Enzyme mutant with improved thermal stability as well as gene and application thereof

Abstract

The invention discloses an enzyme mutant with improved thermal stability and a gene and application thereof, and relates to the technical field of gene engineering and enzyme engineering. The invention provides a mannase mutant Man74s, wherein the amino acid sequence of the mannase mutant Man74s is as shown in SEQ ID NO: 2. The mannase mutant Man74s disclosed by the invention has high thermal stability and has the characteristics of high activity, protease degradation resistance and the like in acidic and neutral ranges at normal temperature, the optimal pH value of the mannase mutant Man74s is 6.0, and the mannase mutant Man74s has relatively high catalytic activity in a pH range of 5.0-7.0; the optimal temperature is 50 DEG C, the residual enzyme activity is still 70% or above after mannase Man74s is treated at 85 DEG C for 3 min, and the mannase Man74s is suitable for rumen animal environments such as cattle and 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, high temperature resistance, wide pH stability, high resistance to pepsin and trypsin, and the like, as well as a gene and application thereof.

[0007] To achieve the above-mentioned application purposes, the technical solutions of the present application are as follows: In one aspect, the present application provides a mannanase mutant Man74s, wherein the amino acid sequence of the mannanase mutant Man74s is shown in SEQ ID NO: 2.

[0008] SEQ ID NO: 2: MNWLAHLPNRTENRVLSAAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLETAKIEDSIDWSCNGDLISYWKNGGIPQISLHLANPAFQSGHFKTPITNDQYKKILDSSTAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFYPGASYVDIVGLDAYFQDWYSINGYDQLTALNKPFAFTEVGPQTANGSFDYSLFINAIKQKYPKTIYFLAWNDEWSPAVNKGASALYHDSWTLNKGEIWSGDSLTPIVE.

[0009] 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; 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; 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; 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; 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; 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; 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; 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.

[0010] 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.

[0011] 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.

[0012] Furthermore, by Bacillus subtilis ( Bacillus subtilis The amino acid sequence of β-mannanase Man74 obtained is shown in SEQ ID NO:1.

[0013] SEQ ID NO:1: MNWLAHLPNRTENRVLSGAFGGYSHDTFSMAEADRIRSATGQSPAIYGCDYARGWLETAKIEDSIDVSCNGDLISYWKNGGIPQISLHLANPAFQSGHFKTPITNDQYKKILDSSTAEGKRLNAMLSKIADGLQELENQGVPVLFRPLHEMNGEWFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFYPGASYVDIVGLDAYFQDAYSINGYDQLTALNKPFAFTEVGPQTANGSFDYSLFINAIKQKYPKTIYFLAWNDEWSPAVNKGASALYHDSWTLNKGEIWSGDSLTPIVE.

[0014] 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.

[0015] In another aspect, the present application provides a gene encoding the above-mentioned mannanase mutant Man74s.

[0016] Specifically, the gene is a mannanase mutant gene man74s , and the nucleotide sequence is shown in SEQ ID NO: 3.

