Modified derived polypeptide and application thereof

Abstract

The invention relates to a modified cellulase derived polypeptide and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. The invention provides a derivative polypeptide of cellulose excision enzyme Cel7A, the cellulose excision enzyme Cel7A is derived from Trichoderma reesei, NCBI Acession No.XM006969162.1, and the amino acid sequence of the cellulose excision enzyme Cel7A is as shown in SEQ ID NO: 1. The invention further provides a preparation method of the derivative polypeptide of the cellulose excision enzyme Cel7A. Wherein the amino acid sequence of the cellulose binding domain CBM is as shown in SEQ ID NO: 2. The derivative polypeptide is a polypeptide of which the amino acid sequence is 70% or more identical to that of a polypeptide shown in SEQ ID NO: 2, can reduce combination with lignin and improve the degradation efficiency of cellulose, so that the saccharification efficiency of cellulase is improved, the post-treatment process cost is greatly saved, and the derivative polypeptide is suitable for industrial production. The method has important industrial application value.

Description

Technical Field

[0001] This invention relates to a modified cellulase-derived polypeptide and its applications, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] Cellulose is a large polysaccharide and the most widely distributed and abundant renewable resource in nature. The degradation of cellulose to obtain high-value biofuels or chemicals has attracted global attention. Developing low-cost, efficient cellulose degradation technologies is key to promoting the industrialization of biomass energy. Compared to the high energy consumption, high cost, and potential environmental pollution problems of physical and chemical methods, enzymatic methods are favored due to their green and low-cost nature. Cellulase is the most commonly used enzyme for degrading cellulose. However, due to the complex structure of lignocellulose, which contains not only cellulose but also lignin and other components, the non-productive adsorption of lignin creates a barrier against cellulose degradation. This not only reduces degradation efficiency but also increases the amount of cellulase required for the degradation process, resulting in persistently high cellulose degradation costs.

[0003] To reduce the inhibitory effect of lignin, methods such as steam explosion, ammonia treatment, acid treatment, or organic solvents are often used to reduce the lignin content in cellulose. However, due to the complex structure of lignocellulose, it is often difficult to completely remove lignin, and the residual lignin after pretreatment still has a certain impact on the degradation of lignocellulose.

[0004] In recent years, researchers have begun to develop novel cellulase-degrading enzymes through targeted design and modification of gene molecules to improve their catalytic performance. Patent CN116970591A describes the expression of an endonuclease mutant using recombinant *Trichoderma reesei* A2H, which significantly enhances enzyme activity and thermostability, playing a crucial role in resisting the degradation barrier of lignocellulose. Patent CN118389469A uses cellulase derived from *Hortaea werneckii* as the parent, and after point mutation modification, expresses it using *Pichia pastoris*. The resulting cellulase mutant exhibits catalytic activity 0.67 times higher than the wild type, significantly improving catalytic efficiency.

[0005] In summary, there is an urgent need to develop novel cellulases with low lignin adsorption and enhanced enzyme activity to increase their ability to degrade cellulose, so as to enable bio-enzymes to catalyze the degradation of cellulose into biomass feedstocks at low cost and high efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a modified cellulase polypeptide, its preparation method, and its application.

[0007] The specific technical solution of the present invention is as follows:

[0008] In a first aspect, this application provides a polypeptide derived from the cellulose exonuclease Cel7A, which is derived from Trichoderma reesei, NCBI Accession No. XM_006969162.1, with an amino acid sequence as shown in SEQ ID NO:1 and an amino acid sequence of its cellulose binding domain CBM as shown in SEQ ID NO:2.

[0009] The derived polypeptide is a polypeptide whose amino acid sequence has 70% or more homology with the polypeptide shown in SEQ ID NO:2, which can reduce the binding with lignin and improve the degradation efficiency of cellulose.

[0010] Preferably, the derived polypeptide is obtained by mutating or substituting one or more amino residues at positions 8, 10-12, and 27 of the polypeptide shown in SEQ ID NO:2.

