Recombinant trichoderma reesei as well as preparation method and application thereof

Abstract

The invention relates to recombinant trichoderma reesei as well as a preparation method and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. The recombinant trichoderma reesei comprises a promoter and a xylanase gene, the promoter is derived from trichoderma reesei, and the xylanase gene is derived from penicillium oxalicum. Trichoderma reesei is transformed by using a xylanase gene expression plasmid, heterologous expression of xylanase from penicillium oxalicum is realized, and feruloyl oligosaccharide is prepared by using the obtained xylanase and taking brewer's grains as a raw material, so that the feruloyl oligosaccharide has high yield.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering and enzyme engineering, and specifically relates to a recombinant Trichoderma reesei and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Brewer's spent grain (BSG) is the most abundant by-product produced after saccharification of barley malt grains in the beer brewing process, accounting for about 85% (w / w) of the total by-products in the beer production process. The structure of BSG is extremely heterogeneous, including husks, pericarps, endosperm fragments and seed coats of original barley grains, rich in cellulose and hemicellulose polysaccharides, lignin and protein, among which cellulose accounts for 20%, hemicellulose polysaccharides account for 24%, lignin accounts for 20%, and protein accounts for about 21%. Among them, hemicellulose polysaccharides are mainly arabinoxylan. The skeleton of arabinoxylan is D-xylopyranose groups connected by β-(1,4)-glycosidic bonds. Its C(O)-2 and C(O)-3 positions are mono- or di-substituted by α-L-arabinofuranosyl groups. At the same time, the O-5 position of α-L-arabinofuranosyl is ester-linked to FA (ferulic acid, erulic Acid, chemical name 4-hydroxy-3-methoxycinnamic acid). The most abundant monosaccharides are xylose, glucose and arabinose, and it contains 0.4% (w / w) FA.

[0004] Feruloyl oligosaccharides (FOs) are composed of FA and arabinoxylan, and also have the physiological functions of ferulic acid and oligoxylose. Due to the presence of hydrophilic ester bonds, FOs are more soluble in water, and have amphiphilic enzymatic and non-ionic properties, strong permeability, and FOs show better physiological activities than free FA, such as antioxidant activity, anti-glycosylation, relief of diabetic syndrome, prebiotic effect, immunomodulation, anti-tumor function, etc. The hemicellulose of spent grain brewer's grain (BSG) contains rich bound ferulic acid, which has the potential to produce FOs, and finds a new way for the rational utilization of BSG.

[0005] At present, the main methods for extracting feruloyl oligosaccharides (FOs) from BSG are acid hydrolysis, high-temperature cooking and biological fermentation. There are some unresolved technical problems when using the above methods to extract FOs, such as the low food safety of FOs prepared by the acid method and the pollution to the environment. The high-temperature cooking method and biological fermentation method have high requirements on equipment and are only suitable for small-scale experiments in the laboratory. The biological fermentation method depends on the activity of the fermentation bacteria and has low process stability. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a recombinant Trichoderma reesei and a preparation method and application thereof, construct a single gene expression cassette for each of the nine xylanase genes of Penicillium oxalicum, and transform Trichoderma reesei using an expression plasmid containing the xylanase gene to achieve heterologous expression of xylanase from Penicillium oxalicum, and use the obtained xylanase to prepare feruloyl oligosaccharides using brewer's grains as raw materials, so that feruloyl oligosaccharides have a high yield.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] In a first aspect, a recombinant Trichoderma reesei comprises a promoter and a xylanase gene;

[0009] The promoter is derived from Trichoderma reesei, and the xylanase gene is derived from Penicillium oxalicum.

[0010] Optionally, the promoter is the promoter Pcdna1 of Trichoderma reesei.

[0011] Optionally, the xylanase gene is one of xyn11A, xyn11B, xyn11C, xyn11D, xyn10A, xyn10B, xyn10C, xyn30A, and xyn30B.

[0012] In a second aspect, the method for preparing the above-mentioned recombinant Trichoderma reesei comprises the following steps:

[0013] S1. preparing protoplasts of Trichoderma reesei QMP strain;

[0014] S2, preparing a recombinant plasmid containing the target xylanase gene;

[0015] S3. Using the recombinant plasmid to transform the protoplasts in S1, culture and obtain a recombinant Trichoderma reesei strain.

