CtXyn10A can be applied to the wine brewing industry, reduces the viscosity of materials, is beneficial to the amylase to act on a starch layer, improves the utilization rate of starch and increases the yield of alcohol. The xylan in the paper industry waste and the agricultural waste can be converted into xylooligosaccharide under the normal temperature condition, and the xylooligosaccharide can be converted into valuable fuel by bacteria, yeast and fungi. Therefore, the application of the xylanase CtXyn10A in the energy industry also shows great potential. The hydrolysate (xylose and xylo-oligosaccharide) CtXyn10A of xylanase can be applied to the food industry and used as a thickening agent, a fat substitute, a probiotic oligosaccharide and an anti-freezing food additive; xylans are used in combination with other substances in the pharmaceutical industry to delay the release of pharmaceutical ingredients. The xylanase and its hydrolysate can be further converted into liquid fuels, solvents and low-calorie sweeteners.

Drawings

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

wherein, lane 1 is deglycosylated purified recombinant xylanase; lane 2 purified recombinant xylanase; lane 3 is a low molecular weight protein Marker.

FIG. 2 is a graph showing the optimum pH determination result of the recombinant xylanase CtXyn 10A.

FIG. 3 is a graph of the results of pH stability determination of recombinant xylanase CtXyn 10A.

FIG. 4 is a graph of the optimum temperature measurement result of the recombinant xylanase CtXyn 10A.

FIG. 5 is a graph of the results of measuring the thermal stability of the recombinant xylanase CtXyn 10A.

Detailed Description

Description of the drawings:

the experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Test materials and reagents:

1. the strain and the vector are Cladosporium tienshanense S L-14 used in the following examples, which is preserved in agricultural strain collection center of China agricultural science institute (No. 12 of south China street in Hakken region, Beijing, and agricultural strain collection center of China agricultural science institute, 100081), the preservation number is ACCC32710, the strain can be purchased from the center by the public, and the Pichia pastoris expression vector pPIC9 and the strain GS115 are purchased from Invitrogen company.

2. Enzymes and other biochemical reagents: the endonuclease was purchased from TaKaRa, and the ligase was purchased from Invitrogen. Zelkova was purchased from Sigma, and others were made by the national laboratory Co., Ltd.

3. Culture medium:

(1) the culture medium Cladosporium tianshanense S L-14 is potato juice culture medium containing 1000m L of potato juice, 10g of glucose, 25g of agar and pH5.0.

(2) Coli culture medium L B (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: the components were identical to BMGY, pH4.0, except that glycerol was replaced with 0.5% methanol (V/V).

Example 1 cloning of genomic DNA of neutral thermophilic xylanase CtXyn10A

Extracting Cladosporium mandshurica Cladosporium Tianshanense S L-14 genome DNA:

filtering mycelium cultured for 3 days by using sterile filter paper, putting the mycelium into a mortar, adding 2m L extracting solution, grinding for 5min, then putting the grinding solution into a 50m L centrifugal tube, cracking for 20min in a 65 ℃ water bath kettle, uniformly mixing every 10min, centrifuging for 5min at 4 ℃ at 10000rpm, taking supernatant, extracting in phenol/chloroform to remove impure proteins, taking supernatant, adding isopyknic isopropanol, standing for 5min at room temperature, centrifuging for 10min at 4 ℃ at 10000rpm, discarding supernatant, washing precipitate with 70% ethanol twice, vacuum drying, adding a proper amount of TE to dissolve, and standing at-20 ℃ for later use.

According to the sequence analysis of the Cladosporium tianshanense S L-14 genome frame diagram, a specific primer Ctxyn10A-F/Ctxyn10A-R is designed and synthesized:

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

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

PCR amplification is carried out by taking total DNA of Cladosporium tienshanense S L-14 as a template and Ctxyn10A-F/Ctxyn10A-R as a specific primer.

The PCR reaction parameters are as follows: denaturation at 94 deg.C for 5 min; then denaturation at 94 ℃ for 30sec, annealing at 60 ℃ for 30sec, elongation at 72 ℃ for 60sec, and heat preservation at 72 ℃ for 10min after 30 cycles.

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

The sequencing result shows that the sequence of the nucleic acid fragment obtained by the PCR amplification has a nucleotide sequence shown as SEQ ID NO.1 in a sequence table, and is 1202bp in total. The nucleic acid fragment with the nucleotide sequence shown in SEQ ID NO.1 in the sequence table is named Ctxyn 10A.

Example 2 obtaining of Gene encoding mature neutral high temperature xylanase CtXyn10A

Extracting total RNA of Cladosporium anisodanense S L-14, obtaining a strand of cDNA by using reverse transcriptase, amplifying the single-strand cDNA by using a primer Ctxyn10A-F/Ctxyn10A-R, recovering the obtained product, and sending the product to Sanbo biotechnology limited company for sequencing.

