Wheat arabinoxylanase, related biological materials thereof, preparation method and application of wheat arabinoxylanase

Abstract

The invention belongs to the technical field of genetic engineering, and discloses wheat arabinoxylanase, a related biological material, a preparation method and application thereof. The invention discloses wheat arabinoxylanase, namely recombinant xylanase PcXyn11A. The amino acid sequence of the wheat arabinoxylanase is as shown in SEQ ID No.1 or is a protein which has at least 80% of identity with the sequence as shown in SEQ ID No.1. The xylanase is derived from brachypodium longibrachiatum, the specific enzyme activity is 409 U / mg, the optimum pH value is 7.5, the optimum temperature is 55 DEG C, the xylanase can be kept stable when the pH value is 4.5-9.0, and products for hydrolyzing zelkova xylan are xylobiose, xylotriose and xylooligosaccharide with the polymerization degree of 4-7. The coding gene of the wheat arabinoxylanase is introduced into pichia pastoris, a constructed recombinant strain is subjected to high-density fermentation, and the enzyme activity of fermentation liquor is as high as 727 U / mL. The method can be applied to the fields of preparation or improvement of flour products, preparation of xylooligosaccharide, food additives or feed additives and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to a xylanase, in particular to a wheat arabinoxylanase, related biological materials, a preparation method and an application thereof. Background Art

[0002] Xylanases are key enzymes that can hydrolyze xylan to produce xylose or xylo-oligosaccharides and are widely used in the food and feed industries. Xylanases are widely distributed in nature, with microorganisms being the main source. Currently, there have been a large number of reports on microbial-derived xylanases, especially from bacteria, such as Bacillus sp. Bacillus sp. ) and Paenibacillus ( Paenibacillus sp. ), while the fungal sources are relatively less studied, mainly from Aspergillus ( Aspergillus sp. )、Penicillium( Penicillium sp. ) and Trichoderma ( Trichoderma sp. ). Genetic engineering technology can be used to increase the production of enzymes to meet the needs of large-scale industrialization. At present, some xylanases have been produced in Escherichia coli ( Escherichia coli )、Bacillus subtilis( Bacillus subtilis ) and Pichia pastoris ( Pichia pastoris ), but the expression levels of most xylanases were still low. Thermostat maritima The xylanase was expressed in Bacillus subtilis, and the enzyme activity after fermentation was only 5 U / mL; Aspergillus sulphurus ( Aspergillus sulphureus ) The expression of xylanase in Pichia pastoris, the enzyme activity after fermentation was 100 U / mL; Trichoderma reesei The xylanase was expressed in Pichia pastoris, and the enzyme activity after fermentation was 130 U / mL. Summary of the invention

[0003] In view of the current situation that the expression level of xylanase in engineered bacteria is generally low in the prior art, the present invention creatively discovered a high-activity wheat arabinoxylanase from Podospora pilosa, which has excellent pH stability and high wheat arabinoxylanase activity.

[0004] Another object of the present invention is to provide the above-mentioned wheat arabinoxylanase-related biological materials, including its encoding gene, a recombinant vector containing the encoding gene and a recombinant strain, so as to achieve the expression of the wheat arabinoxylanase gene and lay the foundation for commercial production; Another object of the present invention is to provide the use of the wheat arabinoxylanase in flour products, preparation of xylo-oligosaccharides, food additives or feed additives.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a wheat arabinoxylanase, namely, a recombinant xylanase PcXyn11A, whose amino acid sequence is shown in SEQ ID No.1 or a protein having at least 80% identity with the sequence of SEQ ID No.1.

[0006] The wheat arabinoxylanase provided by the present invention is named as recombinant xylanase PcXyn11A, and its encoding gene is derived from Podospora longissima ( Podospora comata ), belonging to the glycoside hydrolase (GH) 11 family. The molecular weight of the recombinant xylanase PcXyn11A is 21 kDa, the isoelectric point is 8.59, the specific enzyme activity is 409 U / mg, the optimum pH is 7.5, the optimum temperature is 55°C, and it can remain stable between pH 4.5-9.0. The product of hydrolyzing beechwood xylan is oligosaccharide with a degree of polymerization of 4-7.

[0007] Furthermore, the amino acid sequence of the wheat arabinoxylanase is as shown in SEQ ID No. 2 or a protein having at least 80% identity with the sequence of SEQ ID No. 2.

[0008] The amino acid sequence shown in SEQ ID No. 2 of the present invention consists of 223 amino acid residues, the amino acid residues 1-18 from the amino terminal (N terminal) are signal peptides, and the amino acid residues shown from amino acid positions 19-223 are mature proteins. In the present invention, the amino acid sequence shown in SEQ ID No. 1 is the amino acid residues shown at positions 19-223 in the amino acid sequence shown in SEQ ID No. 2.

[0009] Wherein, the amino acid sequence of SEQ ID No.1 is specifically: vpfnatevfeslqaragtpsstgnhngyyysfwtdnggtvnyqngnggsysvnwqncgnfvggkgwnpgnsnrvinysgqfnpqggngylaiygwtrnplieyy iiesfgtydpssgatrlgtfntdggtytiakstrynqpsiegtktfdqfwsvrtskrvggsvnvgnhfkawadkglrlgshdyqivategyqssgsasitvs.

[0010] The amino acid sequence shown in SEQ ID No.2 is specifically: mvsfkslltvtlatgaaavpfnatevfeslqaragtpsstgnhngyyysfwtdnggtvnyqngnggsysvnwqncgnfvggkgwnpgnsnrvinysgqfnpqgngylaiygw trnplieyyiiesfgtydpssgatrlgtfntdggtytiakstrynqpsiegtktfdqfwsvrtskrvggsvnvgnhfkawadkglrlgshdyqivategyqssgsasitvs.

[0011] The identity in the present invention refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using an identity search site on the Internet, such as the BLAST page on the NCBI homepage website.

[0012] In a second aspect, the present invention provides the above-mentioned wheat arabinoxylanase-related biological material, wherein the biological material is any one of the following (I) to (IV): (I) a nucleic acid molecule encoding the above-mentioned wheat arabinoxylanase; (II) an expression cassette containing the nucleic acid molecule described in (I); (III) a recombinant vector containing the nucleic acid molecule described in (I), or a recombinant vector containing the expression cassette described in (II); (IV) A recombinant strain containing the nucleic acid molecule described in (I), or a recombinant strain containing the expression cassette described in (II), or a recombinant strain containing the recombinant vector described in (III).

