A method for improving the production of cellulase by Trichoderma reesei and its application

Abstract

The present invention discloses a method and application for improving the production of cellulase by Trichoderma reesei, belonging to the field of biotechnology. The recombinant engineered Trichoderma reesei is obtained by introducing the exoglucanase gene LeGH7_3643 in shiitake mushroom into Trichoderma reesei for overexpression, and the nucleotide sequence of LeGH7_3643 is shown in SEQ ID NO: 1, or contains the gene sequence of the sequence shown in SEQ ID NO: 1. It is found through experiments that the activity of cellulase produced by the recombinant engineered Trichoderma reesei fermentation and the amount of protein secretion are significantly improved, and the enzyme liquid secreted by the recombinant engineered Trichoderma reesei can efficiently degrade different types of biomass raw materials, significantly improving the saccharification rate of biomass and the yield of ethanol.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for improving the production of cellulase by Trichoderma reesei and its application. Background Art

[0002] Trichoderma reesei is the most widely used strain in the biotechnology industry. It can produce a variety of enzymes using different types of biomass substrates, mainly including cellulases, hemicellulases and some auxiliary enzymes. Cellulases can be mainly divided into exoglucanase (Exo-1,4-β-D-glucanase, CBH), endoglucanase (Endo-1,4-β-D-glucanase, EG) and β-glucosidase (β-1,4-glucosidase, BG). Among them, CBH accounts for 60-80% of the total cellulase and plays a vital role in the hydrolysis process. EG accounts for about 20%-36%, while BG has the lowest content, accounting for only 1%. At present, the generally low activity of cellulase, the unreasonable composition of the enzyme system and the low hydrolysis efficiency are still the bottlenecks restricting the economic and efficient conversion and utilization of biomass materials.

[0003] The white-rot fungus Lentinula edodes can secrete a large number of lignocellulose-degrading enzymes, including genes of the glycoside hydrolase family related to the degradation of crystalline cellulose. Among them, the glycoside hydrolases of the GH7 family can hydrolyze the β-1,4 glycosidic bonds in cellulose, and some also show certain activity on xylan. However, there are few studies on the functional characteristics and application of the GH7 family cellulase system in Lentinula edodes. If the GH7 family genes in Lentinula edodes are heterologously overexpressed in Trichoderma reesei, it is expected that an enzyme system with high enzyme activity and optimized ratio and high hydrolysis performance can be obtained. Summary of the invention

[0004] The purpose of the present invention is to provide a method and application for improving the production of cellulase by Trichoderma reesei to solve the problems existing in the above-mentioned prior art. The enzyme liquid secreted by recombinant Trichoderma reesei can efficiently degrade different types of biomass raw materials and significantly improve the saccharification rate and ethanol yield of biomass.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a recombinant engineered bacterium of Trichoderma reesei. The recombinant engineered bacterium of Trichoderma reesei is obtained by introducing an exoglucanase gene LeGH7_3643 in shiitake mushroom into Trichoderma reesei for overexpression. The nucleotide sequence of the LeGH7_3643 is shown in SEQ ID NO: 1, or comprises a gene sequence of the sequence shown in SEQ ID NO: 1.

[0007] The sequence shown in SEQ ID NO: 1 is:

[0008]

[0009] Preferably, the Trichoderma reesei includes Trichoderma reesei Rut-C30.

[0010] The present invention also provides a method for improving the production of cellulase by Trichoderma reesei, comprising introducing the exoglucanase gene LeGH7_3643 in shiitake mushroom into Trichoderma reesei for overexpression to obtain recombinant engineered Trichoderma reesei, and using the recombinant engineered Trichoderma reesei to ferment and produce cellulase; wherein the nucleotide sequence of the LeGH7_3643 is as shown in SEQ ID NO: 1, or a gene sequence containing the sequence shown in SEQ ID NO: 1.

[0011] Preferably, the fermentation production conditions are as follows: the fermentation medium includes the following components in amounts: 20 g / L microcrystalline cellulose, 15 g / L KH2PO4, 10 g / L (NH4)SO4, 1 g / L tryptone, 0.3 g / L urea, 0.4 g / L CaCl2·2H2O, 0.3 g / L MgSO4·7H2O, 10 g / L yeast extract and 1 mL / L trace element solution; the trace element solution includes 75 g FeSO4·7H2O, 2 g MnSO4·H2O, 2 g ZnSO4 and 3 g CoCl2, and the distilled water is added to 1 L;

[0012] The fermentation temperature is 28-30°C, the rotation speed is 200rpm, and the fermentation time is 5-7 days.

