A cellobiohydrolase mutant and use thereof

Abstract

The present application discloses a cellobiohydrolase mutant which can provide degradation function in cellulose degradation, and the cellobiohydrolase mutant is a protein of (a) or (b) as follows: aspartic acid at position 259 of the amino acid sequence shown in SEQ ID NO:1 is substituted into other amino acid, the other amino acid is valine or tryptophan, (b) a protein derived from (a) with cellobiohydrolase activity by substituting, deleting or adding one or more amino acids in the amino acid sequence of (a). The present application also discloses the application of the cellobiohydrolase mutant or composition in cellulose hydrolysis. The cellobiohydrolase mutant of the present application has the activity of hydrolyzing cellulose, and the mutant with improved activity is obtained, and the hydrolysis effect on cellulose is improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. More specifically, this invention relates to a cellobiase mutant, an enzyme-containing composition, a DNA molecule, a recombinant vector, a host cell, a method for producing the mutant, and its application in cellulose degradation. Background Technology

[0002] Currently, due to the increasingly severe energy crisis, the development of renewable and clean energy has received widespread attention. Utilizing cellulase to degrade lignocellulose to produce bio-based products, including bioethanol, has significant practical implications for socio-economic development. However, enzyme costs still account for about one-quarter of the cost of biofuels; therefore, engineering methods to improve the hydrolytic efficiency of cellulases is crucial for clean industry. Cellobiose hydrolase is a key enzyme among cellulases, and improving its performance is essential for the overall hydrolytic effect of cellulase. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0004] Another objective of this invention is to provide a cellobiose hydrolase mutant that enhances the hydrolysis of cellulose. A single mutation overcomes both rate-limiting steps of the cellobiose hydrolase, thereby simultaneously improving catalytic efficiency and product yield.

[0005] Another object of the present invention is to provide the application of cellobiose hydrolase mutants in cellulose degradation.

[0006] Therefore, the technical solution provided by this invention is as follows:

[0007] A cellobiose hydrolase mutant, wherein the enzyme (a) or (b) protein:

[0008] (a) Aspartic acid at position 259 of the amino acid sequence shown in SEQ ID NO:1 is replaced with another amino acid, namely valine or tryptophan.

[0009] (b) A protein derived from (a) whose amino acid sequence in (a) has been substituted, deleted or added with one or more amino acids and has cellobiase activity.

[0010] Preferably, in the cellobiose hydrolase mutant, there is a DNA molecule that encodes the cellobiose hydrolase mutant.

[0011] Preferably, in the cellobiose hydrolase mutant, the DNA molecule base sequence is as shown in SEQ ID NO:2.

[0012] Preferably, the cellobiase mutant includes a recombinant expression vector containing the DNA molecule and a regulatory sequence for expression that enables the DNA molecule to be transcribed and translated in large quantities under artificial regulation.

[0013] Preferably, in the cellobiase mutant, the host cell contains the DNA molecule or the recombinant expression vector.

[0014] Preferably, the method for producing the cellobiase mutant includes the following steps: culturing the host cells in a culture medium, and massively replicating a recombinant vector containing the cellobiase mutant gene in the host cells.

[0015] Preferably, the cellobiose hydrolase mutant or the combination thereof is used in cellulose hydrolysis.

[0016] The present invention has at least the following beneficial effects:

[0017] The enzyme provided by this invention has excellent cellulose degradation capabilities, improving the hydrolysis efficiency of cellulose by 30%. This increased hydrolysis efficiency reduces the amount of enzyme required to hydrolyze a certain amount of cellobiose per unit time.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0019] Figure 1 This is a gel electrophoresis image of the cellobiose hydrolase and its mutants expressed according to the present invention.

[0020] Figure 2 This is a bar chart of the cellobiose hydrolase mutant D259V of the present invention hydrolyzing microcrystalline cellulose;

[0021] Figure 3 This is a bar chart showing the hydrolysis of microcrystalline cellulose by the cellobiose hydrolase and its mutants of the present invention. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0023] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not imply the presence or addition of one or more other elements or combinations thereof.

