A highly stable glucanase mutant and its application

Abstract

The present invention belongs to the field of bioengineering technology, and particularly relates to a highly stable glucanase mutant and its application. + An Aspergillus niger mutant strain was obtained by ion implantation mutagenesis breeding technology, and a glucanase mutant with good thermal stability and improved acid and alkali stability was obtained by genetic engineering technology. The amino acid sequence of the mutant is SEQ ID No. 4, and it was efficiently expressed by transgenic technology into Pichia pastoris GS115. The enzymatic properties of the glucanase mutant are suitable for animal gastrointestinal digestion conditions and can be better applied to the feed industry.

Description

Technical field:

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a highly stable glucanase mutant and application thereof. Background technology:

[0002] Glucan is a macromolecular polysaccharide composed of glucose. It is insoluble in water and general organic solvents. It is the main component of plant cell walls and one of the largest renewable biological resources in the world. Plant stems and fruits are used in large quantities in animal feeds, but as a non-starch polysaccharide in feed, glucan is difficult to be digested and absorbed by animals (especially monogastric animals), and can increase the viscosity of digestive tract chyme, resulting in reduced nutrient digestibility and feeding effect in the diet, which limits the application of glucan-containing plant resources in feed. In recent years, with the advancement of biotechnology, we have learned that non-starch polysaccharide enzymes such as glucanase can decompose such polysaccharides and eliminate their anti-nutritional effects.

[0003] Dextranase, also known as endocellulase, is a type of cellulase complex enzyme system. Its function is to randomly hydrolyze the glycosidic bonds in non-crystalline cellulose molecules, causing the viscosity to decrease and the reducing ends to increase. Due to the implementation of the new national feed additive standards and the strict requirements for hormone additives, pure natural bioenzyme preparations as feed additives have been a hot research direction in recent years. Dextranase decomposes non-starch polysaccharides and can eliminate their anti-nutritional effects on animals. However, due to the low activity of microbial fermentation dextranase, the production cost is high, which limits the application of dextranase in the feed industry. At present, there are two main reasons for the low activity of dextranase in the enzyme preparation industry: 1. The original strain has low enzyme production efficiency. 2. The molecular stability of dextranase is poor. The enzyme protein is quickly inactivated and denatured after secretion, resulting in a decrease in yield, which is not suitable for the complex digestive environment in animals. This is a problem that the feed enzyme preparation industry urgently needs to solve.

[0004] Microbial glucanase is mainly fungal glucanase, among which Aspergillus niger is the most studied glucanase-producing strain. Due to its widely recognized safety, it is widely used in the production of enzyme preparations in the food and pharmaceutical industries. + Ion implantation mutagenesis breeding refers to a method of generating a high-energy particle beam through a device and injecting it into an organism, causing permanent changes in the organism's genetic material, and then screening excellent strains from the mutant strains. This application will use this mutagenesis method to induce mutagenesis on Aspergillus niger that produces glucanase, screen out a strain of enzyme-producing bacteria that can produce a glucanase mutant with good stability and enzymatic properties, and further obtain the amino acid and nucleotide sequences of the glucanase mutant through genetic engineering means, and by constructing the corresponding Pichia pastoris engineered bacteria, it can be highly expressed, which is of great significance to meet the needs of feed processing industry production and reduce production costs. Summary of the invention:

[0005] In order to solve the above technical problems, the present invention provides a high-stability glucanase mutant and its application to solve the current demand for high-stability glucanase in the feed processing industry.

[0006] The stability of the enzyme molecule is mainly manifested in good thermal stability and improved acid and alkali stability. The purpose of the present invention can be achieved by the following measures:

[0007] A stable glucanase mutant was obtained by firstly using a wild type Aspergillus niger strain YP preserved in the applicant's laboratory through N + A mutant strain was obtained by ion implantation mutagenesis screening. The isolated glucanase encoding gene was connected to an expression vector and expressed in Pichia pastoris GS115 to obtain an engineered Pichia pastoris with better expression stability.

