Enzyme mutant with improved thermostability, gene thereof and use thereof

Abstract

The present invention relates to the fields of genetic engineering and enzyme engineering. Provided are an enzyme mutant with improved thermostability, a gene thereof and the use thereof. Provided is a β-mannanase mutant. Compared with an amino acid sequence as shown in SEQ ID NO: 2, the β-mannanase mutant contains at least one of the following mutations: V86Y, A248P, and H317N. The β- mannanase mutant has an optimum pH of 6.0, has a relatively good enzymatic activity, pH stability and thermostability at pH 3.5-7.0, and has a relatively good protease resistance. The β-mannanase mutant has broad application prospects in food, feed, papermaking and textiles.

Description

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

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

[0002] Mannan is a kind of non-starch polysaccharide, which is the main component of hemicellulose, and its content in hemicellulose is only second to xylan. According to the structural components of mannan, it can be divided into four subtypes: homogeneous mannan, galactomannan, glucomannan and galactoglucomannan. The four kinds of mannan all have a main chain structure connected by β-1, 4-glycosidic bond of mannose or mannose and glucose. Mannan widely exists in plant food and animal feed, but as an anti-nutritional factor, it can bind a large amount of water in the digestive tract of monogastric animals such as poultry and pigs, thereby increasing the viscosity of the contents in the digestive tract of animals and resisting the peristalsis of the digestive tract of animals. Adding β-mannanase in feed can hydrolyze mannan into mannooligosaccharides, thereby effectively solving the anti-nutritional problem caused by mannan.

[0003] β-mannanase is a general term for a class of enzymes that can degrade mannan into oligomannose and mannose, mainly including β-1, 4-endomannanase, β-1, 4-exomannanase and β-mannosidase. It can degrade mannan into smaller sugar components and is widely used in food, feed, medicine and papermaking industries.

[0004] In recent years, with the in-depth development of the application research of β-mannanase, in the pharmaceutical industry, mannooligosaccharides produced by mannanase hydrolysis are used as antitumor agents. In the food industry, mannanase can be used to improve the clarity of fruit juice and increase the amount of fruit juice. In the feed industry, supplementing β-mannanase in feed in combination with other enzymes can significantly improve the feed conversion efficiency, weight gain and production performance indicators of broilers. In the papermaking industry, mannanase can synergistically act with xylanase to destroy the bond between hemicellulose and lignin, thereby reducing the amount of bleaching agent and chlorine.

[0005] A kind of mannanase is disclosed in Chinese patent CN102994479A, the amino acid sequence of the mannanase is provided in the patent, the enzyme is transformed into Trichoderma reesei and Pichia respectively, and recombinant expression engineering strain is constructed.The present application provides a novel mannanase gene, and recombinant expression engineering bacteria of Pichia and Trichoderma reesei are constructed respectively.The optimal pH of the mannanase produced by the Pichia and Trichoderma reesei engineering bacteria is 4.5, and the optimal temperature is 60 DEG C.The mannanase of the application is acid-resistant, and can be widely used as a feed additive, which can effectively improve the utilization rate of feed, thereby reducing the use amount of feed in breeding, saving grain resources and breeding cost, and increasing production efficiency.But its poor thermal stability limits its application prospect.

[0006] A kind of mannanase and its application are disclosed in Chinese patent CN103667216A, the amino acid sequence of the mannanase is disclosed, and the gene encoding the mannanase and the application of the mannanase in feed are disclosed, the recombinant expression engineering bacteria of Pichia is constructed by introducing the mannanase gene of Bacillus subtilis into Pichia.The Pichia engineering bacteria can efficiently express mannanase, and the optimal pH of the recombinant mannanase is 4, and the optimal temperature is 58 DEG C.Under the condition of reducing the energy value of daily ration, by adding the mannanase of the application in daily ration, the utilization rate of feed can be significantly improved, the daily weight gain and fattening index of broiler chickens are improved, the feed conversion ratio is reduced, the grain resources and breeding cost are saved, and the production efficiency is increased.

[0007] Based on the broad application prospect of mannanase, the research of mannanase has become a hot spot at home and abroad.At present, the neutral mannanase products with good thermal stability in the market are still few, and in the feed industry, the enzyme preparation process often needs to be granulated at high temperature, and the high-temperature processing process will cause most of the enzyme preparations to lose part of the enzyme activity, thereby increasing the production cost.Therefore, it has important industrial significance and application value to develop a new type of neutral mannanase product with high thermal stability.

