Cellulase mutant with high enzyme activity and application thereof

Abstract

The invention discloses a cellulase mutant with high enzyme activity and application thereof, glutamine at the 216th site of an amino acid sequence of wild STCE1 is subjected to site-specific mutagenesis to be changed into any one of glycine, serine, threonine and alanine, and a corresponding mutant gene, amino acid, a recombinant vector and a recombinant strain are obtained. And expressing to obtain the cellulase mutant. According to the invention, mutation sites of the STCE1 gene are enriched, the enzyme activity and the hydrolysis efficiency of cellulose are improved, and the STCE1 gene can be widely applied to industries such as spinning, cellulose hydrolysis, washing and the like.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and enzyme molecule modification technology, specifically relating to a cellulase mutant with high enzyme activity and its application. Background Technology

[0002] Cellulase is a collective term for a group of enzymes that degrade cellulose to produce glucose. It involves the synergistic action of multiple enzyme systems and is a complex enzyme, mainly composed of exo-β-glucanase (EC.3.2.1.91), endo-β-glucanase (EC.3.2.1.4), and β-glucosidase (EC.3.2.1.21), and also includes some xylosidases, xylanases, hemicellulases, etc. Because of its ability to degrade cellulose, cellulase is widely used in feed, textiles, alcohol, food, and other fields. Currently, the cellulases commonly used in industry are mainly derived from fungi, especially molds, such as *Trichoderma harzianum*, *Potassium oxysporum*, *Trichoderma reesei*, etc.

[0003] Cellulases from the *Cyclospora* GH45 family have become major textile cellulases due to their high depilatory activity, widely used in biopolishing and biostone washing. However, the number of enzymes from this family whose properties have been discovered and characterized is far fewer than that of other cellulase families, and most enzymes are purified from the original fungus, resulting in low yields. Furthermore, several GH45 family cellulases with good application results are protected by patents, limiting their use. For example, the STCE1 gene, first protected in 2004 by patent CN1902315B of Meiji Seikoku Pharmaceutical Co., Ltd. of Japan, can be used for clarifying the color of cellulose-containing fibers, reducing pilling, improving skin feel and appearance, localizing color changes, washing for reducing hardening, and fiber processing applications. Novozymes has also made several microelectrical mutations to the STCE1 gene. For example, patent CN108463552 changed one or more positions corresponding to positions 292, 274, 266, 265, 255, 246, 237, 224 and 221, giving it improved biocombination activity compared to the parent GH45 cellulase. CN118530973 describes the substitution of A25G; A32S; S41T; S56A; S77N; S85I; T104K; G114F; G114W; S137E; S137R; S137D; S137K; A146D or A146S; Q147R; S152K; Q156E; S159E; S159D; A162E; Q169Y; D179T; F183V; Q186R; I194L; I194S; K201R and G219W to improve their stability in the presence of proteases and the use of cellulase variants in laundry. Danisco's patent CN108699543 also involves mutations in the STCE1 gene, specifically substitutions of A142D, A142E, A142P, or A142Q, which significantly improve thermal stability and protease tolerance.

[0004] However, the STCE1 gene and its mutants are already protected by patents, which limits our use, and there is still room for further improvement in enzyme activity and cellulose hydrolysis efficiency for sugar production. In addition, there are more possible mutation sites that need to be further explored. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a cellulase mutant with high enzyme activity and its application. A site-directed mutation was performed on the 216th amino acid of the wild-type STCE1 amino acid sequence to obtain the cellulase mutant, which improves enzyme activity and cellulose hydrolysis efficiency.

[0006] To achieve the above objectives, the present invention provides a cellulase mutant with high enzyme activity, wherein the cellulase mutant is obtained by mutating glutamine at position 216 of the wild-type STCE1 amino acid sequence to any one of G, S, T, or A.

[0007] Preferably, the wild-type STCE1 amino acid sequence is SEQ ID NO: 2.

[0008] Preferably, the amino acid sequence of the cellulase mutant is any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or an amino acid sequence that has ≥95% identity with the wild-type STCE1 amino acid sequence and contains a specific amino acid mutation site.