[0008] One of the technical solutions provided by the present invention is a glucanase mutant, wherein the glucanase mutant is based on the wild-type glucanase shown in SEQ ID No. 2, and the amino acids at positions 105 and 111 are mutated, serine (S) at position 105 is mutated to leucine (L), and aspartic acid (D) at position 111 is mutated to alanine (A), and the amino acid sequence of the glucanase mutant is shown in SEQ ID No. 4;

[0009] Furthermore, the nucleotide sequence of the gene encoding the glucanase mutant is shown in SEQ ID No.3.

[0010] The glucanase mutant prepared by the present invention has the following enzymatic properties:

[0011] (1) The optimum reaction temperature is 35°C. The enzyme activity remains unchanged when kept at 60°C for 2 hours, and can still maintain more than 80% of the enzyme activity when kept at 80°C for 2 hours, indicating good thermal stability. Figure 1 , Figure 2 .

[0012] (2) The optimum reaction pH is 6.5. The results showed that the relative enzyme activity remained above 80% in the pH range of 3.0-9.0 when treated for 2 h at pH 3.0-12.0. Figure 3 , Figure 4 .

[0013] The second technical solution provided by the present invention is a recombinant vector or recombinant strain comprising the above-mentioned glucanase mutant encoding gene, reconstructing a recombinant vector with the glucanase mutant encoding gene shown in SEQ ID No. 3, and efficiently expressing it in Pichia pastoris GS115, thereby solving the problem of low enzyme production efficiency of the original strain;

[0014] Furthermore, the expression vector used in the recombinant vector is a pPIC9K plasmid;

[0015] The recombinant engineering strain is obtained by connecting the gene encoding the glucanase mutant shown in SEQ ID No. 3 to the expression vector pPIC9K and inducing expression in Pichia pastoris GS115.

[0016] The third technical solution provided by the present invention is the application of the glucanase mutant shown in SEQ ID No.4, especially in the feed industry. Since the enzymatic characteristics of the high-stability glucanase mutant are suitable for animal gastrointestinal digestion conditions and the stability is improved, it can be better applied to feeds that rely on cellulase additives to enhance the animal's absorption efficiency of cellulose feeds.

[0017] Beneficial effects:

[0018] 1. The present invention provides a glucanase mutant obtained by mutagenesis. The glucanase mutant has stable enzymatic properties, high thermal stability, good acid and alkali resistance, and is not easy to denature and inactivate. It has broad application prospects in the feed enzyme preparation industry.

[0019] 2. The present invention provides an expression vector for a glucanase mutant with higher fermentation activity, and the glucanase liquid biological fermentation activity reaches more than 50,000 U / mL.

[0020] 3. The present invention provides a glucanase mutant that is more suitable for the feed industry, with an optimum reaction temperature of 35°C and an optimum pH of 6.5, which is more in line with the reaction conditions of the animal digestive system. Description of the drawings:

[0021] Figure 1 Optimum reaction temperature curve;

[0022] Figure 2 Thermal stability curve;

[0023] Figure 3 Optimal reaction pH curve;

[0024] Figure 4 pH stability curve graph;

[0025] Figure 5 Enzyme activity standard curve;

[0026] Figure 6 Example of initial screening of flat plate color change

[0027] Among them, a did not add anisidine; b added anisidine;

[0028] Figure 7 Results of shake flask rescreening experiment. Specific implementation method:

[0029] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0030] The amino acid sequence of the wild-type glucanase derived from Aspergillus niger YP of the present invention is shown in SEQ ID NO.2:

[0031] MTDQHIPARADDFSGYGGATAHLLEANDFQHAGPPPGPDTKYTPSPAGPAQPWYNPRGWSLHTKLIAGVVVAVVVAVIVGAVEGTKANRYPDYTPLNYQLVDTYSGTSFLDRFDYYHDEDPTEGFVQYVDRAAANALNNTFVATNPSAFAEAYWEFKSFKVLTYATDSSVVLRVDTSNQNAVKGRQ VSVRLESKTGYDNGLFIFDILHTPYGCGTWPALWLTDTTNWPVNGEIDVLETTNNAPEGNAVTLHTTKGCNMKVRRKQTGSTVYTTCDNSNNGGGVYALELRNAGIRAWMFPRDSIPDDITNSNSSPDPSTWGTALADFPNTDCDIPSHFRNQSIIANIDLCGQLGAQKQFYTEQSQCPGTCYQAP

