CN113151216B - Heat-resistant xylanase with high arabinoxylan activity, and coding gene and application thereof - Google Patents

Abstract

The invention discloses heat-resistant xylanase with high arabinoxylan activity, a coding gene and application thereof. The amino acid sequence of the protein to be protected is the protein of a sequence 1 in a sequence table or the amino acids from 18 th to 222 th positions of the sequence 1, or the protein with the sequence having more than 80 percent of identity and xylanase activity. The protein is derived from thermophilic fungus chaetomium globosum CQ31, has xylanase activity, good heat resistance and pH stability, and has the optimum temperature of 70 ℃ and the optimum pH of 7.0; the enzyme has excellent hydrolysis property and high arabinoxylan activity. The coding gene of the protein is introduced into pichia pastoris, the enzyme activity of the obtained fermentation liquor for expressing the recombinant xylanase reaches 19,000U/mL, the protein content is 10.8mg/mL, the recombinant xylanase can be applied to the bread making process, the aging of bread can be effectively delayed, and the recombinant xylanase has great potential in industries such as food, feed and the like.

Description

Heat-resistant xylanase with high arabinoxylan activity and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to heat-resistant xylanase with high arabinoxylan activity, and a coding gene and application thereof.

Background

Xylan (Xylan) is the main component of hemicellulose, which is extremely abundant in nature, and its content is inferior to cellulose, and is widely present in the cell wall of plant tissues. The complete hydrolysis of xylan requires the participation of various enzymes, Xylanase (Xylanase, EC 3.2.1.8) being among the most critical hydrolases, which degrades xylan into xylose and xylooligosaccharides (patent publication No.: CN11254132A, application date: 20200121). The xylanase has wide application in food, feed and other industries. In the food industry, flour is an important food material, and the arabinoxylan is used as a component in the flour, has a content of about 2% -3% of the flour, and has a significant influence on the quality of flour products due to its high viscosity and strong water absorption capacity. In the bread baking industry, xylanase with high arabinoxylan activity can efficiently degrade arabinoxylan in flour, thereby improving the rheological property of dough, increasing the specific volume of bread, reducing the hardness of bread, delaying the aging of bread and improving the economic value of bread.

Xylanases are widely found in nature, with microorganisms being the most dominant source. Until now, many studies of microbial xylanases have been reported, of which bacterial sources are most reported and mostly belong to the genera Bacillus (Bacillus sp.) and Paenibacillus (Paenibacillus sp.), and fungal sources are relatively few, mostly from the genera Aspergillus (Aspergillus sp.), Penicillium (Penicillium sp.) and Trichoderma (Trichoderma sp.). The xylanase from the thermophilic fungi has good heat stability and is suitable for being applied to food and feed industries, but the xylanase from the thermophilic fungi has few reports. In addition, xylanase having arabinoxylan activity is also less reported, and thus the development of thermostable xylanase genes having arabinoxylan activity is receiving increasing attention.

The yield of the enzyme can be improved by utilizing a genetic engineering means, and the requirement of large-scale industrial application is met. So far, some xylanases have been successfully expressed in escherichia coli (escherichia coli), Bacillus subtilis (Bacillus subtilis) and Pichia pastoris (Pichia pastoris), but the expression level of most xylanases is still low, and in order to reduce the production and application costs of the xylanases, gene discovery and heterologous expression strategies of the xylanases are still in urgent need of research.

Disclosure of Invention

The technical problem to be solved by the invention is how to improve the activity of xylanase or how to prepare heat-resistant xylanase with high arabinoxylan activity.

In order to solve the above technical problems, the present invention provides a protein.

The protein may be a protein of a1), a2), A3), or a4) as follows:

A1) the amino acid sequence is protein of sequence 1 in a sequence table;

A2) the amino acid sequence is protein of 18 th to 222 th amino acid residues of a sequence 1 in a sequence table;

A3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in A1) or A2), has more than 80% of identity with the protein shown in A1) or A2), and has xylanase activity;

A4) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1), A2) or A3).

In the protein, the sequence 1 in the sequence table is composed of 222 amino acid residues. The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 80% or more identity may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

The protein may be derived from Chaetomium sp.

The protein may be xylanase. The substrate of the xylanase can be at least one of beech xylan, birch xylan, wheat arabinoxylan and oat xylan.

In order to solve the above technical problems, the present invention also provides a biomaterial related to the above protein. The biomaterial may be any one of the following B1) -B6):

B1) a nucleic acid molecule encoding a protein as described above;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a nucleic acid molecule that increases or promotes the activity of the protein described above or the expression of a gene encoding the protein described above;

B6) an expression cassette, a recombinant vector or a recombinant microorganism comprising the nucleic acid molecule according to B5).

The microorganism described above may be a yeast or a filamentous fungus. The yeast may be pichia pastoris. The recombinant microorganism described above may be a recombinant pichia pastoris. The filamentous fungus may be aspergillus niger. The recombinant microorganism described above may also be a recombinant A.niger.

In the above biological material, the nucleic acid molecule of B1) is a gene encoding the protein represented by B1), B2) or B3) as follows:

b1) the coding sequence of the coding chain is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b2) the nucleotide of the coding strand is cDNA molecule or DNA molecule of 52 th-699 th nucleotide of sequence 2 in the sequence table;

b3) a cDNA or DNA molecule which hybridizes under stringent conditions with a cDNA or DNA molecule defined under b1) or b2) and encodes a protein having the same function.

The stringent conditions can be hybridization and washing with 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution at 65 ℃ in DNA or RNA hybridization experiments.

As used herein, the term "identity" refers to similarity to a sequence of nucleic acids or amino acids. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above-mentioned biological materials, the expression cassette containing a nucleic acid molecule described in B2) means a DNA capable of expressing the above-mentioned protein in a host cell. The expression cassette may also comprise single-stranded or double-stranded nucleic acid molecules of all the regulatory sequences necessary for expression of the nucleic acid molecule of any of the proteins described above. The control sequences direct the coding sequence to express any of the proteins described above in a suitable host cell under conditions compatible with the control sequences. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. Regulatory sequences with linkers may be provided for the purpose of introducing specific restriction enzyme sites into the vector for ligating the regulatory sequences with the coding region of the nucleic acid sequence encoding the protein. The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell in which the nucleic acid sequence is expressed. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein. The promoter may be any nucleic acid sequence which is transcriptionally active in the host cell of choice, including mutant, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular proteins either homologous or heterologous to the host cell. The control sequence may also be a suitable transcription termination sequence, i.e., a sequence recognized by a host cell to terminate transcription. The termination sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the protein. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protein. Any leader sequence that functions in the host cell of choice may be used in the present invention. The control sequence may also be a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of a protein and directs the encoded protein into the cell's secretory pathway. Signal peptide coding regions which direct the expressed protein into the secretory pathway of the host cell of use may be used in the present invention. It may also be desirable to add regulatory sequences which can regulate the expression of the protein depending on the growth of the host cell. Examples of regulatory systems are those that respond to a chemical or physical stimulus, including in the presence of a regulatory compound, to open or close gene expression. Other examples of regulatory sequences are those which enable gene amplification. In these instances, the nucleic acid sequence encoding the protein should be operably linked to the control sequences.

