An endoglucanase mutant, its encoding gene, and its applications

Abstract

This invention discloses an endoglucanase mutant, its encoding gene, and its applications. The mutant gene was obtained through semi-rational design using alanine scanning mutagenesis and saturation mutagenesis on the wild-type endoglucanase A4 gene, and a recombinant expression vector was constructed. The mutant protein was efficiently expressed by the engineered *Escherichia coli* Rosetta (DE3) after fermentation. This invention establishes a highly efficient expression system through genetic engineering, providing a new technology for cellulose degradation and biomass resource utilization, and has application value in the field of bioenergy. These results provide theoretical insights into mutation-driven enhancement of enzyme activity and stability, guiding future enzyme engineering work.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and genetic engineering, and specifically relates to an endoglucanase A4 mutant obtained by molecular modification, its encoding gene, preparation method, and its application in cellulose degradation and / or biomass conversion. Background Technology

[0002] Cellulose is the most abundant renewable biomass resource on Earth, but its highly crystalline structure and lignin encapsulation limit the efficiency of direct hydrolysis. Endoglucanase A4, a key enzyme, exhibits optimal activity at pH 5.0 and 50°C, but there is still room for improvement in the enzyme activity and thermostability of wild-type A4. Current technologies utilize genetic engineering modifications such as saturation mutagenesis and domain substitution to optimize enzyme performance, but there is a lack of research on the construction of systematic mutants of A4 and the study of its molecular mechanisms. Summary of the Invention

[0003] This invention protects an endoglucanase mutant gene, which is obtained by alanine scanning and saturation mutagenesis of the wild-type A4 gene (sequence shown in SEQ ID NO.1). Key mutants include M87 and M88 (the cDNA sequence of mutant M88 is shown in SEQ ID NO.3), etc. The specific activity of mutant M88 reaches 217% of that of wild type.

[0004] This invention also protects the use of the above-mentioned endoglucanase A4 or the above-mentioned endoglucanase A4 in the decomposition of cellulose.

[0005] The purpose of this invention is to provide an endoglucanase mutant that improves its catalytic efficiency and stability through semi-rational design.

[0006] The present invention also protects the protein encoded by the mutant gene (sequence shown in SEQ ID NO.4), as well as the recombinant expression vector and engineered bacteria containing the gene.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] An endoglucanase mutant is obtained by deleting the dockerin domain of the carbohydrate-binding module from wild-type endoglucanase A4 and fusing the CBM28 domain of the carbohydrate-binding module to the C-terminus of its catalytic domain GH5, while introducing a point mutation of at least one amino acid residue into the catalytic domain GH5 and / or the CBM28 domain. The amino acid sequence of the wild-type endoglucanase A4 is shown in SEQ ID NO. 2.

[0009] The gene sequence encoding a wild-type endoglucanase A4 is shown in SEQ ID NO. 1.

[0010] The amino acid sequence of the endoglucanase mutant is shown in SEQ ID NO.4.

[0011] Genes encoding any of the above-mentioned endoglucanase mutants.

[0012] The encoding gene sequence of the endoglucanase mutant is shown in SEQ ID NO.3.

[0013] A recombinant vector contains the endoglucanase mutant gene described above;

[0014] Preferably, the recombinant expression vector is pCold-TF, the mutant sequence is obtained by whole-genome synthesis, and then transformed into Escherichia coli Rosetta(DE3) for expression.

[0015] The expression vector for the recombinant vector is pCold-TF7820.

[0016] A recombinant strain contains the endoglucanase mutant gene described above or the recombinant vector described above;

[0017] The recombinant strain was Escherichia coli Rosetta (DE3).

[0018] A method for producing an endoglucanase mutant as described above, the method comprising the following steps: constructing a gene encoding the endoglucanase mutant into an expression vector, transferring the expression vector into a host bacterium for expression, and obtaining the endoglucanase mutant from the host bacterium.

[0019] The application of any of the above-mentioned endoglucanase mutants in cellulose degradation and / or biomass conversion.

[0020] Application of the described endoglucanase mutant in the degradation of cellulose and / or biomass conversion under conditions of 45-55℃ and / or acidic pH;

[0021] Preferably, the temperature is 50°C; the acidic pH value is 4.5-5.5; preferably 5.0.