[0032] The amino acid sequence of the glucanase mutant derived from Aspergillus niger YP-106 involved in the present invention is shown in SEQ ID NO.4:

[0033] MTDQHIPARADDFSGYGGATAHLLEANDFQHAGPPPGPDTKYTPSPAGPAQPWYNPRGWSLHTKLIAGVVVAVVVAVIVGAVEGTKANRYPDYTPLNYQLVDTYLGTSFLARFDYYHDEDPTEGFVQYVDRAAANALNNTFVATNPSAFAEAYWEFKSFKVLTYATDSSVVLRVDTSNQNAVKGRQ VSVRLESKTGYDNGLFIFDILHTPYGCGTWPALWLTDTTNWPVNGEIDVLETTNNAPEGNAVTLHTTKGCNMKVRRKQTGSTVYTTCDNSNNGGGVYALELRNAGIRAWMFPRDSIPDDITNSNSSPDPSTWGTALADFPNTDCDIPSHFRNQSIIANIDLCGQLGAQKQFYTEQSQCPGTCYQAP

[0034] The present invention is further explained below by means of specific examples.

[0035] Example 1 Mutagenesis of Glucanase Mutants

[0036] The original strain Aspergillus niger YP is the strain preserved by our company.

[0037] Plate culture medium: PDA medium, natural pH.

[0038] Seed culture medium: glucose 3%, peptone 2.5%, soybean cake powder 2%, sodium chloride 1%, (NH4)2SO42%, MgSO4 0.5%, pH 5.5.

[0039] Liquid fermentation medium: corn starch 1.5%, wood powder 1.5%, yeast powder 0.2%, potassium dihydrogen phosphate 1.7%, magnesium sulfate 0.27%, pH 5.5.

[0040] The original strain YP was activated by plate culture, and the spore suspension was prepared by plate culture and diluted to a concentration of 10 6 -10 8 Take 1 mL of spore suspension aseptically and spread it evenly on a sterile culture dish. Check under a microscope to see if there are no overlapping cells or bacteria. Dry it naturally to form a film. Put it into the target chamber of the ion implanter and control the temperature below 40°C. The ion source emits N + Pulse (30 KeV), dose 10×10 16 N + / cm 2 .

[0041] Screening: Spread the mutagenic spore solution on a plate and culture at 37°C to grow a single colony, then add 5 mg / L anisidine solution. Use the principle that the glucanase-producing strain decomposes anisidine and changes color to screen out the glucanase-producing colonies, such as Figure 6 , a total of 119 glucanase-producing mutagenic strains were screened. The discolored colonies were picked and transferred into 250mL shaking flasks (liquid fermentation medium 30mL) and cultured at 37℃, 150r / min for 72h to obtain enzyme-containing fermentation broth. The enzyme properties were tested by measuring the enzyme activity at pH 6.0, 70℃ for 2h, representing thermal stability, and measuring the enzyme activity at 30℃, pH 3.0, and pH 9.0 for 2h, taking the average value, representing acid-base stability. The original bacteria YP was used as 100% reference, and the results were as follows Figure 7 , and screened out the glucanase mutant with the best stability and the enzyme-producing Aspergillus niger mutant strain YP106.

[0042] The original strain YP and the mutant strain YP106 were cultured in 1L triangular seed bottles containing 100mL seed medium at 37℃ for 16h, respectively, and then inoculated into a fermenter containing 30L liquid fermentation medium, and cultured for 155h at 37℃, pH5.5, and 500r / min. The fermentation broth was centrifuged and the glucanase activity in the supernatant was determined. The fermentation enzyme activity is shown in Table 1.

