Enzyme mutant with improved thermal stability, gene thereof and application thereof

Abstract

The application discloses an enzyme mutant with improved thermal stability and a gene and application thereof, and relates to the technical fields of genetic engineering and enzyme engineering. The application provides a mannanase mutant Man74s, and an amino acid sequence of the mannanase mutant Man74s is shown as SEQ ID NO:2. The mannanase mutant Man74s has high thermal stability, and has high activity in an acidic and neutral range at normal temperature, and other characteristics such as resistance to protease degradation, the optimal pH value of the mannanase mutant Man74s is 6.0, the mannanase mutant Man74s has relatively high catalytic activity in the pH range of 5.0-7.0, the optimal temperature is 50 DEG C, the residual enzyme activity of the mannanase Man74s is still above 70% after being treated at 85 DEG C for 3 min, and the mannanase Man74s is suitable for a rumen animal environment such as a cow or a sheep.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering and enzyme engineering, and particularly relates to an enzyme mutant with improved thermal stability, a gene thereof and application. BACKGROUND

[0002] The discovery of mannanase is derived from the in-depth study of the complex polysaccharide structure of plant cell walls. In the exploration of the composition and function of plant cell walls, researchers found that mannan, as a key component of hemicellulose, is widely present in the cell walls of plants such as corn and soybeans, and therefore is also widely present in animal feed. As an anti-nutritional factor, mannan can absorb a large amount of water after entering the gastrointestinal tract of animals, causing the viscosity of the gastrointestinal contents to increase, inhibiting the peristalsis of the stomach and intestines, and thus causing a series of digestive problems.

[0003] Mannanase is a glycoside hydrolase that can hydrolyze mannan into mannose monomers and mannose oligosaccharides, and can effectively solve the anti-nutritional problem caused by mannan. In addition, the hydrolyzed mannose oligosaccharides are also a functional nutrient that can improve the nutrition of feed. It can improve the utilization rate of feed, promote the proliferation of probiotics such as bifidobacteria and lactobacilli, inhibit the growth of pathogenic bacteria, improve the intestinal microecological environment, significantly improve the immune function of animals, and reduce the use of antibiotics and other chemical drugs in feed, thereby reducing pollution in breeding.

[0004] In recent years, with the in-depth development of application research on β-mannanase, it has been widely used in feed, medicine, papermaking industry and other industries. The addition of mannanase in feed can significantly improve the nutritional value and utilization rate of feed, promote animal growth, enhance the immunity and disease resistance of animals, and reduce breeding costs. For example, the addition of mannanase in broiler feed can improve the average daily gain and feed conversion rate of broilers; the addition in pig feed can enhance the immunity of pigs and reduce the incidence of disease. In the pharmaceutical industry, mannose oligosaccharides produced by mannanase hydrolysis have potential application value in the medical field. In the papermaking industry, mannanase and xylanase can work together to break the bond between hemicellulose and lignin, reduce the viscosity of pulp, improve the bleaching effect of pulp, reduce the amount of bleaching agent and chlorine, reduce the environmental pollution in the papermaking process, and improve the production efficiency and economic benefit of the papermaking industry.

[0005] Based on the broad application prospect of mannanase, its research has become a hot spot in the field of scientific research at home and abroad. At present, there are still few neutral mannanase products with good thermal stability on the market. Especially in the feed industry, the enzyme preparation process often needs to go through high-temperature granulation and other process links, and high-temperature environment can cause part of the enzyme preparation to lose activity, thereby significantly increasing the production cost. Therefore, it is of great industrial significance and broad application value to develop a new type of neutral mannanase product with high thermal stability. SUMMARY

[0006] The present application aims to provide an enzyme mutant with improved thermal stability, which has the advantages of high temperature tolerance, wide pH stability, high resistance to pepsin and trypsin, etc., as well as its gene and application.

[0007] To achieve the above-mentioned application purposes, the technical solutions of the present application are as follows:

[0008] In one aspect, the present application provides a mannanase mutant Man74s, wherein the amino acid sequence of the mannanase mutant Man74s is shown as SEQ ID NO: 2.