[0022] The aforementioned endoglucanase mutant is used to degrade sodium carboxymethyl cellulose (CMC-Na) or treat straw with alkali to generate fermentable sugars.

[0023] Beneficial effects

[0024] This invention constructs a highly efficient mutant using genetic engineering technology, which significantly enhances enzyme activity and improves cellulose degradation efficiency, thus having application value in biofuel production and environmental protection. Attached Figure Description

[0025] Figure 1 Expression, purification and enzymatic properties of endoglucanase A4

[0026] A: SDS-PAGE shows the purified band of the mutant protein (molecular weight approximately 50 kDa).

[0027] B: Mutant enzyme activity curves at different pH levels (highest activity at pH 5.0).

[0028] C: Mutant enzyme activity curves at different temperatures (highest activity at 50°C).

[0029] D: Kinetic curve (relationship between substrate concentration and reaction rate).

[0030] E: Melting curve (Tm value is 42.3°C).

[0031] Figure 2 Mutant molecular docking and structural modeling

[0032] A: The three-dimensional structure of endoglucanase A4 and the electrostatic potential distribution of its substrate binding domain.

[0033] B: Diagram of key local residue interactions between endoglucanase A4 and cellulose hexamer.

[0034] Figure 3 Comparison of enzyme activities between mutant and wild-type

[0035] A: Bar chart of relative activity (mutant vs. wild type).

[0036] B: Statistical chart of screening results for saturated mutants.

[0037] C: Comparison of FTIR product analysis.

[0038] D: Optimal temperature verification curve.

[0039] Figure 4 ITC combined analysis and molecular dynamics simulation results

[0040] A: ITC curves of wild-type A4 and CMC-Na.

[0041] B: ITC curves of mutant GH5(M8)-CBM28 and CMC-Na.

[0042] C: RMSD conformational stability diagram.

[0043] D: Dynamic change in the number of hydrogen bonds.

[0044] Figure 5 Analysis of products from the degradation of various straws using A4

[0045] A: FTIRS analysis of straw degradation products by endoglucanase A4.

[0046] B: FTIRS two-dimensional correlation synchronous spectra of straw degradation products by endoglucanase A4.

[0047] C: FTIRS two-dimensional correlation asynchronous spectra of straw degradation products by endoglucanase A4.

[0048] D: SEM images of various straw products treated with wild-type A4 and mutants. Detailed Implementation

[0049] The present invention will be further described below with reference to the embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out in accordance with known means in the art.

[0050] Example 1: Homology modeling of the A4 functional domain and molecular docking with the substrate

[0051] The three-dimensional structure of the A4 functional domain of endoglucanase was predicted using AlphaFold3. Figure 2 In step B), a protein 1EDG with a sequence similar to A4 was found as a reference using BLASTp search. The predicted structure of A4 was then superimposed and compared with the crystal structure of 1EDG using PyMOL software. For molecular docking, the AutoDock software algorithm was used to dock the predicted model of A4 with a cellohexasaccharide molecule optimized by energy minimization in Avogadro 1.2.0 (force field: MMFF94). Figure 2 A) Five potential binding sites were identified, and the most suitable region was selected based on the binding free energy (ΔG). An interaction map of the binding region was generated using PyMOL 2.5, and the active pocket and nearby amino acid residues were labeled.

[0052] Example 2: Alanine scanning, saturation mutagenesis, and CBM substitution to construct the A4 mutant

[0053] The PyRosetta-4 software package was used for the operation. The A4-cellulose hexasaccharide complex structure obtained from molecular docking in Example 1 was first processed using the pyrosetta.toolbox.cleanATOM tool, and then converted to the Rosetta standard residue type set using SwitchResidueTypeSetMover. Next, the energy of the complex was minimized using the pyrosetta.rosetta.protocols.relax.FastRelax protocol under the ref2015 scoring function to obtain the optimized reference structure. All amino acid residues within a distance ≤5 Å from the ligand molecule were selected as scanning targets. For each target residue (e.g., Arg123), it was mutated to alanine (e.g., Arg123→Ala) while maintaining the conformation of the protein backbone. The binding free energy (ΔGbind) of the wild-type complex and each mutant complex was calculated, and the difference was denoted as ΔΔGbind. Residues with ΔΔGbind > 0.5 kcal / mol were identified as key residues that significantly contribute to substrate binding and were added to the candidate mutation list.