[0008] SUMMARY

[0009] The purpose of the present application is to provide an enzyme mutant with improved thermal stability, as well as its gene and application, which has good pH stability, thermal stability and protease resistance.

[0010] To achieve the above-mentioned purpose of the application, the technical solutions of the present application are as follows:

[0011] In one aspect, the present application provides a beta-mannanase mutant, which contains at least one of the following mutations: V86Y, A248P, H317N, compared with the amino acid sequence of SEQ ID NO: 2.

[0012] Specifically, the beta-mannanase mutant is obtained by replacing a plurality of amino acids in the sequence of SEQ ID NO: 2, and the amino acid replacement is specifically: replacing the valine at position 86 with tyrosine, replacing the alanine at position 248 with phenylalanine, and replacing the histidine at position 317 with asparagine.

[0013] Specifically, the amino acid sequence of SEQ ID NO: 2 is a mature beta-mannanase Man27, which is derived from Bacillus subtilis.

[0014] The theoretical molecular weight of the mature mannase Man27 is 38.3 kDa, and the amino acid sequence is shown in SEQ ID NO: 2.

[0015] SEQ ID NO: 2:

[0016] Preferably, the amino acid sequence of the beta-mannanase mutant is shown in SEQ ID NO: 4.

[0017] SEQ ID NO: 4:

[0018] Specifically, the beta-mannanase mutant of the present application is a beta-mannanase Man27 obtained from Bacillus subtilis, and the amino acid sequence of the beta-mannanase Man27 is SEQ ID NO: 1.

[0019] SEQ ID NO: 1:

[0020] Specifically, the enzyme gene encodes 362 amino acids, and the N-terminal 24 amino acids are its predicted signal peptide sequence "MFKKHTISLLILFLLASAVLAKPI" (SEQ ID NO: 3).

[0021] In another aspect, the present application provides a beta-mannanase mutant gene, which encodes the amino acid sequence shown in SEQ ID NO: 4.

[0022] Specifically, the nucleotide sequence of the beta-mannanase mutant gene is shown as SEQ ID NO: 5.

[0023] SEQ ID NO: 5:

[0024] Specifically, the above-mentioned beta-mannanase mutant gene is called mannanase mutant gene man27-4s.

[0025] The present application synthesizes the mannanase mutant gene man27-4s by a gene synthesis method, and the DNA full sequence analysis result shows that the full length of the mannanase Man27-4S structural gene man27-4s is 1014 bp.

[0026] In another aspect, the present application provides a recombinant vector comprising the above-mentioned beta-mannanase mutant gene.

[0027] Preferably, the vector is ppIC-man27-4s.

[0028] The beta-mannanase gene of the present application is inserted between suitable restriction enzyme cutting sites of an expression vector, so that the nucleotide sequence is operably connected with an expression regulatory sequence. As a preferred embodiment of the present application, the beta-mannanase gene of the present application is inserted between EcoR I and Not I restriction enzyme cutting sites of the plasmid pPIC9, so that the nucleotide sequence is located downstream of the AOX1 promoter and is regulated by the AOX1 promoter, to obtain a recombinant yeast expression plasmid pPIC9-man27-4s.

[0029] In another aspect, the present application provides a recombinant strain comprising the above-mentioned beta-mannanase mutant gene.

[0030] Preferably, the strain is selected from one or more of Escherichia coli, yeast, Bacillus, and Lactobacillus.

[0031] Further preferably, the strain is yeast;

[0032] Still further, the strain is Pichia pastoris GS115.

[0033] Specifically, the recombinant strain is GS115 / man27-4s.

[0034] In another aspect, the present application provides a preparation method of the beta-mannanase mutant, comprising the following steps:

[0035] (1) The beta-mannanase mutant gene is fused with an expression vector to construct a recombinant vector, and the recombinant vector is transformed into a host cell to obtain a recombinant strain;

[0036] (2) cultivating the recombinant strain, inducing expression of the recombinant β-mannanase;

[0037] (3) recovering and purifying the expressed β-mannanase to obtain the β-mannanase mutant.

[0038] Preferably, the expression vector in step (1) is selected from one or more of pPIC9K, pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pHIL-S1, pPICZα, pYAM75P, PNZ8149-usp45.

[0039] Further, the expression vector is pPIC9K.

[0040] Further, the preparation of the recombinant vector comprises a step of enzyme digestion of the expression vector.

[0041] Still further, the enzyme used for enzyme digestion is selected from one or more of EcoRI and NotI.

