A Pichia pastoris engineering bacterium producing β-glucosidase and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a Pichia pastoris engineering bacterium with high yield of β-glucosidase. The amino acid sequence of the β-glucosidase mutant is shown in SEQ ID No. 4. By means of genetic engineering, a vector containing the mutant gene is introduced into a host to obtain a genetically engineered bacterium. Through verification of the fermentation performance, the fermentation enzyme activity of β-glucosidase can reach more than 51,000 U / mL. The optimal pH of the β-glucosidase expressed by the mutant is 4.0, which is 0.5 lower than that before mutation. The optimal temperature is 45 °C. After being treated at pH 3.5 for 24 h, the relative activity still remains about 80%. Compared with that before mutation, the acid resistance is improved, which is more conducive to application in the cellulose bioconversion industry.

Description

Technical Field:

[0002] The present invention relates to the field of biotechnology, and particularly to a Pichia pastoris engineering bacterium with high yield of β-glucosidase. Background Art:

[0004] β-glucosidase (β-D-glucoside hydrolase, EC 3.2.1.21) is a type of cellulase that can catalytically hydrolyze the terminal non-reducing β-D-glycosidic bond from sugar-containing compounds, releasing β-D-glucose and corresponding monosaccharides, oligosaccharides or complex sugars, and is a protein with the function of a biocatalyst. The β-glucosidase gene widely exists in various microorganisms in nature, including bacteria, actinomycetes, yeasts and filamentous fungi, etc., and also exists in some animals. In addition to its important role in degrading cellulose, β-glucosidase is also widely used in other fields, such as food, winemaking, medicine and chemical industry.

[0005] As an important enzyme for cellulose biotransformation, β-glucosidase plays a key role in cellulose saccharification hydrolysis. However, the proportion of β-glucosidase in the cellulase system produced by microorganisms is less than 1%, which makes β-glucosidase a key factor in degrading cellulose into monosaccharides. Industrially, β-glucosidase is added to the cellulose hydrolysis system to improve the hydrolysis efficiency of cellulose.

[0006] Currently, the main source of β-glucosidase products is natural molds. Due to the poor enzyme-producing performance of natural strains, β-glucosidase products cannot meet the market demand. Pichia pastoris is a eukaryotic expression host that has developed rapidly in recent years. It has low nutritional requirements and is suitable for large-scale production methods of high-density fermentation. In particular, Pichia pastoris secretes very little of its own proteins, which is beneficial to the purification of the expression product. Therefore, the present invention transforms a strain of Aspergillus niger that produces β-glucosidase in the company by genetic engineering means to obtain a β-glucosidase mutant with improved acid resistance, and constructs a corresponding Pichia pastoris engineering bacterium to express it, which is of great significance for meeting the needs of industrial production of cellulose biotransformation and reducing production costs. Summary of the Invention:

[0008] The purpose of the present invention is to provide a β-glucosidase mutant with improved acid resistance and its Pichia pastoris engineering bacterium strain, aiming to solve the problems of low enzyme activity of β-glucosidase in industrial production and unsatisfactory application in cellulose biotransformation.

[0009] The present invention is specifically realized through the following technical solutions:

[0010] A β-glucosidase mutant with improved acid resistance, which is obtained from the Aspergillus niger strain preserved in the applicant's laboratory ( Aspergillus nigerPT003 was obtained by screening through the nitrosoguanidine mutagenesis method. After cloning the β-glucosidase mutant coding gene from the mutagenized strain and constructing a recombinant plasmid, it was expressed in Pichia pastoris GS115 to obtain the Pichia pastoris engineering strain.

[0011] One of the technical solutions provided by the present invention: is a β-glucosidase mutant, which is obtained by mutating serine at position 60 to leucine and valine at position 303 to alanine on the basis of the PT003 wild-type β-glucosidase shown in SEQ ID No. 2. The amino acid sequence of the β-glucosidase mutant is as shown in SEQ ID No. 4;

[0012] The present invention also provides the coding gene of the above β-glucosidase mutant;

[0013] Furthermore, the coding gene of the β-glucosidase mutant has the nucleotide sequence shown in SEQ ID No. 3.