[0016] Optionally, in S2, a recombinant plasmid is prepared using the Pcdna1-pyrG plasmid as an expression vector.

[0017] Optionally, in S2, the Pst1 restriction site of the Pcdna1-pyrG plasmid is digested, and then the target xylanase gene is inserted into the restriction site, thereby constructing a recombinant plasmid containing a target gene expression cassette.

[0018] Optionally, in S2, the recombinant plasmid is amplified using the primer pair consisting of M12-F / M12-R as shown in SEQ ID No. 1 and SEQ ID No. 2 as upstream and downstream primers;

[0019] Optionally, in S2, the primers for amplifying the xyn11A gene are shown in SEQ ID No.3 and SEQ ID No.4, the primers for amplifying the xyn11B gene are shown in SEQ ID No.5 and SEQ ID No.6, the primers for amplifying the xyn11C gene are shown in SEQ ID No.7 and SEQ ID No.8, the primers for amplifying the xyn11D gene are shown in SEQ ID No.9 and SEQ ID No.10, the primers for amplifying the xyn10A gene are shown in SEQ ID No.11 and SEQ ID No.12, the primers for amplifying the xyn10B gene are shown in SEQ ID No.13 and SEQ ID No.14, the primers for amplifying the xyn10C gene are shown in SEQ ID No.15 and SEQ ID No.16, the primers for amplifying the xyn30A gene are shown in SEQ ID No.17 and SEQ ID No.18, and the primers for amplifying the xyn30B gene are shown in SEQ ID No.19 and SEQ ID No.20.

[0020] Optionally, in S3, the transformed protoplasts are cultured to obtain a single transformant, and spores produced by the single transformant colony are taken and cultured to obtain a recombinant Trichoderma reesei strain.

[0021] In a third aspect, the use of the above-mentioned recombinant Trichoderma reesei in the production of feruloyl oligosaccharides.

[0022] A fourth aspect is a method for producing feruloyl oligosaccharides, comprising the steps of:

[0023] S4, culturing the recombinant Trichoderma reesei strain using a cellulose fermentation medium, and extracting xylanase from the fermentation supernatant;

[0024] S5. Using the deproteinized brewer's grains as raw material, adding the xylanase obtained in S4, and performing enzymatic hydrolysis to obtain feruloyl oligosaccharides.

[0025] Optionally, in S5, the amount of enzyme added is: the ratio of xylanase activity to BSG is (110-130) U: 1 g.

[0026] One or more of the above technical solutions have the following beneficial effects:

[0027] The structure of cellulosic biomass is a complex structure formed by the close entanglement of cellulose, hemicellulose and lignin. To efficiently degrade cellulosic biomass, the synergistic effect of the cellulase system (including cellulase, hemicellulose, and ligninase) is required. The preparation of FOs by heterologously expressing xylanase in Trichoderma reesei is due to the insufficient content of xylanase in Trichoderma reesei that specifically degrades BSG to produce FOs. By overexpressing xylanases from different genera and species in Trichoderma reesei, it is helpful to screen out xylanases that efficiently degrade BSG to produce FOs. In addition, Trichoderma reesei is the strongest cellulase producer, and the cellulase in its cellulase system can degrade the cellulose in BSG, thereby exposing the hemicellulose chains in BSG, which is conducive to the degradation of BSG by xylanase to produce more FOs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1 It is a schematic diagram of the single gene expression cassette of the xylanase gene in Example 1 of the present invention.

[0030] Figure 2 This is the PCR verification result of the single gene expression cassette in Example 1 of the present invention, wherein the corresponding relationship between the numbers above the pattern and the genes is: 1, Xyn11A; 2, Xyn11B; 3, Xyn11C; 4, Xyn11D; 5, Xyn10A; 6, Xyn10B; 7, Xyn10C; 8, Xyn30A; 9, Xyn30B.