[0013] The present invention also provides an application of a crude enzyme solution prepared by the recombinant engineered Trichoderma reesei or the method described in the degradation of biomass materials, wherein the crude enzyme solution contains cellulase and hemicellulase, and the biomass materials include crop straws.

[0014] The present invention also provides an application of a crude enzyme solution prepared by the recombinant engineered bacteria of Trichoderma reesei or the method described in the production of ethanol, wherein the enzymatic hydrolysis solution is used to hydrolyze biomass materials and produce ethanol through fermentation; the crude enzyme solution contains cellulase and hemicellulase, and the biomass materials include crop straw.

[0015] The present invention also provides a method for degrading biomass materials, which comprises pretreating the biomass materials by using an alkaline solution or steam explosion, and then adding the crude enzyme solution secreted by the recombinant engineered bacteria of Trichoderma reesei for enzymolysis.

[0016] Preferably, the conditions for the alkaline solution pretreatment are: treating the biomass material with 1-2% NaOH by mass concentration, and then placing it on a shaker at 50°C and 150rpm for 2h; the conditions for the steam explosion pretreatment are: treating at 225°C (2.5Mpa) for 3min; and the conditions for the enzymatic hydrolysis are: reacting at 50°C and 150rpm for 48h;

[0017] The biomass material includes crop straw.

[0018] The present invention also provides a method for producing ethanol, comprising obtaining a fermentation broth after saccharification of biomass materials using the method described above, then adding Angel Yeast to the fermentation broth, and culturing at 37° C. for 48 hours.

[0019] Preferably, the final concentration of Angel Yeast is 0.5 g / L.

[0020] The present invention discloses the following technical effects:

[0021] The present invention introduces the shiitake mushroom exoglucanase gene LeGH7_3643 into Trichoderma reesei by Agrobacterium-mediated method, and makes it overexpressed in Trichoderma reesei, and the activity of fermentation and production of cellulase and protein secretion of the recombinant Trichoderma reesei strain are significantly improved. The enzyme liquid secreted by the recombinant Trichoderma reesei can efficiently degrade different types of biomass raw materials, and significantly improve the saccharification rate and ethanol yield of biomass. It can be seen that the present invention provides a method for enhancing the fermentation and production of cellulase by Trichoderma reesei using the shiitake mushroom exoglucanase gene LeGH7_3643, which lays a theoretical foundation for the economical and efficient conversion and utilization of biomass materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, 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.

[0023] Figure 1 is the map of the recombinant plasmid pCZF8X-LeGH7_3643;

[0024] Figure 2 Schematic diagram of the PCR gel electrophoresis results of the mycelial DNA of the recombinant plasmid transformants; A: The identification results of the mycelial DNA of the transformants, M is a 5000bp standard DNA molecule, GH7-1~8 are the recombinants transformed with pCZF8X-LeGH7_3643, blank is the blank control, and CK is the positive control; B: The hygromycin identification results of the mycelial DNA of the transformants, M is a 2000bp standard DNA molecule, GH7-1~8 are the positive transformants, blank is the blank control, and CK is the positive control;

[0025] Figure 3 The results of transformant screening and identification; A: filter paper enzyme activity identification results, B: protein content determination results;

[0026] Figure 4 Determination of enzyme activity and protein concentration in crude enzyme solution of recombinant strains GH7-5 and GH7-6; A: SDS-PAGE analysis; B: filter paper enzyme activity; C: exoglucanase activity; D: endoglucanase activity; E: β-glucosidase activity; F: xylanase activity; G: protein concentration;

[0027] Figure 5 The results of saccharification efficiency of rice, cotton, rapeseed, Miscanthus and corn by crude enzyme solutions of recombinant strains GH7-5 and GH7-6 are shown;

[0028] Figure 6 The effects of the above rice and Miscanthus hydrolysates on the fermentation of bioethanol; A: ethanol yield; B: sugar alcohol conversion rate. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The expression vector pCZF8X involved in the following examples was modified by Xu Chengbao in our laboratory using conventional techniques by replacing the NcoI restriction site in the expression vector pCZF8 with AflII. pCZF8 was donated by Professor Zhao Xinqing of Shanghai Jiaotong University.