[0013] Preferably, the nucleic acid molecule is any one of the following genes (i) to (v): (i) the coding sequence is the 55-669th position from the 5′ end of SEQ ID No. 3; (ii) the nucleotide sequence of which is positions 1 to 669 from the 5′ end of SEQ ID No. 3; (iii) the nucleotide sequence of which is shown in SEQ ID No. 3; (iv) a gene encoding the above-mentioned wheat arabinoxylanase which is hybridized with the gene represented by (i), (ii) or (iii); (v) A gene encoding the above-mentioned wheat arabinoxylanase having an identity of 80% or more to the gene represented by (i), (ii) or (iii).

[0014] SEQ ID No.3 consists of 672 bases, and the coding sequence is the 55th to 669th bases from the 5' end, encoding a protein having the amino acid sequence shown in SEQ ID No.1; the 1st to 54th bases from the 5' end are signal peptide coding sequences, and the 670th to 672nd bases are sequences encoding stop codons. The 1st to 669th bases from the 5' end in SEQ ID No.3 encode a protein having the amino acid sequence of SEQ ID No.2.

[0015] The nucleotide sequence shown in SEQ ID No.3 is specifically: .

[0016] The expression cassette, recombinant vector or recombinant strain containing the above gene all fall within the protection scope of the present invention.

[0017] The nucleic acid molecule encoding the wheat arabinoxylanase is inserted into pPIC9K Carrier or PcAU The recombinant vector is constructed between the multiple cloning sites of the vector; the starting strain of the recombinant strain includes Pichia pastoris GS115, and the recombinant Pichia pastoris is obtained by introducing the above-mentioned recombinant vector into Pichia pastoris; the present invention is described by taking Pichia pastoris GS115 as an example.

[0018] In a third aspect, the present invention provides the use of the above-mentioned wheat arabinoxylanase-related biological materials in preparing wheat arabinoxylanase.

[0019] In a fourth aspect, the present invention provides the use of the wheat arabinoxylanase or the biological material in the preparation or improvement of flour products, the preparation of xylo-oligosaccharides, and food additives or feed additives.

[0020] When the recombinant xylanase PcXyn11A is used in the production process of whole wheat bread, the specific volume of the bread increases by 11.45%, the hardness decreases by 34.68%, and the aging of the bread can be effectively delayed. The wheat arabinoxylanase provided by the present invention has broad application prospects in the flour product industry.

[0021] In a fifth aspect, the present invention also provides a method for preparing the above-mentioned wheat arabinoxylanase, the preparation method comprising the following steps: fermenting and culturing the above-mentioned recombinant strain to obtain wheat arabinoxylanase. The fermentation and culturing comprises the stages of basic culture, glycerol fed-batch fermentation, methanol fed-batch induced expression and mixed fed-batch induced expression.

[0022] Preferably, during the mixed fed-batch induction expression stage, methanol and glycerol are added to the culture medium.

[0023] Preferably, the specific fermentation process of wheat arabinoxylanase is: Preparation of seed solution: inoculate the recombinant strain into BMGY medium or YPD medium, shake and culture at 28 ℃~32 ℃ and 180 rpm~220 rpm for 24 h~30 h, and obtain OD 600 The recombinant strain seed solution is 4.0~6.0; Basic culture stage: Inoculate the recombinant strain seed liquid into a fermenter filled with BSM medium at a 6% to 10% inoculation rate; culture at 28 ℃ to 32 ℃, 180 rpm to 220 rpm, pH 4.8 to 5.2, and dissolved oxygen greater than 20% (specifically 20% to 80%). When the dissolved oxygen DO value rises rapidly, the glycerol is considered to be completely consumed and enters the glycerol fed-batch fermentation stage.

[0024] Glycerol fed-batch fermentation stage: under the culture conditions of 28 ℃~32 ℃, 180 rpm~220 rpm, pH 4.8~5.2 and dissolved oxygen 20%~80%; for every 1L of starting fermentation liquid, add glycerol aqueous solution with a concentration of 480 g / L~520 g / L at a rate of 28.8 mL / h~31.2 mL / h; the addition time is 3.5 h~4.5 h. After the glycerol is completely consumed, stop the addition, starve for 1 h, and enter the methanol fed-batch induced expression stage.

[0025] Methanol feeding induction expression stage: methanol was added at a temperature of 28 ℃~32 ℃, 180 rpm~220 rpm, pH 5.8~6.2, and dissolved oxygen above 20%; the addition dosage was: methanol was added at a rate of 5.8 mL / h~6.2 mL / h for every 1 L of starting fermentation liquid, and the addition of methanol was stopped when the wet weight of the bacteria reached 320 g / L.

[0026] Mixed feed induction expression stage: methanol and glycerol were added to the culture medium at a temperature of 28 ℃~32 ℃, a pH of 5.8~6.2, and a dissolved oxygen content of more than 20%. The dosage of methanol added was: methanol was added at a rate of 4mL / h~5mL / h per 1L of the starting fermentation liquid; the dosage of glycerol added was: glycerol aqueous solution with a concentration of 480 g / L~520 g / L was added at a concentration of 86.5 L / h~93.8 mL / h per 1L of the starting fermentation liquid.

[0027] In the preparation method of the wheat arabinoxylanase provided by the present invention, the enzyme activity of the fermentation broth can reach 727 U / mL and the protein content is 4 mg / mL by high-density fermentation of the recombinant strain and controlling the fermentation parameters at each stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 For the recombinant vector pPIC9K- PcXyn11A The vector map of Figure 2 for PcXyn11A The process of producing recombinant xylanase PcXyn11A by the recombinant strain; Figure 3This is the SDS-PAGE analysis diagram of the mixed feeding induction expression stage during the fermentation process, where: lane M is the marker protein, and lanes 1-7 are the fermentation supernatants of methanol feeding induction at 0, 24, 48, 72, 96, 120, and 132 h, respectively; Figure 4 It is the SDS-PAGE image of the fermentation supernatant of the recombinant xylanase PcXyn11A and the pure enzyme of the recombinant xylanase PcXyn11A; wherein, the lane M is the marker protein, the lane 1 is the fermentation supernatant, and the lane 2 is the pure enzyme solution of the recombinant xylanase PcXyn11A; Figure 5 This is the optimum pH determination curve of the recombinant xylanase PcXyn11A; Figure 6 It is a curve diagram of pH stability determination of recombinant xylanase PcXyn11A; Figure 7 This is the optimum temperature determination curve of the recombinant xylanase PcXyn11A; Figure 8 This is a temperature stability test curve of the recombinant xylanase PcXyn11A; Fig. 9 It is a half-life determination curve of the recombinant xylanase PcXyn11A; Fig.10 It is the result of thin layer chromatography of the hydrolysis product of recombinant xylanase PcXyn11A; wherein, Fig.10 (a) indicates that beechwood xylan is used as substrate. Fig.10 (b) indicates that oligoxylose is used as substrate Fig.11 The results of the effect of different amounts of recombinant xylanase PcXyn11A on bread staling; Fig.12 The scanning electron microscope images of different whole wheat doughs and whole wheat breads are shown in Figure 2. Fig.12 (a) is a blank whole wheat dough without adding recombinant xylanase PcXyn11A. Fig.12 (b) Whole wheat dough with 4 ppm recombinant xylanase PcXyn11A added. Fig.12 (c) is a blank whole wheat bread without the addition of recombinant xylanase PcXyn11A. Fig.12 (d) whole wheat bread supplemented with 4 ppm recombinant xylanase PcXyn11A; Fig.13 This is the DSC graph showing the effect of recombinant xylanase PcXyn11A on bread quality. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] In order to better illustrate the embodiments of the present invention, further examples are given below.

