Acid endoglucanase variants and uses thereof

Abstract

The application discloses an acid endoglucanase variant, which has at least 80% identity with the amino acid sequence SEQ ID NO:2 of cellulase EGII and contains specific acid mutation sites, and the amino acid sequence of the variant is selected from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12. The acid-optimized variant of the sequence has higher enzyme activity under 50 DEG C acid fiber conditions, and compared with a wild type (EGII), the enzyme activity and the hydrolysis efficiency under low pH conditions are significantly improved, and the acid-optimized variant is suitable for SSF process without pH adjustment and reduces the cost of biofuel production.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and enzyme molecular modification technology, specifically relating to an acidic endoglucanase variant and its application. Background Technology

[0002] Cellulase, as a core component of biomass conversion systems, directly impacts the cost of biofuel production due to its catalytic efficiency. Industrial enzymatic hydrolysis processes are typically carried out in an acidic environment with a pH of 4.0-5.0. However, natural endoglucanases (such as Trichoderma reesei EGII) exhibit the following drawbacks under these conditions: significant decrease in specific activity (<50% of initial activity), insufficient thermal stability (half-life <2h at 50℃), and mismatch in substrate-binding domain charge distribution.

[0003] CN105874066A discloses that while the EG2 variant improves thermal stability, it lacks targeted optimization for acidic catalytic sites, resulting in low specific activity at pH 4.0 and necessitating the addition of pH adjusters, thus increasing process costs. Therefore, developing endoglucanase variants with high activity and stability under acidic conditions has significant industrial value. Summary of the Invention

[0004] This invention provides an acidic endoglucanase variant and its applications, which exhibits high activity and stability under acidic conditions.

[0005] To achieve the above objectives, the present invention employs the following technical solution: an acidic endoglucanase variant, which has at least 80% amino acid sequence identity with cellulase EGII (SEQ ID NO:2), and contains a specific acidic mutation site; the mutation site is selected from P49T, G51S, S64A, T127N, N146T, N146P, S151N, S151Y, I152L, S153G, V171I, T195A, S203T, N223D, E233A, A260S, T303V, G309D, G309A, S312A, Q320A, N322G, Q336A, I339E, Q340T, One or more combinations of D341Y, M341L, Q345E, I346V, Q347A, Q351A, G363A, S369T, T370N, V372T, T378N, N382S, W384M, T387Q, S388P, S391A, S392A, A395T and K397S.

[0006] Optionally, the amino acid sequence of the variant is selected from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12.

[0007] The present invention also relates to a nucleic acid that encodes at least one of the above-described acidic endoglucanase variants.

[0008] Optionally, it includes sequences selected from the following: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.

[0009] The present invention also relates to a nucleic acid comprising a selection of the nucleic acids described above.

[0010] The present invention also relates to a host cell comprising nucleic acids selected from those described above.

[0011] The present invention also relates to a host cell comprising a vector selected from those described above.

[0012] Optionally, the host cell is Escherichia coli, Trichoderma reesei, or Pichia pastoris.

[0013] The present invention also relates to an enzyme composition comprising the aforementioned variant and β-glucosidase.

[0014] The present invention also relates to the use of the acidic endoglucanase variant in the hydrolysis of cellulose at pH 3.5-5.5, the production of biofuels and / or textiles.

[0015] The present invention has the following beneficial effects:

[0016] The acidic endoglucanase variant provided by this invention exhibits higher enzyme activity under acidic cellulose conditions at 50°C, with a specific activity generally increased by 54% to 109% compared to the wild type, and also shows better pH tolerance. It is particularly suitable for SSF processes that do not require pH adjustment, thus reducing the cost of biofuel production. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the PCBHI-EGII-TCBHI carrier.

[0018] Figure 2 To verify the nucleic acid electrophoresis gel image for PCR.

[0019] Figure 3 The diagram shows the activity of EGII and mutant CMC hydrolysate produced by Trichoderma reesei fermentation.

[0020] Figure 4 This indicates the temperature tolerance results for EGII and mutants.

[0021] Figure 5 The results indicate the pH tolerance of EGII and mutants. Detailed Implementation

[0022] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention.

[0023] In the following examples, Escherichia coli was derived from Kangti Life competent cells DH5α (KTSM101L); Trichoderma reesei, pET28a vector, and restriction endonucleases were all obtained from commercially available sources.

[0024] The EGII sequence and mutant sequences are shown in Table 1. The mutant sequences were obtained through computer protein simulation and AI evolutionary learning. The synthesis of the specific polypeptide sequences SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12 was commissioned to Suzhou Genewiz Biotechnology Co., Ltd. The amino acid sequences of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12 have ≥80% identity with SEQ ID NO:2 and contain specific acidic mutation sites.