[0035] pCZF8X-LeGH7_3643 contains the Ppdc promoter, hygromycin resistance B gene, CBHI signal peptide, His tag, LeGH7_3643 gene and pdc1 terminator.

[0036] Example 1 Construction of recombinant strains

[0037] 1. Target gene amplification

[0038] The extracted whole genome DNA of Lentinula edodes W1 was used as a template, and LeGH7_3643-F (5'-CTAGCTTAAGATGTTTCGCTCTACTGC-3', SEQ ID NO: 2) and LeGH7_3643-R (5'-TGCTCTAGACTATGCAGTAAAAGTG-3', SEQ ID NO: 3) were used as primers for amplification; the PCR amplification system is shown in Table 1.

[0039] Table 1 Amplification system

[0040]

[0041] The amplification program was pre-denaturation at 95.0°C for 5 min, denaturation at 95.0°C for 30 sec, annealing at 59°C for 30 sec, and extension at 72°C for 10 min.

[0042] 2. Construction of pCZF8X-LeGH7_3643 vector

[0043] The GH7_3643 gene and plasmid pCZF8X amplified above were digested with restriction endonucleases AflⅡ and XbaⅠ at 37℃ for 30min, respectively, and the digested fragments and vectors were purified using a DNA purification kit. The target gene fragment was ligated to the vector using T4 DNA ligase at 16℃ overnight to obtain the recombinant vector pCZF8X-LeGH7_3643 (map see Figure 1 ).

[0044] 3. Conversion

[0045] pCZF8X-LeGH7_3643 was transformed into Escherichia coli DH5α for recombinant screening colony PCR, and the bacterial liquid of the positive recombinants with positive colony PCR results was picked and expanded in liquid LB overnight.

[0046] Table 2 PCR mixed system

[0047]

[0048] The amplification program was pre-denaturation at 95.0°C for 5 min, denaturation at 95.0°C for 30 sec, annealing at 59°C for 30 sec, and extension at 72°C for 10 min.

[0049] 4. Screening of positive transformants

[0050] The recombinant DH5α was cultured in liquid LB medium containing hygromycin resistance. 600 =1.8-2.0, extract the plasmid, transform the extracted pCZF8X-3643 recombinant plasmid into Agrobacterium AGL1, perform colony PCR, and select the positive transformants for Agrobacterium infection.

[0051] 5. Agrobacterium transformation

[0052] Take the Agrobacterium competent cells stored at -80℃ and place them in room temperature or ice water bath for a while to partially melt. When they are in an ice-water mixed state, insert them into ice. Add 1μg of plasmid DNA to every 100μL competent cells, stir the bottom of the tube by hand to mix, and then place them on ice for 5min, in liquid nitrogen for 5min, in a 37℃ water bath for 5min, and in an ice bath for 5min. Add 800μL of LB or 2×YT liquid culture medium without antibiotics and culture at 28℃ for 2-3h. Then centrifuge at 5000rpm for 1min to collect the bacteria, keep about 100μL of supernatant, gently blow and resuspend the bacteria block and apply it on the LB plate containing the corresponding antibiotics, and invert it in a 28℃ incubator for 2-3 days. In order to avoid false positives, it is recommended to use 50μg / mL rifampicin (Rif) and 50μg / mL kanamycin (Kan) for resistance screening at the same time, and culture at 28℃ for 60h.

[0053] Preparation of Trichoderma reesei Rut-C30 spore solution: Inoculate Trichoderma reesei solution on potato-dextrose medium (PDA) and culture at 28° C. for 5-7 days. Gently (to prevent spores from splashing) repeatedly rinse the Trichoderma reesei spores on the PDA medium with 4-5 mL of sterile water to wash out the Trichoderma reesei spores.