[0054] From the above list of candidate mutations, catalytically essential residues and conserved core residues confirmed by multiple sequence alignment were excluded. Saturation mutagenesis was performed on the selected sites (e.g., Leu25), sequentially replacing each site with one of 19 other amino acids (e.g., Leu25→Ala, Leu25→Arg, etc.). For each point mutation, a three-dimensional structural model of the mutant was generated using AlphaFold3, followed by local energy minimization using a 500-step steepest descent method to eliminate spatial conflicts. For each optimized mutant model, the change in binding free energy (ΔΔGbind) with the substrate was calculated using the same method as for the alanine scan. Mutations with ΔΔGbind < -0.5 kcal / mol were classified as "potentially beneficial mutations".

[0055] Using AutoDock software, the original dockerin domain, CBM3, and CBM28 of the A4 gene were molecularly docked with cellohexasaccharide, and their binding free energies (ΔG) were compared. The results showed that the binding affinity of CBM3 and CBM28 was significantly better than that of the dockerin domain. Based on this, the coding sequence for the dockerin domain (413-465) in the A4 gene was removed using gene synthesis technology, while retaining the coding sequence for the GH5 catalytic domain (78-370). Four mutant genes were constructed: CBM3-GH5 (CBM3 at the N-terminus), GH5-CBM3 (CBM3 at the C-terminus), CBM3-GH5-CBM28 (fusion at both ends), and GH5-CBM28 (CBM28 at the C-terminus). The specific construction method was as follows: the sequence encoding the natural linker was precisely truncated near the end of the GH5 catalytic domain. Subsequently, a flexible linker peptide encoding 5-10 amino acids (containing glycine G and serine S) was sequentially ligated after the truncation site, followed by the coding sequence of the target CBM, thereby assembling a complete fusion gene. The expression plasmid pCold-TF7820 of the above mutant was synthesized by Shenzhen BGI Genomics Co., Ltd. The synthesized full sequence of M88 (pCold-TF7820) is shown in SEQ ID NO.5. This vector contains an N-terminal 6×histidine tag, a TF tag to enhance solubility, and a TEV protease cleavage site to facilitate protein purification. The expression plasmid was then transformed into E. coli Rosetta(DE3) (purchased from Beijing Qingke Biotechnology Co., Ltd.) for expression. GH5-CBM28 (CBM28 is located at the C-terminus), which showed the best enzyme activity enhancement, was combined with the point mutant M8 to construct the final mutant named M88. The constructed mutant proteins were expressed and purified according to the method described in Example 3, and their enzyme activities were measured according to the method described in Example 5. The relative activity results of each mutant compared with the wild type are shown below. Figure 3 As shown in Figure A. For saturated mutants at candidate sites, further screening and statistical analysis were performed based on expression patterns and enzyme activity changes, as shown in Figure A. Figure 3 As shown in B. Figure 3 As shown in Figure A, the specific activity of mutant M88 reached 217% of that of wild type. Furthermore, the optimal temperatures of the mutant and wild type were compared and verified, with results as shown in Figure A. Figure 3 As shown in D in the diagram.

[0056] Example 3: Enzyme purification

[0057] E. coli Rosetta(DE3) engineered bacteria containing the recombinant expression vector pCold-TF were fermented, and the bacterial cells were collected by centrifugation and cell disruption. The supernatant was collected by centrifugation to obtain crude enzyme solution. The crude enzyme solution was resuspended in 1 mM PBS buffer (pH 6.0) and initially purified using an NGC Quest 10 Plus chromatography system and a HisSep Ni-NTA 6FF affinity column. All purification processes were performed at a flow rate of 1 ml / min. The target protein was adsorbed onto the column material by the specific binding of the histidine tag to nickel ions, and then eluted with 1 M imidazole solution to obtain the pre-purified protein with the His tag. To further obtain the tag-free protein, TEV protease was added to the pre-purified protein and the tag was removed by enzymatic digestion at 4 °C. The digested mixture was then loaded back onto the nickel column, and the flow-through was collected to obtain high-purity tag-free endoglucanase A4. Finally, the purification effect was verified by SDS-PAGE, and the results are as follows. Figure 1 As shown in Figure A, the molecular weight of the TF-tagged fusion protein is approximately 105 kDa, while the molecular weight of the untagged A4 protein after TEV cleavage is approximately 55 kDa. Both are single, clear bands, indicating that this purification process can efficiently obtain high-purity proteins.