[0032] In the following examples, the experimental methods used are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0033] In the following examples, the enzymatic activity of xylanase was determined with reference to Liu et al. (Liu et al., Biological characterization of a novel xylanase from Paenibacillus barengoltzii and its application in xylooligosaccharides production from corncobs. Food Chemistry, 2018, 264:310-318).

[0034] Definition of enzyme activity: The amount of enzyme required to hydrolyze beech wood xylan to produce 1 μmol of xylose per minute under the above conditions.

[0035] The culture medium involved in the following embodiments is: 1. The composition of BMGY medium is as follows: 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) amino-free yeast nitrogen source YNB, 4×10 -5 % (w / v) biotin, 1% (v / v) glycerol, the balance being 100 mmol / L pH 6.0 phosphate buffer; 2. The composition of the improved BMMY medium is as follows: 1% (w / v) yeast extract, 2% (w / v) peptone, 1.34% (w / v) amino-free yeast nitrogen source YNB, 4×10 -5 % (w / v) biotin, 0.5% (v / v) methanol, the balance being 100 mmol / L pH 6.0 phosphate buffer.

[0036] 3. The composition of YPD medium is as follows: 1% (w / v) yeast extract, 2% (w / v) peptone, 2% (w / v) glucose, 2% (w / v) agar, and the balance is water.

[0037] 4. The composition of MD medium is as follows: 2% (w / v) glucose, 1.34% (w / v) amino-free yeast nitrogen source YNB, 4×10 -5 % (w / v) biotin and 2% (w / v) agar, the balance being water.

[0038] 5. BSM medium: 40 mL of 85% phosphoric acid, 1.4 g of CaSO4, 27.3 g of K2SO4, 22.4 g of MgSO4·7H2O, 6.19 g of KOH and 60 g of glycerol, add distilled water to 1.5 L.

[0039] Example 1 Cloning of wheat arabinoxylanase gene and expression in Pichia pastoris A wheat arabinoxylanase derived from Podospora pilosa, named as recombinant xylanase PcXyn11A in the present invention, has an amino acid sequence as shown in SEQ ID No.1.

[0040] 1. Recombinant vector pPIC9K- PcXyn11A Construction Design upstream primer PcXyn11A F and downstream primers PcXyn11A R, and using the cDNA of Podospora longissima as a template for PCR, amplify the CDS nucleotide sequence (such as the nucleotide sequence shown in SEQ ID No.3) encoding the mature protein containing wheat arabinoxylanase (named PcXyn11A) (whose amino acid sequence is shown in SEQ ID No.1 or SEQ ID No.2). Among them, according to Signal P 4.1 analysis, 18 amino acids at the N-terminus of the amino acid sequence shown in SEQ ID No.2 are signal peptide sequences. In other words, the 1st to 18th amino acids of SEQ ID No.2 are the signal peptide sequence of the PcXyn11A protein, and the corresponding 1st to 54th nucleotide molecules in SEQ ID No.3 are the coding sequence of the signal peptide of the PcXyn11A protein. The amino acid sequence of SEQ ID No.2 from 19 to 223 is the same as SEQ ID No.1.

[0041] The amino acid sequences shown in SEQ ID No.1 or SEQ ID No.2 were compared and analyzed in NCBI, and it was found that PcXyn11A was derived from Aspergillus fumigatus ( Aspergillus fumigatus , B0XXF3.1) G11 family wheat arabinoxylanase has the highest homology of 69.00% with Aspergillus nidulans ( Aspergillus nidulans , P55332.1), Aspergillus clavatus ( Aspergillus clavatus , A1CCU0.1), Aspergillus Fischer ( Aspergillus fischeri , A1DNU5.1), Humicola insolens ( Humicola insolens, P55334.1), Rice blast fungus ( Pyricularia oryzae , G4NA54.1), catenella ( Talaromyces funiculosus The homology of PcXyn11A to GH11 family wheat arabinoxylanase was 67.70%, 66.09%, 62.33%, 61.95%, 58.36% and 59.26% respectively. Therefore, PcXyn11A is a new type of wheat arabinoxylanase of GH11 family.

[0042] The above PCR amplification conditions are: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 20 s, 60℃ annealing for 30 s, 72℃ extension for 60 s, 35 cycles; and finally 72℃ extension for 5 min. The product was recovered by 1% agarose gel electrophoresis. The target gene after gel excision was compared with the expression vector after digestion with the same double restriction endonuclease. pPIC9K The fragments were ligated with T4 DNA ligase overnight at 4°C. E. coli DH5α competent cells were spread on LB plates containing ampicillin (100 μg / mL) and inverted at 37°C for 12-16 h. Transformants were selected for PCR amplification and sequencing using verification primers, and the plasmid of the transformant with correct sequencing was extracted to obtain the recombinant vector pPIC9K- PcXyn11A , in which the recombinant vector pPIC9K- PcXyn11A The vector map of Figure 1 As shown. The recombinant vector pPIC9K- PcXyn11A Used for the next experiment.

[0043] Among them, primer PcXyn11A The nucleotide sequence of F is: 5'-CGGAATTCGTTCCGTTTAATGCAACCG-3'; Primers PcXyn11A The nucleotide sequence of R is: 5'-ATATATATGCGGCCGCGCTAACTGTAATACTCGCG-3'; The amino acid sequence of SEQ ID No.1 is specifically: vpfnatevfeslqaragtpsstgnhngyyysfwtdnggtvnyqngnggsysvnwqncgnfvggkgwnpgnsnrvinysgqfnpqggngylaiygwtrnplieyy iiesfgtydpssgatrlgtfntdggtytiakstrynqpsiegtktfdqfwsvrtskrvggsvnvgnhfkawadkglrlgshdyqivategyqssgsasitvs.

