Endoglucanase mutant and its application

Abstract

The present invention provides an endoglucanase mutant and its application. The endoglucanase mutant includes: 1) a protein having an amino acid sequence mutated at at least one of the following sites: D194, T232 or G247 of SEQ ID NO: 29, and having endoglucanase activity; or 2) a protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the protein in 1), and having endoglucanase activity. Compared to the wild type, the endoglucanase mutant of the present invention has a higher specific activity, which contributes to the wide application of β-endoglucanase in industry.

Description

Technical Field

[0001] The present invention relates to the technical field of protein engineering, in particular to an endoglucanase mutant and application thereof. Background Art

[0002] Cellulase is a complex enzyme system that can degrade cellulose into glucose. According to the action site on cellulose, it can be divided into endoglucanase (EG), exoglucanase (CBH) and β-glucosidase (BG).

[0003] Endoglucanase plays an important role in the hydrolysis of cellulose, and the activity of endoglucanase directly affects its hydrolysis efficiency of cellulose.

[0004] Trichoderma reesei endoglucanase has high activity and stability, and can function in a wide range of pH (3-6) and temperature (20-60°C). Research on its structure and function is of great significance for understanding the mechanism of action of cellulase and improving enzyme performance.

[0005] Trichoderma reesei endoglucanase has broad application prospects in the fields of bioenergy, food industry, and feed industry. For example, in the bioenergy sector, the enzyme can be used to degrade cellulosic biomass and produce renewable energy such as bioethanol. In the food industry, it can be used to prepare low-molecular-weight glucans, improving the taste and nutritional value of food.

[0006] Although Trichoderma reesei has a strong ability to secrete extracellular proteins, when the amount of secreted protein reaches a certain level, modification strategies targeting the transcription level and folding and secretion process of cellulase genes cannot achieve the expected yield increase. At this time, the specific activity of the enzyme becomes one of the bottlenecks limiting the improvement of endoglucanase activity in Trichoderma reesei fermentation broth. Patent application CN105874065A developed peptides such as 11G8, 92A12, 240H12, and 154E4 through "L-shuffling" that have improved endoglucanase activity compared to the endoglucanase activity of the wild-type EG1 protein of Trichoderma reesei. Among them, the 11G8 mutant showed the highest enzyme activity increase, up to 0.7 times.

[0007] However, the specific activity of endoglucanases expressed by Trichoderma reesei in the prior art is low, which increases the production cost of the endoglucanases and limits their application range. Therefore, improving the specific activity of endoglucanases expressed by Trichoderma reesei is an urgent problem to be solved. Summary of the Invention

[0008] The main purpose of the present invention is to provide an endoglucanase mutant and its application, so as to solve the problem of low specific activity of the endoglucanase produced by Trichoderma reesei in the prior art.

[0009] To achieve the above objectives, according to a first aspect of the present invention, an endoglucanase mutant is provided, the endoglucanase mutant comprising: 1) a protein having an amino acid sequence in which at least one of the following sites: D194, T232 or G247 of SEQ ID NO: 29 is mutated, and having endoglucanase activity; or 2) a protein having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to the protein in 1), and having endoglucanase activity.

[0010] Furthermore, in the above 1), the mutated site is selected from at least one of the following sites: D194K or D194L or D194Q, T232W or T232Y or T232L or G247L; wherein the letters before the numbers represent the wild-type amino acid, and the letters after the numbers represent the mutant amino acid.

[0011] Furthermore, the mutation of the above-mentioned endoglucanase mutant includes any one of the following: D194K, D194L, D194Q, T232W, T232Y, T232L or G247L.

[0012] In order to achieve the above object, according to a second aspect of the present invention, a gene is provided, which encodes the above endoglucanase mutant.

[0013] In order to achieve the above object, according to the third aspect of the present invention, an expression cassette is provided, wherein the expression cassette comprises the above gene.

[0014] Furthermore, the above-mentioned expression cassette also includes a promoter, a signal peptide and a terminator for regulating the expression of the above-mentioned gene, wherein the above-mentioned promoter is located upstream of the start codon of the above-mentioned gene, the above-mentioned signal peptide is located between the start codon of the above-mentioned gene and the above-mentioned promoter, and the above-mentioned terminator is located downstream of the stop codon of the above-mentioned gene.