The present invention also relates to recombinant expression vectors comprising a nucleic acid molecule of the invention encoding any of the above proteins, a promoter, and transcriptional and translational stop signals. In preparing an expression vector, a nucleic acid molecule encoding any of the proteins described above can be located in the vector so as to be operably linked to appropriate expression control sequences. The recombinant expression vector may be any vector (e.g., a plasmid or virus) which facilitates recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which it is to be introduced. The vector may be a linear or closed-loop plasmid. The vector may be an autonomously replicating vector, i.e., a complete structure which exists extrachromosomally and can replicate independently of the chromosome, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may comprise any mechanism which ensures self-replication. Alternatively, the vector is one which, when introduced into a host cell, will integrate into the genome and replicate together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that collectively contain the entire DNA to be introduced into the genome of the host cell, or a transposon may be used. The vector contains 1 or more selectable markers that facilitate selection of transformed cells. A selectable marker is a gene the product of which confers resistance to biocides or viruses, resistance to heavy metals, or confers auxotrophy to auxotrophs and the like. Examples of bacterial selectable markers are the dal genes of B.subtilis or B.licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline. The vector contains elements that allow for stable integration of the vector into the host cell genome or that allow for autonomous replication of the vector in the cell independent of the cell genome. In the case of autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the target host cell. The origin of replication may carry a mutation which makes it temperature-sensitive in the host cell (see, for example, fEhrlich,1978, Proc. Natl. Acad. Sci. USA 75: 1433). More than 1 copy of a nucleic acid molecule of the invention encoding any of the above proteins may be inserted into the host cell to increase the yield of the gene product. The copy number of the nucleic acid molecule can be increased by inserting at least 1 additional copy of the nucleic acid molecule into the host cell genome, or inserting an amplifiable selectable marker along with the nucleic acid molecule, and selecting for cells containing amplified copies of the selectable marker gene, and thus additional copies of the nucleic acid molecule, by culturing the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements described above to construct the recombinant expression vectors of the invention are well known to those skilled in the art (see, e.g., Sambrook et al, A molecular cloning laboratory Manual, second edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y., 1989).

The term "operably linked" is defined herein as a conformation in which the control sequences are located at an appropriate position relative to the coding sequence of the DNA sequence such that the control sequences direct the expression of the protein.

The invention also relates to a recombinant cell containing a nucleic acid molecule encoding any of the above proteins. The recombinant cell may be a prokaryotic cell or a eukaryotic cell, such as a bacterial (e.g., E.coli cell) or yeast cell.

The use of the above-described proteins as xylanases is also within the scope of the present invention.

The use of the above-described biological material for the preparation of xylanases is also within the scope of the present invention.

The substrate of the xylanase as described above may be at least one of zelkova xylan, birch xylan, wheat arabinoxylan and oat xylan.

In order to solve the above technical problem, the present invention also provides any one of the following uses of the protein described above and/or the biomaterial described above:

p1, application in preparing flour products;

p2, application in preparing xylo-oligosaccharide;

p3, application in preparing flour food;

p4, application in preparing flour product feed;

p5, use in the preparation of a food additive product;

p6, use in food or feed processing;

p7, use in bread baking;

p8, application in steamed bread making.

Products containing the proteins described above are also within the scope of the invention. The product can be a food additive or a feed additive.

In order to solve the technical problems, the invention also provides a method for preparing xylanase. The method comprises the following steps: the genes encoding the proteins described above are expressed in an organism to obtain the xylanase. The organism may be a microorganism, a plant or a non-human animal. The microorganism may be a yeast or filamentous fungus. The yeast may be pichia pastoris. The filamentous fungus may be aspergillus niger.

The xylanase as described above may have an optimum pH of 7.0. The xylanase is treated for 30min at the pH value of 4.0-10.0, and the residual enzyme activity is above 80%. The xylanase has the optimal reaction temperature of 70 ℃ and can keep better stability at 60 ℃. The xylanase product may be a xylooligosaccharide with a degree of polymerization of 2-6.

In the embodiment of the invention, the coding gene of xylanase derived from chaetomium globosum CQ31 is connected to a universal vector pPIC9K plasmid skeleton to construct a multicopy strong expression recombinant plasmid pPCAU-CsXyn 11A; then the recombinant plasmid is led into pichia pastoris, and the enzyme activity of the recombinant xylanase obtained by the high-density fermentation of the obtained pichia pastoris strain pPCAU-CsXyn11A in a 5L fermentation tank reaches 19,000U/mL, and the protein content is 10.8 mg/mL. The xylanase has the optimum pH of 7.0, is treated for 30min at the pH of 4.0-10.0, and has the residual enzyme activity of more than 80 percent; the optimal reaction temperature is 70 ℃, and the better stability is kept at 60 ℃. The enzyme substrate has specific specificity, high activity on wheat araboxylan, specific enzyme activity of 1850U/mg, and product of hydrolyzed beech xylan, birch xylan, oat xylan and araboxylan is xylooligosaccharide with polymerization degree of 2-6. The xylanase (5ppm) is used in bread making process, and can increase bread specific volume (25.6%) and reduce bread hardness (46%), and also can effectively delay bread aging. The protein provided by the invention has good xylanase enzymology property, good heat resistance and pH stability, excellent hydrolysis characteristic, high araboxylan activity and good application value in industries such as food, feed and the like.

Drawings

FIG. 1 is a vector map of the recombinant vector pPIC9K-CsXyn 11A.

FIG. 2 is a vector map of the recombinant vector pPCAU-CsXyn 11A.

FIG. 3 shows the enzyme production process of recombinant xylanase CsXyn11A Pichia pastoris strain in 5-L fermentation tank (■: enzyme activity; ●: protein concentration;. tangle-solidup-wet weight).

FIG. 4 is an SDS-PAGE electrophoresis of xylanase CsXyn11A in a 5-L fermentor during high density fermentation (panel M: low molecular weight standard protein; panels 1-8: fermentation supernatants inducing 0, 24, 48, 72, 96, 120, 144, 156h, respectively).

FIG. 5 shows the purified electrophoretogram of xylanase CsXyn11A (M: low molecular weight standard protein; 1: crude enzyme solution; 2: pure enzyme solution).

FIG. 6 is a graph showing the optimum pH determination of xylanase CsXyn11A (Glycine-HCl (■, pH 3.0-7.0); Sodium Citrate (●, pH 3.0-6.0); Acetic acid-Sodium acetate (, pH 4.0-6.0); MES (. diamond-solidus., pH 5.5-6.5); MOPS (, 6.5-7.5); Tris-HCl (□, pH 7.0-9.0); CHES (. smalsolidup., pH 8.0-10.0); Glycine-NaOH (. DELTA., 9.0-10.5); Na2HPO4-NaOH (., pH 11.0-12.0)).

FIG. 7 is a graph showing pH stability measurements of xylanase CsXyn11A (Glycine-HCl (■, pH 3.0-7.0); Sodium Citrate (●, pH 3.0-6.0); Acetic acid-Sodium acetate (@ pH 4.0-6.0); MES (. diamond-solid-up., pH 5.5-6.5); MOPS (@ 6.5-7.5); Tris-HCl (□, pH 7.0-9.0); CHES (. smallci., pH 8.0-10.0); Glycine-NaOH (. DELTA., 9.0-10.5); Na ₂ HPO ₄ -NaOH(◇,pH 11.0-12.0))。

FIG. 8 is a graph showing the optimum temperature determination of xylanase CsXyn 11A.

FIG. 9 is a graph showing the temperature stability determination of xylanase CsXyn 11A.

FIG. 10 is a graph showing the half-life determination of xylanase CsXyn 11A. 50 ℃ (■), y ═ 0.001x +4.6292, R ² ＝0.9959；55℃(●),y＝-0.0016x+4.6342,R ² ＝0.9937；60℃(▲),y＝-0.0032x+4.6342,R ² ＝0.9569；65℃(◆),y＝-0.0082x+4.1343,R ² ＝0.9828。

FIG. 11 shows the results of thin layer chromatography analysis of CsXyn11A hydrolyzed beechxylan (a), birchwood xylan (b), oat xylan (c), xylobiose, xylotriose (d), xylotetraose (e), and xylopentaose (f) (X: xylose; X2: xylobiose; X3: xylotriose; X4: xylotetraose; X5: xylopentaose; X6: xylohexaose).

FIG. 12 is a graph showing the application effect of the xylanase CsXyn11A in bread.

