A mutant of an acidophilic endoglucanase

Abstract

The application discloses a mutant of acidophilic endoglucanase, in particular, a mutant of endoglucanase ArCel5A of acidophilic fungi Acidomyces richmondensis . The endoglucanase ArCel5A of acidophilic fungi Acidomyces richmondensis can maintain high activity under harsh conditions such as acidity and high temperature, but the catalytic efficiency still needs to be further optimized to meet the strict requirements of industrial processes on the economy and high efficiency of biological catalysts. After the amino acid mutation of ArCel5A to obtain the mutant Y379C, the specific activity of the mutant is 18.1% higher than that of the wild type, and the enzyme activity is 45.1% higher than that of the wild type under the condition of 70 DEG C water bath.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an acidophilic endoglucanase mutant. Background Technology

[0002] my country has abundant straw resources with huge potential applications in feed, fertilizer, and biofuel. However, its main component, lignocellulose, is difficult to effectively degrade and utilize, leading to a large amount of straw being disposed of through open-air burning, which emits PM2.5. 2.5 Pollutants such as carbon dioxide and CO2 cause serious environmental pollution and resource waste. How to efficiently degrade lignocellulose is a key research area for the development and utilization of straw resources. Lignocellulose consists of a network framework of cellulose, filled with hemicellulose and lignin; its tight and complex structure is the main reason why it is difficult to degrade. Enzymes play a significant role in the degradation of lignocellulose. Studies have shown that enzymes derived from acidophilic fungi... Acidomyces richmondensis The endoglucanase ArCel5A maintains its activity under harsh conditions such as acidity and high temperature, but its catalytic efficiency still needs further optimization to meet the stringent requirements of economic efficiency and high efficiency for biocatalysts in industrial processes. In this invention, ArCel5A was mutated to obtain the mutant Y379C, which showed an 18.1% increase in specific activity compared to the wild type, and a 45.1% increase in enzyme activity under 70°C water bath conditions. Summary of the Invention

[0003] This invention discloses a mutant of an acidophilic endoglucanase, specifically an acidophilic fungus. Acidomyces richmondensis A mutant of the endoglucanase ArCel5A.

[0004] The mutant is characterized in that its amino acid sequence is a; The amino acid sequence shown in SEQ ID NO.2 was mutated at position 379 based on the wild-type SEQ ID NO.1 to obtain the Y379C variant.

[0005] The gene encoding the ArCel5A mutant as described above.

[0006] Expression vectors containing the coding genes described above.

[0007] Recombinant strains carrying the expression vector described above.

[0008] The method for constructing the ArCel5A mutant as described above is characterized by comprising the following steps: A linear expression cassette containing a strong promoter for *Trichoderma reesei*, an ArCel5A coding sequence with a site-directed mutation of Y379C, and a T2A-GFP-hph polycistronic selection marker was constructed using fusion PCR. Mutations were introduced by overlap extension PCR, and the fragments were assembled into a complete expression cassette. After the expression cassette was transformed into *Trichoderma reesei* recipient strains via PEG-mediated protoplast transformation, multiple rounds of selection were performed using hygromycin resistance and GFP fluorescence intensity to obtain positive engineered strains that efficiently expressed the Y379C mutant enzyme.

[0009] For the positive strains expressing the mutant enzyme as described above with high efficiency, enzyme activity assays (DNS method) were performed. The results showed that the specific activity of the Y379C mutant of ArCel5A was 628.77 U / mg, which was 18.1% higher than that of the wild type (532.35 U / mg). Furthermore, thermostability tests (70°C, 0, 5, 10 min) were conducted. The results showed that as the temperature increased to 70°C, the residual enzyme activity of the mutant reached 95.5% at 5 min, which was 20.8% higher than that of the wild type; and at 10 min, the residual enzyme activity of the mutant reached 81.6%, which was 45.1% higher than that of the wild type. Finally, the ArCel5A mutant with significantly improved thermostability and enzyme activity was successfully developed.