[0031] Figure 3 1 is a graph of the SDS-PAGE analysis results in Example 1 of the present invention, wherein (a) is the analysis result of the recombinant strain of the Xyn11A gene, (b) is the analysis result of the recombinant strain of the Xyn11B gene, (c) is the analysis result of the recombinant strain of the Xyn11C gene, (d) is the analysis result of the recombinant strain of the Xyn11D gene, (e) is the analysis result of the recombinant strain of the Xyn11E gene, (f) is the analysis result of the recombinant strain of the Xyn10A gene, (g) is the analysis result of the recombinant strain of the Xyn10B gene, (h) is the analysis result of the recombinant strain of the Xyn10C gene, (i) is the analysis result of the recombinant strain of the Xyn30A gene, (j) is the analysis result of the recombinant strain of the Xyn30B gene, (k) is the analysis result of the Trichoderma reesei QMP strain, and (l) is the analysis result of the Pichia pastoris GS115 strain.

[0032] Figure 4 It is an analysis and statistical diagram of the enzymatic hydrolysis results in Example 2 of the present invention, wherein (a) is a statistical bar graph of the content of free FA and FOs, and (b) is a statistical bar graph of the yield of FOs; wherein the numerical serial numbers on the horizontal axis represent: 0, QMP enzyme solution; 1, Xyn11A; 2, Xyn11B; 3, Xyn11C; 4, Xyn11D; 5, Xyn10A; 6, Xyn10B; 7, Xyn10C; 8, Xyn30A; 9, Xyn30B. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0034] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0035] In the present invention, "expression cassette" refers to an independent component of vector DNA, which consists of the gene and regulatory sequences expressed by the transfected cells. The expression cassette consists of three parts: the promoter sequence, the open reading frame and the 3' untranslated region, which usually contains the polyadenylation site in eukaryotic organisms. Different expression cassettes can be transfected into different organisms such as bacteria, yeast, plants and plants.

[0036] In the present invention, "plasmid" refers to a DNA molecule independent of the host cell chromosome, and in the present invention, it specifically refers to an artificially constructed plasmid. In a specific embodiment of the present invention, a single gene expression cassette is constructed for 9 xylanase genes of Penicillium oxalicum, and the expression plasmids of the 9 xylanase genes of Penicillium oxalicum are transformed into the protoplasts of Trichoderma reesei QMP strain. Except that the gene of xylanase needs to be designed according to the difference of the targeted gene, all other elements in the single gene expression cassette plasmid can use those known to those skilled in the art. For example, the screening marker used in the single gene expression cassette plasmid can be an auxotrophic screening marker, a fluorescent protein screening marker or an antibiotic screening marker. In a specific embodiment of the present invention, the uracil auxotrophic gene pyrG is used.

[0037] In the present invention, Trichoderma reesei selects Trichoderma reesei QMP strain, which has been disclosed in Chinese patent application number 2021110418856 and is a uracil auxotrophic strain.

[0038] In the present invention, the Pcdna1-pyrG plasmid is an expression vector, which is constructed in the early stage of the laboratory and contains the promoter Pcdna1, TtrpC terminator and screening marker gene pyrG of Trichoderma reesei cdna1, and a Pst1 restriction site is designed between the Pcdna1 promoter and the TtrpC terminator; the specific construction method is shown in the technical document: Zhao Q, Zhang Z, Liu Z, et al. A closed-loop strategy for on-site production of saccharolytic enzymes for lignocellulose biorefinery using internal lignocellulosic hydrolysates [J]. Chemical Engineering Journal, 2024, 480: 148272.; wherein, the Escherichia coli competent cell Trans5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0039] In the present invention, the protoplast transformation buffer used includes: transformation solution S1, including 1.2M sorbitol, 0.1M KH2PO4, and the pH is adjusted to 8.0; transformation solution S2, 1M sorbitol, 50mM CaCl2, 10mM Tris-HCl, and the pH is adjusted to 7.5; T1 reagent includes PEG600025g, CaCl20.74g, Tris 0.48g, and HCl is used to adjust the pH to 7.5, and is sterilized for standby use.