FIG. 13 is a vector map of the recombinant vector Blunt-CsXyn 11A-HygB.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, the enzymatic activity of xylanases was determined with reference to Liu et al (Liu et al, Food Chemistry,2018,264: 310-): adding 900 μ L of 1% (1g/100mL) beech xylan solution into a small test tube, preheating at 70 deg.C for 3min, adding 100 μ L of appropriately diluted enzyme solution to be tested (i.e. solution of protein to be tested), using 50mM MOPS with pH 7.0 in the whole reaction system, reacting at 70 deg.C for 10min, adding 1000 μ L of DNS reagent (each 1L of aqueous solution contains 10g of 3, 5-dinitrosalicylic acid, 10g of NaOH and 2g of phenol, and the rest is water), boiling for 15min, adding 1000 μ L of saturated potassium sodium potassium tartrate solution, cooling, and measuring absorbance at 540nm wavelength with xylose as standard. Definition of enzyme activity: the amount of enzyme required to hydrolyze beechwood xylan to 1. mu. mol xylose per minute under the conditions described above.

The media referred to in the following examples:

1. the composition of BMGY medium is as follows: 1 percent of yeast extract, 2 percent of peptone, 1.34 percent of aminofree yeast nitrogen source YNB and 4 multiplied by 10 ^-5 % biotin, 1% glycerol by volume, the balance 100mmol/L pH 6.0 phosphate buffer.

2. The composition of the improved BMMY medium was as follows: 1 percent of yeast extract, 2 percent of peptone, 1.34 percent of aminofree yeast nitrogen source YNB and 4 multiplied by 10 ^-5 % biotin, 0.5% by volume methanol, the balance 100mmol/L pH 6.0 phosphate buffer.

3. The composition of YPD medium was as follows: 1 percent of yeast extract, 2 percent of peptone, 2 percent of glucose, 2 percent of agar and the balance of water.

4. The composition of the MD medium was as follows: 2 percent of glucose by mass, 1.34 percent of nitrogen source YNB without amino yeast by mass, 4 multiplied by 10 ^-5 % of biotin, agar with the mass percentage concentration of 2% and the balance of water.

5. BSM medium: 85% phosphoric acid (85% phosphoric acid is analytically pure,% indicates g/100mL)40mL, CaSO ₄ 1.4g，K ₂ SO ₄ 27.3g，MgSO ₄ ·7H ₂ O22.4 g, KOH 6.19g, glycerin 60g, adding distilled water to 1.5L.

The following examples refer to biological materials:

chaetomium CQ31 was screened in soil and stored in the laboratory (described in "Jiang et al, Food Chemistry,2010,2: 457-" publicly available from the Applicant and only used for duplicate experiments).

The vector pPIC9K is available from Invitrogen corporation, USA.

Birch xylan, zelkova xylan, oat xylan and wheat arabinoxylan are all Sigma products.

The Blunt vector skeleton is a product of Beijing Quanzijin company.

Example 1 cloning and expression of xylanase genes in Pichia and Aspergillus niger

The molecular weight of CsXyn11A mature protein (sequence 1 in a sequence table) from CQ31 of Chaetomium sp (Chaetomium sp.) which is a thermophilic fungus is 23.91kDa, and the isoelectric point is 5.0. Signal P4.1 analyzes that 17 amino acids at the N end are Signal peptide sequences (1-17 th position of sequence 1 in a sequence table). The amino acid sequence of the mature protein was analyzed by NCBI alignment, and the homology of CsXyn11A to xylanase from Achaetometrium sp.Xz-8 GH11 family (AHE13930) was 72% with the highest homology, and to xylanases from GH11 family from Aspergillus nidulans (P55333), Chaetomium sp.CQ31(HM640243) and Humicola grisea (Q9HGE1) with homology of 62%, 60% and 59%, respectively. Therefore, CsXyn11A is a novel GH11 family xylanase.

Firstly, construction of recombinant vectors pPIC9K-CsXyn11A, pPCAU-CsXyn11A and Blunt-CsXyn11A-HygB

1.1 construction of recombinant vector pPIC9K-CsXyn 11A:

an upstream primer CsXyn11AECoRIF and a downstream primer CsXyn11ANOTIR are designed, and PCR is carried out by taking cDNA of thermophilic fungi Chaetomium globosum CQ31 as a template to amplify a CDS nucleotide sequence (sequence 2 in a sequence table) of a mature protein (sequence 1 in the sequence table) of a xylanase gene (named CsXyn11A) of a Glycoside Hydrolase (GH)11 family. The 1 st to 17 th amino acids of the sequence 1 in the sequence table are the signal peptide sequence of CsXyn11A, and the 1 st to 51 th nucleotide molecules of the corresponding sequence 2 are the coding sequence of CsXyn11A protein signal peptide. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 60s, and circulating for 35 times; finally, extension is carried out for 5min at 72 ℃. The product is recovered by 1 percent agarose gel electrophoresis, and is cut by EcoRI and Not I, and the product after the enzyme cutting and the expression vector pPIC9K and T which are cut by the same enzyme are used ₄ DNA ligase was ligated, heat-shocked to transform E.coli DH 5. alpha. competent cells, plated on LB plates containing ampicillin (100. mu.g/mL), and inverted cultured at 37 ℃ for 12-16 h. And selecting transformants which are verified to be positive by colony PCR, extracting plasmids and sequencing. The sequencing result shows that the pPIC9K-CsXyn11A is a recombinant expression vector obtained by replacing the nucleotide sequence of a segment (small segment) between the recognition site of the restriction enzyme EcoRI and the recognition site of the restriction enzyme NotI of pPIC9K with a double-stranded DNA molecule shown at positions 52-669 of the sequence 2 in the sequence table and keeping other nucleotide sequences of the pPIC9K unchanged, and the pPIC9K-CsXyn11A plasmid map is shown in FIG. 1. pPIC9K-CsXyn11A can express protein with amino acid sequence shown in 18 th to 222 th sites of sequence 1 in the sequence table. CsXyn11 AECoRIF: 5' -CATGGAATTCTTCCCCTTCAACGCCACCG-3' (base underlined in the primer represents the EcoRI recognition site sequence);

CsXyn11 ANOTIR: 5'-GAATGCGGCCGCTTACGGCTCGACAGTGATAGAGGAC-3' (base underlined in the primer represents Not I recognition site sequence).

1.2 construction of the recombinant multicopy vector pPCAU-CsXyn 11A:

an upstream primer CsXyn11AF and a downstream primer CsXyn11AR are designed, and a nucleotide sequence for coding a mature protein CsXyn11A is amplified by PCR with cDNA of Chaetomium globosum CQ31 as a template. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 20s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 60s, and circulating for 35 times; finally, extension is carried out for 5min at 72 ℃. The product was recovered by electrophoresis on a 1% agarose gel. An AOX1 promoter is connected in series in vitro by using a Gibbson assembly method, a coding sequence (52 th to 669 th of a sequence 2 in a sequence table) of a CsXyn11A removed signal peptide, a coding gene (a sequence 3 in the sequence table) of an alpha-factor signal peptide, a2 XAOX 1 promoter (a sequence 4 in the sequence table) and an AOX1 terminator (a sequence 5 in the sequence table) are connected to form a CsXyn11A gene single copy expression cassette (figure 2), the three expression cassettes are sequentially connected in series according to cis-trans-cis to obtain a CsXyn11A gene multi-copy expression cassette, the CsXyn11 gene multi-copy expression cassette is connected between enzyme digestion recognition sites EcoRI and CsI on a general vector pPIC9K plasmid skeleton, a multi-copy strong expression plasmid pPCAU-Xyn 11A is constructed, and a plasmid map is shown in figure 2.

E.coli Top10 competent cells were heat-shocked, plated on LB plates containing ampicillin (100. mu.g/mL), and inverted cultured at 37 ℃ for 12-16 h. And selecting transformants, carrying out PCR amplification sequencing by using a verification primer, and extracting transformant plasmids with correct sequencing, wherein the transformant plasmids are named as pPCAU-CsXyn11A and are used for the next experiment. The pPCAU-CsXyn11A can strongly express the xylanase protein with the amino acid sequence shown as 18 th to 222 th sites of the sequence 1 in the sequence table in multiple copies.