[0044] The amino acid sequence shown in SEQ ID No.2 is specifically: mvsfkslltvtlatgaaavpfnatevfeslqaragtpsstgnhngyyysfwtdnggtvnyqngnggsysvnwqncgnfvggkgwnpgnsnrvinysgqfnpqgngylaiygw trnplieyyiiesfgtydpssgatrlgtfntdggtytiakstrynqpsiegtktfdqfwsvrtskrvggsvnvgnhfkawadkglrlgshdyqivategyqssgsasitvs.

[0045] The nucleotide sequence shown in SEQ ID No.3 is specifically: .

[0046] The nucleotide sequence amplified to encode the xylanase PcXyn11A in the present invention is any one of the following genes (i) to (v): (i) the coding sequence is the 55-669th position from the 5′ end of SEQ ID No. 3; (ii) the nucleotide sequence of which is positions 1 to 669 from the 5′ end of SEQ ID No. 3; (iii) the nucleotide sequence of which is shown in SEQ ID No. 3; (iv) a gene that hybridizes with the gene represented by (i), (ii) or (iii) and encodes a wheat arabinoxylanase having an amino acid sequence represented by SEQ ID No. 1 or SEQ ID No. 2; (v) A gene encoding the wheat arabinoxylanase having an amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 2 and having a homology of more than 80% with the gene shown in (i), (ii) or (iii). In this example, the amplification of the nucleotide sequence shown in SEQ ID No. 3 is used as an example for explanation.

[0047] 2. Expression of wheat arabinoxylanase PcXyn11A in Pichia pastoris The competent cells of Pichia pastoris GS115 (Invitrogen) were prepared according to the method described in the instruction manual of Pichia pastoris (Invitrogen). The above recombinant vector pPIC9K- PcXyn11A , and adjust its concentration to 1 μg / μL.

[0048] Take 80 μL of 10 8 CFU / mL of Pichia pastoris GS115 competent cells and 10 μL of linearized recombinant vector pPIC9K- PcXyn11A Mix well, place in a pre-cooled 0.2 cm electroporation cup (Bio-rad), and perform electroporation transformation at 2100 V. After electroporation, different copies of vector pPIC9K-PcXyn11A were obtained, and 1.0 mL of pre-cooled 1 M sorbitol aqueous solution was quickly added to the electroporation cup, and spread on the nutrient deficiency screening plate (MD medium) respectively, and cultured at 30°C for 3 d~4 d. 50 single colonies were selected from the MD plate for enzyme fermentation level comparison. The enzyme fermentation process is as follows: a single colony was inoculated into 25 mL BMGY medium, and cultured at 30°C 200 rpm until OD 600 When the OD was about 12, the cells were collected by centrifugation and transferred to a flask (500 mL) containing 100 mL of improved BMMY medium. 600 After reaching 8, the cells were cultured under the same culture conditions, and methanol was added every 24 hours to a final concentration of 0.5% (volume percentage, i.e., 500 μL of methanol was added every 24 hours to 100 mL BMMY) for 5 days of induction. The enzyme activity was measured to verify whether the protein was successfully expressed. PcXyn11A The constructed Pichia pastoris was used for G418 screening in the next step.

[0049] Collect pPIC9K- PcXyn11AThe collected bacteria on the MD plate were appropriately diluted and coated on the screening plate containing different G418 concentrations (the screening plate was YPD-G418 medium), where the concentrations of G418 were 1 mg / mL, 2 mg / mL, and 4 mg / mL, respectively. They were cultured at 30°C for 3-5 days, and single colonies that grew well under different G418 concentrations were selected for shake flask screening. The strains with high expression levels were selected according to the following method (shake flask fermentation verification): single colonies of Pichia pastoris transformants were selected and inoculated into 25 mL BMGY medium, and cultured at 30°C with shaking at 200 rpm until OD 600 The cells were collected by centrifugation and transferred to a 500 mL Erlenmeyer flask containing 100 mL of improved BMMY medium. 600 When the cell number reached 8, the cells were cultured under the same culture conditions, and methanol was added every 24 h to a final concentration of 0.5% for 5 days of induction.

[0050] After screening, the Pichia pastoris strain with the highest enzyme production was obtained, which was recorded as the PcXyn11A recombinant strain; the PcXyn11A recombinant strain was used for shake flask fermentation verification, and the expression level of the recombinant xylanase PcXyn11A was 5.4 U / mL.

[0051] Example 2 High-density fermentation and purification of recombinant xylanase PcXyn11A 1. High-density fermentation The PcXyn11A recombinant strain obtained in Example 1 was subjected to basic culture, glycerol fed-batch fermentation, methanol fed-batch induced expression and mixed fed-batch induced expression fermentation to obtain recombinant xylanase PcXyn11A. The specific fermentation steps are as follows: Preparation of seed solution: The PcXyn11A recombinant strain obtained in Experimental Example 1 was inoculated into a 500 mL Erlenmeyer flask containing 100 mL BMGY medium or YPD medium (BMGY medium is used as an example in this example), and cultured at 30°C and 200 rpm for 28 h to obtain an OD 600 The PcXyn11A recombinant strain seed solution is 5.0.

[0052] Basic culture stage: 150 mL of PcXyn11A recombinant strain seed liquid was inoculated into a 5 L fermenter (containing 1.5 L of BSM medium). The temperature control system of the fermenter was controlled to 30 ° C, the pH was adjusted to 5.0 with ammonia and phosphoric acid, and the dissolved oxygen was controlled to be greater than 20% by adjusting the rotation speed and air flow (in the present invention, the fermentation conditions were 30 ° C, pH 5.0, and the rotation speed was 600 rpm, which was defined as 100% dissolved oxygen, and the dissolved oxygen in the saturated sodium sulfite solution was 0). In this embodiment, the stirring speed was 600 rpm and the ventilation volume was 2.0 vvm. The dissolved oxygen DO value in the fermented liquid increased by more than 3 times within 1 h, which was recorded as a rapid increase in the dissolved oxygen DO value, and the glycerol was considered to be completely consumed, and then entered the glycerol fed fermentation stage.