[0040] Example 1: Preparation of recombinant Trichoderma reesei and extraction of xylanase

[0041] 1. Preparation of Protoplasts of Trichoderma reesei QMP Strain

[0042] Heat and melt the PDA medium (potato dextrose agar medium) containing 0.1% (w / v) uracil, pour it into a sterile plate, wait for it to solidify to obtain a PDA medium plate, draw an appropriate amount of the spore suspension of Trichoderma reesei QMP strain and inoculate it on the PDA medium plate, culture it at a constant temperature of 30°C for 4-5 days, use a pipette to draw an appropriate amount of 0.9% physiological saline and add it to the plate, then use an inoculation loop to gently scrape off the brown-green spores growing on the surface of the plate, collect the washed spore suspension into a 1.5 mL centrifuge tube, and store it in a 4°C refrigerator.

[0043] Use tweezers to spread a layer of sterile cellophane on the surface of a new PDA culture medium plate, then add about 80 μL of fresh spore suspension on the surface of the cellophane, spread it evenly with a spreader, and incubate it at a constant temperature of 30°C for about 16 hours.

[0044] In a clean bench, weigh 5‰ of the lytic enzyme by mass and fully dissolve it in the transformation solution S1 to obtain the mycelial lysis solution; first add 3mL of lysis solution to a new sterile plate (sterile culture medium plate), then use tweezers to spread the cellophane with mycelium on the sterile plate with the lysis solution, add another 3mL of lysis solution on its surface, and then spread another layer of cellophane; repeat the above operation, add lysis solution between every two pieces of cellophane, and place the plate with the cellophane and lysis solution in a 30°C constant temperature incubator for lysis for 2.5 to 3 hours.

[0045] Use tweezers to remove the cellophane from the lysis solution (try not to take out the mycelium), and use a 5mL scissor tip to blow the lysis solution evenly; filter the lysis solution on ice; use a sterilized funnel with five layers of lens paper to filter and collect the lysis solution, centrifuge at 2500rpm for 10 minutes in a 4℃ centrifuge, and discard the supernatant. Add 5mL of pre-cooled transformation solution S2 to resuspend the precipitate, centrifuge at 2500rpm for 10 minutes in a 4℃ centrifuge, discard the supernatant, and the obtained precipitate is the protoplast of Trichoderma reesei QMP strain; add 200μL of pre-cooled transformation solution S2 to the obtained precipitate, and use the tip of the gun to slowly blow the Trichoderma reesei QMP strain protoplast precipitate evenly, and store it on ice for later use.

[0046] 2. Construction of a single gene expression cassette for the xylanase gene of Penicillium oxalicum

[0047] The Pcdna1-pyrG plasmid was used as an expression vector, and the Pst1 restriction site on the plasmid was digested, and then the target gene and its primers were inserted into this site to construct a recombinant plasmid containing the target gene and its primer expression cassette. II recombination cloning kit was used for construction;

[0048] The specific operation steps include: inserting the target gene and its primer into the Pst1 site of the Pcdna1-pyrG plasmid, and constructing recombinant plasmids containing expression cassettes for expressing different target genes and their primers. Since the Pcdna1-pyrG plasmid itself contains the promoter Pcdna1, TtrpC terminator and selection marker gene pyrG of Trichoderma reesei cdna1, it can form the expression cassette of the target gene and its primer, such as Figure 1 Using these recombinant plasmids as templates and the primer pair consisting of M12-F / M12-R primers as upstream and downstream primers, expression cassettes of different target genes were amplified.

[0049] Among them, the target gene is the xylanase gene of Penicillium oxalicum, specifically one of xyn11A (NCBI No.: EPS27158.1), xyn11B (NCBI No.: EPS27738.1), xyn11C (NCBI No.: EPS29528.1), xyn11D (NCBI No.: EPS30259.1), xyn10A (NCBI No.: EPS33132.1), xyn10B (NCBI No.: EPS27475.1), xyn10C (NCBI No.: EPS25816.1), xyn30A (NCBI No.: EPS28627.1) and xyn30B (NCBI No.: EPS30259.1).

[0050] The primers for constructing different target gene expression cassettes are shown in Tables 1 and 2.