[0009] SEQ ID NO: 2:

[0010] MNWLAHLPNRTENRVLSAAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLETAKIEDSIDWSCNGDLISYWKNGGIPQISLHLANPAFQSGHFKTPITNDQYKKILDSSTAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFYPGASYVDIVGLDAYFQDWYSINGYDQLTALNKPFAFTEVGPQTANGSFDYSLFINAIKQKYPKTIYFLAWNDEWSPAVNKGASALYHDSWTLNKGEIWSGDSLTPIVE.

[0011] According to some embodiments of the present application, the amino acid sequence of the mannanase mutant Man74s has at least 80% homology with the sequence shown in SEQ ID NO: 2;

[0012] According to some embodiments of the present application, the amino acid sequence of the mannanase mutant Man74s has at least 85% homology with the sequence shown in SEQ ID NO: 2;

[0013] According to some embodiments of the present application, the amino acid sequence of the mannanase mutant Man74s has at least 90% homology with the sequence shown in SEQ ID NO: 2;

[0014] According to some embodiments of the present application, the amino acid sequence of the mannanase mutant Man74s has at least 95% homology with the sequence shown in SEQ ID NO: 2;

[0015] According to some embodiments of the present invention, the amino acid sequence of the mannanase mutant Man74s has at least 96% homology with the sequence shown in SEQ ID NO:2;

[0016] According to some embodiments of the present invention, the amino acid sequence of the mannanase mutant Man74s has at least 97% homology with the sequence shown in SEQ ID NO:2;

[0017] According to some embodiments of the present invention, the amino acid sequence of the mannanase mutant Man74s has at least 98% homology with the sequence shown in SEQ ID NO:2;

[0018] According to some embodiments of the present invention, the amino acid sequence of the mannanase mutant Man74s has at least 99% homology with the sequence shown in SEQ ID NO:2.

[0019] Specifically, the mannanase mutant Man74s is a mutant derived from Bacillus subtilis (… Bacillus subtilis It was obtained by site-directed mutation of the gene for β-mannanase Man74.

[0020] Furthermore, the mannanase mutant Man74s is a mannanase mutant Man74 with high temperature resistance and high tolerance to trypsin, generated by replacing multiple amino acids in the amino acid sequence as shown in SEQ ID NO:1. The amino acid substitutions are glycine at position 18 with alanine, valine at position 67 with tryptophan, and alanine at position 229 with tryptophan.

[0021] Furthermore, by Bacillus subtilis ( Bacillus subtilis The amino acid sequence of β-mannanase Man74 obtained is shown in SEQ ID NO:1.

[0022] SEQ ID NO:1:

[0023] MNWLAHLPNRTENRVLSGAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLETAKIEDSIDVSCNGDLISYWKNGGIPQISLHLANPAFQSGHFKTPITNDQYKKILDSSTAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFYPGASYVDIVGLDAYFQDAYSINGYDQLTALNKPFAFTEVGPQTANGSFDYSLFINAIKQKYPKTIYFLAWNDEWSPAVNKGASALYHDSWTLNKGEIWSGDSLTPIVE.

[0024] The enzyme gene shown in SEQ ID NO: 1 encodes 319 amino acids, and the theoretical molecular weight of the mannanase Man74 is 36.2 kDa.

[0025] In another aspect, the present application provides a gene encoding the above-mentioned mannanase mutant Man74s.

[0026] Specifically, the gene is a mannanase mutant gene man74s , and the nucleotide sequence is shown in SEQ ID NO: 3.

[0027] SEQ ID NO: 3:

[0028] ATGAACTGGTTGGCTCACTTGCCAAACAGAACTGAAAACAGAGTTTTGTCAGcTGCTTTTGGTGGTTACTCTCATGATACTTTTTCTATGGCTGAAGCTGATAGAATTAGATCCGCTACTGGTCAATCTCCAGCTATCTACGGTTGTGATTACGCTAGAGGTTGGTTGGAAACTGCTAAGATTGAAGATTCTATTGATtggTCTTGTAACGGAGATTTGATTTCTTACTGGAAGAACGGTGGTATTCCTCAAATTTCTTTGCATTTGGCTAACCCAGCTTTTCAATCTGGTCATTTTAAGACTCCTATTACTAACGATCAATATAAGAAGATTTTGGATTCATCTACTGCTGAAGGTAAAAGATTGAACGCTATGTTGTCTAAGATTGCTGATGGTTTGCAAGAATTGGAAAACCAGGGTGTTCCAGTTTTGTTTAGACCATTGCATGAAATGAACGGTGAATGGTTCTGGTGGGGTTTGACTTCTTACAACCAAAAGGATAACGAAAGAATTTCTTTGTACAAGCAATTGTACAAGAAGATATACCATTACATGACTGATACTAGAGGTCTGGATCATTTGATTTGGGTTTACTCCCCTGATGCTAACAGAGATTTTAAGACTGATTTTTACCCAGGTGCTTCCTACGTTGATATTGTTGGATTGGATGCTTACTTTCAAGATtggTACTCTATTAACGGTTACGATCAACTGACTGCTTTGAACAAGCCATTCGCTTTTACTGAAGTTGGTCCACAAACTGCTAATGGTTCTTTTGATTACTCATTGTTTATTAACGCTATTAAGCAAAAGTACCCTAAGACTATCTACTTCTTGGCTTGGAACGATGAATGGTCTCCAGCTGTTAACAAGGGTGCTTCTGCTTTGTACCATGATTCTTGGACTTTGAACAAGGGTGAAATTTGGTCTGGAGATTCTTTGACTCCAATTGTTGAA.

[0029] Specifically, the application synthesizes the mannanase mutant gene by a gene synthesis method man74s The DNA full sequence analysis result shows that the mannanase Man74S structure gene man74s is 957 bp in full length.

[0030] In another aspect, the application provides a recombinant vector containing the above-mentioned mannanase mutant gene man74s .

[0031] Specifically, the vector is selected from pPIC9K, pET-28a, pET-30a, pUC18, pFastBac or pPICZα.

[0032] Further, the vector is pPIC9K.

[0033] Specifically, the recombinant vector is ppIC . man74s .

[0034] According to some embodiments of the application, the preparation method of the ppIC . man74s is as follows: the expression vector is double enzyme cut, and the gene encoding the mannanase mutant is double enzyme cut, and the gene fragment after enzyme cutting is connected with the vector to obtain the recombinant plasmid containing the mannanase gene man74s . man74s pPIC- man74s .

[0035] Specifically, the double enzyme cutting is EcoR I Not I.

[0036] The mannanase gene of the application is inserted between the suitable restriction enzyme cutting sites of the expression vector, so that the nucleotide sequence is operable and connected with the expression control sequence.

[0037] According to some embodiments of the application, the mannanase gene of the application is inserted between the EcoR I and Not I restriction enzyme cutting sites of the plasmid pPIC9, so that the nucleotide sequence is downstream of the AOX1 promoter and is controlled by the AOX1 promoter, to obtain the recombinant yeast expression plasmid pPIC9- man74s .

[0038] In another aspect, the application provides a recombinant strain containing the above-mentioned mannanase mutant gene man74s .

[0039] Specifically, the strain is Escherichia coli, yeast, Bacillus or Lactobacillus.

[0040] According to some embodiments of the application, the recombinant strain is GS115 / man74s ​.

[0041] In another aspect, the present invention provides a method for preparing the mannanase mutant Man74s, comprising the following steps:

[0042] 1) Transform host cells using a recombinant vector to obtain recombinant bacterial strains;

[0043] 2) Cultivate recombinant strains and induce recombinant mannanase expression;

[0044] 3) The expressed mannanase Man74s was recovered and purified.

[0045] Specifically, the preparation method of the recombinant vector in step 1) is as follows:

[0046] The expression vector was double-digested, and the gene encoding the mannanase mutant was simultaneously extracted. man74s The gene fragments were subjected to double enzyme digestion, and then ligated with the vector to obtain the gene containing mannanase. man74s recombinant plasmid pPIC- man74s .

[0047] Specifically, the double enzyme digestion is EcoR I+ Not I.

[0048] Specifically, in step 1), the host cell is Pichia pastoris cell, brewer's yeast cell, or polymorphonuclear yeast cell.

[0049] Furthermore, in step 1), the host cell is a Pichia pastoris cell.