[0053] Glycerol fed-batch fermentation stage: The culture temperature in this stage is controlled to be 30°C, pH 5.0, and dissolved oxygen greater than 20% (in this embodiment, the dissolved oxygen is controlled to be 20% to 80%). Based on 1 L of the starting fermentation broth, the flow rate of the glycerol aqueous solution (50% w / v, i.e., 500 g / L) is controlled to be 30 mL / h, and the feed is added for 4 h. After the glycerol is completely consumed, the feed is stopped, and the cells are starved for 1 h, and then the methanol fed-batch induction expression stage is entered.

[0054] Methanol feeding induction expression stage: The temperature in this stage is controlled to be 30°C, pH is 6.0, and the dissolved oxygen is above 20% (in this embodiment, the dissolved oxygen is controlled to be 20%-80%). Based on 1 L of the starting fermentation liquid, the methanol flow rate is controlled to be 6 mL / h, and the feeding of methanol is stopped when the wet weight of the bacteria reaches 320 g / L, and the mixed feeding induction expression stage is entered.

[0055] Mixed feed induction expression stage: The temperature of this stage is controlled to be 30°C, pH is 6.0, and the dissolved oxygen is above 20% (in this embodiment, the dissolved oxygen is controlled to be 20% to 80%). Based on 1 L of the starting fermentation liquid, the flow rate of methanol is controlled to be 4.5 mL / h, and the flow rate of the glycerol aqueous solution (50% w / v, i.e., 500 g / L) is controlled to be 90 mL / h. When the enzyme activity is monitored and no longer increases or tends to be stable, the fermentation is terminated to obtain the fermentation liquid.

[0056] The fermentation broth was centrifuged at 10,000 × g for 10 min to obtain the fermentation supernatant, also known as crude enzyme solution.

[0057] The process of producing recombinant xylanase PcXyn11A by the PcXyn11A recombinant strain Figure 2 As shown, the initial time of entering the methanol fed-batch induced expression phase is 0 h, and the time ends at the end of the mixed fed-batch induced expression phase. Figure 2(■) is the enzyme activity of recombinant xylanase PcXyn11A in the fermentation supernatant; (▲) is the protein content in the fermentation supernatant; (●) is the wet weight of the bacteria. Figure 2 It can be seen that the enzyme activity of the recombinant xylanase PcXyn11A reached its maximum at 132 h, with a maximum enzyme activity of 727 U / mL. At the same time, the protein content in the fermentation supernatant reached 4.4 mg / mL, and the wet weight of the bacteria was 365 g / L.

[0058] SDS-PAGE analysis of mixed-batch induction expression during fermentation Figure 3 As shown, the initial time of entering the methanol fed-batch induced expression phase is 0 h, and the time ends at the end of the mixed fed-batch induced expression phase. Figure 3 Middle lane M: Marker protein; Lanes 1-7: fermentation supernatants at 0, 24, 48, 72, 96, 120, and 132 h of methanol feeding induction, respectively.

[0059] 2. Purification of recombinant xylanase PcXyn11A SP-Sepharose FF (SP Fast Flow) ion exchange chromatography: Take 10 mL of the above fermentation broth supernatant, dialyze and balance it into a buffer (the buffer in this embodiment is 20 mM Tris-HCl buffer at pH 6.5, the same below), and then load it onto the SP column balanced with the buffer. Use 5 column volumes of buffer to wash the unbound protein. Then use an AKTA purifier for purification, apply 0-500 mM NaCl linear elution and collect in steps. The flow rate of SP-Sepharose for buffer equilibration is 1 mL / min, the flow rate during loading is 0.5 mL / min, the flow rate of eluting unbound proteins for 5 column volumes is 1 mL / min, and the linear elution rate of the buffer containing 0-500 mM NaCl is 1 mL / min (NaCl linearly increases from 0 to 500 mM within 50 min). The obtained recombinant xylanase PcXyn11A enzyme solutions purified by SP-Sepharose FF column were combined and dialyzed with Tris-HCl buffer at pH 7.0 to obtain a preliminary purified enzyme solution.

[0060] The above-mentioned preliminary purified enzyme solution was purified by SP (Tris-HCl pH 6.5) cation exchange chromatography to obtain electrophoresis grade recombinant xylanase PcXyn11A, which was recorded as recombinant xylanase PcXyn11A pure enzyme. The SDS-PAGE image of the fermentation supernatant of recombinant xylanase PcXyn11A and the recombinant xylanase PcXyn11A pure enzyme is shown in the figure below: Figure 4 Among them, lane M is the marker protein, lane 1 is the fermentation broth supernatant, and lane 2 is the recombinant xylanase PcXyn11A pure enzyme solution.

[0061] Figure 4 In the above analysis, the lane of the pure enzyme solution of recombinant xylanase PcXyn11A showed a single clear band, and the molecular weight of the protein of recombinant xylanase PcXyn11A in a denatured state was measured by SDS-PAGE to be 21 kDa. The statistical table of various parameters before and after purification of recombinant xylanase PcXyn11A is shown in Table 1.

[0062] Table 1 Statistics of parameters of recombinant xylanase PcXyn11A before and after purification

[0063] As shown in Table 1, the specific enzyme activity of the recombinant xylanase PcXyn11A before purification was 258.9 U / mg, and after purification, the specific enzyme activity increased to 409.0 U / mg, the purification factor was 1.58, and the recovery rate was 52.0%. Example 3 Enzymatic properties of recombinant xylanase PcXyn11A 1. Determination of the optimal pH The optimum pH value of the recombinant xylanase PcXyn11A was determined using the following buffer systems with different pH values: Citric acid-Na2HPO4 (i.e., citric acid-disodium hydrogen phosphate), pH 3.0-7.0; Na2HPO4-NaH2PO4, pH 6.0-8.0; Tris-HCl, pH 7.0-9.0; CHES, pH 8.0-10.0. The enzyme activity of the recombinant xylanase PcXyn11A was then determined at 55°C using the standard method, and the enzyme activity measured at each pH was calculated with the highest enzyme activity being 100%.

[0064] The optimum pH determination curve of recombinant xylanase PcXyn11A is shown in Figure 5 As shown. Figure 5 It can be seen that the optimal pH value of the recombinant xylanase PcXyn11A is 7.5.

[0065] 2. Determination of pH stability The recombinant xylanase PcXyn11A pure enzyme solution was diluted with the above buffer solutions of different pH, and the diluted pure enzyme solution was kept in a constant temperature water bath at 40℃ for 30 min, then cooled in an ice water bath for 30 min, and finally the enzyme activity of wheat arabinoxylanase was determined according to the above method at 50℃ and pH 7.5. The enzyme activity of the untreated enzyme solution was used as the control, and the residual enzyme activity of the enzyme solution after treatment under different acid-base conditions was calculated. The relative enzyme activity was calculated as the percentage of the residual enzyme activity to the control enzyme activity.