[0058] Example 4: Preparation of substrates for enzyme activation reaction

[0059] Sodium carboxymethyl cellulose (CMC-Na) is a commonly used cellulose derivative, widely used in enzyme activity assays and related research. In this study, the CMC-Na used in this example is a commercially available analytical grade reagent.

[0060] The preparation method of CMC-Na solution is as follows: Accurately weigh 1 g of CMC-Na powder and place it in a 250 ml Erlenmeyer flask. Add 100 ml of deionized water. Then place the Erlenmeyer flask in a microwave oven and heat it while continuously stirring until the CMC-Na is completely dissolved. Since some water will evaporate during microwave heating, after the CMC-Na is completely dissolved, cool the resulting solution to room temperature and then dilute it to 100 ml with deionized water to obtain a final CMC-Na solution with a concentration of 10 g / L. The prepared CMC-Na solution is used for subsequent enzyme activity reaction experiments.

[0061] Example 5: Determination of optimal reaction conditions for enzymes

[0062] Take 50 μL of diluted enzyme solution containing 1 μM enzyme, add 450 μL of pH 5.0 20 mM acetate-sodium acetate buffer to a 2 mL centrifuge tube, and preheat in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ for 10 min. Add 500 μL of 10 g / L CMC-Na solution (preheated for 10 min at each temperature), mix well, and then place in a water bath at the corresponding temperature for 10 min. Immediately afterward, add 1 mL of 1 mol / L Na2CO3 to terminate the reaction. Incubate at room temperature for 5 min, and measure the absorbance (OD) at 540 nm. Simultaneously, treat the enzyme solution inactivated by heating at 100℃ for 10 min using the same method as a blank control. Three replicates were set for each treatment. Analyze the measured data using graphs and tables. Figure 1 The C) enzyme exhibited the highest activity at 50 °C and maintained high activity in the range of 40-60 °C. Enzyme activity decreased significantly when the temperature exceeded 70 °C.

[0063] Take 50 μL of diluted enzyme solution containing 1 μM of enzyme, add 450 μL of Na₂HPO₄-citric acid buffer (pH 3.0, 4.0, 5.0), Na₂HPO₄-KH₂PO₄ buffer (pH 5.0, 6.0, 7.0), or Tris-HCl buffer (pH 7.0, 8.0, 9.0) to a 2 mL centrifuge tube, preheat in a 50°C water bath for 10 min, add 500 μL of preheated 10 g / L CMC-Na solution, mix well, and then place in a water bath again for 10 min. Immediately afterward, add 1 mL of 1 mol·L⁻¹ enzyme solution. -1 The reaction was terminated with Na₂CO₃, and the solution was incubated at room temperature for 5 min. The absorbance (OD) was measured at 540 nm. Simultaneously, the heat-inactivated enzyme solution was treated in the same way as a blank control. Each treatment was performed in triplicate. The measured data were then analyzed graphically. Figure 1 The B) enzyme exhibits the highest activity at pH 5 and high activity in the pH range of 4-6.

[0064] Example 6: Determination of enzyme Km and Tm

[0065] CMC-Na dilutions with concentrations of 0.2, 0.4, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 g / L were prepared by diluting a 2.0 g / L CMC-Na solution. For each assay, 200 μL of each of these CMC-Na solutions was mixed with 200 μL of pH 5.0 disodium hydrogen phosphate-potassium dihydrogen phosphate buffer and 100 μL of purified A4 enzyme solution (1 g / L). The reaction was carried out at 50°C for 30 seconds, and then terminated by adding an equal volume of DNS reagent. The absorbance was measured at 540 nm using a microplate reader. Km and the maximum reaction rate (Vmax) were calculated using the Michaelis-Menten equation based on a Lineweaver-Burk double reciprocal plot, and the results are shown below. Figure 1 As shown in D in the figure. The purified endoglucanase A4 solution was diluted to a concentration of 0.5 g / L. 70 μL of the enzyme solution was mixed with 0.5 μL of ORANGE dye in a 1.5 mL microcentrifuge tube. After incubating on ice for 30 minutes, 20 μL of the mixture was transferred to a 100 μL eight-tube strip. The melting curve was recorded using qPCR, and Tm was determined as the midpoint of the transformation curve. The results are shown in the figure. Figure 1 The value of E is shown in the figure. The turnover number (kcat) is calculated using the formula kcat = Vmax / [E], where [E] is the molar concentration of the enzyme. Specific activity is expressed in μmol·min⁻¹·mg⁻¹ and is calculated using the same formula.