[0050] According to some embodiments of the present invention, step 1) includes transforming Pichia pastoris cells with a recombinant yeast expression plasmid. Pichia pastoris GS115 was used to obtain a recombinant strain. GS115 / man74s .

[0051] According to some embodiments of the present invention, the culture conditions for culturing the recombinant strain in step 2) are as follows: inoculated into BMGY culture medium, cultured at 30°C and 250 rpm for 48 h with shaking, and the bacterial cells are collected by centrifugation.

[0052] In another aspect, the present invention provides the aforementioned mannanase mutant Man74s and mannanase mutant gene. man74s Applications of recombinant vectors and recombinant strains in feed, food processing, and papermaking.

[0053] Specifically, when applied in the feed industry, it can reduce or eliminate the anti-nutritional effects caused by increased viscosity, and is especially suitable for the rumen environment of cattle and sheep.

[0054] Specifically, when applied in food processing, it can be used as a clarifying agent for fruit juice beverages.

[0055] Specifically, when applied to papermaking, it can cooperate with xylanase to destroy the bond between hemicellulose and lignin, reduce the amount of bleaching agent and chlorine, and thus protect the environment.

[0056] The beneficial effects of the present application are:

[0057] The mannanase mutant Man74s of the present application has high thermal stability, and high activity in the acidic and neutral ranges at room temperature, and resistance to protease degradation. The mannanase mutant of the present application has an optimal pH of 6.0, and has high catalytic activity in the pH range of 5.0-7.0; the optimal temperature is 50℃, and the residual enzyme activity of the mannanase Man74s is still above 70% after being treated at 85℃ for 3min, and is particularly suitable for the rumen environment of cattle, sheep and other animals. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Optimal pH of the recombinant mannanase mutant

[0059] Figure 2 pH stability of the recombinant mannanase mutant

[0060] Figure 3 Optimal temperature of the recombinant mannanase mutant

[0061] Figure 4 Thermal stability of the recombinant mannanase mutant

[0062] Figure 5 Enzyme activity after trypsin treatment

[0063] Figure 6 Enzyme activity after pepsin treatment DETAILED DESCRIPTION

[0064] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the following specific embodiments are further described, but the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the following embodiments, if not otherwise specified, the operation methods used are conventional operation methods, and the equipment used is conventional equipment, and the equipment materials used in each embodiment are the same.

[0065] Test materials and reagents of the present application:

[0066] 1. Strains and vectors: The mannanase mutant gene in the present application man74sThe Pichia pastoris expression vector was synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd. pPIC9 and strains GS115 Purchased from Invitrogen.

[0067] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from TaKaRa, ligases from Invitrogen, mannan from Sigma, and all other reagents were domestically produced (available from general biochemical reagent companies).

[0068] 3. Culture medium

[0069] (1) Yeast culture medium YPD: 1% peptone, 0.5% yeast extract, 1% glucose, 2% agar, pH 7.0.

[0070] (2) Escherichia coli culture medium LB: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0071] (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V / V).

[0072] (4) BMMY medium: except that 0.5% methanol is used instead of glycerol, the other components are the same as BMGY, pH 4.0.

[0073] Note: Molecular biology experimental methods not specifically described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0074] Example 1 Bacillus subtilis Bacillus subtilis Mannanase mutant encoding gene Man74s Synthesis

[0075] This invention utilizes β-mannanase derived from Bacillus subtilis. man74 Using the mature structural gene as a reference, its sequence was mutated as follows (G18A, V67W, A229W), and the sequence was sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for artificial gene synthesis. The amino acid sequence of the artificially synthesized mannanase mutant is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.3.

[0076] Example 2 Mannanase mutant gene man74s Cloning

[0077] Extract the gene vector carrying the mannanase mutant:

[0078] The synthesized gene vector was stored in the form of piercing bacteria, and the piercing bacteria were picked up with a sterile toothpick in a clean bench and placed in an LB shaking tube containing Amp (working concentration: 100 μg / ml) antibiotic, and cultured at 37°C, 220 rpm overnight. The next day, the mutant gene-containing vector was extracted according to the steps of the Kanghao plasmid extraction kit PurePlasmid Mini Kit (CW0500) instruction manual.