[0066] The pH stability test curve of recombinant xylanase PcXyn11A is shown in Figure 6 As shown. Figure 6 It can be seen that the residual enzyme activity is above 80% in the pH range of 4.5-9.0, indicating that the recombinant xylanase PcXyn11A has a wide pH stability range.

[0067] 3. Determination of the optimal reaction temperature The pure enzyme solution of recombinant xylanase PcXyn11A was appropriately diluted in 50 mM Tris-HCl buffer (pH 7.5), and then the enzyme activity of wheat arabinoxylanase was determined at different temperatures from 25°C to 75°C according to the above method. The highest enzyme activity value was taken as 100%, and the relative enzyme activity measured at each temperature was calculated. The optimum temperature determination curve of recombinant xylanase PcXyn11A is shown in the figure below. Figure 7 shown.

[0068] Depend on Figure 7 It can be seen that the activity of recombinant xylanase PcXyn11A first increased and then decreased in the range of 25℃~70℃, and the optimum temperature was 55℃.

[0069] 4. Determination of temperature stability The pure enzyme solution of recombinant xylanase PcXyn11A was appropriately diluted in 50 mM Tris-HCl buffer (pH 7.5), then kept at 25℃~75℃ for 30 min, then cooled in an ice water bath for 30 min, and finally the enzyme activity of xylanase was determined at 55℃ and pH 7.5 according to the above method. The enzyme solution without heat treatment was used as the control, and the residual enzyme activity of the enzyme solution after different heat treatments was calculated. The relative enzyme activity was calculated as the percentage of the residual enzyme activity to the control enzyme activity.

[0070] The temperature stability curve of recombinant xylanase PcXyn11A is shown in Figure 8 As shown. Figure 8 It can be seen that the recombinant xylanase PcXyn11A remains stable at 40°C and the residual enzyme activity can be maintained above 80%.

[0071] 5. Determination of half-life The pure enzyme solution of recombinant xylanase PcXyn11A was appropriately diluted in 50 mM Tris-HCl buffer (pH 7.5), then kept at 40℃~50℃ for 2 h, and samples were taken at different time periods, then cooled in an ice water bath for 30 min, and finally the enzyme activity of wheat arabinoxylanase was determined at 55℃ and pH 7.5 according to the above method. The enzyme solution without heat treatment was used as a control, and the residual enzyme activity of the enzyme solution after different heat treatments was calculated. The half-life of recombinant xylanase PcXyn11A at different temperatures was calculated based on the residual enzyme activity.

[0072] The half-life determination curve of recombinant xylanase PcXyn11A is shown in Fig. 9 As shown. Fig. 9 Calculation showed that the half-lives of the recombinant xylanase PcXyn11A at 40℃, 45℃ and 50℃ were 30 min, 163 min and 34 min, respectively.

[0073] 6. Hydrolysis characteristics analysis A solution of 1% (i.e., 1 g / 100 mL) of oligoxylose or beechwood xylan was prepared with 50 mM Tris-HCl buffer at pH 7.5, and 1 U / mL of recombinant xylanase PcXyn11A was added, and the mixture was kept at 40°C for 12 h. The oligoxylose selected in this embodiment is composed of xylobiose and xylotriose in a mass ratio of 1:1. To monitor the progress of the reaction, samples were taken at different hydrolysis times and kept in a boiling water bath for 5 min to inactivate the enzyme. The hydrolyzate was analyzed by thin layer chromatography (TLC). The TLC results of different hydrolyzates are shown in Figure 2. Fig.10 As shown, Fig.10 (a) indicates that beechwood xylan is used as substrate. Fig.10 (b) indicates that oligoxylose is used as substrate. Fig.10 It can be seen that the products of beech wood xylan or xylooligosaccharides hydrolyzed by the recombinant xylanase PcXyn11A are mainly xylobiose, xylotriose and xylooligosaccharides with a degree of polymerization of 4-7, and no xylose is observed.

[0074] Example 4 Application of recombinant xylanase PcXyn11A in bread baking 1. Bread making process and related parameter determination methods Weigh 240 g whole wheat flour, 24 g sugar, 144 mL water, add a certain amount of recombinant xylanase PcXyn11A enzyme solution (0-5 ppm), commercially available Novozymes wheat arabinoxylanase (addition amount 4 ppm), then add 2.4 g Angel yeast, 2.4 g salt and 9.6 g butter in a dough mixer, stir at low speed for 2 min, stir at high speed for 4 min, stir at low speed again for 2 min, and stir at high speed for 5 min. Proof for 50 min at 38°C and relative humidity of 80%, divide the dough (70 g / piece), shape it, and pack it in boxes. Proof for 15 min under the same proofing conditions, and then bake it in an oven at 160°C on the upper fire and 180°C on the lower fire for 11 min. After baking, cool the whole wheat bread at room temperature for 2 h, seal it in a fresh-keeping bag and set aside.

[0075] Determination of specific volume: The volume of bread was determined by the rapeseed displacement volume method; the mass of bread was weighed using a balance.

[0076] Hardness determination: The bread was cut into uniform slices with a thickness of 20 mm from the middle, and the hardness of the bread core was determined using a texture analyzer (equipped with a P-10 probe). The test speed was 1 mm / s, the compression deformation was 50%, and the induction force was 0.5 N.

[0077] Determination of bread aging: Store the bread at 4°C, measure the bread hardness under the same conditions at the same time intervals, and record the changes in bread hardness.

[0078] The effect of adding recombinant wheat arabinoxylanase PcXyn11A during bread making on the quality of bread is shown in Table 2. The results of the effect of different amounts of recombinant xylanase PcXyn11A on bread staling are shown in Table 2. Fig.11 shown.

[0079] Table 2 Effect of recombinant wheat arabinoxylanase PcXyn11A on bread

[0080] It can be seen from Table 2 that within the scope of the present invention, as the amount of recombinant xylanase PcXyn11A added increases, the specific volume of bread increases first and then stabilizes, and the hardness decreases first and then stabilizes; when the enzyme addition amount is 4 ppm, the specific volume of bread increases by 11.45% and the hardness decreases by 34.68%. Fig.11 It can be seen that PcXyn11A can effectively delay the aging of bread.

[0081] 2. Determination of gelatinization properties of whole wheat flour The gelatinization characteristics of flour with different addition amounts of recombinant xylanase PcXyn11A were determined using a rapid viscosity analyzer. An appropriate amount of flour was weighed and added to an aluminum can, and then distilled water and different amounts of recombinant xylanase PcXyn11A enzyme solution were added for determination.