[0010] Based on the above enzyme test results, further visualization analysis and molecular dynamics simulation were performed on the selected candidate mutants. By using the molecular visualization software PyMOL, the static conformational changes, hydrogen bond networks, and solvent-accessible surface area changes between the wild type and the mutants were compared. Attached Figure Description

[0011] Figure 1 Gel electrophoresis diagram of expression cassette for ArCel5A-Y379C; Figure 2 Protoplasts under a microscope; Figure 3 Image of wild-type flow cytometry cells sorted; Figure 4 Flow cytometry cell sorting diagram of mutant strain; Figure 5 Cysteine ​​highlighted structural diagram (top: wild type, bottom: mutant type); Figure 6 Comparison of hydrogen bond numbers between wild-type and mutant (top: wild-type, bottom: mutant); Figure 7 Van der Rohe view of 379 sites (left: wild type, right: mutant). Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0013] A novel acidophilic and thermostable enzyme for catalyzing β-1,4-glycosidic bonds within cellulose molecules, characterized in that the amino acid sequence of the mutant is a; a. The amino acid sequence shown in SEQ ID NO.2 was mutated at position 379 based on the wild-type SEQ ID NO.1 to obtain the Y379C variant.

[0014] The gene encoding the ArCel5A mutant as described above.

[0015] Expression vectors containing the coding genes described above.

[0016] Recombinant strains carrying the expression vector described above.

[0017] The method for constructing the ArCel5A mutant as described above is characterized by the following specific steps: A linear expression cassette was constructed using fusion PCR technology. Its core consists of a promoter, a target gene coding sequence containing a point mutation, and a fluorescent reporter gene and an antibiotic resistance gene linked by a self-cleaving peptide. The PCR products obtained in each step were size-verified by agarose gel electrophoresis to ensure the correctness of each DNA fragment and the final assembled product. Figure 1 ; Collect and culture mycelia, and prepare protoplasts through enzymatic hydrolysis, such as... Figure 2 ; The purified expression cassette DNA was mixed with protoplasts, and the DNA was introduced into cells via a PEG-mediated method. Finally, the transformation system was plated on regeneration medium and cultured to obtain transformant colonies. The transformant colonies were initially screened for antibiotic resistance carried by the expression cassette; Using co-expressed green fluorescent protein (GFP) as a reporter system, the expression level of the exogenous gene was assessed by quantitatively detecting fluorescence intensity. Multiple rounds of screening were then conducted to ultimately select positive clones with the highest expression levels for subsequent research. Figure 3 , Figure 4 .

[0018] Enzyme activity assay (DNS method) and thermal stability test (70℃, 0, 5, 10 min) were performed to determine the performance of the final mutant scheme and the difference from the wild-type mutant, and to verify the reliability of this mutant.