[0015] Furthermore, the promoter includes the cbh1 promoter; the signal peptide includes the cbh1 signal peptide; and the terminator includes the cbh1 terminator.

[0016] Furthermore, the nucleotide sequence of the cbh1 promoter is SEQ ID NO: 32; the nucleotide sequence of the gene encoding the cbh1 signal peptide is SEQ ID NO: 33; and the nucleotide sequence of the cbh1 terminator is SEQ ID NO: 34.

[0017] In order to achieve the above object, according to a fourth aspect of the present invention, a plasmid is provided, wherein the plasmid comprises the above gene or the above expression cassette.

[0018] Furthermore, the above-mentioned plasmid includes: pAN7-1, pPTRⅡ or pTRUC.

[0019] Furthermore, the above plasmid also contains the selection marker gene pyr4.

[0020] Furthermore, the selection marker gene pyr4 is derived from Trichoderma reesei, Aspergillus nidulans or Aspergillus niger.

[0021] In order to achieve the above object, according to the fifth aspect of the present invention, a host cell is provided, wherein the host cell is a non-animal or plant cell; the non-animal or plant cell comprises the above gene or the above expression cassette or the above plasmid.

[0022] Furthermore, the non-animal and plant cells are selected from any one of the following: Trichoderma reesei TU-6, Trichoderma reesei QM6a or Trichoderma reesei QM9414.

[0023] Furthermore, the above-mentioned non-animal and plant cells are uracil auxotrophic strains.

[0024] In order to achieve the above-mentioned purpose, according to the sixth aspect of the present invention, a method is provided for applying the above-mentioned endoglucanase mutant, the above-mentioned gene, the above-mentioned expression cassette or the above-mentioned plasmid or the above-mentioned host cell in the fields of bioenergy, food industry and / or feed industry.

[0025] The technical solution of the present invention is applied, based on a wild-type endo-β-1,4-glucanase from Trichoderma reesei (a cellulase having the amino acid sequence shown in SEQ ID NO: 29), to provide: 1) a protein having an amino acid sequence in which at least one of the following positions of SEQ ID NO: 29, D194, T232 or G247, is mutated, and having endoglucanase activity; or 2) a protein having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to the protein in 1), and having endoglucanase activity.

[0026] Compared to the wild-type endoglucanase expressed by Trichoderma reesei, the endoglucanase mutant expressed by Trichoderma reesei has a higher specific activity, which is 1.07 to 1.29 times that of the wild-type endoglucanase. This helps reduce the cost of producing endoglucanase by Trichoderma reesei fermentation and broaden its application range. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0028] Figure 1 The three-dimensional structure diagram of the wild-type endoglucanase EG1 according to an embodiment of the present invention is shown.

[0029] Figure 2 The acid and alkali resistance of the wild-type endoglucanase EG1 and mutants according to the examples of the present invention are shown.

[0030] Figure 3 The temperature resistance of the wild-type endoglucanase EG1 and mutants according to the examples of the present invention is shown. DETAILED DESCRIPTION

[0031] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0032] As mentioned in the background, prior art shows low specific activity for endoglucanases produced by fermentation with Trichoderma reesei. The inventors of the present invention attempted to rationally design a wild-type endo-β-1,4-glucanase (a cellulase with the amino acid sequence set forth in SEQ ID NO: 29) derived from Trichoderma reesei. By docking amino acids in the enzyme's active pocket region with small molecules of the cellotetraose substrate, they screened for a series of mutants. These mutant structures were able to reduce the energy required for enzyme-substrate binding, thereby improving the enzyme's specific activity. Consequently, a series of protection schemes were proposed for the present invention.

[0033] In a first typical embodiment of the present invention, an endoglucanase mutant is provided, and the endoglucanase mutant includes: 1) a protein having an amino acid sequence in which at least one of the following sites: D194, T232 or G247 of SEQ ID NO: 29 is mutated, and having endoglucanase activity; or 2) a protein having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homologous to the protein in 1), and having endoglucanase activity.