[0079] According to the sequence of the mannase mutant gene, the following primers were designed and synthesized:

[0080] P1: 5'-GAATTCATGAACTGGTTGGCTCACTTGC-3' (SEQ ID NO: 4);

[0081] P2: 5'-GCGGCCGCTTCAACAATTGGAGTCAAAGAATCT-3' (SEQ ID NO: 5).

[0082] The extracted vector was used as a template for PCR amplification. The PCR reaction parameters were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1 min, 30 cycles, and then 72°C for 10 min. A fragment of about 971 bp was obtained, which was recovered and connected to pMD19 vector and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 36.2 kDa.

[0083] According to the nucleotide sequence obtained by sequencing, the obtained nucleotide sequence was compared with the man74 sequence by DNAMan software, and it was confirmed that the mutations at positions G18A, V67W, and A229W were correct.

[0084] Example 3 Preparation of recombinant mannase

[0085] The expression vector pPIC9 was double-digested Eco R I+ Not I), and the gene man74s encoding the mannase mutant was also double-digested Eco R I+ Not I). pPIC9 The gene fragment encoding the mature mannase was digested and connected to the expression vector man74s to obtain a recombinant plasmid pPIC- man74s containing the mannase gene GS115 . GS115 man74s ​​

[0086] The recombinant plasmid containing GS115 The strain with the recombinant plasmid and the control strain (i.e. the strain GS115 / man74 ) were inoculated into 300 mL of BMGY culture solution and cultured at 30°C with 250 rpm shaking for 48 h, and then the bacterial cells were collected by centrifugation. Then the bacterial cells were resuspended in 150 mL of BMMY culture medium and cultured at 30°C with 250 rpm shaking. After 72 h of induction, the supernatant was collected by centrifugation, and the activity of the recombinant mannanase was determined.

[0087] Example 4: Activity analysis of the recombinant mannanase Man74S

[0088] DNS method: The specific method is as follows: 1 mL of the reaction system includes 100 μL of the appropriate diluted enzyme solution, 900 μL of the substrate, and the reaction is performed at 37°C and pH 7.0 for 10 min, 1.5 mL of DNS is added to terminate the reaction, and boiling water is boiled for 5 min. After cooling, the OD value is determined at 540 nm.

[0089] Definition of the activity unit of β-mannanase: under certain conditions, the amount of enzyme required to decompose 1 μmol of β-mannan to produce reducing sugar per minute is 1 activity unit (U). The expression amount of the recombinant mannanase is 3500 U / mL, and the expression amount of the control group mannanase is 3700 U / mL. The SDS-PAGE result shows that the recombinant mannanase is expressed in Pichia pastoris.

[0090] Example 5: Property determination of the recombinant mannanase Man74S

[0091] The recombinant high-temperature-resistant mannanase Man74S and the unmutated acid protease Man74 were subjected to enzymatic property determination and comparison. Meanwhile, the mutant recombinant acid protease Man74S2 was added for comparison of the enzymatic properties, and the sequence of Man74S2 is based on SEQ ID NO. 1 with V67Y and A229F mutations. The preparation method of the Man74S mutant and the Man74S2 recombinant enzyme is as described above.

[0092] 1. The determination method of the optimum pH and pH stability of the recombinant mannanase Man74S is as follows:

[0093] The purified mannanases Man74, Man74S and Man74S2 were subjected to enzymatic reactions under different pH conditions to determine the optimum pH. The buffer solutions used were KCl-HCl buffer solution with pH 0.5-2.2, citric acid-disodium hydrogen phosphate buffer solution with pH 2.2-8.0, and Tris-HCl buffer solution with pH 8.0-10.0. The results are as follows: Figure 1) showed that the optimum pH of the three enzymes were all 6.0, and they all had high enzyme activity in the pH range of 5.0-7.0. The mutant Man74S still had relatively high enzyme activity at pH 4.0, and the optimum pH range was improved to some extent.