[0082] The effect of the amount of recombinant xylanase PcXyn11A added on the gelling properties of bread during the production of whole wheat bread is shown in Table 3.

[0083] Table 3 Effects of different amounts of recombinant xylanase PcXyn11A on the gelatinization properties of whole wheat flour Enzyme addition amount / ppm Enzyme type Peak viscosity / Pa·s Valley viscosity / Pa·s Attenuation value / Pa·s Final viscosity / Pa·s Regeneration value / Pa·s Peak time / min Gelatinization temperature / ℃ 0 / / 916.67±5.51 452.00±3.60 464.67±5.86 1081.67±5.86 629.67±2.31 5.40±0.00 89.62±0.03 2 Recombinant xylanase PcXyn11A 910.00±2.65 454.33±3.11 455.67±1.53 1078.67±5.86 624.33±3.05 5.40±0.00 89.33±0.51 3 Recombinant xylanase PcXyn11A 903.33±4.73 449.00±1.73 454.33±3.79 1070.00±2.65 621.00±2.00 5.42±0.04 89.35±0.52 4 Recombinant xylanase PcXyn11A 888.00±10.44 444.00±6.25 444.00±8.19 1058.33±15.50 614.33±9.29 5.47±0.00 90.47±0.06 5 Recombinant xylanase PcXyn11A 905.00±7.81 450.00±3.61 455.00±11.36 1072.67±0.58 622.67±3.21 5.42±0.04 89.63±0.55 4 Novozymes wheat arabinoxylanase 900.33±3.23 447.30±1.43 453.03±3.79 1068.00±2.65 620.7±2.00 5.42±0.04 89.33±0.52 As shown in Table 3, within the experimental range of the present invention, with the increase in the amount of recombinant xylanase PcXyn11A added, the peak viscosity, valley viscosity and final viscosity of the flour all showed a trend of first decreasing and then increasing. When the amount of recombinant xylanase PcXyn11A added exceeded 4 ppm, the viscosity values ​​showed an upward trend. This shows that the addition of an appropriate amount of recombinant xylanase PcXyn11A makes whole wheat flour easier to gelatinize.

[0084] 3. Scanning electron microscope (SEM) microstructure observation Blank whole wheat dough without recombinant xylanase PcXyn11A, blank whole wheat bread without recombinant xylanase PcXyn11A, whole wheat dough with 4 ppm recombinant xylanase PcXyn11A, and whole wheat bread samples with 4 ppm recombinant xylanase PcXyn11A were prepared. The preparation process of whole wheat dough and whole wheat bread was as described in the first part of this embodiment. After freeze-drying and gold spraying, the microstructure was observed by scanning electron microscopy. The scanning electron microscopy images of whole wheat dough and whole wheat bread with different additions are shown in FIG. Fig.12 As shown, Fig.12 (a) is a blank whole wheat dough without adding recombinant xylanase PcXyn11A. Fig.12 (b) Whole wheat dough with 4 ppm recombinant xylanase PcXyn11A added. Fig.12 (c) is a blank whole wheat bread without the addition of recombinant xylanase PcXyn11A. Fig.12 (d) Whole wheat bread supplemented with 4 ppm recombinant xylanase PcXyn11A.

[0085] Depend on Fig.12 It can be seen that when whole wheat dough and whole wheat bread are enlarged by 12000× and 50× respectively, Fig.12 (a) There are more starch granules exposed and scattered; Fig.12 (b) When 4 ppm of recombinant xylanase PcXyn11A was added, the aggregation of starch granules in the dough was reduced, and some of them were exposed or wrapped in the gluten structure. Fig.12 (c) the bread has a dense texture, uneven structure, and rough texture; Fig.12 (d) The network structure of the gluten matrix in whole wheat bread with 4 ppm recombinant xylanase PcXyn11A was more continuous, the internal structure of the bread was finer, and the meshes were more numerous and more uniform in size. The above shows that the addition of an appropriate amount of recombinant xylanase PcXyn11A can effectively improve the organizational structure of fermented dough and bread.

[0086] 4. Prediction of the effect of recombinant xylanase PcXyn11A on bread staling by differential scanning calorimetry (DSC) The aging of whole wheat bread was evaluated by differential scanning calorimeter (DSC). The whole wheat bread was cut into uniform slices with a thickness of 10 mm from the middle. The same mass of bread crumbs at the same position in the bread slices were placed in the sample crucible for the experiment, and an empty crucible was used as a blank control. The DSC program was set to equilibrate at 20°C, cool to -40°C, keep at -40°C for 15 min, and then heat to 110°C. The melting temperature (starting temperature, peak temperature, and ending temperature) and the integrated dissolution enthalpy (J / g dry starch) were recorded during the process. The DSC graph of the effect of recombinant xylanase PcXyn11A on bread quality is shown in Figure 2. Fig.13 shown.

[0087] Depend on Fig.13 It can be seen that compared with the blank control group, the melting onset temperature, peak temperature and termination temperature of the bread group added with recombinant xylanase PcXyn11A decreased, and the integrated enthalpy value of the differential scanning calorimetry graph decreased from 49.14 J / g in the blank control group to 32.31 J / g. The decrease in the integrated enthalpy value indicates that the recombinant xylanase PcXyn11A inhibited the staling of bread.

[0088] 5. Effect of recombinant xylanase PcXyn11A on whole wheat dough rheology The moisture content of whole wheat flour was measured by MB120 moisture analyzer and was 9%. The water absorption rate of the whole wheat flour without adding recombinant xylanase was 51.4%, which can meet the instrument operation conditions. The effect of recombinant xylanase PcXyn11A on the rheology of whole wheat dough was investigated, and the results are shown in Table 4.