[0019] In heterologous expression: The experimental strain selected was the Trichoderma reesei mutant strain A2H; Reagents related to protoplast transformation: Protoplast washing buffer (S1): containing 100 mM sodium citrate and 1 M NaCl, pH adjusted to 6.0 with hydrochloric acid or sodium hydroxide solution; Protoplast resuspension buffer (S2): containing 10 mM Tris-HCl and 1 M sorbitol, adjusted to pH 7.5; Protoplast transformation buffer (S3): containing 10 mM Tris-HCl, 1 M sorbitol and 10 mM CaCl2, adjusted to pH 7.5; Protoplast transformation solution (T1): containing 25% (w / v) PEG 6000, 10 mM Tris-HCl and 50 mM CaCl2, adjusted to pH 7.5; Protoplast transformation solution (T2): containing 60% (w / v) PEG 4000, 10 mM Tris-HCl and 50 mM CaCl2, adjusted to pH 7.5; Mycelial lysis buffer: a 0.3 M aqueous solution of sodium hydroxide; Mycelial neutralization solution: 12.5 mM Tris-HCl buffer, pH 8.0; The experimental reagents are listed in the table below: The main instruments are shown in the table below: Enzymatic verification in progress: The experimental materials are as follows: The DNS solution is prepared as follows: Accurately weigh 3.15 g of 3,5-dinitrosalicylic acid and dissolve it in 500 mL of water. Heat the solution in a 45°C water bath with continuous stirring. While stirring, slowly add 100 mL of sodium hydroxide solution (concentration 200 g / L) until the solid is completely dissolved. Then add 91.0 g of potassium sodium tartrate tetrahydrate, 2.50 g of phenol, and 2.50 g of sodium sulfite sequentially, stirring until completely dissolved. Stop heating and allow the solution to cool to room temperature. Dilute to 1000 mL with distilled water, mix well, and filter. Store the filtrate in a brown plastic bottle, protected from light. It can be used after standing at room temperature for 24 hours. The shelf life of this solution is six months. Citric acid-sodium citrate buffer (0.1 M, pH 4.8): Prepare 0.1 M citric acid solution and 0.1 M sodium citrate solution separately. When using, mix the two solutions at a volume ratio of 35:15 to obtain the buffer solution with the desired pH. Sodium carboxymethyl cellulose (CMC-Na) substrate solution (1.5%, w / v): Accurately weigh 1.5 g of sodium carboxymethyl cellulose, dissolve it in an appropriate amount of the above citrate-sodium citrate buffer (pH 4.8), stir thoroughly until completely dissolved, and then bring the volume to 100 mL with the same buffer solution. Mix well and set aside. The experimental reagents are shown in the table below: The main instruments are shown in the table below: The enzyme activity assay used the DNS method to plot a glucose standard curve. A 10 g / L glucose standard stock solution was prepared using deionized water. The calculation formula is as follows: C1V1 = C2V2; In the formula: C1 is the concentration of glucose standard stock solution (10 g / L). V1 is the volume (mL) of the stock solution to be transferred. C2 represents the target glucose working solution concentration (g / L). V2 is the final volume of the working solution (1.5 mL). The preparation of glucose standard solutions at different concentrations is shown in the table below: Test tube number 1 2 3 4 5 6 7 Target concentration (g / L) 0.00 0.80 1.00 1.20 1.40 1.60 1.80 Glucose stock solution (10 g / L) Volume (mL) 0 1.12 1.15 0.18 0.21 0.24 0.27 Deionized water volume (mL) 1.50 1.38 1.35 1.32 1.29 1.26 1.23 The test results show that: The specific activity of the Y379C mutant of ArCel5A was 628.77 U / mg, which was 18.1% higher than that of the wild type (532.35 U / mg). As the temperature rose to 70°C, the residual enzyme activity of the mutant reached 95.5% after 5 minutes, an increase of 20.8% compared to the wild type; At 10 min, the residual enzyme activity of the mutant was as high as 81.6%, which was 45.1% higher than that of the wild type. The results show that the method successfully screened and developed the ArCel5A mutant with improved thermal stability and catalytic efficiency.

[0020] To further explore the mechanism by which mutations enhance enzyme performance, the selected candidate mutants were subjected to visual analysis.

[0021] Mutant analysis based on structural visualization: To elucidate how the Y379C mutation simultaneously improves the thermal stability and catalytic activity of ArCel5A, this study used pymol software to conduct a detailed comparative analysis of the three-dimensional structures of wild-type and mutant types, including the calculation and visualization of hydrogen bonds, disulfide bonds, and solvent accessible surface area (SASA).