[0009] The present invention also provides a gene encoding a cellulase mutant with high enzyme activity, wherein the nucleotide sequence of the gene is any one of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 or a nucleotide sequence having ≥95% identity with the wild-type STCE1 gene and containing a specific mutation site.

[0010] The present invention also provides a recombinant expression vector comprising the gene of claim 4.

[0011] The present invention also provides a recombinant strain comprising the gene described in claim 4.

[0012] Preferably, the original strain of the recombinant strain is any one of Escherichia coli, Trichoderma reesei, and Pichia pastoris.

[0013] The present invention also provides a method for improving cellulase activity, the method comprising mutating the 216th amino acid of the wild-type STCE1 amino acid sequence shown in SEQ ID NO: 2 to any one of G, S, T, or A.

[0014] This invention also provides a method for preparing high-enzyme-activity cellulase, the method comprising the following steps: (1) Transform the host strain with the recombinant expression vector containing the gene of claim 4 to obtain the recombinant strain; (2) Inducing recombinant strains to express cellulase; (3) Separate and purify to obtain cellulase with high enzyme activity.

[0015] The present invention also provides the application of a cellulase mutant in hydrolyzed cellulose, textile processing, detergents or papermaking.

[0016] The beneficial effects of this invention are as follows: Site-directed mutagenesis was performed on amino acid position 216 of the wild-type STCE1 cellulase, resulting in four cellulase mutants. Compared with the wild-type STCE1 cellulase, these mutants exhibited 45-130% increased enzyme activity and improved the conversion rate of cellulose in corn cobs by approximately 12%, with the highest conversion rate reaching 87.9%. This invention enriches the mutation sites of the STCE1 gene, breaks the blockade imposed by foreign companies, and improves enzyme activity, making it widely applicable in the textile, cellulose hydrolysis, and detergent industries. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the expression plasmid structure in Example 2.

[0018] Figure 2 This is the PCR verification result of the coli transformant in Example 2.

[0019] Figure 3 This is a bar chart showing the enzyme activity of cellulase produced by different strains during fermentation in Example 5.

[0020] Figure 4 The graph shows the relative enzyme activity of different cellulases at different temperatures in Example 6. Detailed Implementation

[0021] The technical solution of the present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention should be determined by the contents of the claims. Modifications and substitutions made by those skilled in the art to the technical solution of the present invention without creative effort all fall within the scope of protection of the present invention.

[0022] Strains and reagents: Competent cells DH5α (KTSM101L): purchased from Kangti Life; Trichoderma reesei: provided by Yichang Dongyangguang Biochemical Pharmaceutical Co., Ltd.; PrimeSTAR® High Fidelity Enzymes: Purchased from TakaRa Corporation; SacII and SmaI restriction endonucleases: purchased from TakaRa. T4 ligase: purchased from TakaRa. STC solution: 1.2M sorbitol, 50mM calcium chloride, 10mM Tris-HCl, pH 7.5, 121℃ for 20min.

[0023] PTC solution: 25% (w / v) PEG6000, 100mM calcium chloride, 0.6M potassium chloride, 10mM Tris-HCl pH7.5, filtered for sterilization.

[0024] DNS reagent: Weigh 9.45g of 3,5-dinitrosalicylic acid and slowly add it to 1500mL of purified water while stirring constantly. In a 45℃ water bath, gradually add 300mL of solution A (accurately weigh 60.0g of sodium hydroxide, dissolve it in purified water, and bring the volume to 300mL). Stir constantly until the solution is clear and transparent (during the addition of sodium hydroxide solution, the solution temperature should not exceed 48℃). Then gradually add 273.0g of potassium sodium tartrate tetrahydrate, 7.5g of phenol, and 7.5g of anhydrous sodium sulfite. Continue heating at 45℃ while adding 900mL of water. Stir constantly until all substances are completely dissolved. After cooling to room temperature, bring the volume to 3000mL.

[0025] All other reagents not listed can be obtained from regular commercial channels.