[0011] Preferably, the derived polypeptide is selected from one of the following substitution schemes of the polypeptide shown in SEQ ID NO:2:

[0012] 1) The amino acid at position 12 is replaced by D instead of G, thus obtaining polypeptide C1;

[0013] 2) The 10th amino acid of polypeptide C1 is replaced by D instead of G, thereby obtaining polypeptide C2;

[0014] 3) The amino acid at position 11 of polypeptide C1 is replaced by D instead of I, thereby obtaining polypeptide C3;

[0015] 4) The amino acid at position 12 of polypeptide C2 is replaced by D instead of G, thereby obtaining polypeptide C4;

[0016] 5) The amino acid at position 27 of polypeptide C2 is replaced by D instead of V, thus obtaining polypeptide C5;

[0017] 6) The amino acid at position 12 of polypeptide C2 is replaced by D with G, and the amino acid at position 27 is replaced by D with V, thereby obtaining polypeptide C6.

[0018] 7) The amino acid at position 8 of polypeptide C2 is replaced by D instead of C, thus obtaining polypeptide C7;

[0019] 8) The amino acid at position 8 of polypeptide C3 is replaced by D instead of C, thus obtaining polypeptide C8;

[0020] 9) The amino acid at position 17 of polypeptide C3 is replaced by D instead of T, thus obtaining polypeptide C9.

[0021] Secondly, this application provides a polynucleotide encoding any one of the derivative polypeptides C1-C9 described in the first aspect above.

[0022] Thirdly, this application provides a recombinant vector containing the encoding genes of the derived polypeptides C1-C9 described in the second aspect.

[0023] The vector, preferably, can be a variety of commercially available kits containing transforming plasmids and other related nucleotides. Any isolated and / or purified nucleotides can be further manipulated to produce other nucleotides for transfecting cells, integrating into relevant vectors to infect organisms for expression, etc. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters that can be used to regulate the expression of specific target nucleotides; the vector optionally includes a universal expression cassette.

[0024] In one specific embodiment, the expression cassette contains all the elements necessary for the expression of the derived polypeptide C1-C9, including elements required for transcription and translation in the host cell. For example, the expression cassette includes a promoter and a terminator, which are not particularly limited and can be promoters and terminators known in the art that enable the expression of the polypeptide or its mutants.

[0025] Fourthly, this application provides a Trichoderma reesei gene-modified cell that, relative to the starting strain, has: (1) reduced non-productive adsorption activity for lignin; and (2) increased cellulose degradation activity.

[0026] Preferably, the starting strain of the *Trichoderma* gene-modified cell is *Trichoderma* strain.

[0027] In a preferred embodiment, the original strain of the Trichoderma reesei gene-modified cell is Trichoderma reesei T1, which is derived from the China Center for Type Culture Collection (CCTCC) with the strain accession number M2015804 and contains the gene encoding Cel7A (XM_006969162.1).

[0028] Preferably, the *Trichoderma reesei* gene-modified cells have the following characteristics:

[0029] (1) The ligase gene (XM_006963830.1) of *Trichoderma reesei* was knocked out.

[0030] (2) The gene (XM_006969162.1) encoding Cel7A in Trichoderma reesei was knocked out.

[0031] Fifthly, a transformed host cell is provided, which is transformed with the polynucleotide described in the second aspect or the recombinant vector described in the third aspect.

[0032] This application also provides the use of the Trichoderma reesei gene-modified cells described in the fourth aspect and the transformed host cells described in the fifth aspect in the preparation of the derived polypeptides C1-C9 described in the first aspect.

[0033] Sixthly, this application provides a method for expressing the polypeptide described in the first aspect and its derivative polypeptides, comprising:

[0034] (1) Culture Trichoderma mareesei gene-modified cells as described in the fourth aspect or host cells transformed as described in the fifth aspect under conditions suitable for expressing the polypeptide; and

[0035] (2) The polypeptide is recovered.