[0025] Table 1

[0026]

[0027] The method for producing the endoglucanase variant specifically involves transforming host cells with an expression vector encoding a polypeptide, culturing the host cells under conditions that enable the expression of the polypeptide, and optionally recovering and purifying the polypeptide variant.

[0028] Example 1: Expression of EGII and EGII mutant Escherichia coli

[0029] After synthesizing the EGII sequence SEQ ID NO:2 and the mutant sequences SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12, the synthesized gene was ligated into the pET28a vector and transformed into BL21 competent cells. Transformants were selected and subjected to shake-flask fermentation with IPTG induction. After induction, the cells were collected and the cell walls were broken up using a high-pressure homogenizer to collect intracellular proteins for enzyme activity detection. Enzyme activity was detected using the DNS method: diluted enzyme solution was placed in a test tube, preheated at 50℃ for 5 min, reacted for 15 min, then 2 mL of DNS reagent was added and mixed thoroughly. After boiling in a water bath for 5 min, the absorbance was measured at 540 nm, and the corresponding enzyme activity was calculated. After induction expression, the enzyme activity of the EGII sequence was 43 U / mL, while the enzyme activity of the mutant sequence was between 45 and 72 U / mL. The enzyme activity expressed in Escherichia coli by the mutant sequences SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12 was increased to varying degrees compared with the enzyme activity expressed by the original EGII sequence.

[0030] Example 2: Expression of Trichoderma reesei in EGII and EGII mutant sequences

[0031] The gene encoding the acidic cellulase EGII and its mutant sequence provided by this invention is inserted between suitable restriction enzyme sites on the expression vector, making its nucleotide sequence operably linked to the expression regulatory sequence. Specifically, the synthetic genes (SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12) are double-digested with SacII and SmaI and ligated into the PCBHI-TCBHI vector, so that the gene is located downstream of and regulated by the PCBHI promoter, resulting in the recombinant vector PCBHI-EGII-TCBHI. See details. Figure 1 .

[0032] The constructed vector was cultured in *E. coli* to extract plasmids, which were then transformed into *Trichoderma reesei* cells using protoplast transformation. The transformation method was as follows: *Trichoderma reesei* spore glycerol tubes were inoculated into 50 ml of PDB medium and cultured at 28℃ and 220 rpm for approximately 18-20 h. Cells were collected using a sintered glass funnel, and an appropriate amount was transferred to a 2 ml EP tube. 1.5 ml of mycelial lysis buffer was added, and the cells were incubated at 30℃ and 90 rpm for 50-60 min. A suitable amount of sterile absorbent cotton was placed in the empty cartridge of a 5 ml disposable sterile syringe. The enzymatic digest was transferred to the cotton, and the stopcock was gently pressed to filter the digest into a 2 ml EP tube. The tube was centrifuged at 3000 rpm for 3 min, the supernatant was discarded, and the protoplasts were resuspended in 1 ml of STC solution. This process was repeated once, with each step involving resuspending the protoplasts in 1 ml of STC solution. The tubes were then stored on ice. Protoplasts were diluted to 1*10⁷ cells / ml with STC solution. 50 μL of the diluted protoplasts were transferred to a new 2 ml EP tube, and 50 μL of PTC solution and 10 μg of plasmid were added. STC solution was then added to bring the volume to 150 μL. The mixture was inverted and mixed thoroughly, then incubated on ice for 20 min. 285 μL of PTC solution was added, and the mixture was inverted and mixed thoroughly, then incubated at room temperature for 5 min. 565 μL of STC solution was added, and the mixture was inverted and mixed thoroughly. 200 μL of the mixture was spread evenly on an antibiotic-free CMAS plate and then air-dried in a clean bench at 28°C. Genomic DNA was extracted from the transformed individuals, and the target band was verified by PCR. The results are shown below. Figure 2 As shown, the current fragment size is 1.6k, and the correct transformant is selected for plate culture.

[0033] Example 3: Cellulase Production in Shake Flask Fermentation

[0034] The transformants obtained in Example 2 were cultured on potato plates for sporulation. An appropriate amount of mycelium was inoculated into a seed bottle and cultured for one day before being inoculated into a fermentation bottle containing 2% glucose, 10% lactose, 2.5% corn steep liquor, 0.5% potassium dihydrogen phosphate, and 0.5% disodium hydrogen phosphate. The mixture was cultured in a shaker at 180 rpm and 28°C for 7 days. After centrifugation at 8000 rpm for 5 min, the supernatant was collected and subjected to SDS-PAGE electrophoresis. The presence of a protein band of approximately 50 kDa indicated that both EGII and the mutant sequence were successfully expressed.