[0026] Example 1 Site-directed mutagenesis (1) Based on the mutation site and STCE1 Gene sequence information is used to design primers for site-directed mutagenesis; among which... STCE1 The nucleotide sequence of the gene is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO:2; Table 1. Primer sequence listing for site-directed mutagenesis

[0027] (2) Genomic DNA of Trichoderma reesei was extracted using a rapid fungal extraction method, and then amplified using primer sequences P1 and P2 shown in Table 1. STCE1 Wild-type gene; then amplify the gene containing the mutation site separately. STCE1 Gene front sequence and those containing mutation sites STCE1 The gene posterior sequence and primer combinations are shown in Table 2, the amplification system is shown in Table 3, and the amplification program is shown in Table 4. Table 2 Primer Compositions and Amplification Fragments

[0028] Table 3 PCR amplification program

[0029] Table 4 PCR amplification system

[0030] (3) Fusion PCR technology was used to fuse the front and back sequences of 5 mutation sites respectively. The amplification system of fusion PCR is shown in Table 5. The amplification program is the same as that in Table 4, except that the pre-denaturation time is 2 min. The DNA template is derived from the front and back sequences amplified and recovered in step (2). 2 μL of each sequence is added to the PCR system at a volume ratio of 1:1 as a template. Table 5. Amplification system for fusion PCR

[0031] (6) The fusion fragment containing the mutation site was recovered by gel extraction to obtain the STCE1 gene containing the mutation site (sequence information is shown in Table 6).

[0032] Table 6 Before and after the mutation STCE1 Gene sequence information

[0033] Example 2: Construction of Recombinant Vector (1) The plasmid backbone pUC18 and the amplified and fused plasmid obtained in Example 2 containing mutation sites STCE1 Genes and STCE1 Wild-type genes were subjected to double enzyme digestion, and the enzyme digestion reaction conditions are shown in Table 7. The prepared enzyme digestion reaction system was reacted at 37℃ for 1 h. (2) The plasmid and gene fragments after double enzyme digestion were recovered and ligated using T4 DNA ligase at 16℃ for 1 h. The plasmid map is shown below. Figure 1 As shown, the ligation reaction solution was then transferred into *E. coli* DH5α competent cells and incubated overnight at 37°C inverted. Colonies were picked for PCR verification, and the target band was approximately 1248 bp. The results are shown below. Figure 2 As shown, strains containing recombinant vectors were obtained through screening.

[0034] Table 7 Double enzyme digestion reaction system

[0035] Example 3: Expression of the mutant gene in Trichoderma reesei (1) Preparation of Trichoderma reesei protoplasts: Inoculate Trichoderma reesei spore glycerol tubes into 50 mL PDB medium and incubate at 28℃ and 220 rpm for 20 h; collect the cells through a sand core funnel, pick an appropriate amount of cells into a 2 mL EP tube, add 1.5 mL of hyphal lysis buffer (1.5% (w / v) yatalase, dissolved in STC solution, filtered for sterilization), and enzymatically hydrolyze at 30℃ and 90 rpm for 50-60 min; take an appropriate amount of sterile defatted cotton into a 5 mL disposable sterile syringe empty tube, transfer the enzymatic hydrolysate onto the sterile defatted cotton, gently press the stopcock to filter the enzymatic hydrolysate into a 2 mL EP tube, centrifuge at 3000 rpm for 3 min and discard the supernatant; add 1 mL of STC solution to resuspend the protoplasts, centrifuge at 3000 rpm for 3 min and discard the supernatant; repeat once, add 1 mL of STC solution to resuspend and obtain Trichoderma reesei protoplasts; (2) Protoplast transformation: Trichoderma reesei protoplasts were added to STC solution and diluted to 1*10. 7Cells / mL, take 50 μL of diluted protoplasts into a new 2 mL EP tube, add 50 μL of PTC solution, 10 μg of the recombinant plasmid constructed in Example 2, and add STC solution to make up to 150 μL; mix by inversion, and place in an ice bath for 20 min; add 285 μL of PTC solution, mix by inversion, and let stand at room temperature for 5 min; add 565 μL of STC solution, mix by inversion; take 200 μL of the mixture and spread it evenly on an antibiotic-free CMAS solid plate (2% (w / v) glucose, 2% (w / v) malt extract powder, 0.1% (w / v) soybean extract, 2% (w / v) Agar, 1M sorbitol), let it air dry in a clean bench, and culture at 28℃ for 3 days. Cover with a top medium containing 200 ug / mL hygromycin and continue to culture for 3-5 days to obtain transformants containing recombinant plasmids; (3) Extract genomic DNA from transformants, verify by PCR, and screen transformants containing recombinant plasmids. The PCR verification primers are 5CBHI and 3CBHI.