[0024] This invention provides a cellobiose hydrolase mutant, wherein the enzyme is a protein as shown in (a) or (b) below:

[0025] (a) The 259th amino acid of the amino acid sequence shown in SEQ ID NO:1 is replaced with another amino acid, said other amino acid being valine or tryptophan.

[0026] (b) A protein derived from (a) having one or more amino acids substituted, deleted, or added to the amino acid sequence in (a) and possessing cellobiose hydrolase activity; said cellobiose hydrolase mutant provides degradation function in cellulose degradation. It can be used alone to degrade cellulose, or it can work in conjunction with other cellobiose hydrolases to hydrolyze cellulose, thereby improving the hydrolysis efficiency of cellulose.

[0027] In the above-described scheme, as a preferred embodiment, the cellobiose hydrolase mutant and its application provide a DNA molecule that encodes the cellobiose hydrolase mutant.

[0028] In one embodiment of the present invention, preferably, the cellobiose hydrolase mutant and its application, wherein the mutant base sequence of the amino acid sequence shown in SEQ ID NO:1, in which aspartic acid at position 259 is mutated to valine, is shown in SEQ ID NO:4, and the amino acid sequence encoded therein is shown in SEQ ID NO:3.

[0029] In one embodiment of the present invention, preferably, the cellobiase mutant and its application provide a recombinant vector containing the DNA molecule and a regulatory sequence for expression that enables the DNA molecule to be transcribed and translated in large quantities under artificial regulation. The expression vector used in the present invention is pPICZαA (Invitrogen, Carlsbad, CA).

[0030] In one embodiment of the present invention, preferably, the cellobiose hydrolase mutant and its application also provide a host cell containing the DNA molecule or the recombinant vector. The host cell is a Pichia pastoris cell.

[0031] In one embodiment of the present invention, as a preferred embodiment, the cellobiase mutant and its application also provide a method for producing the cellobiase mutant, comprising the following steps: culturing the host cells in a culture medium, and massively replicating a recombinant vector containing the cellobiase mutant gene in the host cells.

[0032] In one embodiment of the present invention, preferably, the cellobiose hydrolase mutant and its application are further provided, and the application of the cellobiose hydrolase mutant or the composition thereof in cellulose hydrolysis is also provided.

[0033] To illustrate the effectiveness of the present invention, the inventors have provided the following comparative experiments:

[0034] Amino acid auxotrophic culture medium (YPDS) is used for yeast hybridization and screening of genetic mutants. The culture medium used in this invention was purchased from zeocin (Thermo Fisher Scientific, Runcorn, Cheshire, UK), and its formula is: 1% yeast extract, 2% peptone, 2% glucose, 1M sorbitol, and 2% agar.

[0035] A buffered glycerol-complex medium, controlled at pH 6.0, was used to optimize the expression of the target protein. The formulation consisted of 1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% amino acid-free nitrogen source, and 1% glycerol.

[0036] Buffered Minimal Methanol (BMMH) medium is a buffered basal medium containing methanol, used for methanol-induced protein expression in recombinant Pichia pastoris strains. The formulation is: 100 mM potassium phosphate, pH 6.0, 1.34% amino acid-free nitrogen source, and 0.5% methanol.

[0037] Wild-type cellobiase, derived from cellobiase (TeCel7A) secreted by *Talaromyces emersonii*, has the DNA molecular sequence shown in SEQ ID NO:2 and the encoded amino acid sequence shown in SEQ ID NO:1.

[0038] The cellobiose hydrolase mutants provided by this invention were obtained by screening two mutants with the highest number of hydrogen bonds, namely D259V (SEQ ID NO:3) and D259W (SEQ ID NO:5), using hydrogen bond network analysis and virtual saturation mutagenesis. They were then obtained by site-directed mutagenesis on the DNA sequence shown in SEQ ID NO:2.