CsXyn11AF：5′-GCTGAAGCTTACGTAGAATTCCAGATTCCCCTTCAACGCCACCG-3' (base underlined in the primer represents the EcoRI recognition site sequence);

CsXyn11AR：5′-AAGGCGAATTAATTCGCGGCCGCTTACGGCTCGACAGTGATAGAGGAC-3' (base underlined for primer represents Not I recognition site sequence).

1.3 construction of the recombinant vector Blunt-CsXyn 11A-HygB:

the DNA fragment containing the CsXyn11A gene target fragment (sequence 6 in the sequence table) is connected to the recognition sites Kpn I and Xba I on the Blunt expression plasmid by using the Gibbson assembly method to obtain the recombinant vector Blunt-CsXyn11A-HygB (shown in FIG. 13). Wherein, the DNA sequence shown by the 7 th-1006 th nucleotide of the sequence 6 in the sequence table is an Aspergillus niger promoter sequence Pgla; the DNA sequence shown by nucleotides 1007-1060 of the sequence 6 is a saccharifying enzyme signal peptide sequence; the DNA sequence shown by 1061-1678 th nucleotides of the sequence 6 is a CsXyn11A gene target fragment; the DNA sequence shown by the 3359-4384 nucleotide of the sequence 6 in the sequence table is a hygromycin resistance gene sequence.

The constructed expression vector Blunt-CsXyn11A-HygB is transformed into a cloning host E.coli DH5 alpha, colony PCR verification and sequencing are carried out on a positive escherichia coli transformant, and verification primers are ZD-Pgla-F (5'-GTTGATGCATGTGCTTCTTCCTTCAG-3') and ZD-Pgla-R (5'-CGGATTAATAATCATCCACTGCACCTC-3').

Sequencing results show that Blunt-CsXyn11A-HygB contains the CsXyn11A gene shown in 52 th to 669 th positions of a sequence 2 in a sequence table, and can express the CsXyn11A protein shown in 18 th to 222 th positions of a sequence 1 in the sequence table.

Extracting plasmids from escherichia coli with correct sequencing result for double enzyme digestion, wherein the 400 mu L enzyme digestion system is as follows: 356. mu.L of the recombinant plasmid Blunt-CsXyn11A-HygB, 40. mu.L of Cutsmart buffer, 2. mu.L of Kpn I and 2. mu.L of Xba I were digested at 37 ℃ overnight. Carrying out alcohol precipitation and recovery on the plasmid after enzyme digestion (namely the linearized plasmid Blunt-CsXyn 11A-HygB).

Expression of recombinant xylanase in Pichia pastoris and Aspergillus niger

2.1 expression of recombinant xylanase in Pichia pastoris

Pichia pastoris GS115 (Invitrogen) competent was prepared according to the method described for Pichia pastoris (Invitrogen). The above recombinant vector plasmids pPIC9K-CsXyn11A and pPCAU-CsXyn11A were linearized with the restriction enzyme SalI and adjusted to respective concentrations of 1. mu.g/. mu.L. 80. mu.L of yeast competent cells were taken, mixed with 10. mu.L of linearized pPIC9K-CsXyn11A or pPCAU-CsXyn11A plasmid, and placed in a precooled 0.2cm cuvette (Bio-rad Co.) for electroporation transformation. After electric shock, 1.0mL of precooled 1M sorbitol aqueous solution is rapidly added, the mixture is respectively coated on an auxotrophic screening plate (MD) and cultured for 3-4d, and 50 single colonies (pPIC9K-CsXyn11A recombinant pichia pastoris strain) or pPCAU-CsXyn11A transformed pichia pastoris GS11 are respectively selected from the MD plate of pPIC9K-CsXyn11A transformed pichia pastoris GS11550 single colonies (pPCAU-CsXyn11A recombinant Pichia pastoris strain) were picked on MD plates of 5 for comparison of the levels of enzyme production fermentation as follows: inoculating single colony in 25mL BMGY medium, shaking and culturing at 30 ℃ and 200rpm to OD of fermentation broth _600nm 12 (BMGY medium as blank control), the cells were collected by centrifugation and transferred to a 500mL Erlenmeyer flask containing 100mL of modified BMMY medium to increase the OD of the broth _600nm 8 (using the improved BMMY culture medium as a blank control), culturing under the same culture conditions, and supplementing methanol to the final concentration of 0.5% every 24h (volume percentage content, namely 100mL of the improved BMMY is supplemented with 500 mu L of methanol every 24 h), and inducing for 5 d.

TABLE 1 comparison of enzyme activities of xylanase CsXyn11A of Pichia pastoris transformants transformed with different plasmids

Table 1 shows the xylanase activity measured by the respective pPIC9K-CsXyn11A and pPCAU-CsXyn11A plasmid representative transformants, and it can be seen from the table that the pPCAU-CsXyn11A recombinant Pichia pastoris strain obtained by transforming Pichia pastoris GS115 with the multi-copy strong expression plasmid pPCAU-CsXyn11A constructed in the step one can be 2-3 times higher than the pPIC9K-CsXyn11A recombinant Pichia pastoris strain obtained by transforming Pichia pastoris GS115 with the recombinant plasmid pPIC9K-CsXyn11A, so that the recombinant Pichia pastoris constructed by pPCAU-CsXyn11A is selected for the next G418 screening.

Collecting thallus on an MD plate of pichia pastoris GS115 transformed by pPCAU-CsXyn11A with sterile water, properly diluting the collected thallus, coating the diluted thallus on G418 screening plates with different concentrations, wherein the concentration of G418 is 1mg/mL, 2mg/mL, 3mg/mL and 4mg/mL respectively, selecting single colonies with good growth under different G418 concentrations respectively, and carrying out shake flask rescreening. Strains with high expression levels were selected as follows (shake flask fermentation validation): selecting single colony of Pichia pastoris transformant, inoculating to 25mL BMGY medium, and performing shaking culture at 30 ℃ and 200rpm until OD is reached _600nm 12 (BMGY medium as blank), the cells were collected by centrifugation and transferred to a 500mL Erlenmeyer flask containing 100mL of modified BMMY medium to OD _600nm To 8 (in)Modified BMMY media was blank), cultured under the same culture conditions, supplemented with methanol every 24h to a final concentration of 0.5% (volume percent, i.e., 100mL of modified BMMY supplemented with 500 μ L of methanol every 24 h), and induced for 5 d.

Through screening, the recombinant pichia pastoris pPCAU-CsXyn11A with the highest enzyme yield is obtained, and the recombinant pichia pastoris pPCAU-CsXyn11A is adopted to be verified by shake flask fermentation, so that the expression quantity of the recombinant xylanase is 190U/mL. And subsequently, carrying out high-density fermentation verification on the fermentation tank by adopting the recombinant pichia pastoris pPCAU-CsXyn 11A.

2.2 expression of recombinant xylanase in Aspergillus niger

Preparation and purification of aspergillus niger protoplast: aspergillus niger (a. niger) (biowind, strain No. as3.3289) GA 3d was cultured in an air shaker at 30 ℃ using DPY liquid medium. And centrifuging the cracked protoplast at 4 ℃ for 10min under 1000g, discarding the supernatant, adding 20mL of precooled STC solution to wash the protoplast, repeatedly blowing and resuspending a gun head, and centrifuging for 15min at 4 ℃ under 1000 g. The above steps are repeated for 1 time, and then 0.6mL of STC is added for resuspension and standby.