[0036] Host cells can be cultured in a suitable medium for peptide production using methods known in the art. For example, cells can be cultured by shake-flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-by-feed fermentation, or solid-state fermentation) in a laboratory or industrial fermenter in a suitable medium and under conditions that allow for peptide expression and / or isolation. The culture occurs using procedures known in the art in a suitable medium containing carbon and nitrogen sources and inorganic salts. Suitable media can be purchased commercially or prepared according to publicly available compositions.

[0037] The polypeptide can be recovered from the culture using methods known in the art. For example, variants can be recovered from nutrient media through a variety of routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0038] In addition, peptides can be purified by a variety of procedures known in the art to obtain substantially pure peptides, including but not limited to chromatographic methods (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, gel filtration chromatography), electrophoretic procedures (e.g., preparative isoelectric point focusing), differential solubility methods (e.g., ammonium sulfate precipitation), SDS-PAGE, salting out, and combinations thereof; more preferably, purification can be carried out by Ni column affinity chromatography.

[0039] The derived polypeptide has enhanced cellulase activity.

[0040] The derived polypeptide can reduce lignin binding.

[0041] In a seventh aspect, this application provides an enzyme preparation containing one or more of the derivative polypeptides described in the first aspect.

[0042] Eighthly, this application provides the use of the derived polypeptides described in the first aspect and the enzyme preparations described in the seventh aspect in the degradation or conversion of cellulose-containing materials.

[0043] The application uses microcrystalline cellulose as a reaction substrate and decomposes it into cellobiose under the catalysis of the derivative polypeptide described in the first aspect or the enzyme preparation described in the seventh aspect.

[0044] The application described in this application can be carried out in the presence of lignin.

[0045] The application uses microcrystalline cellulose as a reaction substrate, which is decomposed into cellobiose under the catalysis of the modified cellulase-derived polypeptide.

[0046] Preferably, the microcrystalline cellulose is selected from Avicel and purchased from Shanghai McLean Biotech Co., Ltd., China.

[0047] Preferably, in the application, the reaction system may also contain lignin or β-glucosidase (Shanghai Maclean Biochemical Technology Co., Ltd., China).

[0048] Preferably, in the application, the reaction system further contains an antibacterial agent, and preferably, the antibacterial agent is selected from sodium azide.

[0049] Preferably, in the application, the pH of the reaction system is 4.5, and preferably the pH of the reaction system is controlled by a citrate buffer.

[0050] Beneficial effects

[0051] 1. This application provides a series of cellulase Cel7A-derived polypeptides C1-C9, which can efficiently and accurately bind to cellulose during cellulose degradation, avoiding non-productive adsorption with lignin, thereby improving the saccharification efficiency of cellulase, significantly reducing post-processing costs, and are suitable for industrial production, thus having important industrial application value.

[0052] 2. This application utilizes recombinant Trichoderma reesei to express the derived polypeptide. Compared to bacteria, the cellulase expressed by Trichoderma reesei is mainly an extracellular enzyme, which is easy to isolate and extract and has high stability. In addition, compared to enzymes produced by Escherichia coli and yeast, it does not produce excessive glycosylation, ensuring that the cellulase polypeptide is closer to the natural structure.

[0053] In summary, the cellulase-derived polypeptides provided by this invention have great potential value in the field of low-cost biotransformation of cellulose. Detailed Implementation

[0054] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; all materials and reagents are commercially available unless otherwise specified.

[0055] The present invention will be further described in detail below through embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Modifications or substitutions to the details and form of the technical solutions of the present invention without departing from the concept and scope of use of the present invention shall fall within the protection scope of the present invention.