[0042] Still further, the expression vector is double-digested using EcoRI and NotI.

[0043] Preferably, the host cell in step (1) is selected from any one or more of E. coli cell, B. subtilis cell, Pichia pastoris cell, Saccharomyces cerevisiae cell, Hansenula polymorpha cell, and mammalian cell.

[0044] Further preferably, the host cell is selected from any one or more of Pichia pastoris cell, Saccharomyces cerevisiae cell, and Hansenula polymorpha cell.

[0045] Further preferably, the host cell is Pichia pastoris GS115.

[0046] Still further preferably, the recombinant strain obtained in step (1) is GS115 / man27-4s.

[0047] Preferably, the culture conditions in step (2) are 20-35°C, 100-300 rpm shaking culture for 24-76 h.

[0048] Further, the culture conditions in step (2) are 30°C, 250 rpm shaking culture for 48 h.

[0049] Preferably, the expression induction conditions in step (2) are 20-35°C, 100-300 rpm shaking culture for 48-96 h.

[0050] Further, the expression inducing condition in step (2) is 30℃, 250rpm shaking culture for 72h.

[0051] In yet another aspect, the present application provides the use of the above-mentioned β-mannanase mutant, β-mannanase mutant gene, recombinant vector, recombinant strain or the β-mannanase mutant prepared by the above-mentioned preparation method in food, feed, papermaking or textile.

[0052] Specifically, the β-mannanase mutant can be applied in the feed industry to reduce or eliminate the anti-nutritional effects caused by viscosity increase.

[0053] Specifically, the β-mannanase mutant can be used as a clarifying agent for fruit juice beverages.

[0054] Specifically, the β-mannanase can be used in cooperation with xylanase to destroy the bond between hemicellulose and lignin, thereby reducing the amount of bleaching agent and chlorine.

[0055] The present application has the following advantages:

[0056] (1) The β-mannanase mutant provided by the present application has an optimal pH of 6.0 and good enzyme activity at pH 3.5-7.0, and strong pH stability;

[0057] (2) The β-mannanase mutant of the present application has an optimal reaction temperature of 60℃, and after being treated at 85℃ for 5min, the residual enzyme activity is still above 70%, and has good thermal stability;

[0058] (3) The β-mannanase mutant of the present application has good protease resistance. BRIEF DESCRIPTION OF DRAWINGS

[0059] In the following drawings, CK is the control group of mannanase Man27, and Man27-4S is the recombinantly expressed mannanase Man27-4s.

[0060] Figure 1 is a protein electropherogram of mannanase shake flask fermentation at different time periods.

[0061] Figure 2 is the optimal pH of the recombinant β-mannanase mutant.

[0062] Figure 3 is the pH stability of the recombinant β-mannanase mutant.

[0063] Figure 4 is the optimal temperature of the recombinant β-mannanase mutant.

[0064] Figure 5 is a graph of the thermal stability detection results of the recombinant β-mannanase mutant. DETAILED DESCRIPTION

[0065] The application will be further described in connection with the following specific examples, which are not intended to limit the application. The experimental methods used in the following examples are as follows unless otherwise specified. The experimental methods not specified in the examples are generally performed under conventional conditions. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0066] Test materials and reagents

[0067] 1. Strains and vectors: The mannanase mutant gene man27-4s in the application was synthesized by Beijing Ruibo Xingke Biotechnology Co., Ltd. The Pichia pastoris expression vector pPIC9 and the strain GS115 were purchased from Invitrogen Co.

[0068] 2. Enzymes and other biochemical reagents: Endonuclease was purchased from TaKaRa Co., and ligase was purchased from Invitrogen Co. Mannan was purchased from Sigma Co., and the others were domestic reagents (all of which can be purchased from ordinary biochemical reagent companies).

[0069] 3. Culture medium:

[0070] (1) YPD medium for yeast culture: 1% peptone, 0.5% yeast extract, 1% glucose, 2% agar, pH 7.0.

[0071] (2) LB medium for E. coli culture: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0072] (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (V / V).

[0073] (4) BMMY medium: 0.5% methanol instead of glycerol, and the other components are the same as BMGY, pH 4.0.

[0074] The molecular biology experimental methods not specifically described in the following examples were performed according to the specific methods listed in the book "Molecular Cloning Laboratory Guide" (third edition) by J. Sambrook, or according to the reagent kit and product instructions.