Aspergillus niger protoplast PEG-mediated transformation: 3 EP tubes of 2mL were labeled for the control group, experimental group and regeneration group, respectively. Control group: 160 μ L protoplasts +100 μ L STC +60 μ L PEG; experimental groups: 160. mu.L protoplasts + 100. mu.L linearized plasmid (linearized Blunt-CsXyn11A-HygB plasmid obtained in step 1.3) + 60. mu.L PEG; regeneration group: 160 μ L protoplasts +100 μ L STC +60 μ L PEG. Flicking three groups uniformly, carrying out ice bath for 30min, flicking once every 10min, and then adding 1mL of PEG into each EP tube to stand at room temperature for 25 min.

Pouring culture: adding 3mL of STC buffer solution and 6mL of sucrose CD hypertonic soft agar into a 15mL centrifuge tube for uniform mixing, respectively transferring a control group, an experimental group and a regeneration group into the 15mL centrifuge tube, adding HygB with the final concentration of 300 mu g/mL into the experimental group and the control group, respectively taking 6mL after being turned upside down and uniformly mixed, pouring the 6mL into a sucrose CD hypertonic agar plate (the control group and the experimental group contain 300 mu g/mL of HygB), uniformly distributing and culturing at 30 ℃ for 3-4 d.

And (3) transformant identification: and carrying out subculture twice on single colonies growing in the hypertonic plate of the experimental group, then carrying out genome extraction, designing and verifying primers ZD-Pgla-F (5'-GTTGATGCATGTGCTTCTTCCTTCAG-3') and ZD-Pgla-R (5'-CGGATTAATAATCATCCACTGCACCTC-3'), and carrying out amplification and sequencing verification. The PCR reaction system was the same as above.

And (3) carrying out shake flask fermentation culture on the transformant with the correct genotype, culturing for 120h at the conditions of 34 ℃ and 200rpm in a constant temperature shaking table, and taking the centrifuged fermentation supernatant to detect xylanase activity and carrying out SDS-PAGE analysis.

The xylanase activity of the positive transformation strain Blunt-CsXyn11A-HygB is 1200U/mL through determination.

Example 2 high Density fermentation and purification of recombinant xylanase CsXyn11A

High-density fermentation of recombinant xylanase CsXyn11A

Seed culture: the recombinant Pichia pastoris strain pPCAU-CsXyn11A obtained in Experimental example 1 was inoculated into a 500mL Erlenmeyer flask containing 100mL of BMGY or YPD medium, and shake-cultured at 30 ℃ and 200rpm for 24-30 hours to obtain OD _600nm About 2.0 to about 6.0.

And (3) a glycerol fermentation stage: the seed solution was inoculated into a 5L fermentor (containing 1.5L of BSM medium). In the process, the temperature is 30 ℃, the pH value is adjusted to 5.0 by ammonia water and phosphoric acid, the dissolved oxygen is controlled to be more than 20 percent by adjusting the rotating speed and the air flow (the relative value is determined by the invention according to the fermentation condition of 30 ℃, the pH value of 5.0 and the rotating speed of 600rpm as 100 percent, and the dissolved oxygen of saturated sodium sulfite solution is 0, the same below) (for example, the stirring rotating speed is 600rpm, and the ventilation volume is 2.0 vvm). After the glycerol is completely consumed (the DO value of dissolved oxygen is rapidly increased), the glycerol feeding fermentation stage is started.

And (3) glycerol feeding and fermenting stage: the flow rate of the aqueous glycerol solution (50% w/v, i.e., 500g/L of the aqueous glycerol solution) was 30mL/h/L of the starting fermentation broth, and the addition was carried out for 4 hours. Stopping feeding after the glycerol is completely consumed, starving for 1h, and then entering a methanol feeding induced expression stage. The culture temperature of the glycerol feeding fermentation stage is 30 ℃, the pH value is 5.0, and the dissolved oxygen is controlled to be more than 20%.

Methanol feeding induction expression stage: adjusting pH to 6.0 at 30 deg.C, controlling methanol flow rate to be about 6mL/h/L, stopping adding methanol until wet weight of thallus reaches 320g/L, and maintaining dissolved oxygen at above 20%, if not above 20%, properly reducing the adding speed.

And (3) mixed feeding induction expression stage: the temperature is kept at 30 ℃, the pH is kept at 6.0, the flow rate of methanol is controlled to be about 4-5mL/h/L of the initial fermentation broth, meanwhile, the flow rate of glycerol aqueous solution (50% w/v, namely 500g/L of glycerol aqueous solution) is controlled to be about 90mL/h/L of the initial fermentation broth till the end of the fermentation, and the dissolved oxygen is kept above 20% in the stage, if the dissolved oxygen cannot be kept above 20%, the feeding speed of glycerol and methanol is properly reduced.

The fermentation process is shown in figure 3, (■) is enzyme activity of xylanase in fermentation supernatant (supernatant obtained by centrifuging fermentation liquor for 10min under 10000 Xg after fermentation is finished); (●) protein content in the fermentation supernatant; (. tangle-solidup): the wet weight of the cells. The enzyme activity reaches the maximum on the 7 th day, the highest enzyme activity reaches 19000U/mL, the protein content in the fermentation supernatant reaches 10.8mg/mL, and the wet weight of the thallus is 520 g/L. SDS-PAGE analysis of the fermentation history is shown in FIG. 4, lane M: a low molecular weight standard protein; lanes 1-8: induction of protein content of fermentation supernatants 0, 24, 48, 72, 96, 120, 144, 156h, respectively.

Secondly, purifying the recombinant xylanase CsXyn11A

Q-Sepharose FF (Q-Sepharose FF) ion exchange chromatography: the supernatant of the fermentation broth (supernatant obtained by centrifuging the fermentation broth at 10000 Xg for 10min after the end of fermentation, i.e., crude enzyme solution in Table 2) in a 10mL fermenter was dialyzed and equilibrated to 20mM Tris-HCl buffer solution pH7.5, and then applied to a Q-Sepharose FF column equilibrated with 20mM Tris-HCl buffer solution pH 7.5. After washing unbound proteins with 5 column volumes of buffer (20mM pH7.5Tris-HCl buffer), the column was eluted linearly for 50min (NaCl increased linearly from 0 to 500mM within 50 min) with 0-500mM NaCl in buffer (NaCl added to 20mM Tris-HCl pH7.5 buffer) and collected in fractions. The flow rate for buffer equilibration of Q-Sepharose was 1mL/min, the flow rate for loading was 0.5mL/min, the flow rate for elution of unbound protein was 1mL/min for 5 column volumes, and the linear elution rate for buffer containing 0-500mM NaCl was 1mL/min (NaCl increased linearly from 0 to 500mM within 50 min). The pure enzyme, namely the recombinant xylanase CsXyn11A is combined and dialyzed by MOPS buffer solution with the pH value of 7.0 for standby.

The recombinant protein CsXyn11A was further purified by Q-Sepharose (Tris-HCl pH 7.5) anion exchange chromatography to obtain electrophoresis-grade pure enzyme, the recovery rate of the enzyme was 72%, the purification fold was 1.1 times, and the specific enzyme activity of the pure enzyme was 1920U/mg (Table 2). SDS-PAGE of the crude enzyme solution of the recombinant Pichia strain and the purified product (CsXyn11A) obtained therefrom is shown in FIG. 5. Wherein, Lane M is the molecular weight standard, Lane 1 is the supernatant of the fermentation broth, Lane 2 is the purified enzyme purified by Q-Sepharose column chromatography. The results in FIG. 5 show that the recombinant protein CsXyn11A has a size of 20 kDa.

TABLE 2 purification of xylanases Table

Example 3 Properties of recombinant xylanase CsXyn11A

Determination of optimum pH of CsXyn11A

The optimum pH value of the enzyme is determined by the following buffer systems with different pH values respectively: (Glycine-hydrochloric acid) Glycine-HCl (pH 3.0-7.0); (citric acid-Sodium Citrate) Sodium Citrate (pH 3.0-6.0); (acetate) Acetic acid-Sodium acetate (pH 4.0-6.0); MES (pH 5.5-6.5); MOPS (6.5-7.5); Tris-HCl (pH 7.0-9.0); CHES (pH 8.0-10.0); (Glycine-sodium hydroxide) Glycine-NaOH (9.0-10.5); na (Na) ₂ HPO ₄ NaOH (pH 11.0-12.0). Then, the xylanase enzyme activity is measured by adopting a standard method at 70 ℃, and the enzyme activity measured under each pH is respectively calculated by taking the highest enzyme activity as 100%.