[0051] Table 1 Primers used to amplify target genes and target gene expression cassettes

[0052]

[0053]

[0054] That is, the primers for amplifying the xyn11A gene are shown in SEQ ID No.3 and SEQ ID No.4, the primers for amplifying the xyn11B gene are shown in SEQ ID No.5 and SEQ ID No.6, the primers for amplifying the xyn11C gene are shown in SEQ ID No.7 and SEQ ID No.8, the primers for amplifying the xyn11D gene are shown in SEQ ID No.9 and SEQ ID No.10, the primers for amplifying the xyn10A gene are shown in SEQ ID No.11 and SEQ ID No.12, the primers for amplifying the xyn10B gene are shown in SEQ ID No.13 and SEQ ID No.14, the primers for amplifying the xyn10C gene are shown in SEQ ID No.15 and SEQ ID No.16, the primers for amplifying the xyn30A gene are shown in SEQ ID No.17 and SEQ ID No.18, and the primers for amplifying the xyn30B gene are shown in SEQ ID No.19 and SEQ ID No.20.

[0055] Table 2 Other primers used to construct different target gene expression cassettes

[0056]

[0057]

[0058] The constructed target gene expression cassettes were verified by PCR. Figure 2 As shown, it was shown that the single gene expression cassettes of xyn11A, xyn11B, xyn11C, xyn11D, xyn10A, xyn10B, xyn10C, xyn30A and xyn30B were successfully constructed.

[0059] 3. Transformation of Single Gene Expression Cassette into Protoplasts of Trichoderma reesei QMP Strain

[0060] Prepare the transformation system on ice, slowly and evenly add 12 μL of gene cassette expression to 200 μL of protoplasts, immediately add 60 μL of T1 reagent, shake gently to mix, and let stand on ice for 20 min; continue to add 2 mL of T1 reagent, shake gently to mix, and let stand at room temperature for 5 min; then add 4 mL of transformation solution S2 to terminate the reaction, and obtain the protoplasts of Trichoderma reesei QMP strain transformed with a single gene expression cassette, which are the transformants.

[0061] The upper culture medium was heated to melt, and when cooled to 40°C, it was slowly added to the system after the termination reaction and mixed evenly, and then evenly distributed to the plate with the lower culture medium. After solidification, it was sealed with a sealing film and placed in a 30°C incubator for 2-3 days. A single transformant with diffuse hyphae growing on the transformation plate was picked out and placed on a spore culture medium, and cultured at 30°C for 5-7 days. After spores were produced, they were purified.

[0062] Note: All the above operations must be performed in a sterile environment, and all equipment and reagents used must be sterile.

[0063] 4. Isolation and Purification of Transformants

[0064] Prepare a sterile 1.5mL EP tube, and fill each tube with 200μL sterile saline; sterilize the inoculation loop over the flame of an alcohol lamp, and insert it into the EP tube filled with saline to form a layer of water film on the inoculation loop; use the water film on the inoculation loop to pick up spores produced by a single transformant colony, and transfer the water film with spores to the EP tube filled with saline; seal the EP tube and shake it in an oscillator to evenly distribute the spores in the saline; use the inoculation loop to pick up a small amount of spore suspension and streak it on a spore culture plate; seal the spore culture plate with a sealing film and place it in a 30℃ incubator for static culture to obtain the isolated and purified transformants.

[0065] The purified spore colonies were picked into physiological saline, shaken, and 50 μL of spore suspension was added to a 1.5 mL centrifuge tube containing 600-800 μL of mycelial growth medium, and cultured at 30°C, 200 rpm for 24 h; the supernatant was discarded after centrifugation, 500 μL of genome extraction buffer and an appropriate amount of quartz sand were added, and then placed in a tissue cell disruptor (BULLET BLENDERTM), oscillated at level seven for 1 min, and water bathed at 65°C for 10 min; after the water bath, 200 μL of 7.5 M ammonium acetate solution was added, mixed by inversion, and placed on ice for 10 min; the sample was centrifuged at 4°C, 12,000 rpm for 10 min; 700 μL of supernatant was taken to a new 1.5 mL centrifuge tube, and 350 μL of pre-cooled isopropanol was added, and after thorough inversion and mixing, it was placed at -20°C for 20 min; 4°C, 12,000 rpm, centrifuged for 10 min, the supernatant was discarded, and 1 mL 70% ethanol, mix by inversion; centrifuge at 4°C, 12,000rpm for 2 minutes, discard the supernatant, centrifuge for 30 seconds to centrifuge the liquid on the wall of the centrifuge tube to the bottom of the tube, use a 200μL pipette to suck away the remaining liquid, open the lid and let it air-dry for 10 minutes at room temperature, add 30μL of distilled water to dissolve the genome precipitate after the residual ethanol evaporates, and store it at -20°C for later use. The extracted genome was then used for verification to obtain the correct transformant.