[0014] The enzymatic properties of the β-glucosidase mutant are as follows:

[0015] (1) Optimal pH: The enzyme activity is stable at pH 3.5 - 5.5, and the optimal acting pH is 4.0;

[0016] (2) Optimal temperature: The enzyme activity is stable at 35°C - 55°C, and the optimal acting temperature is 45°C;

[0017] (3) Acid resistance: The enzyme retains more than 80% of its enzyme activity after 24 hours under the condition of pH 3.5.

[0018] One of the technical solutions provided by the present invention: is a recombinant vector or recombinant strain containing the above β-glucosidase mutant coding gene. The above β-glucosidase mutant coding gene is reconstructed into a recombinant vector and highly expressed in Pichia pastoris to obtain a recombinant strain producing high-activity β-glucosidase, and high-activity β-glucosidase is obtained through technologies such as fermentation and extraction;

[0019] Furthermore, the host cell for expressing the β-glucosidase mutant is Pichia pastoris GS115;

[0020] Furthermore, the expression vector for expressing the β-glucosidase mutant is the pPIC9K plasmid;

[0021] Preferably, the recombinant strain is obtained by ligating the β-glucosidase mutant coding gene shown in SEQ ID No. 3 to the expression vector pPIC9K and expressing it in Pichia pastoris GS115.

[0022] The fourth technical solution provided by the present invention is the application of the recombinant vector or recombinant strain described in the third technical solution, especially the application in the fermentation production of the β-glucosidase mutant shown in SEQ ID No. 4.

[0023] The fifth technical solution provided by the present invention is the application of the β-glucosidase mutant shown in SEQ ID No. 4, especially the application in catalyzing the hydrolysis of glycosidic bonds, and more particularly the application in the cellulose bioconversion industry.

[0024] The following definitions are adopted in the present invention:

[0025] 1. Nomenclature of amino acid and DNA nucleic acid sequences

[0026] The generally recognized IUPAC nomenclature of amino acid residues is used in the form of three-letter or single-letter codes. The DNA nucleic acid sequences adopt the generally recognized IUPAC nomenclature.

[0027] 2. Identification of β-glucosidase mutants

[0028] The mutated amino acids in the mutants are represented by "the amino acid replaced at the original amino acid position". For example, S60L means that the amino acid at position 60 is replaced from the wild-type Ser to Leu, and the position number corresponds to the amino acid sequence number of the wild-type β-glucosidase in SEQ ID No. 2. In the present invention, G-1 represents the wild-type β-glucosidase, and G-2 represents the β-glucosidase mutant. The information is as shown in the following table.

[0029]

[0030] Beneficial effects:

[0031] The present invention discloses a brand-new β-glucosidase mutant, which has the characteristics of high enzyme activity and high acid resistance, and the enzyme activity of the mutant fermentation broth can reach more than 51000 U / mL.

[0032] The optimal reaction pH of the β-glucosidase obtained in the present invention is the highest at 4.0. Compared with the β-glucosidase of the wild-type PT003, the optimal reaction pH is reduced by 0.5, indicating an increase in acid resistance.

[0033] The β-glucosidase obtained in the present invention still retains more than 80% of its enzyme activity after 24 h under the condition of pH 3.5. Compared with the β-glucosidase of the wild-type PT003, the acid resistance is significantly improved. Description of the drawings:

[0035] Figure 1 Optimal pH curve;

[0036] Figure 2Optimal temperature curve;

[0037] Figure 3 Acid tolerance curve. Specific implementation manner:

[0039] The present invention will be described in more detail through specific examples, which are only for illustrative purposes and not for limiting the scope of implementation of the present invention. For those skilled in the art, improvements that can be made based on the principle of the present invention should also be regarded as within the scope of protection of the present invention. For the molecular biology experimental methods not specifically described in this example, reference can be made to "Molecular Cloning Experiment Guide".