[0054] Agrobacterium infection of Trichoderma reesei: Take 20 μL of PCR-verified Agrobacterium culture and inoculate it into 20 mL LB liquid medium (with 40 μL kanamycin stock solution and 20 μL rifampicin stock solution added), and culture it at 220 r / min and 28°C overnight. 600After reaching 0.6-0.8, place the bacterial solution on ice, take an appropriate amount of bacterial solution and add it to a sterile cryopreservation tube, centrifuge at 7000r / min for 3 minutes, gently suspend the centrifuged bacterial solution with an appropriate amount of sterile water, centrifuge at 7000r / min for 3 minutes, wash once with sterile water, and centrifuge at 7000r / min for 3 minutes. Take 1mL of culture medium from 9.6mL of IM liquid culture medium to suspend the centrifuged bacteria, add the suspended bacteria to IM liquid culture medium, take 0.4mL of MES stock solution and 10μL of acetosyringone (AS) stock solution and add them to IM liquid culture medium (be careful to avoid light), 220r / min, and culture at 28℃ in the dark for 6h. When the bacterial solution OD 600 After reaching 0.5-0.8, take 100 μL of bacterial solution and add 10 6 spores, mix gently and spot on the cellophane of IM solid medium, then spread evenly with a glass rod, blow dry in a clean bench and culture at 24℃ in the dark. Two days later, when hyphae grow slightly on the edge of the cellophane, use sterile tweezers to transfer the membrane to a 2×Mandels resistance plate and culture at 28℃.

[0055] The formula of IM liquid culture medium is as follows: 5 mL / L glycerol, 1 g / L glucose, 0.5 g / L MgSO4·7H2O, 1.45 g / L KH2PO4, 2.05 g / L K2HPO4, 0.5 g / L (NH4)2SO4, 0.15 g / L NaCl, 0.05 g / L CaCl2·2H2O and 1 mL / L trace elements, wherein the trace element components include FeSO4·7H2O (2.5 g), MnSO4·H2O (1.6 g), ZnSO4 (1.4 g), CuSO4·5H2O (0.25 g), H3BO3 (0.1 g), Na2MoO4·2H2O (0.1 g), and distilled water is added to 1 L. 2-N-morpholine-ethylsulfonic acid (MES) and acetosyringone (AS) are added before use. MES (21.325 g) was adjusted to pH 5.7 with KOH, dissolved in 100 mL ddH2O, and sterilized by filtration with a 0.22 μm pore size. AS (0.392 g) was dissolved in 10 mL dimethyl sulfoxide, and sterilized by filtration with a 0.22 μm pore size.

[0056] The formula of 2×Mandels medium is: yeast extract 0.6g / L, KH2PO44g / L, (NH4)2SO42.8g / L, Tryptone3g / L, Urea0.6g / L, CaCl2·2H2O0.5g / L, MgSO4·7H2O0.6g / L, glucose 20g / L and trace elements 1mL / L. The components of the trace element solution are: FeSO4·7H2O10g, MnSO4·H2O3.2g, ZnSO42.8g, CoCl220g. The solid culture medium is Agar with 15%-20%, pH=5.5, and distilled water is added to 1L.

[0057] 6. Screening of recombinant Trichoderma reesei strains with high enzyme activity

[0058] (1) Take 0.3 g of fresh mycelium of recombinant Trichoderma reesei, extract recombinant Trichoderma reesei DNA by CTAB method, and use the extracted DNA as template for PCR amplification. The primers used are Hyg primer and target gene specific primer. The results are shown in Figure 2 A and B.

[0059] The primers are:

[0060] pCZF8X-LeGH7_3643-F (SEQ ID NO: 4): 5'-CGGCTTAAGATGTTTCGCTCTACTGCTCT-3';

[0061] pCZF8X-LeGH7_3643-R (SEQ ID NO: 5): 5'-TGCTCTAGATTAGTGGTGATGGTGGTGGTGCTCGAGTGCAGTAAAAGTGGT-3';

[0062] Hgy-F (SEQ ID NO: 6): 5'-TGTCCTGCGGGTAAATAGC-3';

[0063] Hgy-R (SEQ ID NO: 7): 5'-TGCTCCATACAAGCCAACC-3'.