[0089] Table 4 Effect of recombinant xylanase PcXyn11 on the rheology of whole wheat dough Type and amount of enzyme added 0 PcXyn11APcXyn11A2ppm2ppm PcXyn11APcXyn11A3ppm3ppm PcXyn11APcXyn11A4ppm4ppm PcXyn11APcXyn11A5ppm5ppm Novozymes Wheat Arabinoxylanase 4ppm C1 / Nm 1.112±0.030 1.074±0.022 1.076±0.004 1.074±0.016 1.078±0.001 1.075±0.014 C2 / Nm 0.463±0.004 0.441±0.007 0.435±0.006 0.422±0.006 0.438±0.006 0.430±0.106 C3 / Nm 1.516±0.014 1.493±0.002 1.492±0.004 1.483±0.018 1.491±0.001 1.490±0.004 C4 / Nm 1.264±0.041 1.229±0.032 1.260±0.004 1.244±0.013 1.270±0.010 1.255±0.060 C5 / Nm 2.201±0.090 2.134±0.084 2.132±0.045 2.077±0.034 2.116±0.069 2.122±0.115 C5-C4 / Nm 0.937±0.050 0.905±0.052 0.873±0.042 0.833±0.047 0.846±0.085 0.853±0.032 Formation time / min 4.280±0.115 4.52±0.141 4.240±0.509 4.250±0.028 4.360±0.269 4.245±0.500 Stabilization time / min 7.30±0.173 7.70±0.283 7.60±0.424 7.45±0.071 7.75±0.212 7.50±0.420 The decrease in dough C1 value represents the decrease in dough water absorption and dough viscosity. The decrease in C3 and C4 values ​​represents the decrease in starch gelatinization rate and starch gelatinization stability. The decrease in C5 value and C5-C4 value represents the decrease in starch recovery rate. As can be seen from Table 4, with the increase in the amount of recombinant xylanase PcXyn11A added, the torque value of the dough continues to decrease, and the torque value of the dough is the lowest when the addition amount is 4 ppm. As the addition amount of recombinant xylanase PcXyn11A continues to increase, the dough torque value gradually increases, indicating that 4 ppm is the optimal enzyme addition amount. The above shows that the addition of an appropriate amount of recombinant xylanase PcXyn11A can delay the aging of the dough.

[0090] In summary, the present invention constructs a PcXyn11A recombinant strain by introducing the coding gene of the xylanase PcXyn11A from the long-haired podospora into Pichia pastoris. Further, the constructed PcXyn11A recombinant strain is subjected to high-density fermentation. By optimizing the fermentation process, the enzyme activity of the obtained fermentation supernatant reaches 727 U / mL and the protein content is 4 mg / mL. The enzymatic properties of the recombinant xylanase PcXyn11 were studied, and it was found that the enzyme molecular weight is 21 kDa, the optimum pH is 7.5, and it remains stable at pH 4.5-9.0; the optimum temperature is 55 ℃, and it maintains a high enzyme activity below 40 ℃; its specific enzyme activity is 409U / mg, and the products of hydrolyzing beechwood xylan are xylobiose, xylotriose and oligoxylose with a degree of polymerization of 4-7. The recombinant xylanase PcXyn11 is applied to the whole-wheat bread making process to increase the specific volume of the whole-wheat bread and reduce the hardness of the whole-wheat bread, and can also effectively delay the aging of the bread. The recombinant xylanase PcXyn11 provided by the present invention has good application prospects in the preparation or improvement of flour products, the preparation of oligoxylose, food additives or feed additives and other industries.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A wheat arabinoxylanase, characterized in that: The amino acid sequence of the wheat arabinoxylanase is shown in SEQ ID No. 1 or a protein having at least 80% identity with the sequence of SEQ ID No.

1.

2. The wheat arabinoxylanase according to claim 1, characterized in that: The amino acid sequence of the wheat arabinoxylanase is shown in SEQ ID No. 2 or a protein having at least 80% identity with the sequence shown in SEQ ID No.

2.

3. The wheat arabinoxylanase-related biological material according to claim 1 or 2, characterized in that: The biological material is any one of the following (I) to (IV): (I) a nucleic acid molecule encoding the wheat arabinoxylanase according to claim 1 or 2; (II) an expression cassette containing the nucleic acid molecule described in (I); (III) a recombinant vector containing the nucleic acid molecule described in (I), or a recombinant vector containing the expression cassette described in (II); (IV) A recombinant strain containing the nucleic acid molecule described in (I), or a recombinant strain containing the expression cassette described in (II), or a recombinant strain containing the recombinant vector described in (III).

4. The wheat arabinoxylanase-related biological material according to claim 3, characterized in that: The nucleic acid molecule is any one of the following genes (i) to (v): (i) the coding sequence is the 55-669th position from the 5′ end of SEQ ID No. 3; (ii) the nucleotide sequence of which is 1 to 669 from the 5′ end of SEQ ID No. 3; (iii) the nucleotide sequence of which is shown in SEQ ID No. 3; (iv) a gene which hybridizes with the gene represented by (i), (ii) or (iii) and encodes the wheat arabinoxylanase according to claim 1 or 2; (v) A gene encoding the wheat arabinoxylanase according to claim 1 or 2 which has 80% or more identity with the gene represented by (i), (ii) or (iii).

5. The wheat arabinoxylanase-related biological material according to claim 3, characterized in that: Inserting the nucleic acid molecule encoding the wheat arabinoxylanase according to claim 1 or 2 into the multiple cloning site of the pPIC9K vector or the pPCAU vector to construct the recombinant vector; and / or The starting strain of the recombinant strain includes Pichia pastoris.

6. Use of the wheat arabinoxylanase-related biological material according to claim 3 or 4 in the preparation of wheat arabinoxylanase.

7. Use of the wheat arabinoxylanase according to claim 1 or 2, or the biomaterial according to claim 3 or 4, in the preparation or improvement of flour products, the preparation of xylo-oligosaccharides, or in food additives or feed additives.

8. A method for preparing wheat arabinoxylanase according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: fermenting and culturing the recombinant strain described in claim 5 to obtain wheat arabinoxylanase; The fermentation culture comprises the following stages which are carried out in sequence: a basic culture stage, a glycerol fed-batch fermentation stage, a methanol fed-batch induced expression stage and a mixed fed-batch induced expression stage.

9. The method for preparing wheat arabinoxylanase according to claim 8, characterized in that: During the mixed fed-batch induction expression stage, methanol and glycerol are added to the culture medium.

10. The method for preparing wheat arabinoxylanase according to claim 8 or 9, characterized in that: In the glycerol fed-batch fermentation stage, the dosage of glycerol added is: for every 1 L of the starting fermentation liquid, a glycerol aqueous solution is added at a rate of 28.8 mL / h to 31.2 mL / h, and the concentration of the glycerol aqueous solution is 480 g / L to 520 g / L; and / or During the methanol fed-batch induction expression stage, the dosage of methanol added is: based on 1 L of the starting fermentation liquid, methanol is added at a rate of 5.8 mL / h to 6.2 mL / h; and / or During the mixed feed induction expression stage, the dosage of methanol added is: based on 1 L of starting fermentation liquid, the speed of methanol added is 4 mL / h~5 mL / h; the dosage of glycerol added is: based on 1 L of starting fermentation liquid, a glycerol aqueous solution with a concentration of 480 g / L~520 g / L is added at a speed of 86.5 L / h~93.8 mL / h.

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