[0040] The present invention provides a β-glucosidase mutant with improved acid tolerance, which is obtained by screening the Aspergillus niger strain ( Aspergillus niger ) PT003 preserved in the applicant's laboratory after mutagenesis with nitrosoguanidine. The mutated sites obtained by gene sequencing are that serine at position 60 is mutated to leucine, and valine at position 303 is mutated to alanine. The present invention first obtains the coding gene of the β-glucosidase mutant, connects the mutant coding gene with the pPIC9K vector to construct a recombinant plasmid, transfers it into the corresponding host bacterium GS115 for heterologous expression, and ferments to obtain the corresponding β-glucosidase of the mutant. This β-glucosidase has high enzyme activity and is suitable for industrial production and the cellulose bioconversion industry.

[0041] 1. Experimental materials and reagents:

[0042] Experimental strains and vectors: Gene source strain: Aspergillus niger PT003 preserved by the company; Expression host bacterium and vector: GS115 and pPIC9K are both purchased from Invitrogen Corporation, USA; Host bacterium: DH5α.

[0043] Main reagents: DNA polymerase, T4 DNA ligase, DNA gel recovery kit, plasmid extraction kit, RNA extraction kit, pNPG, DNA marker, agarose, ampicillin, IPTG, X-gal: all purchased from Sangon Biotech, Shanghai; Various restriction endonucleases: purchased from NEB Company.

[0044] Experimental instruments: Gel imaging system (Bio-Rad), Protein electrophoresis apparatus (Bio-Rad), Nucleic acid electrophoresis apparatus (Bio-Rad), PCR amplifier (Bio-Rad), High-speed centrifuge (Eppendorf).

[0045] 2. The method for measuring the activity of β-glucosidase adopted by the present invention:

[0046] (1) Take 200 μL of 0.2 M acetic acid - sodium acetate buffer (pH 5.0). Add 50 μL of appropriately diluted fermentation supernatant, i.e., enzyme solution, to the sample tube, and add 50 μL of buffer to the blank control. Preheat and equilibrate in a water bath at 50 °C for 5 min;

[0047] (2) At the same time, preheat and equilibrate the pNPG solution (5.0 mM) at 50 °C;

[0048] (3) Add 250 μL of preheated and equilibrated pNPG substrate solution to the pre - equilibrated solution of the enzyme solution sample to form a 500 μL enzyme reaction system. React at 50 °C in a constant - temperature water bath for 10 min;

[0049] (4) Immediately add 500 μL of 1.0 M Na 2 CO 3 solution to enhance the color development and terminate the reaction. After mixing evenly, let it stand at room temperature for 5 min;

[0050] (5) Measure the absorbance A405 of p - Nitrophenol at 405 nm using an enzyme - linked immunosorbent assay reader. Each sample is measured in triplicate.

[0051] Definition of enzyme activity unit: Using pNPG as the substrate, under the assay conditions (unless otherwise specified, the assay conditions are pH 5.0, 50 °C), the amount of enzyme required to hydrolyze the substrate to produce 1 μmoL of p - Nitrophenol per minute is defined as one enzyme activity unit (U / mL). That is:

[0052] Enzyme activity unit (U / mL) = (C × N) / T

[0053] Wherein, N: dilution factor of the original enzyme solution; T: reaction time (min); C: concentration of p - Nitrophenol corresponding to the absorbance A405 on the standard curve (μmol / mL).