[0064] The amplification reaction system is shown in Table 3:

[0065] Table 3 Amplification reaction system

[0066]

[0067] (2) Screening of recombinant Trichoderma reesei strains with high enzyme activity

[0068] Referring to the standard method of IUPAC (Ghose 1987), the specific method is as follows: roll 50 mg Whatman No. 1 filter paper into a paper roll and place it in a 15 mL centrifuge tube. Take 1 mL of the enzyme solution to be tested into a 15 mL centrifuge tube containing filter paper, add 3 mL of pH 4.8 citric acid buffer, and react in a shaker at 50°C and 150 r / min for 1 hour. After the enzymatic hydrolysis reaction is completed, kill the enzyme activity at 100°C for 10 minutes. After cooling to room temperature, centrifuge at 3000g for 5 minutes, take an appropriate amount of diluted supernatant, and determine the reducing sugar content in the enzymatic hydrolyzate by DNS method. The amount of enzyme required to hydrolyze the substrate for 1 minute to produce 1 μmol of glucose is defined as one filter paper enzyme activity unit (FPU).

[0069] Bradford method for protein concentration determination: Preparation of standard curve: Take 1 mg / mL BSA stock solution and dilute it to final concentrations of 0, 20, 40, 60, and 80 μg / mL; Take 1 mL of BSA of each concentration in a 10 mL colorimetric tube, add 3 mL of Coomassie Brilliant Blue colorimetric solution, react at room temperature for 10 minutes; measure the absorbance at 595 nm; Take 1 mL of the crude enzyme solution to be tested, add 3 mL of colorimetric solution, react at room temperature for 10 minutes, and measure the absorbance at a wavelength of 595 nm. Calculate the protein concentration of the crude enzyme solution based on the standard curve.

[0070] The results are as follows Figure 3 As shown, among the 8 recombinants, GH7-5 and GH7-6 showed the highest filter paper enzyme activity, which were 0.63FPU / mL and 0.53FPU / mL, respectively, which were 112-156% higher than Rut-C30. At the same time, the protein secretion was also the highest, which was 0.36mg / mL and 0.34mg / mL, which was 94-107% higher than Rut-C30.

[0071] Example 2 Analysis of enzyme activity produced by fermentation of recombinant strains

[0072] 1. Enzyme activity assay

[0073] (1) The recombinant Trichoderma reesei strains GH7-5 and GH7-6 were inoculated on conidia plates and cultured at 30°C for 5-7 days; the conidia plates were composed of 30 g / L malt extract powder, 20 g / L agar powder, and distilled water to 1 L.

[0074] (2) Wash the spores with sterile distilled water and dilute them to ensure that the concentration of the inoculated spore solution is 10 7 Pieces / mL;

[0075] (3) Inoculate 500 μL of spore liquid into every 30 mL of enzyme fermentation medium and culture at 30°C and 200 rpm in a shaking table for 7 days. Centrifuge the fermentation liquid and the supernatant after centrifugation is the crude enzyme liquid. Measure the enzyme activity and protein concentration respectively. The composition of the enzyme fermentation medium is as follows: microcrystalline cellulose 20 g / L, KH2PO4 15 g / L, (NH4)SO4 10 g / L, trypsin 1 g / L, urea 0.3 g / L, CaCl2·2H2O 0.4 g / L, MgSO4·7H2O 0.3 g / L, yeast extract 10 g / L and trace element liquid 1 mL / L; wherein, the components of the trace element liquid are: FeSO4·7H2O 75 g, MnSO4·H2O 2 g, ZnSO4 2 g and CoCl2 3 g, and distilled water is used to make the volume to 1 L.

[0076] (4) Determination of filter paper enzyme activity (FPase): Refer to the standard method of IUPAC (Ghose 1987). The specific method is as follows: Roll a 50 mg Whatman No. 1 filter paper strip into a paper roll and place it in a 15 mL centrifuge tube. Take 1 mL of the enzyme solution to be tested into a 15 mL centrifuge tube containing filter paper, add 3 mL of pH 4.8 citric acid buffer, and react in a shaker at 50°C and 150 r / min for 1 hour. After the enzymatic hydrolysis reaction is completed, kill the enzyme activity at 100°C for 10 minutes. After cooling to room temperature, centrifuge at 3000g for 5 minutes, take an appropriate amount of diluted supernatant, and determine the reducing sugar content in the enzymatic hydrolyzate by DNS method. The amount of enzyme required to hydrolyze the substrate for 1 minute to produce 1 μmol of glucose is defined as one filter paper enzyme activity unit (FPU).