[0034] The amino acid sequence of the wild-type protease of the present invention is SEQ ID NO: 29. Based on this amino acid sequence and according to common knowledge in the art, the nucleotide sequence of the gene encoding it can be determined. In a preferred embodiment of the present invention, the gene has the nucleotide sequence set forth in SEQ ID NO: 30. Compared to the wild-type endoglucanase, the endoglucanase exhibits a higher specific activity, which helps reduce the cost of endoglucanase use and thus expands the application areas of the endoglucanase.

[0035] The amino acid sequence of wild-type endoglucanase EG1 is SEQ ID NO: 29:

[0036] MAPSVLTLPLTTAILAIARLVAAQQPGTSTPEVHPKLTTYKCTKSGGCVAQDTSVVLDWNYRWMHDANYNSCTVNGGVNTTLCPDEATCGKNCFIEGVDYAASGVTTSGSSLTMNQ YMPSSSGGYSSVSPRLYLLDSDGEYVMLKLNGQELSFDVDLSALPCGENGSLYLSQMDENGGANQYNTAGANYGSGYCDAQCPVQTWRNGTLNTSHQGFCCNEMDILEGNSRANA LTPHSCTATACDSAGCGFNPYGSGYKSYYGPGDTVDTSKTFTIITQFNTDNGSPSGNLVSITRKYQQNGVDIPSAQPGGDTISSCPSASAYGGLATMGKALSSGMVLVFSIWNDN SQYMNWLDSGNAGPCSSTEGNPSNILANNPNTHVVFSNIRWGDIGSTTNSTAPPPPPASSTTFSTTRRSSTTSSSPSCTQTHWGQCGGIGYSGCKTCTSGTTCQYSNDYYSQCL.

[0037] The nucleotide sequence of the gene encoding the wild-type endoglucanase EG1 is SEQ ID NO: 30:

[0038]

[0039] In a preferred embodiment of the present invention, in 1) above, the mutated site is selected from at least one of the following: D194K, D194L, D194Q, T232W, T232Y, T232L, or G247L; wherein the letters before the numbers represent the wild-type amino acids, and the letters after the numbers represent the mutant amino acids. Mutating the wild-type cellulase EG1 at these sites may increase the specific activity of the endoglucanase.

[0040] Based on this application, while retaining the mutations at the above sites, the remaining sites are mutated to obtain a protein having a certain homology with the protein in 1) and the same endoglucanase activity.

[0041] It should be noted that homology in the present invention refers to the "sequence identity" between two amino acid sequences, that is, the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, it can be determined using the BLAST program in the NCBI database. For determination of sequence identity, see, for example, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primers, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0042] The above proteins have 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) homology with the mutants shown in 1) and have endoglucanase activity, and their active sites, active pockets, activity mechanisms, protein structures, etc. are most likely the same as those of the corresponding proteins provided in 1).

[0043] Amino acid residues can be represented by the standard three-letter or one-letter amino acid code commonly known and agreed upon in the art. Herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0044] Conservative amino acid substitutions or replacements are well known in the art. For example, conservative amino acid substitutions preferably involve replacing one amino acid residue from the following groups (1)-(5) with another amino acid from the same group: (1) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (2) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (3) polar positively charged residues: His, Arg, and Lys; (4) larger aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys; and (5) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln or Glu; Met is substituted by Leu, Tyr or Ile; Phe is substituted by Met, Leu or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; and Val is substituted by Ile or Leu.

[0045] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0046] In a preferred embodiment of the present invention, the mutation of the endoglucanase mutant includes any one of the following: D194K, D194L, D194Q, T232W, T232Y, T232L or G247L. The mutant obtained by performing the above mutation at the above sites of the wild-type endoglucanase has the advantage of higher specific activity.

[0047] In a second exemplary embodiment of the present invention, a gene is provided that encodes the aforementioned endoglucanase mutant. The endoglucanase mutant expressed by the gene encoding the aforementioned endoglucanase has a higher specific activity than the wild-type endoglucanase, thereby helping to broaden the application areas of the endoglucanase.

[0048] In a third typical embodiment of the present invention, an expression cassette is provided, wherein the expression cassette comprises the above gene, and the use of the above expression cassette has the beneficial effect of expressing endoglucanase at a high level.