[0066] Example 7: Molecular dynamics simulations reveal the substrate binding mechanism of endoglucanase A4 and its mutants

[0067] To elucidate the interaction mechanism between endoglucanase A4 and its mutants and substrates, we conducted molecular dynamics simulations. After the enzyme-substrate complex was equilibrated, three independent 100 ns simulations were performed on both the wild-type (WT) and mutant (Mut) systems. The dynamic stability of the system was assessed by analyzing parameters such as root mean square deviation (RMSD) and root mean square fluctuation (RMSF), and the binding free energy (ΔGbind) and its difference (ΔΔGbind) were calculated using the MM / PBSA method. (See attached...) Figure 4 A and appendix Figure 4 The isothermal titration calorimetry (ITC) test results shown in Figure B indicate that, compared with wild-type endoglucanase A4, the A4 mutant obtained in this invention exhibits a different binding heat effect during substrate interaction, suggesting that the binding ability or binding mode between the mutant and the substrate has been altered. Further, [the following text appears to be incomplete and requires further context: "further binding to..."] Figure 3The FTIR analysis results of product C in Figure 1 show that the change in binding characteristics leads to differences in the degree of substrate degradation or the effect of the mutant on the wild-type A4 under the same reaction conditions. These results further validate the improved catalytic performance of the A4 mutant obtained in Example 2 from the perspectives of enzyme-substrate interaction and product structure. The mutation alters the binding affinity between the enzyme and the substrate by affecting the interaction network of key residues and the conformational kinetics of the substrate binding pocket, providing molecular-level insights into its functional differences.

[0068] Example 8: Product analysis of A4 wild-type and mutant degradation of various straws

[0069] To analyze the functional group changes of straw substrates before and after enzyme treatment, FTIR analysis was performed. Two mg of alkaline-treated wheat straw sample after A4 enzyme hydrolysis was ground evenly with 200 mg of potassium bromide in an agate mortar, dried in an infrared rapid dryer for 3 minutes, and then compressed into tablets. The FTIR spectrometer was used in the range of 800-4000 cm⁻¹. -1 Spectra were collected within the range, with a resolution of 4 cm⁻¹. -1 128 scans were performed. FTIR results showed that after A4 processing, the image was 1595 cm⁻¹. -1 The significantly enhanced absorption peak at the -COONa group at the reduced end of the aldonic acid indicates that a sugar acid derivative with a reduced end was generated during enzymatic hydrolysis. Figure 5 (A) Two-dimensional infrared correlation spectrum at 3300 cm⁻¹ -1 The automatic peak at that location further confirms the functional group changes, proving that A4 can effectively degrade cellulose components. Figure 5 (B and C in the text).

[0070] To visually evaluate the enzymatic hydrolysis effect, SEM observation was performed on straw samples before and after enzyme treatment. The solid residue after enzymatic hydrolysis was washed three times with 0.1M phosphate buffer (pH 7.0), freeze-dried at -80℃ for 48 hours, and then sputter-coated with gold. Field emission scanning electron microscopy was used for observation at an accelerating voltage of 5.0 kV. The results showed that samples treated with mutant M88, especially steam-explosion pretreated straw, exhibited significant erosion and porous structures on the fiber surface, indicating a stronger fiber destructive ability. Wild-type A4 treated samples showed only slight surface roughening, while the untreated control group had a smooth and intact fiber structure. This example, through multi-dimensional product analysis, confirms that mutant M88 exhibits superior catalytic efficiency compared to wild-type A4 in degrading various pretreated straws, with significantly improved product characteristics and substrate destructive effects, providing experimental evidence for its practical application in biomass conversion. Figure 5 (D in the middle).