[0066] 5. Fermentation of recombinant strains of Trichoderma reesei, enzyme activity and protein content determination

[0067] Spore suspensions were prepared from transformants of various constructed xylanase recombinant strains, and then 500 μL of fresh spore suspension was inoculated into 50 mL of glucose fermentation medium, with 3 parallels for each strain; the culture was carried out in a 2% glucose fermentation medium at 30°C and 200 rpm in a shake flask for 24 h, and the fermentation supernatant was taken for enzyme activity determination and SDS-PAGE analysis.

[0068] The recombinant strain of Trichoderma reesei expressing 9 xylanases and the Trichoderma reesei QMP strain were inoculated into cellulose fermentation medium, respectively, and cultured at 30°C and 200 rpm for 7 days. The fermentation supernatant was taken for enzyme activity determination and SDS-PAGE analysis. Figure 3 As shown: Among them, Figure 3 (a) is the analysis result of the recombinant strain of Xyn11A gene. Figure 3 (b) is the analysis result of the recombinant strain of Xyn11B gene. Figure 3 (c) in the figure is the analysis result of the recombinant strain of Xyn11C gene. Figure 3 (d) in the figure is the analysis result of the recombinant strain of Xyn11D gene. Figure 3 (e) in the figure is the analysis result of the recombinant strain of Xyn11E gene. Figure 3(f) in the figure is the analysis result of the recombinant strain of Xyn10A gene. Figure 3 (g) in the figure is the analysis result of the recombinant strain of Xyn10B gene. Figure 3 (h) in the figure is the analysis result of the recombinant strain of Xyn10C gene. Figure 3 (i) is the analysis result of the recombinant strain of Xyn30A gene, Figure 3 (j) in the figure is the analysis result of the recombinant strain of Xyn30B gene. Figure 3 (k) in the figure is the analysis result of Trichoderma reesei QMP strain, Figure 3 (l) in the figure is the analysis result of Pichia pastoris GS115 strain.

[0069] Example 2, Preparation of Feruloyl Oligosaccharide

[0070] 1. Preparation of feruloyl oligosaccharides using heterologously expressed xylanase from brewer's grains

[0071] The brewer's grains with a water content of 80% are dried at 70°C in an electric blast drying oven, crushed with a blade crusher for 1-2 minutes, and passed through a 60-80 mesh sieve to complete the mechanical pretreatment of the brewer's grains; 500mL of 0.1mol / L pH7.0 phosphate buffer is prepared, and 50g of the brewer's grains that have completed the mechanical pretreatment are added; 2.4g of neutral protease (2400U / g BSG) is added, and the reaction is continuously stirred at 50°C for 2h with a magnetic stirrer, and then the enzyme is treated at 100°C for 10min to inactivate it; the supernatant is discarded after filtering, and the solid residue is repeatedly washed with hot water at 60-70°C until the suspension is clear, and filtered after cooling; the solid residue is placed in an oven for drying to obtain deproteinized brewer's grains (deproteinized BSG).

[0072] Accurately weigh 0.5 g of deproteinized BSG into a 50 mL conical flask, add 6.25 mL of 0.2 M sodium acetate buffer at pH 4.8, then add enzyme solution, and then add distilled water to make the total system 20 mL, and perform enzymolysis at 50 ° C and 200 rpm for 10 h; determine the content of FOs in the system; wherein the enzyme solution is the fermentation supernatant obtained in step 5 of Example 1, and the enzyme activity of xylanase in the enzyme solution is 120 IU / mL.

[0073] Analyze the enzymatic hydrolysis results, such as Figure 4 As shown, Figure 4 (a) in the figure shows the statistical bar graph of the content of free FA and FOs. Figure 4 (b) in the figure shows the statistical bar chart of the yield of FOs.