[0049] In a preferred embodiment of the present invention, the above-mentioned expression cassette further includes a promoter, a signal peptide and a terminator for regulating the expression of the above-mentioned gene, wherein the above-mentioned promoter is located upstream of the start codon of the above-mentioned gene, the above-mentioned signal peptide is located between the start codon of the above-mentioned gene and the above-mentioned promoter, and the above-mentioned terminator is located downstream of the stop codon of the above-mentioned gene.

[0050] In a preferred embodiment of the present invention, the promoter comprises the cbh1 promoter; the signal peptide comprises the cbh1 signal peptide; and the terminator comprises the cbh1 terminator. In a more preferred embodiment of the present invention, the nucleotide sequence of the cbh1 promoter is SEQ ID NO: 32; the nucleotide sequence of the gene encoding the cbh1 signal peptide is SEQ ID NO: 33; and the nucleotide sequence of the cbh1 terminator is SEQ ID NO: 34. Using these elements can achieve the beneficial effects of high transcription and secretion levels of the target protein.

[0051] In a fourth exemplary embodiment of the present invention, a plasmid is provided, comprising the aforementioned gene or expression cassette. When introduced into a specific host cell, the plasmid comprising the aforementioned gene or expression cassette is capable of expressing an endoglucanase mutant having increased specific activity compared to the wild-type endoglucanase in the host cell. In a preferred embodiment of the present invention, the plasmid comprises pAN7-1, pPTRⅡ, or pTRUC. In a preferred embodiment of the present invention, the plasmid further comprises the selection marker gene pyr4. In a preferred embodiment of the present invention, the selection marker gene pyr4 is derived from Trichoderma reesei, Aspergillus nidulans, or Aspergillus niger. In a preferred embodiment of the present invention, the nucleotide sequence of the selection marker gene pyr4 is SEQ ID NO: 31. pAN7-1, pPTRⅡ, and pTRUC are commonly used vectors in the art for overexpressing heterologous genes.

[0052] It should be noted that the pyr4 gene is a selectable marker gene. The pyr4 gene encodes orotidine-5′-monophosphate decarboxylase, a key enzyme in the de novo uracil synthesis pathway. Under normal growth conditions, cells containing the pyr4 gene are able to synthesize uracil, allowing them to survive in uracil-deficient culture medium. In uracil-deficient culture medium, only cells containing the pyr4 gene can synthesize uracil through the de novo synthesis pathway, allowing them to survive and grow. Cells lacking the pyr4 gene, however, are unable to synthesize uracil and die.

[0053] In a fifth typical embodiment of the present invention, a host cell is provided, wherein the host cell is a non-animal or plant cell; the non-animal or plant cell comprises the above-mentioned gene, the above-mentioned expression cassette, or the above-mentioned plasmid. In a preferred embodiment of the present invention, the non-animal or plant cell is selected from any one of the following: Trichoderma reesei TU-6, Trichoderma reesei QM6a, or Trichoderma reesei QM9414. In a more preferred embodiment of the present invention, the non-animal or plant cell is a uracil auxotrophic strain. Trichoderma reesei TU-6, Trichoderma reesei QM6a, and Trichoderma reesei QM9414 are all common host fungi used for heterologous expression in the art.

[0054] The non-animal and plant cells can produce endoglucanase mutants after induced expression, and the specific activity of the endoglucanase mutants is higher than that of the wild-type endoglucanase. The present invention uses Trichoderma reesei TU-6 as an example to construct a variety of genetically engineered bacteria that can produce endoglucanases.

[0055] In a sixth typical embodiment of the present invention, a method is provided for applying the above-mentioned endoglucanase mutant, the above-mentioned gene, the above-mentioned expression cassette or the above-mentioned plasmid or the above-mentioned host cell in the fields of bioenergy, food industry and / or feed industry.

[0056] The present invention is further described in detail below with reference to specific examples. These examples should not be construed as limiting the scope of protection claimed in the present invention.

[0057] The methods for determining the enzymatic activity and protein content of the endoglucanase and calculating the specific activity of the enzyme in the embodiments of the present invention are as follows:

[0058] (1) Determination of endoglucanase activity

[0059] With reference to "NY / T912-2020 Determination of Cellulase Activity in Feed Additives - Spectrophotometric Method", this standard stipulates the determination of cellulase activity by reducing sugar colorimetry.