[0094] The purified mannanases Man74, Man74S and Man74S2 were treated in the above-mentioned buffers of different pH at 37°C for 60 min, and then the enzyme activity was determined in a buffer system at pH 6.0 at 37°C to study the pH resistance of the enzymes. The results (Fig. 3) showed that the recombinant mannanase Man74s was very stable between pH 4.0 and 10.0, and still maintained more than 70% of the enzyme activity after being treated for 60 min in this pH range, indicating that the enzyme had good pH stability. Compared with the control group mannanases Man74 and Man74S2, the pH stability of the recombinant mannanase Man74s in the pH range of 4.0-10.0 was improved to some extent. Figure 2

[0095] 2. The optimum temperature and thermal stability of the mannanase were determined as follows:

[0096] The determination of the optimum temperature of the mannanase was to determine the enzyme activity of the recombinant mannanase Man74s and the control group mannanases Man74 and Man74S2 at different temperatures (20-85°C) at pH 6.0. The optimum reaction temperature of the three recombinant mannanases was 50°C (Fig. 4). Figure 3

[0097] The determination of the temperature resistance was to treat the mannanase at 85°C for 3 min, and then determine the enzyme activity at 37°C. The temperature resistance experiment showed that the residual enzyme activity of the recombinant mannanase Man74s was still more than 70% after being treated at 85°C for 3 min (Fig. 5), while the control group mannanases Man74 and Man74S2 lost most of the enzyme activity after being treated at 85°C for 3 min, indicating that the thermal stability of the recombinant mannanase Man74s was improved significantly compared with the control group, and it could adapt to the high-temperature granulation process. Figure 4

[0098] 3. The determination of the anti-pepsin and trypsin ability of the recombinant mannanase Man74s was as follows:

[0099] ​​​0.1 mg / mL pepsin was prepared with pH 2.0 KCl-HCl buffer, 0.1 mg / mL trypsin was prepared with pH 7.0 Tris-HCl buffer. 0.5 mL purified enzyme solution diluted with pH 2.0 KCl-HCl buffer was mixed with 0.5 mL pepsin, 0.5 mL purified enzyme solution diluted with pH 7.0 Tris-HCl buffer was mixed with 0.5 mL trypsin, the ratio of protease / mannase (w / w) was about 0.1, and the mixture was incubated at 37℃ for 60 and 120 min, and then sampled, and the enzyme activity was determined at pH 7.0 and 60℃.

[0100] The experimental results show that (see Table 1) Figure 5- Figure 6 ), after 120 min of pepsin and trypsin treatment, the enzyme activity of Man74S did not decrease compared with that before treatment (the enzyme activity before treatment was 100%). The enzyme activity of the control group of mannase after trypsin treatment was 85% of that before treatment, and the enzyme activity after pepsin treatment was 80% of that before treatment. The above results show that the recombinant mannase Man74S after mutation has improved resistance to pepsin and trypsin, and has good resistance to pepsin and trypsin.

[0101] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A mannanase mutant Man74s, characterized in that, The amino acid sequence of the mannanase mutant Man74s is shown as SEQ ID NO:

2.

2. A gene encoding the mannanase mutant Man74s of claim 1.

3. The gene of claim 2, wherein The gene is a mannanase mutant gene man74s The nucleotide sequence is shown as SEQ ID NO:

3.

4. A recombinant vector comprising the gene of any one of claims 2-3.

5. The recombinant vector of claim 4, wherein, The vector is selected from pPIC9K, pET-28a, pET-30a, pUC18, pFastBac or pPICZα.

6. The recombinant vector of claim 5, wherein, The vector is pPIC9K.

7. The recombinant vector of claim 6, wherein, The recombinant vector is ppIC - man74s The aforementioned ppIC - man74s The preparation method is as follows: the expression vector is double-digested, and the gene encoding the mannanase mutant is simultaneously added. man74s The gene fragments were subjected to double enzyme digestion, and then ligated with the vector to obtain the gene containing mannanase. man74s recombinant plasmid pPIC- man74s .

8. A recombinant strain comprising the gene of any one of claims 2-3.

9. A method for preparing the mannanase mutant Man74s of claim 1, characterized by, The method comprises the following steps: 1) transforming a host cell with the recombinant vector to obtain a recombinant strain; 2) culturing the recombinant strain to induce expression of the recombinant mannanase; 3) recovering and purifying the expressed mannanase mutant Man74s.

10. Use of the mannanase mutant Man74s of claim 1, the gene of any one of claims 2-3, the recombinant vector of any one of claims 4-7, or the recombinant strain of claim 8 in feed, food processing, papermaking.

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