[0077] (5) Determination of exoglucanase activity (pNPCase): 500 μL of appropriately diluted crude enzyme solution was mixed with 250 μL of 1 mg / mL p-nitrophenyl cellobioside (pNPC) solution, and the mixture was incubated at 50°C for 30 min. 750 μL of 10% Na2CO3 was added to terminate the reaction, and the absorbance was measured at 420 nm. The crude enzyme solution inactivated by boiling was used as a control.

[0078] (6) Determination of endoglucanase activity (CMCase): Take 1 mL of 1% (w / v) sodium carboxymethyl cellulose (CMC) solution (dissolved in 0.05 mol / L, pH 4.8 sodium citrate buffer) in a 15 mL centrifuge tube, add 1.5 mL of sodium citrate buffer (0.05 mol / L, pH 4.8) and 500 μL of crude enzyme solution, incubate in a 50°C water bath for 30 min, and inactivate in a boiling water bath for 10 min. After cooling to room temperature, centrifuge at 4000 r / min for 5 min, take the supernatant, and determine the reducing sugar content in the enzymatic hydrolyzate by DNS method. (Under the conditions of pH 4.8 and temperature 50°C, the amount of enzyme required to hydrolyze the substrate for 1 min to produce 1 μmol of glucose is defined as one unit of enzyme activity).

[0079] (7) Determination of β-glucosidase activity (BGLase): Take 1 mL of 1% salicin solution (dissolved in 0.05 mol / L, pH 4.8 sodium citrate buffer) in a 15 mL centrifuge tube, add 1.5 mL of sodium citrate buffer (0.05 mol / L, pH 4.8) and 500 μL of crude enzyme solution, incubate in a 50°C water bath for 30 min, and inactivate in a boiling water bath for 10 min. After cooling to room temperature, centrifuge at 4000 r / min for 5 min, take the supernatant, and determine the reducing sugar content in the enzymatic hydrolyzate by the DNS method. (Under the conditions of pH 4.8 and temperature 50°C, the amount of enzyme required to hydrolyze the substrate for 1 min to produce 1 μmol of glucose is defined as one unit of enzyme activity).

[0080] (8) Xylanase activity determination: Take 1.5 mL of 1% xylan (dissolved in 0.05 mol / L, pH 4.8 sodium citrate buffer) in a 15 mL centrifuge tube, add 1.5 mL of sodium citrate buffer (0.05 mol / L, pH 4.8) and 500 μL of crude enzyme solution, incubate in a 50°C water bath for 30 min, and inactivate in a boiling water bath for 10 min. After cooling to room temperature, centrifuge at 4000 r / min for 5 min, take the supernatant, and determine the reducing sugar content in the enzymatic hydrolyzate by the DNS method. (Under the conditions of pH 4.8 and temperature 50°C, the amount of enzyme required to hydrolyze the substrate for 1 min to produce 1 μmol of xylose is defined as one unit of enzyme activity).

[0081] 2. Determination of protein concentration by Bradford method

[0082] Preparation of standard curve: Take 1 mg / mL BSA stock solution and dilute it to final concentrations of 0, 20, 40, 60, and 80 μg / mL; take 1 mL of BSA of each concentration in a 10 mL colorimetric tube, add 3 mL of Coomassie Brilliant Blue colorimetric solution, react at room temperature for 10 minutes; measure the absorbance at 595 nm; take 1 mL of the crude enzyme solution to be tested, add 3 mL of colorimetric solution, react at room temperature for 10 minutes, and measure the absorbance at a wavelength of 595 nm. Calculate the protein concentration of the crude enzyme solution based on the standard curve.

[0083] The results are as follows Figure 4 As shown, compared with the wild type, the filter paper enzyme activity of the crude enzyme solution secreted by GH7-5 and GH7-6 increased by 118-120%, the exocellulase activity increased by 133-241%, the endocellulase activity increased by 119-140%, the β-glucanase activity increased by 77-113%, the xylanase activity increased by 87-196%, and the protein concentration in the crude enzyme solution increased by 13%.