[0060] DNS reagent: Weigh 3.15 g of 3,5-dinitrosalicylic acid, add 500 mL of water and stir, heat the mixture to 45°C in a water bath, slowly add 100 mL of sodium hydroxide solution (200 g / L) while stirring continuously until completely dissolved, then gradually add 91.0 g of potassium sodium tartrate tetrahydrate, 2.5 g of phenol, and 2.5 g of sodium sulfite, stir until dissolved, stop heating, cool to room temperature, add water to make up to 1000 mL, shake well, filter, and store in dark.

[0061] 0.1 M sodium acetate buffer (pH = 5.5): Weigh 23.14 g of sodium acetate trihydrate, dissolve it in water, add 1.70 mL of glacial acetic acid, dilute to 2000 mL with water, and adjust the pH to 5.5 with acetic acid solution or sodium acetate solution.

[0062] Glucose solution: Weigh 0.5 g of anhydrous glucose (accurate to 0.001 g) and place it in a 50 mL volumetric flask. Dissolve it in sodium acetate buffer and dilute to the mark. Shake well.

[0063] Drawing of the standard curve

[0064] (1) Take 4.0 mL of 0.1 M sodium acetate buffer (pH = 5.5), add 5.0 mL of DNS reagent, and heat in a boiling water bath for 5 min. Cool to room temperature with water. Make up to 25.0 mL with water to prepare a standard blank.

[0065] (2) Take 1.00 mL, 2.00 mL, 3.00 mL, 4.00 mL, 5.00 mL, 6.00 mL and 7.00 mL of 10 g / L glucose solution respectively, dilute to 100 mL with sodium acetate buffer, and prepare glucose standard solutions with concentrations of 0.10 mg / mL to 0.70 mg / mL.

[0066] (3) Pipette 2.00 ml of each glucose standard solution (do two replicates) into a graduated test tube. Then, add 2 ml of water and 5 ml of DNS reagent, respectively. Electromagnetic vibration is applied for 3 seconds, and the tube is heated in a boiling water bath for 5 minutes. Then, cool the tube to room temperature with tap water, make up the volume to 25 ml with water, adjust the volume to zero using a standard blank as a control, and measure the absorbance OD value at 540 nm.

[0067] (4) Draw a standard curve with glucose concentration as the Y-axis and absorbance OD value as the X-axis. The standard curve needs to be redrawn each time the DNS reagent is newly prepared. Specific method: Take 2 mL of sodium carboxymethyl cellulose solution (1.5%), add 2 mL of appropriately diluted enzyme solution, and mix. After reacting at 37℃ for 30 minutes, add 5 mL of DNS reagent, vortex and shake for 3 seconds to stop the enzymatic reaction, add 1 mL of 0.3 mg / mL glucose solution, heat in a boiling water bath for 5 minutes, add distilled water to 25 mL, vortex and shake to mix, and measure the absorbance at 540 nm. The blank control is to add DNS first and then the enzyme solution.

[0068] Definition of endoglucanase activity (cellulase activity): At 37°C and pH 5.5, the amount of enzyme required to produce 1 μmol of reducing sugar per minute from a 7.5 mg / mL sodium carboxymethyl cellulose solution is defined as one enzyme activity unit (U).

[0069] (B) Protein content was determined by Coomassie Brilliant Blue G-250 staining (Bradford).

[0070] (3) Calculation of specific activity

[0071] Specific activity refers to the number of enzyme activity units per unit weight of protein, typically expressed as U / mg protein. The formula for calculating specific activity is: Specific activity (U / mg) = enzyme activity (U / mL) / protein content (mg / mL).

[0072] (IV) Experimental materials and reagents

[0073] 1. Strains: The starting strain used was Trichoderma reesei TU-6, a uracil auxotrophic mutant with ATCC accession number MYA-256. Escherichia coli strain TOP10 was used for the construction of the recombinant plasmid and was purchased from Takara.

[0074] 2. High-fidelity Phanta Max Super-Fidelity DNA Polymerase was purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0075] 3. NEBuilder HiFi DNA Assembly Cloning Kit was purchased from NEB.

[0076] 4. Plasmid vector pAN7-1.1-pyr4 was constructed in our laboratory and was modified from vector pAN7-1 (manufacturer: Beina Biotechnology; model number BNCC357701). It carries the orotidine 5'-phosphate decarboxylase gene pyr4 (nucleotide sequence of the gene is SEQ ID NO: 31) as a selection marker.