As shown in FIG. 6, the optimum pH value of the recombinant xylanase CsXyn11A of example 2 was 7.0.

II, determination of pH stability of CsXyn11A

Respectively diluting the CsXyn11A enzyme solution with the buffer solutions with different pH values in the step one, preserving the temperature of the diluted enzyme solution in a constant-temperature water bath kettle at 60 ℃ for 30min, then cooling the enzyme solution in an ice water bath for 30min, finally measuring the enzyme activity of the xylanase according to the method in the step one under the conditions of 70 ℃ and pH 7.0, and respectively calculating the residual enzyme activity of the enzyme solution after being treated under different acid-base conditions by taking the enzyme activity of the untreated enzyme solution as a reference. The relative enzyme activity is calculated by the percentage of the residual enzyme activity in the reference enzyme activity.

The result is shown in figure 7, the recombinant xylanase CsXyn11A of example 2 has a wide pH stability range, and the residual enzyme activity is above 80% when the pH is 2.0-9.0.

Determination of optimum reaction temperature of CsXyn11A

Properly diluting CsXyn11A in 50mM MOPS buffer solution (pH 7.0), then respectively measuring the enzyme activity of the xylanase at different temperatures of 30-85 ℃ according to the method in the step one, and respectively calculating the relative enzyme activity measured at each temperature by taking the highest value of the enzyme activity as 100%.

As shown in FIG. 8, the optimum temperature of the recombinant xylanase CsXyn11A of example 2 was 70 ℃.

Temperature stability determination of CsXyn11A

Properly diluting CsXyn11A in 50mM MOPS buffer solution (pH 7.0), then respectively preserving the temperature for 30min at 30-85 ℃, then placing in an ice water bath for cooling for 30min, finally measuring the enzyme activity of the xylanase according to the method under the conditions of 70 ℃ and pH 7.0, and respectively calculating the residual enzyme activity of enzyme solutions after different heat treatments by taking the enzyme solution without heat treatment as a control. The relative enzyme activity is calculated by the percentage of the residual enzyme activity in the reference enzyme activity.

The result is shown in figure 9, the recombinant xylanase CsXyn11A of the example 2 is stable at 60 ℃, and the residual enzyme activity is more than 80%.

Half-life determination of CsXyn11A

The recombinant xylanase CsXyn11A of example 2 was diluted appropriately in 50mM MOPS buffer (pH 7.0), then incubated at 50-65 ℃ for 4h, sampled at different time periods, then placed in an ice-water bath to cool for 30min, finally the enzyme activity of the xylanase was determined according to the above method at 70 ℃ and pH 7.0, and the residual enzyme activity of the enzyme solutions after different heat treatments was calculated with the enzyme solution without heat treatment as a control. And calculating half-lives of CsXyn11A at different temperatures according to the residual enzyme activity.

The results are shown in FIG. 10, the half-life of the recombinant xylanase CsXyn11A at 50 ℃, 55 ℃, 60 ℃ and 65 ℃ is 717, 450, 182 and 27min respectively.

Sixthly, substrate specificity

The substrate specificity of CsXyn11A was determined using various glycans and artificially synthesized p-nitrophenol-glycoside (pNP-glycoside) as substrates. The glycan comprises: birch xylan, beechwood xylan, oat xylan, wheat arabinoxylan, barley glucan, CMC, colloidal chitin, chitosan, soluble starch, tamarind gum, and locust bean gum. pNP-glycosides include: pNP-. beta. -xylopyranoside, pNP-. beta. -glucopyranoside and pNP-. beta. -galactopyranoside.

The specific determination method is as follows:

glycan substrate: the concentration was 1% (w/v, 1g/100mL) in 50mM MOPS buffer pH 7.0, and the concentration was measured according to the xylanase activity assay.

pNP-glycoside substrate: the different synthetic glycoside substrates were prepared to a concentration of 5mM with 50mM MOPS buffer pH 7.0, reacted at 70 ℃ for 10min, and OD _540nm Absorbance is measured and the enzyme activity units (U) define the amount of enzyme required to hydrolyze the substrate to release 1. mu. mol pNP per minute under the reaction conditions described above.

As shown in Table 3, CsXyn11A showed high activity on beech xylan, birch xylan, wheat arabinoxylan and oat arabinoxylan, and showed no activity on other substrates, confirming that the substrate specificity of the enzyme was specific.

TABLE 3 substrate specificity of recombinant xylanase CsXyn11A

Kind of substrate	Specific enzyme activity (U/mg)	Relative enzyme activity (%)
			Beech xylan	1920	100
Birch xylan	1800	94
			Wheat arabinoxylan	1850	96
Oat xylan	2540	132
			Barley glucan	-
CMC	-
			Colloidal chitin	-
Chitosan
			Tamarind gum	-
Soluble starch
			pNP-β-xylopyranoside	-
pNP-β-glucopyranoside	-

"-" indicates no detectable activity

Hydrolysis characteristics of seven, CsXyn11A

1% (i.e., 1g/100mL) of different substrates (xylobiose-xylohexaose, beechwood xylan, birch xylan, oat xylan and wheat arabinoxylan) were prepared in 50mM MOPS buffer pH 7.0 with an enzyme addition of 10U/mL and incubated at 50 ℃ for 12 h. In order to monitor the reaction process, samples are taken for 0min, 15min, 30min, 1h, 2h, 4h, 8h and 12h respectively, and enzyme is inactivated in boiling water bath for 10 min. The hydrolysate was analyzed by Thin Layer Chromatography (TLC) (spreading agent: n-butanol/acetic acid/water 2:1:1 (vol.); developer: sulfuric acid/methanol 5/95 (vol.)).

The results of thin layer chromatography are shown in FIG. 11. It can be seen that CsXyn11A can hydrolyze wheat arabinoxylan and oat xylan, and the hydrolysis products are mainly xylobiose and xylotriose. The products of hydrolysis of beechwood xylan and birch xylan are mainly xylobiose, xylotriose, pentasaccharide and hexasaccharide. The CsXyn11A hydrolyzes the hydrolysis products of xylotetraose and xylopentaose into xylobiose and xylotriose, and the minimum hydrolysis product is xylotriose.

Example 4 application of recombinant xylanase CsXyn11A in bread baking

Bread making process and related parameter measuring method

700g of flour was weighed and 8.4g of dried yeast powder was added thereto. 70g of white granulated sugar and 7.0g of salt are weighed and dissolved in 406mL of purified water, and a certain amount of pure enzyme solution (0-6ppm, 1ppm, namely 1mg of enzyme is added to 1 kg) is added. Pouring the flour and the mixed solution into a dough mixer, stirring at low speed for 2min and at high speed for 3min, adding 28g of butter, continuing stirring at low speed for 2min, and stirring at high speed for 3 min. Taking out the dough after stirring, and standing for 5 min. Dividing the dough into 150 g/piece, kneading until the surface is smooth and mellow, standing for 15min, rolling bubbles, shaping, boxing, and fermenting at 38 deg.C and 80% relative humidity for 90 min. Baking the proofed dough in an oven with the upper fire of 180 ℃ and the lower fire of 200 ℃ for 20 min. After baking, the bread is cooled for 2 hours at the temperature of 25 ℃, and then is packaged into a fresh-keeping bag for sealing.

Specific volume measurement: measuring the volume of the bread by a rapeseed row volume method; the bread mass was weighed using a balance.