[0074] 2. Determination of enzyme dosage for preparation of feruloyl oligosaccharides

[0075] Add according to xylanase activity: other conditions are the same as when adding according to the total protein content of the crude enzyme solution, the enzyme dosage is controlled to be 0, 30, 60, 120, 180, 240U / g BSG, and the FOs content at different enzyme dosages is measured after enzymatic hydrolysis at 200rpm for 10 hours to determine the optimal enzyme dosage for preparing FOs; the results are: 120U / g BSG, the yield of FOs is 23-31%, of which the yield of FOs produced by Xyn10B degrading BSG is 30%.

[0076] Compared with Trichoderma reesei, budding Aureobasidium is not a major industrial cellulase producer, and its cellulase production capacity is relatively weak. Relying solely on the action of xylanase from budding Aureobasidium cannot effectively degrade the xylan chains surrounded by cellulose chains to produce FOs.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant Trichoderma reesei, characterized in that contains a promoter and a xylanase gene; The promoter is derived from Trichoderma reesei, and the xylanase gene is derived from Penicillium oxalicum; The promoter is the promoter Pcdna1 of Trichoderma reesei; The xylanase gene is one or more of xyn11A, xyn11B, xyn11C, xyn11D, xyn10A, xyn10B, xyn10C, xyn30A, and xyn30B.

2. A method for preparing the recombinant Trichoderma reesei according to claim 1, characterized in that: The following steps are involved: S1. preparing protoplasts of Trichoderma reesei QMP strain; S2, preparing a recombinant plasmid containing the target xylanase gene; S3. Using the recombinant plasmid to transform the protoplasts in S1, culture and obtain a recombinant Trichoderma reesei strain.

3. The method for preparing recombinant Trichoderma reesei according to claim 2, characterized in that: In S2, the recombinant plasmid was prepared using the Pcdna1-pyrG plasmid as an expression vector.

4. The method for preparing recombinant Trichoderma reesei according to claim 3, characterized in that: In S2, the Pst1 restriction site of the Pcdna1-pyrG plasmid is digested, and then the target xylanase gene is inserted into the restriction site, thereby constructing a recombinant plasmid containing the target gene expression cassette.

5. The method for preparing recombinant Trichoderma reesei according to claim 4, characterized in that: In S2, the recombinant plasmid is amplified using the primer pair consisting of M12-F / M12-R as shown in SEQ ID No. 1 and SEQ ID No. 2 as upstream and downstream primers.

6. The method for preparing recombinant Trichoderma reesei according to claim 4, characterized in that: In S2, the primers for amplifying the xyn11A gene are shown in SEQ ID No.3 and SEQ ID No.4, the primers for amplifying the xyn11B gene are shown in SEQ ID No.5 and SEQ ID No.6, the primers for amplifying the xyn11C gene are shown in SEQ ID No.7 and SEQ ID No.8, the primers for amplifying the xyn11D gene are shown in SEQ ID No.9 and SEQ ID No.10, the primers for amplifying the xyn10A gene are shown in SEQ ID No.11 and SEQ ID No.12, the primers for amplifying the xyn10B gene are shown in SEQ ID No.13 and SEQ ID No.14, the primers for amplifying the xyn10C gene are shown in SEQ ID No.15 and SEQ ID No.16, the primers for amplifying the xyn30A gene are shown in SEQ ID No.17 and SEQ ID No.18, and the primers for amplifying the xyn30B gene are shown in SEQ ID No.19 and SEQ ID No.

20.

7. The method for preparing recombinant Trichoderma reesei according to claim 2, characterized in that: In S3, the transformed protoplasts are cultured to obtain a single transformant, and spores produced by the single transformant colony are taken and cultured to obtain a recombinant Trichoderma reesei strain.

8. Use of the recombinant Trichoderma reesei as claimed in claim 1 in producing feruloyl oligosaccharides.

9. A method for producing feruloyl oligosaccharides, characterized in that: Includes steps: S4, culturing the recombinant Trichoderma reesei strain according to claim 1 using a cellulose fermentation medium, and extracting xylanase from the fermentation supernatant; S5. Using the deproteinized brewer's grains as raw material, adding the xylanase obtained in S4, and performing enzymatic hydrolysis to obtain feruloyl oligosaccharides.

10. The method for producing feruloyl oligosaccharides according to claim 9, wherein In S5, the amount of enzyme added is: the ratio of xylanase activity to BSG is (110-130) U: 1 g.

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