[0077] 5. PDA medium: 200 g / L potato juice, 20 g / L glucose, 1.8% agar powder.

[0078] 6. PDB culture medium was purchased from Beijing Solebow Technology Co., Ltd.

[0079] 7. MM medium: 2% glucose, 0.5% ammonium sulfate, 1.5% potassium dihydrogen phosphate, 0.06% calcium chloride, 0.06% magnesium sulfate, 0.2% peptone, 0.0037 g / L CoCl2·6H2O, 0.005 g / L FeSO4·7H2O, 0.0014 g / L ZnSO4·7H2O, 0.0016 g / L MnSO4·H2O, 1.8% agar powder, pH 4.5-5.5.

[0080] 8. MMS-soft medium: MM medium (liquid), 1 M sorbitol, 0.7% agarose, pH 5.6.

[0081] 9. Seed culture medium (2-8% glucose, 0.2-2% yeast powder, 0.05-0.6% dipotassium hydrogen phosphate, 0.05-0.6% potassium dihydrogen phosphate, 0.02-0.18% magnesium sulfate, 0.02-0.1% calcium chloride).

[0082] 10. Fermentation medium (0.1-2% glucose, 1-3% lactose, 0.8-3% corn steep liquor, 0.1-1% ammonium sulfate, 0.3-0.8% dipotassium hydrogen phosphate, 0.3-0.6% potassium dihydrogen phosphate, 0.02-0.18% magnesium sulfate, 0.02-0.1% calcium chloride).

[0083] Example 1 Rational Design of Mutation Sites in Endoglucanase EG1

[0084] Based on the rational design of protein structure, the structure of Trichoderma reesei endoglucanase EG1 (which has high cellulase activity and can function in a wide pH range and temperature range, and has an amino acid sequence of SEQ ID NO: 29) was analyzed (see the structure). Figure 1 ), its β-1,4 glucanase structure conforms to the characteristics of the GH16 family, and the catalytic active region is composed of four groups of antiparallel β folds ( Figure 1 The yellow portion (shown in yellow) forms the substrate-binding pocket region of the enzyme, combined with the surrounding irregular flexible loop region, for catalytic function. The amino acids in the active pocket region of the enzyme were docked with the cellotetraose substrate small molecule. Virtual mutagenesis was performed on the complex conformation using Discovery Studio, with saturation mutations performed on amino acids within 5 Å of the small molecule. Based on energy scores, mutants with high mutation energy rankings were selected for experimental verification. Ultimately, mutants D194L, D194K, G247L, D194Q, A244F, T232W, T232Y, T232L, N164K, and A230L were selected. It is hypothesized that these mutant structures can reduce the energy required for enzyme-substrate binding, thereby enhancing the enzyme's catalytic activity.

[0085] Example 2 Construction of recombinant expression vectors of EG1 and its mutants

[0086] Using genomic DNA from Trichoderma reesei QM6a as a template, the cbh1 promoter (including the cbh1 signal peptide), cbh1 terminator, and EG1 gene fragments were generated. Using the pAN7-1-pyr4 vector as a template, the vector backbone pAN-his6-pyr4 was generated; his represents a purification tag. These four fragments were assembled in vitro using Gibson assembly to generate the recombinant plasmid pAN-EG1-pyr4, which overexpresses the EG1 gene.

[0087] The mutant expression vector was constructed using pAN-EG1-pyr4 as a template and site-directed mutagenesis was performed by PCR. The mutant expression plasmid was constructed in the same manner as above. The primers used in the experiment are shown in Table 1.

[0088] Table 1. Primers used

[0089]

[0090] Example 3 Construction of genetically engineered strains of EG1 and its mutants

[0091] Protoplast preparation:

[0092] Trichoderma reesei Tu-6 was inoculated into PDA medium and cultured at 30°C for 4 days to produce sufficient spores.

[0093] The spores were gently scraped off with sterile water and inoculated into 50 ml of PDB medium containing uracil. The final concentration of spores was 10 5 Cultivate at 30°C and 180 rpm on a shaker for 24-36 hours.