[0071] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

[0072] sequence list

[0073] SEQ ID NO.1:

[0074]

[0075] SEQ ID NO.2:

[0076] YDASLIPNLQIQQKNIPNNDAMNFVKGLRLGWNLGNTFDAFNGTNITNELDYETSWSGIKTTKQMIDTIKQKGFNTVRIPVSWHPHVSGSDYKISDAWMNRVQEVVNYCIDNKMYVILNIHHDVDKVKGYFPSSQYMTSSKKYITSVWAQIAAKFANYDEHLIFEGMNEPRLVGHANEWWPDLTNSDVIDSINCINQLNQDFVNTVRAAGGKNASRYLMCPGYVASPDGATNDYFRMPNDSGNNNKIIVSVHAYVPWGFAGLAMADGGTNAWNINDSKDQSEVTWFMDNIYNKYTSRGIPAIIGECGAVDKNNLKTRVEYMSYYVAQAKARGILCVLWDNNNFSGTGELFGFFDRRSCQFKFPEIIDGMVKYAFEAQTDPDPVIVYGDYNNDGSVDALDFAGLKKYIMAADHAYVKNLDVNLDNEVNAFDLAILKKFLLGMVSKLPSN

[0077] SEQ ID NO.3:

[0078]

[0079] SEQ ID NO.4:

[0080] YDASLIPNLQIQQKNIPNNDAMNFVKGLRLGWNLGNTFDAFNGTNITNELDYETSWSGIKTTKQMIDTIKQKGFNTVRIPVSWHPHVSGSDYKISDAWMNRVQEVVNYCIDNKMYVILNIHHDVDKVKGYFPSSQYMTSSKKYITSVWAQIAAKFANYDEHLIFEGMNEPRLVGHANEWWPDLTNSDVIDSINCINQLNQDFVNTVRAAGGKNASRYLMCPGYVASPDGATNDYFRMPNDSGNNNKIIVSVHAYVPWGFAGLAMADGGTNAWNINDSKDQSEVTWFMDNIYNKYTSRGIPAIIGECGAVDKNNLKTRVEYMSYYVAQAKARGILCVLHDRNNFSLWADNQLTTSGQYVRARIKGAYYATPVDPVTNQPTAPKDFSSGFWDFNDGTTQGFGVNPDSPITAINVENANNALKISNLNSKGSNDLSEGNFWANVRISADIWGQSINIYGDTKLTMDVIAPTPVNVSIAAIPQSSTHGWGNPTRAIRVWTNNFVAQTDGTYKATLTISTNDSPNFNTIATDAADSVVTNMILFVGSNSDNISLDNIKFT

[0081] SEQ ID NO.5:

[0082]

Claims

1. An endoglucanase mutant, characterized in that, The amino acid sequence of the endoglucanase mutant is shown in SEQ ID NO.

4.

2. The gene encoding the endoglucanase mutant of claim 1, characterized in that, The gene sequence is shown in SEQ ID NO.

3.

3. A recombinant vector, characterized in that, The recombinant vector contains the endoglucanase mutant gene as described in claim 2, and the expression vector of the recombinant vector is pCold-TF.

4. A recombinant bacterial strain, characterized in that, The recombinant strain contains the endoglucanase mutant gene as described in claim 2 or the recombinant vector as described in claim 3, and the recombinant strain is Escherichia coli Rosetta (DE3).

5. A method for producing the endoglucanase mutant according to claim 1, characterized in that, The production method includes the following steps: constructing the gene encoding the endoglucanase mutant of claim 1 into an expression vector, transferring the expression vector into a host bacterium for expression, and obtaining the endoglucanase mutant from the host bacterium.

6. The application of the endoglucanase mutant according to claim 1 in cellulose degradation.

7. The application according to claim 6, characterized in that, The application of the described endoglucanase mutant in the degradation of cellulose under acidic conditions at a temperature of 45-55℃.

8. The application according to claim 7, characterized in that, The temperature is 50°C; the pH value of the acidic conditions is 4.5-5.

5.

9. The application according to any one of claims 6-8, characterized in that, The aforementioned endoglucanase mutant is used to degrade sodium carboxymethyl cellulose or treat straw with alkali to generate fermentable sugars.

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