[0084] Example 3 Analysis of sugar production efficiency of different biomass materials by enzymatic hydrolysis of crude enzyme solution of recombinant strain

[0085] The NaOH pretreatment conditions were: adding NaOH (w / v) solution of corresponding concentration, 50°C, 150 rpm shaking for 2 h; the steam explosion pretreatment conditions were: 225°C (2.5 MPa) for 3 min. Steam explosion reaction parameters: 2.5 MPa, 3 min. The treated residues were collected, air-dried, crushed with a small powder sample machine, sieved with 40 mesh, and dried at 50°C to constant weight to obtain the experimental materials after steam explosion; 0.3000g of straw powder was weighed in 15mL centrifuge tubes, and rice (0.5%, 1% NaOH), cotton, rapeseed (1% NaOH), Miscanthus (2% NaOH) and corn (1% NaOH and steam explosion) were pretreated accordingly; after the treatment, 10mL dH2O was added to wash until neutral, and then washed once with 0.2mol / L pH4.8 phosphate buffer, and an equal volume of 3mL of crude enzyme solution fermented by recombinant Trichoderma reesei strains GH7-5 and GH7-6 was added, and enzymolysis was carried out at 50°C and 150rpm for 48h; after the enzymolysis was completed, the supernatant was centrifuged and the content of trisaccharides and pentoses was determined by colorimetry.

[0086] The results are as follows Figure 5 As shown in the figure, the crude enzymes secreted by the recombinant strains GH7-5 and GH7-6 had significantly higher sugar production efficiency for the five biomasses than the wild type, among which the largest increase was in cotton straw pretreated with 1% NaOH, reaching 107.9%. The best performance of the recombinant Trichoderma reesei enzyme was in rice straw pretreated with 1% NaOH, with an enzymatic hydrolysis efficiency of 97.1%, followed by corn straw pretreated with steam explosion, with an enzymatic hydrolysis efficiency of 88.2%.

[0087] Example 4 Analysis of the efficiency of fermentation of ethanol production by different biomass materials using crude enzyme solution of recombinant strains

[0088] 1. Fermentation to produce ethanol

[0089] The pH of the fermentation supernatant of 1% NaOH pretreated rice and 2% NaOH pretreated Miscanthus with the highest enzymatic sugar production efficiency in Example 3 was adjusted to 4.8, and sterilized at 121°C for 20 min; 3 mL of crude enzyme solution secreted by recombinant engineering strains GH7-5 and GH7-6 were added respectively, and the volume was adjusted to 6 mL with 0.2 mol / L pH 4.8 phosphate buffer, and the mixture was placed in a shaker for enzymatic hydrolysis for 48 h; Angel yeast was added to the enzymatic hydrolysis system in an ultra-clean workbench for fermentation ethanol production analysis; the final concentration of Angel yeast was 0.5 g / L, and the mixture was cultured in a 37°C incubator for 48 h. After the fermentation was completed, the fermentation broth was transferred to a 250 mL distillation flask with an equal volume of dH2O as the fermentation broth, and an equal volume of ethanol as the fermentation broth was distilled out at 100°C, and the ethanol concentration was determined by the K2Cr2O7 method.

[0090] The results are as follows Figure 6As shown in Figure A, the ethanol yield of rice straw increased from 4.08% to 5.05% and 7.93%, respectively, with an increase of 23.7% and 94.3%, respectively. The ethanol yield of Miscanthus straw increased from 6.11% to 8.37% and 8.34%, respectively, with an increase of 37.0% and 36.5%, respectively.

[0091] 2. Determination of ethanol yield

[0092] Preparation of standard curve for ethanol content determination: Prepare 20% (v / v) ethanol solution, take 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, 6.0mL, 8.0mL in a 100mL volumetric flask, dilute to 100mL with distilled water, and prepare standard solutions with concentrations (%, v / v) of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.6, respectively. Take 1.0mL of the above standard solutions, add 2mL K2Cr2O7 solution, shake and mix, and then boil in a boiling water bath for 10min, measure the absorbance at 600nm, and draw a standard curve using absorbance and ethanol concentration; use the absorbance and ethanol concentration standard curve to calculate the ethanol concentration in the fermentation broth.