[0094] Collect the mycelia by filtration with nylon cloth, take an appropriate amount of mycelia and add 10 mg / mL lytic enzyme, 10 mg / mL lysozyme and 5 mg / mL cellulase, and treat at 30℃ for 1.5h-2h. Collect the prepared protoplasts by centrifugation, resuspend and dilute to 10 7 pieces / mL.

[0095] PEG-CaCl2-mediated protoplast transformation:

[0096] Transform the recombinant plasmid into Trichoderma reesei Tu-6 via PEG / CaCl2-mediated protoplast transformation. Add 5 μg of the recombinant plasmid and pre-chilled PEG solution to 200 μL of the protoplasts, mix gently, and incubate on ice for 20 minutes. Add 1 mL of PEG solution and incubate at 25°C for 5 minutes. Then, slowly add 2 mL of Solution II and mix gently. Finally, mix the transformed product with MMS-soft medium and pour it onto the underlying MM medium. Allow it to cool and solidify, then incubate inverted at 28°C for 5 days.

[0097] Example 4 Screening and Verification of Recombinant Strain Transformants

[0098] After transformants have grown on the plates, transfer them to PDA solid medium and culture at 28°C for 5 days. Transfer a portion of the grown mycelium to PDB medium and culture in a shaker at 28°C and 200 rpm for 24 hours. Collect the mycelium using nylon cloth and filter into a 2 ml centrifuge tube. Dry the tube and grind it using a tissue grinder. Genome extraction was performed using the Omega® SP Fungi DNA kit.

[0099] The above transformant genome was used as a template and primers P9 and P10 were used for genomic PCR verification. At the same time, the constructed expression vector plasmid was used as a template as a positive control, and the genomic DNA of the starting strain TU-6 was used as a template as a negative control. The PCR products with the correct band size were sequenced.

[0100] Example 5 Shake flask fermentation of recombinant transformants

[0101] (1) Spore collection: Inoculate the correctly sequenced transformants onto PDA solid medium for spore production and incubate at 28°C for 5 days. Gently scrape the spores from the solid medium with sterile water, collect the spore suspension, and count the number of spores per milliliter using a hemocytometer.

[0102] (2) Seed culture: inoculate the spore suspension into 50 ml of seed culture medium. The final concentration of the inoculated spore suspension is 10 6 / ml, 30℃, 200rpm for 24h until the mycelium morphology matures.

[0103] (3) Shake flask fermentation: The above seed liquid was inoculated into the fermentation medium at an inoculum rate of 10%, and shake flask fermentation was carried out at 30°C and 220 rpm. The supernatant was collected on the 7th day of shake flask fermentation. According to the instructions for use of GE's 25mL nickel ion affinity chromatography filler and AKAT purifier, the supernatant was loaded onto a pre-equilibrated nickel column, and then the impurities and target protein were eluted with 5mM imidazole, 40mM imidazole, 100mM imidazole, 250mM imidazole, and 500mM imidazole prepared with 50mM Tris-HCl buffer solution (pH 8.0) containing 300mM NaCl. The target protein was eluted with 250mM imidazole, and the resulting eluate was concentrated 20 times by 10KDa ultrafiltration membrane. The concentrate was the pure enzyme of Trichoderma reesei EG1 and its mutants, and was stored at -20°C.

[0104] (4) Referring to the "NY / T 912-2020 Determination of Cellulase Activity in Feed Additives - Spectrophotometric Method" and the protein concentration detection method (Bradford method for determining protein concentration), the specific activity and relative specific activity of the pure enzyme of wild-type endoglucanase EG1 and its mutants were measured as shown in Table 2.

[0105] Table 2

[0106]

[0107] It should be noted that the above relative specific activity is calculated as: specific activity of the mutant / specific activity of wild-type endoglucanase EG1 * 100%. The specific activities of mutants D194K, T232W, and G247L were 751.0 U / mg, 677.3 U / mg, and 632.2 U / mg, respectively, representing increases of 29%, 17%, and 9%, respectively, compared to wild-type endoglucanase EG1 (581.2 U / mg).

[0108] Example 6 Enzymatic properties of wild-type EG1 and its mutants

[0109] To investigate the acid and alkali resistance of the wild-type endoglucanase EG1 and the mutants, the enzyme solution was appropriately diluted and placed at 30°C and within a pH range of 3 to 9 (e.g., 3, 4, 5, 6, 7, 8, and 9) for enzymatic reaction. Other procedures were the same as those in "(I) Determination of Endoglucanase Activity" of the present invention. The experimental results are shown in Figure 2 .