[0056] It should be noted that the terminology used herein is for descriptive purposes only and is not intended to limit the exemplary embodiments of the invention. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the invention is for describing specific embodiments and not for limiting the scope of protection of the invention. Experimental methods in the following specific embodiments, unless specific conditions are specified, are generally performed according to conventional methods and conditions in molecular biology within the art, and such techniques and conditions are fully explained in the literature. See, for example, the techniques and conditions described in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, or according to the conditions recommended by the manufacturer.

[0057] In the following examples, cellobiose and glucose were analyzed using high performance liquid chromatography (HPLC) under the following conditions: column type: Aminex HPX-87H, mobile phase: 5mM dilute sulfuric acid, column temperature: 45℃, injection volume: 10μL, refractive index detector (Hitachi, Japan), flow rate: 1.0mL / min.

[0058] Example 1. Construction of recombinant Trichoderma reesei T1-1 strain.

[0059] The starting strain was *Trichoderma reesei* T1, obtained from the China Center for Type Culture Collection (CCTCC), with accession number M2015804. The construction of the recombinant *Trichoderma reesei* strain included the following steps:

[0060] (1) Knockout of the Trichoderma reesei ligase (XM_006963830.1): The ligase gene (XM_006963830.1) in the Trichoderma reesei strain was knocked out using homologous recombination technology. The knockout cassette contained a thiamine resistance gene, a 1500bp upstream fragment of the ligase gene, and a 1500bp downstream fragment of the ligase gene. The knockout cassette was amplified and purified using PCR technology. PCR conditions: 94℃ for 5 min; 98℃ for 10 s, 58℃ for 60 s, 68℃ for 4 min, 30 cycles; 68℃ for 10 min, 4℃ for storage. Trichoderma reesei protoplasts were prepared using the PEG / CaCl2 method. The knockout cassette was mixed with the protoplasts, and the transformants were selected by three consecutive passages in a medium containing pyridoxine hydrobromide and uracil to obtain strains with the ligase gene deletion.

[0061] (2) Using homologous recombination technology, the Cel7A gene (XM_006969162.1) in *Trichoderma reesei* T. T1 strain was knocked out based on the above-mentioned ligase gene-deficient strain. The knockout cassette contained a geneticin resistance gene, a 1500bp upstream fragment encoding the Cel7A gene, and a 1500bp downstream fragment encoding the ligase gene. The knockout cassette was amplified and purified using the same PCR technique as above. *Trichoderma reesei* protoplasts were prepared using the PEG / CaCl2 method. The knockout cassette was mixed with the protoplasts, and the transformants were selected by three consecutive passages in a geneticin-containing medium to obtain *Trichoderma reesei* gene-modified cells T. reesei T1-1.

[0062] Example 2. Expression and purification of cellulose exonuclease Cel7A and its derived polypeptides.

[0063] (1) The genes of peptides C1-C9 were integrated into the gene-modified Trichoderma reesei T1-1 cells: Transformed Trichoderma reesei host cells were constructed using ribonucleoprotein (RNP) mediated CRISPR / Cas technology.

[0064] The knockout cassette contains the hygromycin resistance gene, a 1500bp upstream fragment encoding the CBM gene, and a 1500bp downstream fragment encoding the ligase gene. sgRNA targeting the 46-66bp region of the geneticin resistance gene sequence was designed, amplified by PCR, and purified. *Trichoderma reesei* protoplasts were prepared using the PEG / CaCl2 method. The purified sgRNA, knockout cassette, and Cas9 protein (Suzhou Nearshore Protein Co., Ltd., China) were mixed with the protoplasts. Transformants were selected by three consecutive passages in hygromycin-containing medium to obtain *Trichoderma reesei* transformants containing wild-type Cel7A and the C1-C9 polypeptide genes.