[0036] PCR results showed that when 5CBHI and 3CBHI were used as primers for verification, bands of the corresponding size were successfully amplified, indicating that the transformation into Trichoderma reesei was successful.

[0037] Example 4 Cellulase Production (1) Take the transformant containing the recombinant plasmid prepared in Example 3 and inoculate it into potato plate medium (24g potato glucose powder dissolved in 1L water, 20g agar powder). Incubate at 28℃ for 7 days until green spores appear on the white mycelium and cover the entire plate. (2) Take an appropriate amount of mycelium and inoculate it into a seed bottle. After culturing at 28℃ and 200rpm for 1 day, the seed liquid is obtained. The formula of the culture medium in the seed bottle is 24g potato glucose powder dissolved in 1L water. (3) Transfer all the seed culture to a fermentation bottle and culture at 28℃ and 180rpm for 7 days. Take 2mL of fermentation broth daily, centrifuge at 8000rpm for 5min, and take the supernatant for enzyme activity detection during the fermentation process. The culture medium formula in the fermentation bottle is: glucose 3%, lactose 12%, corn steep liquor 1%, potassium dihydrogen phosphate 0.5%, and disodium hydrogen phosphate 1.0%. (4) After 7 days of fermentation, take all the fermentation liquid, centrifuge at 8000 rpm for 5 min, and take the supernatant for evaluation of enzymatic properties and application effects.

[0038] Example 5: Cellulase Activity Detection The supernatant of fermentation broth from different fermentation cycles obtained in Example 4 was diluted with 0.1M acetate-sodium acetate buffer solution (pH=6.0), and 2 mL was added to a CMC solution (concentration of 8 mg / mL). The pH was adjusted to 7.0, and the mixture was incubated at 50℃ for 15 min to detect cellulase activity.

[0039] The results are as follows Figure 3 As shown, the cellulase activity produced by the four mutants was increased to varying degrees compared with the wild type, with the increase ranging from 45% to 130%. Among them, Q216A showed a 105% increase in cellulase activity compared with the wild type STCE1, and Q216G achieved a shake-flask activity of 388 U / mL, which was 130% higher than the wild type STCE1 activity of 168 U / mL.

[0040] Example 6 Temperature Tolerance Test (1) Take 2 mL of the supernatant of the fermentation broth obtained in Example 4 and dilute it with 0.1 M acetate-sodium acetate buffer at pH 6.0 to an enzyme concentration of 100 U / mL to obtain a cellulase dilution solution; (2) Take 4.5 mL of pH 6.0 buffer solution into a 10 mL colorimetric tube, preheat it in a water bath at 80℃, 85℃, and 90℃ for 5 min, add 0.5 mL of cellulase dilution solution (100 U / mL, pH 6.0) to the colorimetric tube, vortex quickly to mix, and time accurately for 3 min; (3) Immediately after the end of the treatment, place the graduation tube in an ice-water bath to terminate the heat resistance treatment. After cooling, dilute to 10 mL and then determine the remaining enzyme activity at 50 °C according to the method described in Example 5.

[0041] The results are as follows Figure 4 As shown, both the wild-type cellulase (i.e., wild-type STCE1 protein) and its mutant exhibit excellent heat resistance. No significant loss of enzyme activity was observed after treatment at 80℃ for 3 minutes, while a 6% loss of activity was observed after treatment at 90℃ for 3 minutes. This indicates that mutating glutamine at position 216 did not affect the enzyme's temperature tolerance, and it remains suitable for use under high-temperature conditions.