[0043] Enzyme activity detection method The widely used DNS method was used to detect enzyme activity. 1 mL of reaction substrate 1% β-glucan, 1 mL of enzyme solution, 3 mL of pH 6.0 acetic acid buffer solution, placed in a 35°C water bath for 5 min, 2 mL of 3,5-dinitrosalicylic acid reagent was added, and the mixture was placed in a boiling water bath for 10 min. After cooling, the volume was adjusted to 10 mL and the absorbance was measured at a wavelength of 520 nm. The standard curve ( Figure 5 ) Calculate the amount of reducing sugar produced (mg).

[0044] Definition of unit glucanase activity: It is defined as the amount of enzyme that produces 1 μg of glucose per minute in 1 mL of fermentation broth under certain reaction conditions (if not otherwise specified, the reaction conditions are 35°C, pH 6.0).

[0045] Glucanase activity (U / mL) = C×B×1000 / (T×N)(1)

[0046] In formula 1), C: the amount of reducing sugar produced (mg);

[0047] 1000: Conversion factor from mg to μg;

[0048] B: represents the dilution multiple of the enzyme solution;

[0049] T: enzyme catalytic reaction time (min);

[0050] N: amount of enzyme used (mL).

[0051] Table 1.30L small tank liquid fermentation test results

[0052]

[0053] The amino acid sequence of the glucanase protein molecules produced by the original strain and the mutant strain was analyzed and detected (Beijing Biotech Biotechnology Co., Ltd.), and the amino acid sequence of the glucanase from the wild-type Aspergillus niger YP was obtained as shown in SEQ ID NO. 2, and the amino acid sequence of the glucanase mutant from the mutagenic strain Aspergillus niger YP106 was shown in SEQ ID NO. 4. The nucleotide sequence of the gene encoding the glucanase mutant was obtained by gene comparison and software analysis as shown in SEQ ID NO. 3.

[0054] Shenzhen BGI Co., Ltd. was commissioned to synthesize the glucanase nucleotide sequence SEQ ID NO.1 and the glucanase mutant nucleotide sequence SEQ ID NO.3 for subsequent experiments.

[0055] Example 2 Transformation and screening of Pichia pastoris engineered bacteria GS115

[0056] Pichia pastoris GS115 and vector pPIC9K plasmid were purchased from Novagen.

[0057] Introduced genes: nucleotide sequence of wild-type glucanase gene SEQ ID NO.1, nucleotide sequence of mutant glucanase gene SEQ ID NO.3.

[0058] Plate culture medium: YPD medium.

[0059] Shake flask culture medium: BMGY culture medium (purchased from Shanghai Harling Biotechnology Co., Ltd.).

[0060] MD screening medium: 10 μg / mL ampicillin, 1.34% YNB, 4×10 -5 % biotin, 1.0% glucose.

[0061] Electroporation conditions: voltage 1.5 kV; capacitance 25 μF; resistance 200 Ω; shock time 4 to 10 msec.

[0062] Competent cell preparation and transformation steps:

[0063] (1) Enrichment of bacteria: Pick a single colony of yeast GS115 on the YPD plate and inoculate it into a 50 mL Erlenmeyer flask containing 5 mL BM GY medium, and culture it at 30°C and 220 r / min for 10-16 h; take 200 μL of the culture and inoculate it into a 200 mL Erlenmeyer flask containing 50 mL fresh medium, and culture it at 28230°C and 250-300 r / min for about 20 h until the biomass OD reaches 0. 600 Reach 1.3 or above;

[0064] (2) Collecting and resuspending the cells: centrifuge the cell culture at 4°C, 1500 g for 5 minutes, and resuspend the cell pellet with 50 mL of ice-cold sterile water; centrifuge for the second time, and resuspend the cell pellet with 25 mL of ice-cold sterile water; centrifuge for the third time, and resuspend the cell pellet with 2-5 mL of 1 M ice-cold sorbitol solution; centrifuge for the fourth time, and resuspend the cell pellet with 160 μL of ice-cold 1 M sorbitol solution, and the final volume is about 240 μL;

[0065] (3) Enzyme digestion of target gene and plasmid: 2 μL of the synthetic fragment containing the mutant glucanase gene Byp106 (SEQ ID NO.3) and 1 μL of the expression vector pPIC9K were digested with 1 μL of restriction endonucleases EcoRⅠ and Not I, respectively, in a 37°C water bath overnight. After 1% gel electrophoresis and purification with a gel recovery kit, 30 μL of the mutant genome Byp106 and 20 μL of the linearized vector pPIC9K were obtained.