[0054] The nucleotide sequence of the wild - type β - glucosidase G - 1 encoding gene described in the present invention and its embodiments is shown as SEQ ID No.1:

[0055]

[0056] The amino acid sequence of the wild-type β-glucosidase G-1 described in the present invention and the embodiments is shown in SEQ ID No. 2 of the sequence listing:

[0057] MGSATASTLPPDFLWGFATASYQIEGAVTEDGRGPSIWDTFCKIPGKIAGGANGDVACDSYHRTAEDIALLKECGAQAYRFSISWSRIIPLGGRNDPINDKGVQHYVKFVDDLLAAGITPLVTLFHWDLPDALDKRYGGLLNKEEFVADFANYARVMFRALGSKVKHWITFNEPWCSSVLGYNVGQFAPGRTSDRSKSAEGDSSRECWIVGHNILVAHGAAVKIYREEFKSRDGGEIGITLNGDWAEPWDPENPADIEACDRKIEFAISWFADPIYHGRYPDSMIKQLGDRLPSWTAEDIALVHGSNDFYGMNHYCANYIKAKTGEADPNDTAGNLEILLKNKKGEFIGPETQSAWLRPYALGFRKLLKWLSDRYGQPKIYVTENGTSLKGENDLPVEELLKDEFRTQYFRDYIAAMADA

[0058] YTLDGVNVRAYMAWSLMDNFEWAEGYETRFGSTYVDYEHGQKRIPKDSAKQIGQIFSQYIEKK

[0059] The nucleotide sequence of the coding gene of the β-glucosidase mutant G-2 described in the present invention and the embodiments is shown in SEQ ID No. 3:

[0060]

[0061] The amino acid sequence of the β-glucosidase mutant G-2 described in the present invention and its embodiments is shown in Sequence Listing SEQ ID No. 4:

[0062] MGSATASTLPPDFLWGFATASYQIEGAVTEDGRGPSIWDTFCKIPGKIAGGANGDVACDLYHRTAEDIALLKECGAQAYRFSISWSRIIPLGGRNDPINDKGVQHYVKFVDDLLAAGITPLVTLFHWDLPDALDKRYGGLLNKEEFVADFANYARVMFRALGSKVKHWITFNEPWCSSVLGYNVGQFAPGRTSDRSKSAEGDSSRECWIVGHNILVAHGAAVKIYREEFKSRDGGEIGITLNGDWAEPWDPENPADIEACDRKIEFAISWFADPIYHGRYPDSMIKQLGDRLPSWTAEDIALAHGSNDFYGMNHYCANYIKAKTGEADPNDTAGNLEILLKNKKGEFIGPETQSAWLRPYALGFRKLLKWLSDRYGQPKIYVTENGTSLKGENDLPVEELLKDEFRTQYFRDYIAAMADA

[0063] YTLDGVNVRAYMAWSLMDNFEWAEGYETRFGSTYVDYEHGQKRIPKDSAKQIGQIFSQYIEKK

[0064] The present invention will be further explained and illustrated through specific embodiments below.

[0065] Example 1 Obtaining the encoding gene of β-glucosidase mutant G-2

[0066] A strain of Aspergillus niger producing β-glucosidase ( Aspergillus niger ) PT003 preserved by our company was mutagenized with nitrosoguanidine and screened to obtain a strain with acid-resistant β-glucosidase activity. PCR primers were designed according to the nucleotide sequence of the wild-type β-glucosidase G-1 encoding gene, with a restriction enzyme site added to the 5' end Xho I and a restriction enzyme site added to the 3' end Not I. The encoding gene of mutant G-2 was obtained by PCR, and its nucleotide sequence was SEQ ID No. 3 through sequencing, and the corresponding amino acid sequence was SEQ ID No. 4.

[0067] In the present invention, through the comparison of the nucleotide sequences of the wild type and the mutant, the mutation site information is as shown in the following table.