[0039] Engineered strains of wild-type cellobiose hydrolase and its mutants were constructed, and cellobiose hydrolase mutants D259V (SEQ ID NO:3) and D258W (SEQ ID NO:5) were constructed, namely pPICZαA-D259V and pPICZαA-D259W, respectively. The expression vector used was pPICZαA. Wild-type and mutant cellobiose hydrolysate cells containing the constructed recombinant vectors were transformed into Pichia pastoris competent cells, and positive clones were selected to obtain engineered strains containing the mutant gene of this invention.

[0040] The wild-type and mutant strains of cellobiase from positive clones were expressed and their proteins were purified.

[0041] The obtained wild-type cellobiose hydrolase strain and its mutant strains were plated into an amino acid-deficient medium containing 100 μg / ml bleomycin and cultured at 30°C for 3 days. Positive colonies were then picked and inoculated into a buffered complex liquid medium containing glycerol, and cultured overnight at 240 rpm and 30°C with shaking. When the bacterial concentration reached OD600 = 2-6 (using a UNICOUV2102 UV-Vis spectrophotometer, with the glycerol-containing buffered complex medium as a blank control), the bacterial culture was centrifuged at 4000g, and the supernatant was resuspended in buffered minimum methanol (BMMH) medium. Subsequently, 0.5% methanol was added every 12 hours, and the culture was continued at 240 rpm and 30°C with shaking for 3 days.

[0042] The crude enzyme solution was purified by Ni-NTA column chromatography, eluting with 300 mM imidazole eluent for 10 column volumes. The obtained protein was then analyzed by SDS-PAGE to determine its purity. Cellobiose hydrolase protein (amino acid sequence SEQ ID NO:1) and cellobiose hydrolase mutants D259V and D259W (amino acid sequences SEQ ID NO:3 and SEQ ID NO:5) were obtained using the above method.

[0043] The assay, using microcrystalline cellulose as a substrate, determined the activity and hydrolysis rate of cellobiose hydrolase mutants D259V (amino acid sequence SEQ ID NO:3) and D259W (amino acid sequence SEQ ID NO:5). The specific methods and steps are as follows:

[0044] Preheat 1% (w / v) microcrystalline cellulose in a 60°C constant-temperature metal bath for 5-10 min, then add 1.33 μM enzyme dilution. Place the well-mixed enzyme containing the mutant and the control group simultaneously in a 60°C constant-temperature metal bath and react for 60 h. At the end of the reaction, boil the reactants in a boiling water bath for 5 min to inactivate the enzyme.

[0045] Table 1 Enzyme kinetics experiment

[0046]

[0047] Enzyme kinetic experiments showed that D259V had the best effect, K cat It increased by 78%. And in the subsequent hydrolyzed microcrystalline cellulose experiment, it increased the cellobiose yield by 30%.

[0048] The number of modules and processing scale described herein are for simplification. Applications, modifications, and variations of the cellobiose hydrolase mutant of the present invention and its applications will be readily apparent to those skilled in the art.

[0049] As described above, according to the present invention, since enzymes and their compositions with enhanced enzyme activity are provided, the hydrolysis efficiency of cellobiase per unit time can be increased, the amount of enzyme used can be reduced, and the effect of balancing the enzyme system and improving the overall hydrolysis rate in enzyme system compounding can be enhanced, which has broad application prospects.

[0050] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Other modifications can be readily made by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A cellobiose hydrolase mutant, characterized in that, The amino acid sequence of the cellobiose hydrolase mutant is shown in SEQ ID NO:

3.

2. A DNA molecule encoding a cellobiase mutant as described in claim 1.

3. The DNA molecule of claim 2, wherein the nucleotides of the DNA molecule are as shown in SEQ ID NO:

4.

4. A recombinant expression vector comprising the DNA molecule of claim 2 and a regulatory sequence operatively linked to the DNA molecule for expression.

5. A host cell containing the DNA molecule of claim 2 or the recombinant expression vector of claim 4, wherein the host cell is a non-animal or plant cell.

6. A method for producing a cellobiase mutant, comprising the following steps: culturing the host cell as described in claim 5 in a culture medium, and massively replicating a recombinant vector containing the cellobiase mutant gene in the host cell.

7. The application of the cellobiose hydrolase mutant according to claim 1 in cellulose hydrolysis.

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