Measurement of hardness: the bread was cut into uniform slices with a thickness of 20mm from the middle, and the hardness of the core-spun was measured using a texture analyzer (equipped with an R-36A cylindrical probe). The test speed is 1mm/s, the compression deformation amount is 40 percent, and the induction force is 0.5N.

Determination of bread aging: the bread was stored at 4 ℃ and the bread hardness was measured under the same conditions at the same time intervals and the change in bread hardness was recorded.

The effect of adding recombinant xylanase (CsXyn11A) on bread quality during bread making is shown in table 4. The results show that the specific volume of bread is increasing and the hardness is decreasing with the increasing amount of CsXyn11A, and when the amount of enzyme is 5ppm, the specific volume of bread is increased by 25.6% and the hardness is decreased by 46%. In addition, CsXyn11A can effectively delay the aging of bread (fig. 12)

TABLE 4 Effect of recombinant xylanase (CsXyn11A) on bread

Enzyme addition amount	Specific volume of bread	Increase of bread specific volume	Hardness of	Reduction in hardness
					(ppm)	(mL/g)	Percent (%)	(N)	Percent (%)
0	4.96±0.13	0	5.17±0.03	0
					1	5.35±0.06	7.9	3.77±0.12	27.1
2	5.65±0.15	13.9	3.26±0.02	36.9
					3	5.80±0.10	16.9	2.99±0.10	42.2
4	6.14±0.06	23.8	2.89±0.03	44.1
					5	6.23±0.09	25.6	2.79±0.02	46.0
6	6.16±0.08	24.2	2.74±0.11	47.0

Example 5 application of recombinant xylanase CsXyn11A in steamed bread making

Steamed bun making process and related parameter measuring method

500g of flour is weighed, and 2.5g of dried yeast powder and a certain amount of pure enzyme solution (2-5ppm) are added into the flour. Taking the control group without enzyme solution, stirring at low speed for 5min, and stirring for 3 min. Taking out the dough, covering with film at room temperature, standing for 10min, dividing into 100 g/dough, kneading, shaping, placing into a proof box with temperature of 38 deg.C and relative humidity of 80%, proof for 40min, and steaming in a steamer for 15 min. After the steaming is finished, the steamed buns are placed at room temperature for cooling for 1 hour and then are put into a self-sealing bag, and the specific volume and the hardness are measured.

Measurement of specific volume: measuring the volume of the steamed bun by a rapeseed volume displacement method; the steamed bun mass was weighed using a balance.

Measurement of hardness: the bread was cut into uniform slices with a thickness of 20mm from the middle, and the hardness of the steamed bread was measured using a texture analyzer (equipped with an R-36A cylindrical probe). The test speed is 1mm/s, the compression deformation amount is 30 percent, and the induction force is 0.5N.

Measurement of aging: storing the steamed buns at 4 ℃, measuring the hardness of the steamed buns at the same time interval and under the same conditions, and recording the hardness change of the steamed buns.

The effect of adding recombinant xylanase (CsXyn11A) on steamed bun quality during steamed bun preparation is shown in table 5. The result shows that the specific volume of the steamed bread is increased and the hardness is reduced along with the increase of the addition amount of CsXyn11A, and when the addition amount of the enzyme is 4ppm, the specific volume of the steamed bread is increased by 29.3 percent and the hardness is reduced by 41 percent. In addition, the CsXyn11A can effectively delay the aging of the steamed bread.

TABLE 5 Effect of recombinant xylanase (CsXyn11A) on steamed bread

Enzyme addition amount	Specific volume of steamed bread	Increase of specific volume of steamed bread	Hardness of	Reduction in hardness
					(ppm)	(mL/g)	Percent (%)	(N)	Percent (%)
0	2.08±0.09	0	27.43±0.03	0
					2	2.30±0.01	10.6	22.17±0.12	19.1
3	2.44±0.05	17.3	18.29±0.02	33.3
					4	2.69±0.04	29.3	16.19±0.10	40.9
5	2.71±0.03	30.2	16.09±0.03	41.3

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> university of agriculture in China

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gccctgtctt aaaccttttt ttttatcatc attattagct tactttcata attgcgactg 660

gttccaattg acaagctttt gattttaacg acttttaacg acaacttgag aagatcaaaa 720

aacaactaat tattcgaaat tggagctcgc tcattccaat tccttctatt aggctactaa 780

caccatgact ttattagcct gtctatcctg gcccccctgg cgaggttcat gtttgtttat 840

ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag ggctttctga 900

gtgtggggtc aaatagtttc atgttcccca aatggcccaa aactgacagt ttaaacgctg 960

tcttggaacc taatatgaca aaagcgtgat ctcatccaag atgaactaag tttggttcgt 1020

tgaaatgcta acggccagtt ggtcaaaaag aaacttccaa aagtcgccat accgtttgtc 1080

ttgtttggta ttgattgacg aatgctcaaa aataatctca ttaatgctta gcgcagtctc 1140

tctatcgctt ctgaaccccg gtgcacctgt gccgaaacgc aaatggggaa acacccgctt 1200

tttggatgat tatgcattgt ctccacattg tatgcttcca agattctggt gggaatactg 1260

ctgatagcct aacgttcatg atcaaaattt aactgttcta acccctactt gacagcaata 1320

tataaacaga aggaagctgc cctgtcttaa accttttttt ttatcatcat tattagctta 1380

ctttcataat tgcgactggt tccaattgac aagcttttga ttttaacgac ttttaacgac 1440

aacttgagaa gatcaaaaaa caactaatta ttcgaa 1476

<210> 5

<211> 247

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt 60

ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc 120

ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa 180

tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta 240

agtgaga 247

<210> 6

<211> 5390

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggtacccctc tcgtatgcag aggaaatctc ccctgatctt ccgaactggt cgtacctggc 60