[0066] (4) Ligation: 12 μL of Byp106 fragment and 3 μL of linearized pPIC9K vector plasmid were mixed, T4 DNA ligase was added, and the reaction was carried out at 16°C for 15 h to obtain the target gene and plasmid ligation product.

[0067] (5) Transformation verification: Add 5 μL of the ligated product to 50 μL of DH5α competent cells, mix thoroughly, and place on ice for 30 minutes, then place in a 42°C water bath for 90 seconds. After another 5-minute ice bath, add 500 μL of LB medium, culture at 37°C, 150 rpm shaker for 1 hour, spread on an LB plate containing ampicillin (final concentration 25 μg / mL), and culture at 37°C for 12 hours. Select a single colony on the plate and culture it in ampicillin LB liquid medium. After 12 hours, until the OD reaches 0. 600 At about 1.3, the bacteria were obtained by centrifugation, and samples were taken for inspection. After PCR verification and comparison with sequencing results (Shenzhen BGI Co., Ltd.), positive clones that met the sequence shown in the nucleotide sequence SEQ ID NO.3 were selected. The positive clones contained the correct recombinant plasmid pPIC9K-Byp106.

[0068] (6) Extract the recombinant expression vector pPIC9K-Byp106: Select the correct positive single colony on the plate and culture it in LB liquid medium until OD 600 At about 1.2-1.5, the bacteria were obtained by centrifugation, and the recombinant expression vector pPIC9K-Byp106 was obtained using a plasmid extraction kit.

[0069] (7) Transformation: The obtained recombinant expression vector pPIC9K-Byp106 was digested with Sal I to obtain a linearized plasmid, and the linearized expression vector was mixed with the obtained GS115 Pichia pastoris cells, transferred to an ice-precooled electroporation transformation cup, and ice-bathed for 5 minutes before electroporation; after the transformation was completed, 1 mL of 1 M ice-precooled sorbitol solution was added to mix the cells, and placed in a 30°C shaker for low-speed culture for 1 hour.

[0070] (8) Screening positive clones: The obtained bacterial solution was spread on MD medium plates, and positive colonies were selected and sent for sequencing. The sequence that matched the sequence described in SEQ ID NO. 3 was the correct recombinant strain GS115-YP106.

[0071] The same scheme was used to obtain the recombinant bacteria GS115-YP expressing the wild-type glucanase gene SEQ ID NO.1.

[0072] Example 3 Production of glucanase by liquid fermentation of Pichia pastoris and its extraction and preparation

[0073] Bacterial strains: recombinant strain GS115-YP106 (containing mutant gene); recombinant strain GS115-YP (containing wild-type gene).

[0074] Seed medium formula: 1.0% yeast extract, 2.5% peptone, 1.0% diammonium phosphate, 1.34% YNB, 4×10 -5 % biotin, 1.0% glycerol, pH 6.0.

[0075] Fermentation medium formula: 1.5% yeast extract, 2.5% peptone, 1.0% diammonium phosphate, 0.5% dipotassium phosphate, 1.34% YNB, 4×10 -5 % biotin, 1.5% methanol, pH 6.0.

[0076] The recombinant Pichia pastoris was inoculated into a 1L Erlenmeyer flask containing 100 mL of seed culture medium and cultured at 30°C and 220 r / min until the OD 600 The temperature is about 8-10, 100 mL of seed liquid is added to a 30 L fermentation medium fermentation tank, and the culture is continued for 155 h at 30 ° C, pH 5.0, 500 r / min. After the fermentation liquid is centrifuged, the glucanase activity in the supernatant is determined.

[0077] Two recombinant Pichia pastoris and culture medium were used for fermentation. Table 2 shows the fermentation cycle and enzyme activity results of the fermentation broth for 6 batches of fermentation. The fermentation enzyme activity of GS115-YP106 reached more than 50,000 U / mL, which was much higher than that of the mutagenic Aspergillus niger strain YP106 and the wild genotype recombinant strain GS115-YP.