[0022] In structural visualization of mutant analysis: The mutant and wild-type structures were loaded and superimposed, with all cysteine ​​(Cys) residue side chains highlighted in yellow stick models, as shown. Figure 5 As shown; Execute the distance measurement command to automatically calculate the spatial distance between the sulfur atom of the 379th Cys residue and the sulfur atoms of all other Cys residues in the structure, and display the connecting dashed lines only for atom pairs with a distance of less than 8.0 Å; The results showed that there was no possibility of disulfide bonds forming within the current 8.0 Å range, ruling out the possibility that the Y379C mutation stabilizes the protein directly by forming new intramolecular disulfide bonds. The visualization and quantitative detection of local hydrogen bond interactions at mutation sites were performed. The target residues were displayed using a stick model. Distance objects were created for wild type and mutant type respectively (the cutoff value was set to 3.5 Å), and potential hydrogen bonds were marked with yellow dashed lines. like Figure 6 As shown, both wild-type and mutant types formed the same number of hydrogen bonds in their respective structural environments, ruling out the speculation that hydrogen bonds caused the mutation. Calculate the solvent accessible surface area (SASA) of residue 379 and its local hydrophobic microenvironment in wild type and mutant. The calculation results show that the SASA of residue 379 itself decreased significantly from 67.79 Ų in the wild type to 53.40 Ų in the mutant (ΔSASA = -14.39 Ų, a decrease of about 21%). This significant change indicates that when residue 379 is mutated from a bulky tyrosine (Y) to a smaller, hydrophobic cysteine ​​(C), its side chain is buried deeper inside the protein, and the hydrophobic exposed area is greatly reduced. To visually verify the above quantitative results, a detailed three-dimensional structural visualization analysis was conducted. like Figure 7 As shown, in the wild-type structure, the long side chain of Tyr-379 is partially exposed to the solvent interface due to its terminal hydrophilic hydroxyl group, and there are certain gaps in the stacking of its aromatic ring and the surrounding hydrophobic residues. After mutation, Cys-379 is shorter, and the completely hydrophobic side chain can be embedded more deeply and closely into the "pocket" formed by the surrounding hydrophobic residues (shown as gray van der Waals spheres). By using cross-sectional display and van der Waals sphere model, it can be clearly observed that in the mutant, the side chain of Cys-379 is surrounded more tightly and uniformly by the surrounding hydrophobic residues, forming an optimized local stacking. In summary, the Y379C mutation achieves a dual improvement in thermal stability and catalytic activity without the presence of new hydrogen / disulfide bonds by significantly increasing the hydrophobic burial of the residue at position 379 and optimizing the local hydrophobic environment.

[0023] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it; although The present invention has been described in detail with reference to the foregoing embodiments. Those skilled in the art should understand that it can still be applied to... Modifications may be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features therein; These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and essence of the technical solutions in the embodiments of this invention. scope.

Claims

1. A mutant of an eosinophilic endoglucanase, characterized in that, The amino acid sequence of the mutant is a; a. The amino acid sequence shown in SEQ ID NO.2 was mutated at position 379 based on the wild-type SEQ ID NO.1 to obtain the Y379C variant.

2. The gene encoding the mutant as described in claim 1.

3. An expression vector containing the encoding gene as described in claim 2.

4. A recombinant strain carrying the expression vector as described in claim 3.

5. The method for constructing a mutant as described in claim 1, characterized in that: Includes the following steps: A linear expression cassette containing a strong promoter for *Trichoderma reesei*, an ArCel5A coding sequence with a site-directed mutation of Y379C, and a T2A-GFP-hph polycistronic selection marker was constructed using fusion PCR. Mutations were introduced by overlap extension PCR, and the fragments were assembled into a complete expression cassette. After the expression cassette was transformed into *Trichoderma reesei* recipient strains via PEG-mediated protoplast transformation, multiple rounds of selection were performed using hygromycin resistance and GFP fluorescence intensity to obtain positive engineered strains that efficiently expressed the Y379C mutant enzyme.

6. An enzymatic test is performed on the positive engineered strain of the mutant enzyme according to claim 5, characterized in that, Includes the following steps: Enzyme activity assay (DNS method) and thermal stability test (70℃ water bath, 0, 5, 10 min) were conducted to determine the performance of the final mutant scheme and the difference from the wild-type mutant, and to verify the reliability of the mutant.

7. Mechanistic analysis of the mutant described in claim 5, characterized in that, Includes the following steps: The mechanism by which mutations enhance enzyme performance was analyzed using the molecular visualization software PyMOL.

Images