[0035] Example 4: Cellulase Activity Detection

[0036] Cellulase can degrade sodium carboxymethyl cellulose (CMC) into oligosaccharides and monosaccharides. Oligosaccharides with reducing ends and monosaccharides with reducing groups react with DNS reagent in a boiling water bath. The intensity of the reaction color is directly proportional to the amount of reducing sugar produced by enzymatic hydrolysis, and the amount of reducing sugar produced is directly proportional to the cellulase activity in the reaction solution. Cellulase activity can be calculated by spectrophotometric determination of the absorbance of the reaction solution. Cellulase activity was detected in EGII protein and its mutants. The hydrolytic activity of the variants against CMC was measured at pH 4.0 and 50℃. The results are as follows: Figure 3 As shown, the enzyme activities of the mutants were significantly increased compared to EGII, ranging from 50% to 110%, with EG-TB-4 showing a 109% increase in enzyme activity compared to EGII. All mutant endoglucanase variants exhibited at least a 50% increase in activity at pH 4 compared to EGII of SEQ ID NO:2.

[0037] Example 5: Temperature Tolerance Test

[0038] Cellulase applications require high-temperature conditions, making it crucial to maintain high activity under these conditions. Temperature tolerance of EGII protein and its mutants was investigated. Samples were weighed and diluted to 100 U / mL with pH 4.8 buffer. 4.5 mL of pH 4.8 sodium acetate buffer was added to a 10 mL colorimetric tube. The tubes were preheated in water baths at 70℃, 75℃, and 80℃ for 5 min. 0.5 mL of the enzyme solution (100 U / mL, pH 4.8) was added to the tube, and the mixture was rapidly vortexed and timed for 3 min. Residual enzyme activity was then measured, and the results are shown in Table 2 below. Figure 4 .

[0039] Table 2

[0040]

[0041] It is evident that the thermal stability of the mutants is improved compared to EGII at different temperatures, with EG-TB-4 exhibiting better stability at 75℃ and 80℃.

[0042] Example 6: pH tolerance

[0043] The application scenarios for acidic cellulase are mainly concentrated below pH 5, therefore, its ability to tolerate different pH values ​​is crucial for its application. EGII and EG-TB-4 mutant cellulases were diluted to 10 U / mL with buffers at pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. 10 mL of each was then placed in test tubes and incubated at 50°C for 30 min. The solutions were then diluted to the specified concentration with buffer at pH 4.8, and enzyme activity was measured at 50°C. Specific results are shown in [link to results]. Figure 5The EGII and EG-TB-4 mutant cellulases exhibit good tolerance within the pH range of 3 to 9, with enzyme activity remaining at over 80%.

[0044] Example 7: Cellulase hydrolysis of furfural residue

[0045] Cellulase mutant EG-TB-4 and wild-type EGII were combined with the same mass of glucose transglycosylase (β-glucosidase) to form complexes. 10% furfural residue was hydrolyzed at 50℃. After 96 h of reaction, the contents of glucose, reducing sugar, and xylose in the hydrolysate were measured. The results are shown in Table 3.

[0046] Table 3

[0047]

[0048] When the same mass of EGII and EG-TB-4 cellulase and glucose transglycosylase hydrolyze furfural residue, at different reaction time points, the EG-TB-4 complex enzyme can hydrolyze to produce more glucose, reducing sugar and xylose, indicating that the hydrolysis is more complete and more conducive to the later fermentation to produce ethanol.

[0049] The specific embodiments of the present invention have been described in detail above, but are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. An acidic endoglucanase variant, characterized in that, The amino acid sequence of the acidic endoglucanase variant is selected from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:

12.

2. A nucleic acid, characterized in that, It encodes at least one acidic endoglucanase variant of claim 1, selected from the following sequences: SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:

11.

3. A host cell, characterized in that: It contains the nucleic acid as described in claim 2.

4. The host cell according to claim 3, characterized in that: The host cells mentioned are Escherichia coli, Trichoderma reesei, or Pichia pastoris.

5. An enzyme composition, characterized in that: It is a complex comprising the acidic endoglucanase variant of claim 1 and β-glucosidase.

6. Use of the acidic endoglucanase variant of claim 1 in the hydrolysis of cellulose and / or the production of biofuels at pH 3.5-5.5.

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