[0078] Table 2.30L small tank fermentation test results

[0079]

[0080] It can be seen from the above table that the fermentation level of recombinant Pichia pastoris GS115-YP106 is relatively stable, and the fermentation enzyme activity has reached more than 50,000 U / mL.

[0081] After fermentation, the glucanase from the fermentation broth of the two groups of recombinant bacteria was extracted by adding 40% water and 0.3% calcium chloride according to the volume of the fermentation broth, adding 3% perlite filter aid, and filtering was performed. The filtrate was concentrated by ultrafiltration to obtain liquid glucanase preparations of the two recombinant bacteria, which were stored at room temperature (25°C).

[0082] Example 4 Optimum reaction temperature

[0083] The glucanase product prepared in Example 3 was taken, and the enzyme activity was measured at 20, 25, 30, 35, 40, 45, 50, 55, and 60°C at pH 6.0, and the relative enzyme activity was calculated, with the highest enzyme activity in the experimental group as 100% control. The results are shown in FIG. Figure 1 As shown, the optimal reaction temperature of the enzymes produced by GS115-YP106 (containing mutant genes) and GS115-YP (containing wild-type genes) is 35°C, but the catalytic efficiency of GS115-YP enzyme is lower under other temperature conditions. The comparison shows that the optimal temperature range of the mutant glucanase is wider.

[0084] Example 5 Thermal Stability

[0085] The glucanase product prepared in Example 3 was taken and the enzyme solution was placed at 60, 65, 70, 75, and 80°C for 2 hours. After the insulation, the enzyme activity was measured and the relative enzyme activity was calculated, with the enzyme activity before treatment as 100% control. The experimental results are shown in Figure 2. Figure 2 As shown in the figure, the activity of the mutant glucanase produced by GS115-YP106 remained basically unchanged at 60℃ for 2h, and it could still maintain more than 80% of the activity at 80℃ for 2h. The activity of the glucanase produced by GS115-YP dropped to 80% at 60℃ for 2h, and the activity decreased rapidly with the increase of the insulation temperature. At 80℃ for 2h, the activity could only maintain 40%.

[0086] Example 6 Optimal reaction pH

[0087] The glucanase product prepared in Example 3 was taken, and the activity of the glucanase was measured at pH values ​​of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 at a temperature of 35°C according to the enzyme activity determination method described in Example 2, and the relative enzyme activity was calculated, with the highest enzyme activity in the experimental group as 100% control. The determination results are as follows: Figure 3 As shown, the activity of the mutant glucanase produced by GS115-YP106 was the highest at pH 6.5, and the activity was maintained above 80% under the conditions of pH 5.0-8.5. The optimum pH for the glucanase produced by GS115-YP was 7.0, and the activity reached above 60% under the reaction conditions of pH 5.0-7.5.

[0088] Example 7 Acid and alkali resistance

[0089] The finished glucanase prepared in Example 3 was taken and the pH of the enzyme solution was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 with 0.05 M NaOH or 0.05 M HCl, respectively. The enzyme activity was measured after standing at room temperature for 2 h, with the enzyme activity before treatment being 100% as the control. The measurement results are as follows: Figure 4 As shown. After being treated for 2 hours at pH 3.0-9.0, the relative enzyme activity of the mutant glucanase produced by GS115-YP106 still remained above 80%. The range of pH 5.0-8.0 for the glucanase produced by GS115-YP to maintain more than 80% of its enzyme activity was poor compared to the mutant's acid-base stability wild-type glucanase.

[0090] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A glucanase mutant, characterized in that: The amino acid sequence of the glucanase mutant is shown in SEQ ID No.

4.

2. A gene encoding the glucanase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID No.

3.

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

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

6. The recombinant strain according to claim 4, characterized in that The host bacteria used was Pichia pastoris GS115.

7. Use of the recombinant vector or recombinant strain according to claim 4 in producing the glucanase mutant according to claim 1.

8. Use of the glucanase mutant according to claim 1, characterized in that: It is used in feed industry.

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