[0017] SEQ ID NO: 3: ATGAACTGGTTGGCTCACTTGCCAAACAGAACTGAAAACAGAGTTTTGTCAGcTGCTTTTGGTGGTTACTCTCATGATACTTTTTCTATGGCTGAAGCTGATAGAATTAGATCCGCTACTGGTCAATCTCCAGCTATCTACGGTTGTGATTACGCTAGAGGTTGGTTGGAAACTGCTAAGATTGAAGATTCTATTGATtggTCTTGTAACGGAGATTTGATTTCTTACTGGAAGAACGGTGGTATTCCTCAAATTTCTTTGCATTTGGCTAACCCAGCTTTTCAATCTGGTCATTTTAAGACTCCTATTACTAACGATCAATATAAGAAGATTTTGGATTCATCTACTGCTGAAGGTAAAAGATTGAACGCTATGTTGTCTAAGATTGCTGATGGTTTGCAAGAATTGGAAAACCAGGGTGTTCCAGTTTTGTTTAGACCATTGCATGAAATGAACGGTGAATGGTTCTGGTGGGGTTTGACTTCTTACAACCAAAAGGATAACGAAAGAATTTCTTTGTACAAGCAATTGTACAAGAAGATATACCATTACATGACTGATACTAGAGGTCTGGATCATTTGATTTGGGTTTACTCCCCTGATGCTAACAGAGATTTTAAGACTGATTTTTACCCAGGTGCTTCCTACGTTGATATTGTTGGATTGGATGCTTACTTTCAAGATtggTACTCTATTAACGGTTACGATCAACTGACTGCTTTGAACAAGCCATTCGCTTTTACTGAAGTTGGTCCACAAACTGCTAATGGTTCTTTTGATTACTCATTGTTTATTAACGCTATTAAGCAAAAGTACCCTAAGACTATCTACTTCTTGGCTTGGAACGATGAATGGTCTCCAGCTGTTAACAAGGGTGCTTCTGCTTTGTACCATGATTCTTGGACTTTGAACAAGGGTGAAATTTGGTCTGGAGATTCTTTGACTCCAATTGTTGAA.

[0018] 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.

[0019] In another aspect, the application provides a recombinant vector comprising the above-mentioned mannanase mutant gene man74s .

[0020] Specifically, the vector is selected from pPIC9K, pET-28a, pET-30a, pUC18, pFastBac or pPICZα.

[0021] Further, the vector is pPIC9K.

[0022] Specifically, the recombinant vector is ppIC . man74s .

[0023] 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, the gene fragment after enzyme cutting is connected with the vector to obtain the recombinant plasmid containing the mannanase gene man74s . man74s pPIC- man74s .

[0024] Specifically, the double enzyme cutting is EcoR I Not I.

[0025] 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 operably connected with the expression control sequence.

[0026] 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 on 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 .

[0027] In another aspect, the application provides a recombinant strain comprising the above-mentioned mannanase mutant gene man74s .

[0028] Specifically, the strain is Escherichia coli, yeast, Bacillus or Lactobacillus.

[0029] According to some embodiments of the application, the recombinant strain is GS115 / man74s ​.

[0030] In another aspect, the present invention provides a method for preparing the mannanase mutant Man74s, comprising the following steps: 1) Transform host cells using a recombinant vector to obtain recombinant bacterial strains; 2) Cultivate recombinant strains and induce recombinant mannanase expression; 3) The expressed mannanase Man74s was recovered and purified.

[0031] Specifically, the preparation method of the recombinant vector in step 1) is as follows: 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 .

[0032] Specifically, the double enzyme digestion is EcoR I+ Not I.

[0033] Specifically, in step 1), the host cell is Pichia pastoris cell, brewer's yeast cell, or polymorphonuclear yeast cell.

[0034] Furthermore, in step 1), the host cell is a Pichia pastoris cell.

[0035] According to some embodiments of the present invention, step 1) includes transforming a recombinant yeast expression plasmid into Pichia pastoris cells (…). Pichia pastoris GS115 was used to obtain a recombinant strain. GS115 / man74s .

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Specifically, when applied in food processing, it can be used as a clarifying agent for fruit juice beverages.

[0040] 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.

[0041] The beneficial effects of the present application are: 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 treatment at 85℃ for 3min, and is particularly suitable for the rumen environment of cattle, sheep and other animals. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Optimal pH of the recombinant mannanase mutant.

[0043] Figure 2 pH stability of the recombinant mannanase mutant.

[0044] Figure 3 Optimal temperature of the recombinant mannanase mutant.

[0045] Figure 4 Thermal stability of the recombinant mannanase mutant.

[0046] Figure 5 Enzyme activity after trypsin treatment.

[0047] Figure 6 Enzyme activity after pepsin treatment. DETAILED DESCRIPTION

[0048] 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 to illustrate the present application, 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.