[0068]

[0069] Example 2 Construction of the recombinant vector pPIC9K-G-2

[0070] The coding gene of the mutant G-2 (SEQ ID No.3) and the plasmid pPIC9K were respectively Xho I and Not I digested, the products were recovered, the recovered G-2 coding gene and pPIC9K were mixed in proportion, and ligated overnight at 16 °C with T4 ligase. The ligation product was transformed into competent cells of Escherichia coli DH5α, and the transformed product was spread on an LB (containing ampicillin) solid plate and cultured overnight at 37 °C in an inverted position. Single colonies were picked into an LB liquid medium and cultured at 37 °C. The target sequence was obtained by colony PCR, and the sequencing alignment showed that it was the nucleotide sequence shown in SEQ ID NO.3, that is, the obtained recombinant vector contained the correct mutant gene, and the obtained recombinant vector was named pPIC9K-G-2.

[0071] Example 3 Transformation of the recombinant plasmid into Pichia pastoris

[0072] 1. Preparation of competent cells of Pichia pastoris GS115

[0073] (1) Pick a single colony from the Pichia pastoris plate and inoculate it into 5 mL of YPD medium. Incubate overnight at 30 °C with shaking at 220 r / min;

[0074] (2) Take 0.5 mL of the overnight cultured bacterial solution and inoculate it into 50 mL of freshly prepared YPD medium. Incubate at 30 °C with shaking at 220 r / min until the OD 600 value reaches 1.3 - 1.5;

[0075] (3) Take the above culture solution and centrifuge it at 4 °C at 3000 r / min for 5 min;

[0076] (4) Discard the supernatant, add 50 mL of sterile water pre-cooled on ice, and resuspend the cells by shaking;

[0077] (5) Centrifuge at 4 °C at 3000 r / min for 5 min, discard the supernatant, dry the residual liquid on the tube wall, add 25 mL of sterile water pre-cooled on ice, and resuspend the cells by shaking;

[0078] (6) Centrifuge at 4 °C at 3000 r / min for 5 min, discard the supernatant, dry the residual liquid on the tube wall, add 10 mL of sterile 1 mol / L sorbitol solution pre-cooled on ice, and resuspend the cells;

[0079] (7) At 4 °C, centrifuge at 3000 r / min for 5 min, discard the supernatant, dry the residual liquid on the tube wall, add 1 mL of sterile sorbitol solution (previously added with glycerol to a final concentration of 15%) pre-cooled on ice, and mix well by oscillation;

[0080] (8) Aliquot 100 μL per tube into sterile EP tubes and store frozen at -70 °C in a refrigerator (freshly prepared competent cells have better effects).

[0081] 2. Transformation of linearized plasmid

[0082] The positive clone obtained in Example 2 was extracted to obtain the recombinant plasmid pPIC9K-G-2, which was digested with Sal I to obtain a linearized plasmid. Take freshly prepared (or stored at -70 °C) GS115 competent cells and place them in an ice bath to completely thaw.

[0083] (1) Transfer 100 μL of competent cells to a new sterile EP tube, add 10 μL of linearized plasmid, gently blow and mix well, aspirate and transfer to a 0.2 cm type electroporation cuvette;

[0084] (2) Place the cuvette in an ice bath for 5 - 10 min to maintain low temperature;

[0085] (3) Electroporation transformation conditions: 1500 V, 200 Ω, 25 μF, discharge time about 5 ms, one-time electroporation;

[0086] (4) Immediately after electroporation, add 1 mL of 1 mol / L sorbitol solution pre-cooled at 4 °C to the electroporation cuvette, pipette and mix evenly, and place in an ice bath;

[0087] (5) Under sterile operation in a laminar flow hood, spread MD medium (1.34% YNB; 4×10 -5 % biotin; 2% glucose plate), 150 μL per plate, and incubate the spread plates upside down at 30 °C for 3 - 4 days;

[0088] (6) Two recombinant strains were screened on the MD plate. The target sequence was obtained by colony PCR, and sequencing alignment showed that both were the nucleotide sequence shown in SEQ ID NO.3, that is, the obtained recombinant strains contained the correct mutant gene. The two recombinant strains were named N-01 and N-02 respectively.