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

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

By comparing the genomic sequence of the nucleic acid fragment determined in example 1 with the sequencing results of the PCR amplification products described above, it was found that the gene had 2 introns, the cDNA length was 1059bp, had the nucleotide sequence of SEQ ID NO.2 of the sequence Listing, had the amino acid sequence shown by SEQ ID NO.3 of the coding sequence Listing, and had a stop codon. The protein with the amino acid sequence shown by SEQ ID NO.3 in the sequence table has 352 amino acid residues in total, 18 amino acids at the N end are predicted signal peptide sequences, and the protein with the amino acid sequence shown by SEQ ID NO.4 in the sequence table. The 141 th-199 th nucleotide sequence and the 512 th-595 th nucleotide sequence of SEQ ID NO.1 in the sequence table are intron sequences of 59bp and 84bp respectively

The sequencing result of the PCR amplification product shows that the nucleic acid fragment obtained by the PCR amplification comprises EcoRI + NotI double enzyme cutting sites, and the nucleic acid fragment with the nucleotide sequence from 55 th site to 1059 th site of SEQ ID NO.2 in a sequence table is arranged between the two enzyme cutting sites.

The nucleic acid fragment with the 55 th-1059 th nucleotide sequence of SEQ ID NO.2 in the sequence table is a nucleic acid fragment of a Ctxyn10A gene encoding mature protein part, encodes an amino acid sequence shown in SEQ ID NO.5 in the sequence table, and the nucleic acid fragment of the Ctxyn10A gene encoding mature protein part is subjected to homologous comparison with a xylanase gene sequence on GenBank, so that the highest consistency is 74 percent and the highest consistency of the amino acid sequence is 72 percent, and the xylanase encoding gene obtained by separation and cloning from Cladosporium japonicum shanense S L-14 is proved to be a new gene.

Example 3 preparation of neutral thermophilic xylanase CtXyn10A

The pichia pastoris expression vector pPIC9 is subjected to double enzyme digestion (EcoRI + NotI), and meanwhile, the nucleic acid fragment (EcoRI + NotI) for coding the mature protein prepared in the embodiment 2 is subjected to double enzyme digestion, and the gene fragment for coding the mature protein is cut out and connected with the expression vector pPIC9 subjected to double enzyme digestion.

Sequencing verifies the correctness of the sequence. The sequence of the exogenous gene inserted into the obtained recombinant plasmid is the 55 th to 1059 th nucleotides of SEQ ID NO.2, and the recombinant plasmid is named as pPIC-Ctxyn 10A.

Transforming the recombinant plasmid pPIC-Ctxyn10A into pichia pastoris GS 115; and (3) extracting plasmid from the positive recombinant bacteria, sequencing and verifying the correctness of the sequence, and naming the recombinant pichia pastoris strain containing the recombinant plasmid pPIC-Ctxyn10A with correct sequencing as GS115/Ctxyn 10A.

Inoculating the recombinant strain GS115/Ctxyn10A into 300m L BMGY culture solution, carrying out shake culture at 30 ℃ and 250rpm for 48h, centrifuging to collect thalli, then carrying out heavy suspension on 100m L BMMY culture medium, carrying out shake culture at 30 ℃ and 250rpm, inducing for 72h, centrifuging to collect supernatant, purifying protein, and determining the activity of xylanase Ctxyn 10A.

The SDS-PAGE result is shown in figure 1, and the result in figure 1 shows that the xylanase CtXyn10A is expressed in the Pichia pastoris recombinant strain GS115/CtXyn 10A. After the expressed xylanase CtXyn10A is purified, the content of target protein reaches more than 90% of total protein, and the electrophoretic purity is achieved. After the ENDO-H deglycosylation, the molecular weight of the purified protein is consistent with the theoretical value and is 37.4 kDa.

Example 4 determination of enzyme Activity of neutral high temperature xylanase CtXyn10A

The activity of the xylanase CtXyn10A prepared in the above example 3 is determined by a DNS method, wherein a reaction system of 1m L comprises a proper diluted enzyme solution of 100 mu L and a substrate of 900 mu L at the temperature of 70 ℃ and the pH of 6.5, the reaction is performed for 10min, 1.5m L DNS is added to terminate the reaction, the reaction is boiled in water for 5min, the OD value is determined at 540nm after cooling, 1 enzyme activity unit (U) is defined as the enzyme quantity which releases 1 mu mol of reducing sugar per minute under the given conditions, and the enzyme activity of the neutral high-temperature xylanase CtXyn10A prepared in the example 3 is determined to be 160U/m L.

Example 5 determination of the Properties of the neutral thermophilic xylanase CtXyn10A

1. The method for determining the optimal pH and pH stability of the recombinant xylanase CtXyn10A is as follows:

the recombinant xylanase purified in example 3 is subjected to enzymatic reaction at different pH values to determine the optimum pH., the substrate xylan is dissolved by 0.1 mol/L citric acid-disodium hydrogen phosphate buffer solutions with different pH values, and the xylanase activity is determined at 30 ℃, the result is shown in figure 2, the optimum pH value of the xylanase CtXyn10A is 6.0-7.0, the enzyme activity is maintained to be more than 80% of the maximum enzyme activity within the range of pH5.0-8.0, the xylanase is treated in the buffer solutions with different pH values at 37 ℃ for 60min, and then the enzyme activity is determined in a buffer solution system with pH6.5 at 70 ℃ to study the pH tolerance of the enzyme, the result is shown in figure 3, the xylanase CtXyn10A is stable within the range of pH 4.0-12.0, and the enzyme activity is more than 80% after the treatment within the pH range for 60min, which indicates that the enzyme has better pH stability.