[0049] Test materials and reagents of the present application: 1. Strains and vectors: The mannanase mutant gene in the present application man74s was synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd., and the Pichia pastoris expression vector pPIC9 and strain GS115 were purchased from Invitrogen Company.

[0050] 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).

[0051] 3. Culture medium (1) Yeast culture medium YPD: 1% peptone, 0.5% yeast extract, 1% glucose, 2% agar, pH 7.0.

[0052] (2) Escherichia coli culture medium LB: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0053] (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V / V).

[0054] (4) BMMY medium: except that 0.5% methanol is used instead of glycerol, the other components are the same as BMGY, pH 4.0.

[0055] 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.

[0056] Example 1 Bacillus subtilis Bacillus subtilis Mannanase mutant encoding gene Man74s Synthesis 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.

[0057] Example 2 Mannanase mutant gene man74s Cloning Extract the gene vector carrying the mannanase mutant: The synthesized gene vector was preserved as a puncture culture. The puncture culture was picked up with a sterile toothpick in a clean bench and placed in an LB shaker containing antibiotic Amp (working concentration: 100 μg / ml). It was cultured overnight at 37°C and 220 rpm. The next day, the vector containing the mutant gene was extracted according to the instructions of the Kangwei Century Plasmid Extraction Kit PurePlasmid Mini Kit (CW0500).

[0058] Based on the mannanase mutant gene sequence, the following primers were designed and synthesized: P1:5'-GAATTCATGAACTGGTTGGCTCACTTGC-3' (SEQ ID NO:4); P2: 5'-GCGGCCGCTTCAACAATTGGAGTCAAAGAATCT-3' (SEQ ID NO: 5).

[0059] PCR amplification was performed using the extracted vector as a template. The PCR reaction parameters were: denaturation at 94℃ for 5 min; followed by denaturation at 94℃ for 30 sec, annealing at 55℃ for 30 sec, extension at 72℃ for 1 min, and 30 cycles, followed by incubation at 72℃ for 10 min. A fragment of approximately 971 bp was obtained. This fragment was recovered, ligated into the pMD19 vector, and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 36.2 kDa.

[0060] Based on the nucleotide sequences obtained from sequencing, the DNAMan software was used to compare the obtained nucleotide sequences with... man74 Sequence alignment confirmed that the mutations at the three positions G18A, V67W, and A229W were correct.

[0061] Example 3 Preparation of recombinant mannanase expression carrier pPIC9 Double enzyme digestion ( Eco R I+ Not I) Simultaneously, the gene encoding the mannanase mutant will be... man74s Double enzyme digestion ( Eco R I+ Not I) The gene fragment encoding mature mannanase and its expression vector were digested using enzymes. pPIC9 Linkage was performed to obtain the mannanase gene. man74s recombinant plasmid pPIC- man74s And transform Pichia pastoris GS115 Recombinant Pichia pastoris strain was obtained GS115 / man74s .

[0062] Take the sample containing the recombinant plasmid. GS115 Strains and control strains (i.e., unmutated strains)GS115 / man74 ), and after 48 h of cultivation at 30°C and 250 rpm, the cells were collected by centrifugation. Then the cells were resuspended in 150 mL of BMMY medium and cultivated at 30°C and 250 rpm. After 72 h of induction, the supernatant was collected by centrifugation and the activity of the recombinant mannanase was determined.

[0063] Example 4 Activity analysis of recombinant mannanase Man74s DNS method: The specific method is as follows: 1 mL of reaction system includes 100 μL of appropriately diluted enzyme solution, 900 μL of substrate, reaction 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.

[0064] Definition of β-mannanase activity unit: under certain conditions, the amount of enzyme required to decompose 1 μmol of 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.