[0089] Example 4 Induced expression of yeast containing recombinant plasmid pPIC9K-G-2

[0090] BMGY medium formulation: 1.5% yeast extract, 2.5% peptone, 0.1 mol / L phosphate buffer at pH 6.0, 1.34% YNB, 4×10 -5 % biotin, 1% glycerol, and the rest is water.

[0091] BMMY medium formulation: 1.5% yeast extract, 2.5% peptone, 0.1 mol / L phosphate buffer at pH 6.0, 1.34% YNB, 4×10 -5 % biotin, 0.6% methanol, and the rest is water.

[0092] The recombinant strains N-01, N-02, and the recombinant strain ND-01 constructed by the same method as in Example 3 using the wild-type β-glucosidase encoding gene (the nucleotide sequence shown in SEQ ID NO.1) were respectively inoculated into Erlenmeyer flasks containing 30 mL of BMGY medium and cultured at 30 °C and 220 r / min until the OD 600 was about 10. The cells were collected by centrifugation, resuspended with 35 mL of BMMY induction medium, and continued to be cultured at 30 °C and 220 r / min for 50 h. After centrifugation of the fermentation broth, the β-glucosidase activity in the supernatant was measured, and the results are shown in the following table.

[0093]

[0094] Example 5 Verification of the fermentation performance of recombinant strain N-02

[0095] Using the recombinant strain N-02 obtained in Example 3 as the production strain.

[0096] Seed tank medium formulation: 3.8% glycerol, 1.4% ammonium dihydrogen phosphate, 0.7% potassium dihydrogen phosphate, 0.5% magnesium sulfate, 0.6% potassium sulfate, 0.08% calcium sulfate, 0.4% potassium hydroxide, and the rest is water, pH 4.5;

[0097] Fermentation tank medium formulation: 3.8% glycerol, 1.4% ammonium dihydrogen phosphate, 0.7% potassium dihydrogen phosphate, 0.5% magnesium sulfate, 0.6% potassium sulfate, 0.08% calcium sulfate, 0.4% potassium hydroxide, and the rest is water, pH 4.5;

[0098] Carbon source: 50% glycerol;

[0099] Methanol: pure methanol;

[0100] Seed tank culture: The culture temperature is 30 °C, the initial rotation speed is 200 r / min, and the initial air volume is 2 m 3 / h, ventilation and agitation culture, pH 4.5, dissolved oxygen maintained at 30 - 40%. When the dissolved oxygen is below 30%, it is controlled by increasing the rotation speed and air volume. Transfer the culture when the wet weight increases to 85 g / L;

[0101] Fermenter culture: Culture temperature 30°C, initial rotation speed 200 r / min, initial air volume 2 m 3 / h, ventilation and agitation culture, inoculation amount 10%, pH 4.5. In the 0 - 22 h cycle, add 50% glycerol as the carbon source at a flow rate of 600 g / h. Dissolved oxygen is maintained at 30 - 40%. When it is below 30%, it is controlled by increasing the rotation speed and air volume. Cultivate the bacteria until the wet weight reaches 230 g / L. After the 22 h cycle, stop supplementing the carbon source, and the dissolved oxygen rebounds to above 80% and is maintained for 0.5 h. Then add methanol at a speed of 220 g / h, and the dissolved oxygen is maintained at 20 - 30%. Cultivate until the total fermentation cycle is 160 h and the fermentation ends.

[0102] Perform a 50 L fermenter scale-up verification experiment using the above fermentation method. The fermentation cycle is 160 h. The enzyme production of 3 batches is as shown in the following table. The average enzyme production level is 52106 U / mL, indicating that the strain not only has high production of acidic β-glucosidase, but also has certain stability in its fermentation performance and the enzyme activity of the β-glucosidase it produces.