[0065] (2) The Trichoderma reesei transformants containing wild-type Cel7A and polypeptide C1-C9 genes constructed in (1) above were cultured and expressed. First, the transformed Trichoderma reesei host cells were cultured in bran medium (1g bran, 1g corn cob, 1g glucose, 1g peptone, 0.2g ammonium sulfate, 0.3g potassium dihydrogen phosphate, 0.09g magnesium sulfate heptahydrate, 0.5g calcium carbonate, 100mL sterile purified water, and appropriate trace elements) for 5-7 days until sporulation occurred. Then, the cells were inoculated into seed culture medium for further culture. After 3 days, the bacterial culture from the seed culture stage was inoculated into enzyme-producing medium (6g corn cob, 1.2g microcrystalline cellulose, 1g peptone, 0.2g urea, 0.8g ammonium sulfate, 0.6g potassium dihydrogen phosphate, 0.18g magnesium sulfate heptahydrate, 0.4g sodium nitrate, 1g calcium carbonate, 100mL sterile purified water, and appropriate trace elements) at a ratio of 1:10 (v / v) for induction culture. The enzyme solution was collected after 7 days of enzyme production culture.

[0066] (3) After centrifugation, the crude enzyme solution obtained above was used to remove impurities, and the supernatant containing the enzyme was collected. The collected supernatant was concentrated in a concentration tube and centrifuged at 5000g at 4℃ until the enzyme solution was concentrated to about 3mL. The concentrated enzyme solution was transferred to a 1.5mL centrifuge tube and centrifuged at 12000rpm for 10min to remove insoluble matter. The supernatant enzyme solution was transferred to a 1.5mL centrifuge tube and placed on ice for later use. 2mL of the concentrated enzyme solution was taken with a 2.5mL syringe and loaded onto a molecular sieve S-400 (pre-washed with 50mM Tris-HCl buffer). The elution peak protein was collected, and the protein was detected by SDS-PAGE electrophoresis. Proteins with appropriate band size and high purity were selected for electrophoresis, and the protein buffer was replaced with a desalting column. Citrate buffer (pH 4.8) was used for elution, and the purified enzyme was collected.

[0067] Example 3. Determination of non-productive adsorption of lignin by cellulose exonuclease Cel7A and its derived polypeptides.

[0068] 1. Reaction system configuration:

[0069] (1) Avicel reaction system:

[0070] The solution contained 0.06 mg / mL Cel7A or its derivative peptides, 30 mg / mL microcrystalline cellulose (Avicel, Shanghai Maclean Biotech Co., Ltd., China), 0.02 mg / mL β-glucosidase (Shanghai Maclean Biotech Co., Ltd., China), and 0.02% sodium azide as antibacterial agents, with a final volume of 1 mL. The buffer solution was 50 mM citrate buffer with a pH of 4.5.

[0071] (2) Mixture reaction system:

[0072] In addition to adding 30 mg / mL of mixed microcrystalline cellulose (Avicel), 30 mg / mL of lignin was added, while the rest remained unchanged.

[0073] (3) CCR reaction system:

[0074] The mixture included 0.06 mg / mL Cel7A or its derivative peptides, 30 mg / mL microcrystalline cellulose (Avicel, Shanghai Maclean Biotechnology Co., Ltd., China), 0.02 mg / mL β-glucosidase (Shanghai Maclean Biotechnology Co., Ltd., China), 150 mg / mL delignified corn cob (CCR, Hebei Yigao Biotechnology Co., Ltd.), and 0.02% sodium azide as an antibacterial agent. The final volume was 1 mL, and the buffer was a 50 mM citrate buffer with a pH of 4.5.

[0075] 2. Test Methods: The “λ” values ​​of the Mixture and Avicel reaction systems were tested using the method described in the reference (Wang, M., Han, L., Liu, S., Zhao, X., Yang, J., Loh, SK, Sun, X., Zhang, C., & Fang, X. (2015). A Weibull statistics-based lignocellulosesaccharification model and a built-in parameter accurately predict lignocellulose hydrolysis performance. Biotechnology Journal, 10(9), 1424–1433. (https: / / doi.org / 10.1002 / biot.201400723). The “λ” value represents the time required to enzymatically hydrolyze 62.3% of the cellulose substrate.