2. The method for measuring the optimum temperature and the heat stability of the xylanase comprises the following steps:

the optimum temperature of xylanase was determined by performing enzymatic reactions in a citrate-disodium phosphate buffer (pH6.5) buffer system at various temperatures. The temperature tolerance is determined by treating xylanase at different temperatures for different times and then determining the enzyme activity at pH6.5 and 70 ℃. The result of the measurement of the optimum temperature of the enzyme reaction is shown in FIG. 4, the optimum temperature of the xylanase CtXyn10A is 70 ℃, and the specific activity is 461U/mg; the activity is higher in the high temperature range of 50-75 ℃; the enzyme activity is more than 70 percent at the temperature of 60-75 ℃; the enzyme activity is 10 percent at the temperature of 0 ℃; the results of the enzyme heat stability test are shown in FIG. 5 (the result is shown in FIG. 5 at 80 ℃ and is 10 minutes with the substrate reaction time; the result is shown in FIG. 4 at 80 ℃ and is shown in the state that the enzyme activity is measured after the substrate-free 80 ℃ warm bath for a certain time), the xylanase CtXyn10A has very good stability at 60 ℃, and the enzyme activity is completely lost after the temperature is kept for 60 minutes at 70 ℃.

In addition, the enzyme activity of the xylanase CtXyn10A at 90 ℃ and pH9.0 is also determined, and the result shows that the enzyme activity is kept by more than 20%.

The experimental result shows that the xylanase CtXyn10A is neutral high-temperature enzyme and has the characteristic of neutral high temperature.

3. The Km value of xylanase is determined as follows:

the xylanase CtXyn10A has the Km value of 0.64mg/m L, the maximum reaction speed Vmax of 450 mu mol/min mg, the kcat value of 281/s and the kcat/Km value of 434 ml/mg.s at 70 ℃, and the test result shows that the affinity of the xylanase CtXyn10A and the substrate is stronger, the catalytic efficiency is higher under the high temperature condition, the enzymological property is greatly superior to that of fungus xylanase FoXyn10A with similar sequence (the optimum pH is 7.0, the optimum temperature is 45 ℃, Km 0.8mg/m L, Vmax1.22 mu mol/min, Chos and kosyl/38:. kappa. 1997,302. the optimum pH is 7.0)

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

different metal ions and chemical reagents with different concentrations are added into an enzymatic reaction system, the influence of various chemical reagents on the activity of the xylanase CtXyn10A is researched, the final concentration of various substances is 5 mmol/L, the enzyme activity is measured at 70 ℃ and pH 6.5. the result is shown in table 1, most of the ions and the chemical reagents have no obvious influence on the activity of the recombinant xylanase CtXyn10A when the concentration is 5mmol, SDS has partial inhibition effect, and the activity of the recombinant enzyme can be increased to 1.32 times when the concentration is 5 mmol.

5. Substrate specificity of xylanase CtXyn10A

Adding different substrates into an enzymatic reaction system, and researching the substrate specificity of the xylanase CtXyn 10A. The enzyme activity was measured at 70 ℃ and pH 6.5. The results are shown in table 2, and the xylanase CtXyn10A has the activities of xylanase, cellulase and glucanase simultaneously.

The experimental result shows that the xylanase CtXyn10A has wide substrate specificity, can act on xylan, and can catalyze the degradation of various substrates such as cellulose, glucan and the like, so the xylanase has wider application prospect in the industrial field.

TABLE 1

Table 26, analysis of the product of degrading beech xylan by xylanase CtXyn10A is as follows:

adding 100 mu L purified enzyme liquid into 500 mu L% of xylan, preserving the temperature for 12h at the optimum temperature, precipitating enzyme protein by using absolute ethyl alcohol, using a 2500 chromatograph for supernate, and using a high-efficiency anion exchange chromatography-pulse amperometric (HPAEC-PAD) detection method to analyze the sugar types in the product.

Sequence listing

<110> feed institute of Chinese academy of agricultural sciences

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

<160>5

<210>1

<211>1202

<212>DNA

<213> Cladosporium Tianshanense S L-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 ctgccacact 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

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 S L-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 S L-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 S L-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 S L-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

1. A xylanase, wherein the amino acid sequence of the xylanase is as shown in SEQ ID No: 3 or SEQ ID NO: 5, respectively.

2. A xylanase gene encoding the xylanase of claim 1.

3. A recombinant expression vector comprising the xylanase gene of claim 2.

4. A method for producing a xylanase, comprising the steps of:

(1) introducing the recombinant expression vector of claim 3 into a host cell;

(2) inducing expression of xylanase;

(3) isolating and purifying the xylanase.

5. Use of a xylanase according to claim 1 for the hydrolysis of xylan.