[0103] Enzyme production of 3 batches of genetically engineered bacteria

[0104]

[0105] Example 6 Enzymatic properties of β-glucosidase

[0106] (1) Optimal action pH

[0107] Using the supernatant of the N-02 fermentation broth obtained in Example 4 as the mutant β-glucosidase sample, and the supernatant of the ND-01 fermentation broth as the wild-type control sample, adopt the β-glucosidase activity determination method used in the present invention. Under the condition of 50°C, measure the relative enzyme activities under different pH conditions of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, with the highest β-glucosidase enzyme activities measured for the two enzymes as the benchmark. From Figure 1 It can be seen that the β-glucosidase mutant of the present invention has stable enzyme activity in the pH range of 3.5 - 5.0, and the optimal action pH is 4.0, and its optimal pH is 0.5 lower than that of the control. The above results show that compared with before mutation, the mutant β-glucosidase of the present invention has higher enzyme activity under higher acidic conditions, and its action pH range is wider, which is more suitable for the cellulose bioconversion industry.

[0108] (2) Optimal action temperature

[0109] Using the supernatant of the N-02 fermentation broth obtained in Example 4 as the mutant β-glucosidase sample and the supernatant of the ND-01 fermentation broth as the wild-type control sample, and adopting the β-glucosidase activity assay method used in the present invention, the enzyme activities were measured at different temperatures of 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C under the condition of pH 4.0. Based on the highest β-glucosidase activity measured for each of the two enzymes, the relative enzyme activity was calculated. The results are as Figure 2 shown. The β-glucosidase mutant of the present invention has stable enzyme activity at 35 - 55 °C, and the optimal working temperature is 45 °C. Compared with the wild-type β-glucosidase, the optimal reaction temperature remains unchanged, but the optimal temperature range has increased.

[0110] (3)Acid resistance

[0111] Using the supernatant of the N-02 fermentation broth obtained in Example 4 as the mutant β-glucosidase sample and the supernatant of the ND-01 fermentation broth as the wild-type control sample, with the β-glucosidase activity of each without treatment taken as the 100% benchmark, the two samples were respectively incubated at 45 °C under the condition of pH 3.5. Every 2 h, the enzyme activity was measured under the conditions of a temperature of 50 °C and a buffer pH of 5.0, and the remaining enzyme activity was calculated. From Figure 3 this, it can be seen that after 24 h, the relative activity of the β-glucosidase mutant of the present invention still remains above 80%. Compared with the wild-type β-glucosidase, the acid resistance of the mutant β-glucosidase has been greatly improved, indicating that the β-glucosidase mutant of the present invention has good acid resistance. The above results show that compared with before the mutation, the improved acid resistance makes the mutated β-glucosidase more suitable for application in the cellulose bioconversion industry.

[0112] The above-described embodiments only represent several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of this patent, several deformations, combinations, and improvements can be made to the above-mentioned implementation modes, and these all belong to the protection scope of this patent. Therefore, the protection scope of this patent should be based on the claims.

Claims

1. A β-glucosidase mutant, characterized in that the amino acid sequence of the β-glucosidase mutant is as shown in SEQ ID NO.

4.

2. The coding gene of the β-glucosidase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that the nucleotide sequence of the coding gene is as shown in SEQ ID NO.

3.

4. A recombinant vector or recombinant strain containing the coding gene according to claim 2.

5. The recombinant vector according to claim 4, characterized in that the expression vector is the pPIC9K plasmid.

6. The recombinant strain according to claim 4, characterized in that the host cell used is Pichia pastoris GS115.

7. The recombinant strain according to claim 4, characterized in that the recombinant strain is obtained by ligating the β-glucosidase mutant coding gene shown in SEQ ID No.3 to the expression vector pPIC9K and expressing it in Pichia pastoris GS115.

8. Use of the recombinant vector or recombinant strain according to claim 4 in the production of the β-glucosidase mutant according to claim 1.

9. Use of the β-glucosidase mutant according to claim 1, characterized in that specifically, it is the use of the β-glucosidase mutant in catalyzing the hydrolysis of glycosidic bonds or in the cellulose bioconversion industry.

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