[0076] 3. Calculation formula: “α”=(λ1-λ0) / λ1×100%;

[0077] Where λ1 represents the time required for the Mixture reaction system to enzymatically hydrolyze 62.3% cellulose;

[0078] λ0 represents the time required for the Avicel reaction system to enzymatically hydrolyze 62.3% of cellulose.

[0079] “α” describes non-productive adsorption; the lower the value, the lower the non-productive adsorption of lignin.

[0080] The calculation results are recorded in Table 1.

[0081] Table 1. Results of non-productive adsorption tests of lignin on wild-type polypeptides and their derived polypeptides.

[0082] polypeptide "α”(％) Cel7A 72.65 C1 56.09 C2 60.73 C3 54.34 C4 61.38 C5 54.42 C6 53.33 C7 53.58 C8 50.47 C9 65.18

[0083] Example 4. Cellulose degradation experiments using cellulose exonuclease Cel7A and its derived polypeptides.

[0084] The cellulose used in this experiment was Avicel, purchased from Maclean's Biochemical Technology Co., Ltd.; the non-grain biomass used was CCR, Hebei Yigao Biotechnology Co., Ltd.; Cel7A and its mutant peptides were purified using an AKTA avant chromatography system (GE Healthcare, US) and a HIPREP 16 / 60 SEPHACRYL S-300HR column (Cytiva, US). The mobile phase for purification was 50 mM Tris-HCl buffer (containing 50 mM sodium chloride), pH 7.0. β-glucosidase was purchased from Maclean's Biochemical Technology Co., Ltd.; protein concentration was determined using Bradford Biotechnology Co., Ltd. of China Sangon Biotech Co., Ltd., with bovine serum albumin (BSA) as the protein standard.

[0085] This embodiment includes three reaction systems:

[0086] (1) Avicel reaction system: 0.06 g / L Cel7A or its mutant protein, 30 g / mL microcrystalline cellulose (Avicel), 0.02 g / L β-glucosidase, and 0.02% sodium azide as antibacterial agents, with a final volume of 1 mL. The buffer is 50 mM citrate buffer with pH 4.5. The reaction is carried out at 45℃ for 72 h. Samples are taken every 12 h for the first 24 h, and every 24 h from 24 h to 72 h. Three parallel tests are performed.

[0087] (2) Mixture reaction system: In addition to adding 30 g / L microcrystalline cellulose (Avicel), 30 g / L lignin was added separately, and the rest remained unchanged;

[0088] (3) CCR reaction system: The moisture content of CCR was determined using a halogen moisture analyzer XF-110MA (Xiamen Xiongfa Instrument Co., Ltd.), and its cellulose, hemicellulose, and lignin composition was determined according to the method of NREL (National Energy Laboratory). The results showed that the moisture content of CCR was 60%, cellulose accounted for 40% of the dry weight, and lignin accounted for 60% of the dry weight. To ensure that the cellulose content was consistent with that in Example 3, 150 mg of CCR was added in the subsequent reaction. The remaining operations were the same as in Example 3. Wild-type Cel7A and the C1-C9 mutant peptide obtained in the above experiment were selected as the peptides. After the reaction, the reaction solution was kept at 100°C in water for 10 min to inactivate the protein. The supernatant was collected by centrifugation, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC. The mobile phase was 5 mM dilute sulfuric acid.

[0089] In comparing the glycation rates of Cel7A and its mutant peptides, this invention converts the residual cellobiose into an equivalent amount of glucose, and then compares the differences in glucose equivalent. One molecule of cellobiose (molecular weight 342) contains two molecules of glucose, since two molecules of glucose (molecular weight 180) release one water molecule when synthesizing one cellobiose.

[0090] Define "Glucose equivalent" to describe the amount of glucose released:

[0091] Calculation formula:

[0092] Glucose equivalent (mg / mL) = glucose concentration (mg / mL) + cellobiose concentration (mg / mL) / 342 × 180 × 2;

[0093] Formula for calculating saccharification rate:

[0094] Glycation rate (%) = (Glucose equivalent (mg / mL) / 30 (mg / mL)) × 100%.