gacctatgac tatggcaccc cagttctggg gaccttccac ggaagtgacc tgctgcaggt 120

gttctatggg atcaagccaa actatgcagc tagttctagc cacacgtact atctgagctt 180

tgtgtatacg ctggatccga actccaaccg gggggagtac attgagtggc cgcagtggaa 240

ggaatcgcgg cagttgatga atttcggagc gaacgacgcc agtctcctta cggatgattt 300

ccgcaacggg acatatgagt tcatcctgca gaataccgcg gcgttccaca tctgatgcca 360

ttggcggagg ggtccggacg gtcaggaact tagccttatg agatgaatga tggacgtgtc 420

tggcctcgga aaaggatata tggggatcat gatagtacta gccatattaa tgaagggcat 480

ataccacgcg ttggacctgc gttatagctt cccgttagtt atagtaccat cgttatacca 540

gccaatcaag tcaccacgca cgaccgggga cggcgaatcc ccgggaattg aaagaaattg 600

catcccaggc cagtgaggcc agcgattggc cacctctcca aggcacaggg ccattctgca 660

gcgctggtgg attcatcgca atttcccccg gcccggcccg acaccgctat aggctggttc 720

tcccacacca tcggagattc gtcgcctaat gtctcgtccg ttcacaagct gaagagcttg 780

aagtggcgag atgtctctgc aggaattcaa gctagatgct aagcgatatt gcatggcaat 840

atgtgttgat gcatgtgctt cttccttcag cttcccctcg tgcagatgag gtttggctat 900

aaattgaagt ggttggtcgg ggttccgtga ggggctgaag tgcttcctcc cttttagacg 960

caactgagag cctgagcttc atccccagca tcattacacc tcagcaatgt cgttccgatc 1020

tctactcgcc ctgagcggcc tcgtctgcac agggttggca ttccccttca acgccaccga 1080

ggtgagggag ctcgtgggcc gcgccggaac ccccagcggt actggcaccc acgacggctt 1140

cttctactcg ttctggaccg acaacggcgg cactgtctgg tacgagaacg gccctggcgg 1200

ctcgtacagc gtcaactggg agaactgcgg caactttgtc ggcggcaagg gctggaaccc 1260

cggctcggac cggaccatca actactcggg ccagttcaac ccgtcgggca acggctacct 1320

ggccatctac ggctggacgc gcaacccgct ggtcgagtac tacatcgtcg aggcgttcgg 1380

caactacgac ccctcgtcgc aggccgaggt gctgggcacg gtcgagacgg acggcagcac 1440

gtacaccatc gccaagagca cgcggtacaa cgcgccgtcg atcgagggcg actcgagcac 1500

gttcgaccag tactggtcgg tccgccacaa ccaccggtcc agcggctccg tcaacgtcgg 1560

caaccacttc cgcgcctggg ccgagagggg cctcaacctc ggctcgcacg actaccagat 1620

cgtcgccacc gagggctacc agagcagcgg ctcgtcctct atcactgtcg agccgtaaga 1680

tccacttaac gttactgaaa tcatcaaaca gcttgacgaa tctggatata agatcgttgg 1740

tgtcgatgtc agctccggag ttgagacaaa tggtgttcag gatctcgata agatacgttc 1800

atttgtccaa gcagcaaaga gtgccttcta gtgatttaat agctccatgt caacaagaat 1860

aaaacgcgtt tcgggtttac ctcttccaga tacagctcat ctgcaatgca ttaatgcatt 1920

ggacctcgca accctagtac gcccttcagg ctccggcgaa gcagaagaat agcttagcag 1980

agtctatttt cattttcggg agacgagatc aagcagatca acggtcgtca agagacctac 2040

gagactgagg aatccgctct tggctccacg cgactatata tttgtctcta attgtacttt 2100

gacatgctcc tcttctttac tctgatagct tgactatgaa aattccgtca ccagcccctg 2160

ggttcgcaaa gataattgca ctgtttcttc cttgaactct caagcctaca ggacacacat 2220

tcatcgtagg tataaacctc gaaaatcatt cctactaaga tgggtataca atagtaacca 2280

gaataagatg gtggagagct tataccgagc tcccaaatct gtccagatca tggttgaccg 2340

gtgcctggat cttcctatag aatcatcctt attcgttgac ctagctgatt ctggagtgac 2400

ccagagggtc atgacttgag cctaaaatcc gccgcctcca ccatttgtag aaaaatgtga 2460

cgaactcgtg agctctgtac agtgaccggt gactctttct ggcatgcgga gagacggacg 2520

gacgcagaga gaagggctga gtaataagcg ccactgcgcc agacagctct ggcggctctg 2580

aggtgcagtg gatgattatt aatccgggac cggccgcccc tccgccccga agtggaaagg 2640

ctggtgtgcc cctcgttgac caagaatcta ttgcatcatc ggagaatatg gagcttcatc 2700

gaatcaccgg cagtaagcga aggagaatgt gaagccaggg gtgtatagcc gtcggcgaaa 2760

tagcatgcca ttaacctagg tacagaagtc caattgcttc cgatctggta aaagattcac 2820

gagatagtac cttctccgaa gtaggtagag cgagtacccg gcgcgtaagc tccctaattg 2880

gcccatccgg catctgtagg gcgtccaaat atcgtgcctc tcctgctttg cccggtgtat 2940

gaaaccggaa aggccgctca ggagctggcc agcggcgcag accgggaaca caagctggca 3000

gtcgacccat ccggtgctct gcactcgacc tgctgaggtc cctcagtccc tggtaggcag 3060

ctttgccccg tctgtccgcc cggtgtgtcg gcggggttga caaggtcgtt gcgtcagtcc 3120

aacatttgtt gccatatttt cctgctctcc ccaccagctg ctcttttctt ttctctttct 3180

tttcccatct tcagtatatt catcttccca tccaagaacc tttatttccc ctaagtaagt 3240

actttgctac atccatactc catccttccc atcccttatt cctttgaacc tttcagttcg 3300

agctttccca cttcatcgca gcttgactaa cagctacccc gcttgagcag acatcaccat 3360

gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 3420

cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 3480

aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 3540

ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 3600

ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 3660

agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc 3720

gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 3780

cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 3840

ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 3900

gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 3960

caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 4020

gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg 4080

tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg 4140

gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 4200

caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 4260

cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 4320

tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga 4380

atagacaatc aatccatttc gctatagtta aaggatgggg atgagggcaa ttggttatat 4440

gatcatgtat gtagtgggtg tgcataatag tagtgaaatg gaagccaagt catgtgattg 4500

taatcgaccg acggaattga ggatatccgg aaatacagac accgtgaaag ccatggtctt 4560

tccttcgtgt agaagaccag acagacagtc cctgatttac ccttgcacaa agcactagaa 4620

aattagcatt ccatccttct ctgcttgctc tgctgatatc actgtcattc aatgcatagc 4680

catgagctca tcttagatcc aagcacgtaa ttccatagcc gaggtccaca gtggagcagc 4740

aacattcccc atcattgctt tccccagggg cctcccaacg actaaatcaa gagtatatct 4800

ctaccgtcca atagatcgtc ttcgcttcaa aatctttgac aattccaaga gggtccccat 4860

ccatcaaacc cagttcaata atagccgaga tgcatggtgg agtcaattag gcagtattgc 4920

tggaatgtcg gggccagttg gcccggtggt cattggccgc ctgtgatgcc atctgccact 4980

aaatccgatc attgatccac cgcccacgag gcgcgtcttt gctttttgcg cggcgtccag 5040

gttcaactct ctctgcagct ccagtccaac gctgactgac tagtttacct actggtctga 5100

tcggctccat cagagctatg gcgttatccc gtgccgttgc tgcgcaatcg ctatcttgat 5160

cgcaaccttg aactcactct tgttttaata gtgatcttgg tgacggagtg tcggtgagtg 5220

acaaccaaca tcgtgcaagg gagattgata cggaattgtc gctcccatca tgatgttctt 5280

gccggctttg ttggccctat tcgtgggatg cgatgccctc gctgtgcagc agcaggtact 5340

gctggatgag gagccatcgg tctctgcacg caaacccaac ttcctctaga 5390

Claims (10)

1. A protein which is a protein of a1), a2) or A3) as follows:

A1) the amino acid sequence is protein of sequence 1 in a sequence table;

A2) the amino acid sequence is protein of 18 th to 222 th amino acid residues of a sequence 1 in a sequence table;

A3) a fusion protein obtained by connecting protein tags at the N terminal or/and the C terminal of A1) or A2).

2. The biomaterial related to the protein of claim 1, which is any one of the following B1) -B4):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

3. The biomaterial of claim 2, wherein: B1) the nucleic acid molecule is a coding gene of the protein shown in the following b1) or b 2):

b1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of a sequence 2 in a sequence table;

b2) the nucleotide of the coding chain is cDNA molecule or DNA molecule of 52 th-699 th nucleotide of sequence 2 in the sequence table.

4. Use of a protein according to claim 1 as a xylanase.

5. Use of a biomaterial as claimed in claim 2 or 3 in the preparation of a xylanase.

6. Use according to claim 4 or 5, characterized in that: the substrate of the xylanase is at least one of beechwood xylan, birch xylan, wheat araboxylan and oat xylan.

7. Use of a protein as claimed in claim 1 and/or a biomaterial as claimed in claim 2 or 3 in any one of the following applications:

p1, application in preparing flour products;

p2, application in preparing xylo-oligosaccharide;

p3, application in preparing flour food;

p4, application in preparing flour product feed;

p5, use in the preparation of a food additive product;

p6, use in food or feed processing;

p7, use in bread baking;

p8, application in steamed bread making.

8. A product comprising a protein according to claim 1, wherein: the product is food additive or feed additive.

9. A method of making a xylanase, comprising: expressing a gene encoding the protein of claim 1 in an organism to obtain xylanase; the organism is a microorganism, a plant or a non-human animal.

10. The method of claim 9, wherein: the microorganism is a yeast or filamentous fungus.

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