[0042] Example 7 pH tolerance (1) Take 2 mL of the supernatant of the fermentation broth obtained in Example 4 and dilute it to 10 U / mL with acetate-sodium acetate buffer solution at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 respectively to obtain cellulase dilution solution; (2) Take 10 mL of cellulase dilution solution and place it in a 50°C constant temperature water bath for 30 min. After taking it out and cooling it rapidly, dilute it with a buffer solution of pH=6.0. Then, determine the remaining enzyme activity at 50°C according to the method described in Example 5.

[0043] Table 8. Relative enzyme activities after different pH treatments

[0044] The results showed that wild-type cellulase (i.e. wild-type STCE1 protein) and its mutants had good tolerance in the pH range of 3-9, and the residual enzyme activity was above 94% (Table 8).

[0045] Example 8: Application of Cellulase (1) Mix corn cob powder with water to prepare a corn cob powder solution with a mass concentration of 10% (w / v) and place it in an Erlenmeyer flask. Adjust the pH value to 5.2 using 5 mol / L NaOH solution, add 0.1% (w / v) benzoisothiazolinone, mix well and heat to 50℃. (2) While shaking at 200 rpm, add the same mass of the supernatant of the fermentation broth of wild-type cellulase and its mutant obtained in Example 4. After mixing evenly, seal with a breathable membrane and enzymatically hydrolyze for 96 h to obtain the enzymatic hydrolysate. (3) Centrifuge the enzyme hydrolysate at 4000 rpm for 100 min, take the supernatant and inactivate it at 100℃, and then determine the glucose content.

[0046] Table 9. Glucose and reducing sugar content and conversion rate

[0047] The formula for calculating the conversion rate is: , In the formula, C represents the glucose content, in g / mL; V 总 M represents the total volume of the liquid, in mL; 干物料 R represents the initial dry mass of the material at the start of hydrolysis, in grams. 纤维素 The percentage represents the cellulose content in the dry material.

[0048] As shown in Table 9, the wild-type cellulase had a strong hydrolytic ability when hydrolyzing corn cobs, with a conversion rate of 78.48%. However, the cellulase obtained by mutating the 216th amino acid in the amino acid sequence of STCE1 had a better hydrolytic efficiency. Among them, Q216G had the best hydrolytic efficiency, reaching 87.9%, which was 12% higher than that of the wild-type cellulase.

Claims

1. A cellulase mutant with high enzyme activity, characterized in that: The cellulase mutant was obtained by mutating glutamine at position 216 of the wild-type STCE1 amino acid sequence to any one of G, S, T, or A.

2. The cellulase mutant with high enzyme activity according to claim 1, characterized in that: The amino acid sequence of the wild-type STCE1 is SEQ ID NO:

2.

3. A cellulase mutant with high enzyme activity according to claim 1, characterized in that: The amino acid sequence of the cellulase mutant is any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or an amino acid sequence that has ≥95% identity with the wild-type STCE1 amino acid sequence and contains a specific amino acid mutation site.

4. A gene encoding a cellulase mutant with high enzyme activity as described in any one of claims 1-3, characterized in that: The nucleotide sequence of the gene is any one of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or a nucleotide sequence that has ≥95% identity with the wild-type STCE1 gene and contains a specific mutation site.

5. A recombinant expression vector comprising the gene of claim 4.

6. A recombinant strain comprising the gene of claim 4.

7. The recombinant strain according to claim 6, characterized in that: The original strain of the recombinant strain is any one of Escherichia coli, Trichoderma reesei, and Pichia pastoris.

8. A method for increasing cellulase activity, characterized in that: The method includes mutating the 216th amino acid of the wild-type STCE1 amino acid sequence shown in SEQ ID NO: 2 to any one of G, S, T, or A.

9. A method for preparing high-enzyme-activity cellulase, characterized in that: The method includes the following steps: (1) Transform the host strain with the recombinant expression vector containing the gene of claim 4 to obtain the recombinant strain; (2) Inducing recombinant strains to express cellulase; (3) Separate and purify to obtain cellulase with high enzyme activity.

10. The use of a cellulase mutant as described in claim 1 in hydrolyzed cellulose, textile processing, detergents, or papermaking.

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