[0093] The results are as follows Figure 6 As shown in B, the sugar alcohol conversion rate of the cellulase produced by the recombinant Trichoderma reesei GH7-6 on rice and Miscanthus was 74.32% and 56.18%, respectively, with increases of 52.5% and 5%, respectively. The sugar alcohol conversion rate of the cellulase produced by the recombinant Trichoderma reesei GH7-5 on Miscanthus reached 60.27%, with an increase of 12.2%.

[0094] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant engineered bacterium of Trichoderma reesei, characterized in that The recombinant engineering bacteria of Trichoderma reesei is a gene encoding the exoglucanase in Lentinula edodes. LeGH7_3643 The Trichoderma reesei is introduced for overexpression to increase the activity of the Trichoderma reesei in producing cellulase. LeGH7_3643 The nucleotide sequence is shown in SEQ ID NO:

1.

2. The recombinant engineered bacterium of Trichoderma reesei according to claim 1, characterized in that The Trichoderma reesei includes Trichoderma reesei Rut-C30.

3. A method for improving the production of cellulase by Trichoderma reesei, characterized in that: Including the exoglucanase gene from Lentinula edodes LeGH7_3643 Introducing Trichoderma reesei for overexpression, improving the activity of Trichoderma reesei in producing cellulase, obtaining recombinant engineered Trichoderma reesei, and fermenting with the recombinant engineered Trichoderma reesei, centrifuging the fermentation broth, and obtaining a crude enzyme solution containing the produced cellulase; wherein, LeGH7_3643 The nucleotide sequence is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that The fermentation production conditions are as follows: the fermentation medium includes the following components in amounts: 20 g / L microcrystalline cellulose, 15 g / L KH2PO4, 10 g / L (NH4)SO4, 1 g / L tryptone, 0.3 g / L urea, 0.4 g / L CaCl2·2H2O, 0.3 g / L MgSO4·7H2O, 10 g / L yeast extract and 1 mL / L trace element solution; the trace element solution includes: 75 g FeSO4·7H2O, 2 g MnSO4·H2O, 2 g ZnSO4 and 3 g CoCl2, and distilled water is added to 1L; The fermentation temperature is 28-30°C, the rotation speed is 200 rpm, and the fermentation time is 5-7 days.

5. Use of a crude enzyme solution prepared by using the recombinant engineered bacteria of Trichoderma reesei according to any one of claims 1-2 or the method according to any one of claims 3-4 in degrading biomass materials, characterized in that: The crude enzyme solution contains cellulase and hemicellulase, and the biomass material is rice straw, corn straw, Miscanthus, rape straw or cotton straw.

6. Use of a crude enzyme solution prepared by using the recombinant engineered bacteria of Trichoderma reesei according to any one of claims 1-2 or the method according to any one of claims 3-4 in the production of ethanol, characterized in that: The crude enzyme solution is used to enzymolyze biomass materials to produce ethanol through fermentation; the crude enzyme solution contains cellulase and hemicellulase, and the biomass materials are rice straw, corn straw, Miscanthus, rape straw or cotton straw.

7. A method for degrading biomass materials, characterized in that: The biomass material is pretreated by alkaline solution or steam explosion, and then the crude enzyme solution secreted by the recombinant engineered bacteria of Trichoderma reesei according to claim 1 or 2 is added for enzymolysis; the biomass material is rice straw, corn straw, Miscanthus, rape straw or cotton straw.

8. The method according to claim 7, characterized in that The conditions for alkaline solution pretreatment are: treating the biomass material with 1-2% NaOH by mass concentration, and then placing it on a shaker at 50°C and 150 rpm for 2 h; the conditions for steam explosion pretreatment are: treating at 225°C and 2.5 Mpa for 3 min; and the conditions for enzymatic hydrolysis are: reacting at 50°C and 150 rpm for 48 h.

9. A method for producing ethanol, characterized in that: The method comprises obtaining a fermentation liquid after saccharification of a biomass material by the method of claim 7 or 8, then adding Angel Yeast to the fermentation liquid, and culturing at 37° C. for 48 hours; the biomass material is rice straw or Miscanthus.

10. The method according to claim 9, characterized in that The final concentration of Angel Yeast was 0.5 g / L.

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