[0065] Example 5 Property determination of recombinant mannanase Man74s The enzymatic properties of the recombinant high-temperature-resistant mannanase Man74S and the non-mutated acid protease Man74 were determined and compared. At the same time, the mutant recombinant acid protease Man74S2 was added for comparison of enzymatic properties. The sequence of Man74S2 is based on SEQ ID NO. 1 and has V67Y and A229F mutations. The preparation method of the Man74S mutant and the Man74S2 recombinant enzyme is as described above.

[0066] 1. The determination method of the optimum pH and pH stability of the recombinant mannanase Man74s is as follows: The purified mannanases Man74, Man74S and Man74S2 were subjected to enzymatic reaction under different pH conditions to determine the optimum pH. The buffer used was KCl-HCl buffer with pH 0.5-2.2, citric acid-disodium hydrogen phosphate series buffer with pH 2.2-8.0, and Tris-HCl series buffer with pH 8.0-10.0. The results (Table 1) show that the optimum pH of the three is 6.0, and they all have high enzyme activity under the condition of 5.0-7.0. The mutant Man74S still has relatively high enzyme activity under the condition of pH 4.0, and the optimum pH range is improved to a certain extent. Figure 1

[0067] ​Purified mannanases Man74, Man74S, and Man74S2 were treated at 37°C for 60 min in various buffer solutions with different pH values, and then their activity was measured at 37°C in a pH 6.0 buffer system to study the enzyme's pH tolerance. Results ( Figure 2 The results showed that the recombinant mannanase Man74s was very stable between pH 4.0 and 10.0, maintaining more than 70% of its activity even after 60 min of treatment within this pH range, indicating that the enzyme has good pH stability. Compared with the control mannanases Man74 and Man74S2, the pH stability of the recombinant mannanase Man74s was improved to a certain extent under pH 4.0-10.0 conditions.

[0068] 2. The optimal temperature and thermal stability determination method for mannanase are as follows: The optimal temperature for mannanase was determined by measuring the enzymatic activity of recombinant mannanase Man74s and control mannanases Man74 and Man74S2 at different temperatures (20-85℃) under pH 6.0 conditions. The optimal reaction temperature for the three recombinant mannanases was 50℃. Figure 3 ).

[0069] The thermostability test involved treating the mannanase at 85℃ for 3 min, followed by enzyme activity measurement at 37℃. The thermostability experiment showed that after treatment at 85℃ for 3 min, the recombinant mannanase Man74s still retained over 70% of its original enzyme activity. Figure 4 The control group of mannanases Man74 and Man74S2 lost most of their enzyme activity after being treated at 85℃ for 3 minutes, indicating that the thermal stability of recombinant mannanase Man74s was significantly improved compared with the control group, and it can adapt to high-temperature granulation and other processing processes.

[0070] 3. The anti-pepsin and anti-trypsin capabilities of recombinant mannanase Man74s were determined as follows: Prepare 0.1 mg / mL pepsin using pH 2.0 KCl-HCl buffer and 0.1 mg / mL trypsin using pH 7.0 Tris-HCl buffer. Add 0.5 mL of pepsin to 0.5 mL of purified enzyme solution diluted in pH 2.0 KCl-HCl buffer, and add 0.5 mL of trypsin to 0.5 mL of purified enzyme solution diluted in pH 7.0 Tris-HCl buffer. The pepsin / mannanase (w / w) ratio is approximately 0.1. Incubate at 37℃ for 60 and 120 min, and then measure enzyme activity at pH 7.0 and 60℃.

[0071] Experimental results show that ( Figure 5- Figure 6After 120 min of treatment with pepsin and trypsin, the enzyme activity of Man74S did not decrease compared with the untreated enzyme (100% of the untreated enzyme). The enzyme activity of the control mannanase was 85% of the untreated enzyme after treatment with trypsin and 80% of the untreated enzyme after treatment with pepsin. The above results show that the mutant recombinant mannanase Man74S has improved resistance to pepsin and trypsin and has good resistance to pepsin and trypsin.

[0072] The above merely describes the preferred embodiments of the present application and is not used to limit the present application. 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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