[0110] To investigate the temperature resistance of the wild-type endoglucanase EG1 and the mutants, the enzyme solution was appropriately diluted and placed under different temperatures (30°C, 40°C, 50°C, 55°C, 60°C, 65°C, and 70°C) and a pH of 7 for enzymatic reaction. Other conditions were the same as those in "(I) Determination of Endoglucanase Activity" of the present invention. The experimental results are shown in the table. Figure 3 .

[0111] According to the experimental results, there are certain differences in pH tolerance between the mutant and the wild-type EG1, but they are basically consistent in temperature tolerance.

[0112] Figure 3 The remaining enzyme activity in the assay = endoglucanase activity under current conditions (i.e., different temperatures or different pH values) / endoglucanase activity under standard conditions, where the standard conditions are set at 30°C and pH 7.0.

[0113] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: by performing site-directed mutagenesis on adjacent amino acids in the spatially proximal β-sheet structure of the wild-type endo-β-1,4-glucanase EG1 from Trichoderma reesei, the present invention recombinantly constructed multiple mutants with enhanced specific activity. The specific activities of the mutants D194K, T232W, and G247L were 751.0 U / mg, 677.3 U / mg, and 632.2 U / mg, respectively, which are 29%, 17%, and 9% higher than the specific activity of the wild-type EG1 enzyme (581.2 U / mg). This characteristic is beneficial for improving the production efficiency of β-endoglucanase in actual industrial production, reducing production costs, and promoting the widespread application of β-endoglucanase in industry.

[0114] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An endoglucanase mutant, characterized in that The endoglucanase mutant is a mutant having any one of the following mutations based on the amino acid sequence shown in SEQ ID NO: 29: D194K, D194L or D194Q.

2. A gene, characterized in that The gene encodes the endoglucanase mutant according to claim 1.

3. An expression cassette, characterized in that The expression cassette comprises the gene of claim 2.

4. The expression cassette according to claim 3, characterized in that The expression cassette further comprises a promoter, a signal peptide and a terminator for regulating the expression of the gene, wherein the promoter is located upstream of the start codon of the gene, the signal peptide is located between the start codon of the gene and the promoter, and the terminator is located downstream of the stop codon of the gene.

5. The expression cassette according to claim 4, characterized in that The promoter includes a cbh1 promoter; the signal peptide includes a cbh1 signal peptide; and the terminator includes a cbh1 terminator.

6. The expression cassette according to claim 5, characterized in that The nucleotide sequence of the cbh1 promoter is SEQ ID NO: 32; the nucleotide sequence of the gene encoding the cbh1 signal peptide is SEQ ID NO: 33; and the nucleotide sequence of the cbh1 terminator is SEQ ID NO:

34.

7. A plasmid, characterized in that The plasmid comprises the gene of claim 2 or the expression cassette of any one of claims 3 to 6.

8. The plasmid according to claim 7, characterized in that The plasmid includes: pAN7-1, pPTRⅡ or pTRUC.

9. The plasmid according to claim 8, characterized in that The plasmid also contains the selection marker gene pyr4.

10. The plasmid according to claim 9, characterized in that The screening marker gene pyr4 is derived from Trichoderma reesei, Aspergillus nidulans or Aspergillus niger.

11. A host cell, characterized in that The host cell is a non-animal or plant cell; the non-animal or plant cell comprises the gene according to claim 2 or the expression cassette according to any one of claims 3 to 6 or the plasmid according to any one of claims 7 to 10.

12. The host cell according to claim 11, characterized in that The non-animal or plant cell is selected from any one of the following: Trichoderma reesei TU-6, Trichoderma reesei QM6a or Trichoderma reesei QM9414.

13. The host cell according to claim 12, characterized in that The non-animal or plant cell is a uracil auxotrophic strain.

14. Use of the endoglucanase mutant according to claim 1, the gene according to claim 2, the expression cassette according to any one of claims 3 to 6, the plasmid according to any one of claims 7 to 10, or the host cell according to claims 11 to 13 in the fields of bioenergy, food industry and / or feed industry.

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