[0095] The calculation results are recorded in Table 2.

[0096] Table 2. Results of saccharification of cellulose catalyzed by wild-type polypeptides and their derivatives in different reaction systems.

[0097]

[0098] As can be seen from the table above, although the Mixture and CCR reaction systems showed some inhibition of cellulase activity after the addition of lignin compared to the Avicel reaction system, the saccharification rate of the mutant C1-C9 peptide-catalyzed reaction was improved compared to the wild-type Cel7A. This indicates that the derived peptide C1-C9 can reduce lignin adsorption and has enhanced cellulase activity.

[0099] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A polypeptide derived from cellulose exonuclease Cel7A, wherein the derived polypeptide is a polypeptide whose amino acid sequence has 70% or more homology with the polypeptide shown in SEQ ID NO:2; Preferably, the derived polypeptide is obtained by mutating or substituting one or more amino residues at positions 8, 10-12, and 27 of the polypeptide shown in SEQ ID NO:

2.

2. The derived polypeptide of claim 1, selected from one of the following substitution schemes of the polypeptide shown in SEQ ID NO:2: 1) The amino acid at position 12 is replaced by D instead of G, thus obtaining polypeptide C1; 2) The 10th amino acid of polypeptide C1 is replaced by D instead of G, thereby obtaining polypeptide C2; 3) The amino acid at position 11 of polypeptide C1 is replaced by D instead of I, thereby obtaining polypeptide C3; 4) The amino acid at position 12 of polypeptide C2 is replaced by D instead of G, thereby obtaining polypeptide C4; 5) The amino acid at position 27 of polypeptide C2 is replaced by D instead of V, thus obtaining polypeptide C5; 6) The amino acid at position 12 of polypeptide C2 is replaced by D with G, and the amino acid at position 27 is replaced by D with V, thereby obtaining polypeptide C6. 7) The amino acid at position 8 of polypeptide C2 is replaced by D instead of C, thereby obtaining polypeptide C7; 8) The amino acid at position 8 of polypeptide C3 is replaced by D instead of C, thus obtaining polypeptide C8; 9) The amino acid at position 17 of polypeptide C3 is replaced by D instead of T, thereby obtaining polypeptide C9.

3. A polynucleotide encoding any one of the derivative polypeptides C1-C9 of claim 1.

4. A recombinant vector comprising the encoding gene of the derived polypeptides C1-C9 of claim 1.

5. A gene-modified cell of *Trichoderma reesei*, having the following characteristics: (1) The ligase gene (XM_006963830.1) of *Trichoderma reesei* was knocked out. (2) The gene (XM_006969162.1) encoding Cel7A in Trichoderma reesei was knocked out.

6. A transformed host cell, which is transformed with the polynucleotide of claim 3 or the recombinant vector of claim 4.

7. The method for expressing the derived polypeptide according to claim 1, comprising: (1) Cultivate the Trichoderma reesei of claim 5 under conditions suitable for expressing the polypeptide. (Trichoderma reesei) gene-modified cells or host cells transformed as described in claim 6; and (2) The polypeptide is recovered.

8. An enzyme preparation comprising one or more of the derivative polypeptides described in any one of claims 1 or 2.

9. The use of the derived polypeptide of any one of claims 1-2 or the enzyme preparation of claim 8 in the degradation or conversion of cellulose-containing materials. Preferably, the application uses microcrystalline cellulose as a reaction substrate, and decomposes it into cellobiose under the catalysis of the derived polypeptide of claim 1 or 2, or the enzyme preparation of claim 8.

10. The use of the Trichoderma reesei gene-modified cell of claim 5 or the transformed host cell of claim 6 in the preparation of the derivative polypeptide of any one of claims 1 or 2.

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