[0075] Example 1 Synthesis of Bacillus subtilis mannanase mutant gene Man27-4s

[0076] The mature structure gene sequence of the beta-mannanase man27 from Bacillus subtilis is mutated (V86Y, A248P, H317N), and EcoRI and NotI restriction enzyme cutting sites are added to the 5' end and 3' end of the mutated sequence respectively, and the sequence is sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for artificial synthesis of gene. The amino acid sequence of the artificially synthesized mannanase mutant is shown in SEQ ID NO: 4, and the nucleotide sequence is shown in SEQ ID NO: 5.

[0077] Example 2 Cloning of mannanase mutant gene Man27-4s

[0078] Extract the gene vector carrying the mannanase mutant:

[0079] The synthesized gene vector is stored in the form of puncture bacteria, and the sterile toothpick is used to pick the puncture bacteria in the super-clean bench, and placed in the LB shaking tube containing Amp (working concentration: 100 μg / ml) antibiotic, 37℃, 220 rpm overnight culture, the next day according to the steps of the plasmid extraction kit PurePlasmid Mini Kit (CW0500) instruction manual. Extract the vector containing the mutant gene.

[0080] According to the sequence of the mannanase mutant gene, the following primers are designed and synthesized:

[0081] P1: 5'-GCTGAATTCAGATCCCGGTCAGAAGCGCATACTGTGTCGCC-3' (SEQ ID NO: 6);

[0082] P2: 5'-ATTCGCGGCCGCTCACTCAACGATTGGCGTTAAAGAAT-3' (SEQ ID NO: 7).

[0083] PCR amplification is carried out with the extracted vector as the template. The PCR reaction parameters are: denaturation at 94℃ for 5min; then denaturation at 94℃ for 30sec, annealing at 55℃ for 30sec, extension at 72℃ for 1min, 30 cycles of 72℃ for 10min. A about 1050bp fragment is obtained, which is recovered and connected with pMD19 vector and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing, and the predicted protein molecular weight is 38.3kDa.

[0084] According to the nucleotide sequence obtained by sequencing, the obtained nucleotide sequence is compared with the man27 sequence by DNAMan software, and it is confirmed that the mutations of V86Y, A248P and H317N at three positions are correct.

[0085] Example 3 Preparation of recombinant mannanase

[0086] The expression vector pPIC9 was double-digested (EcoR I + Not I), and the gene encoding the mannanase mutant Man27-4s was double-digested (EcoR I + Not I) to obtain a gene fragment encoding mature mannanase, which was then ligated to the expression vector pPIC9 to obtain a recombinant plasmid pPIC-Man27-4s containing the mannanase gene Man27-4s. The recombinant plasmid was then transformed into Pichia pastoris GS115 to obtain a recombinant Pichia pastoris strain GS115 / Man27-4s.

[0087] The GS115 strain containing the recombinant plasmid and the control strain (i.e., the unmutated strain GS115 / man27, which was prepared in the same manner as the GS115 / Man27-4s) were inoculated into 300 mL of BMGY culture solution and cultured at 30°C with shaking at 250 rpm for 48 h, after which the bacterial cells were collected by centrifugation. The bacterial cells were then resuspended in 150 mL of BMMY culture medium and cultured at 30°C with shaking at 250 rpm. After 72 h of induction, the supernatant was collected by centrifugation, and the activity of the mannanase was determined.

[0088] Example 4 Activity analysis of the recombinant mannanase Man27-4s

[0089] DNS method: 1 mL of the reaction system included 500 μL of diluted enzyme solution, 500 μL of mannan, and was reacted for 10 min at 37°C and pH 7.0. Then, 1.5 mL of DNS was added to terminate the reaction, and the mixture was boiled in water for 5 min. After cooling, the OD value was determined at 540 nm.

[0090] Definition of the activity unit of β-mannanase: the amount of enzyme required to decompose 1 μmol of reducing sugar from β-mannan per minute under certain conditions was defined as 1 activity unit (U).

[0091] The expression amount of the recombinant mannanase was 3200 U / mL, and the expression amount of the mannanase in the control group was 3000 U / mL. The SDS-PAGE results (Figure 1) showed that the recombinant mannanase was expressed in Pichia pastoris.

[0092] Example 5 Property determination of the recombinant mannanase Man27-4s

[0093] 1. The determination method of the optimum pH and pH stability of the recombinant mannanase Man27-4s was as follows:

[0094] The control group of mannanase Man27 (CK group) and the recombinant expressed mannanase Man27-4s were subjected to enzymatic reaction under different pH conditions to determine the optimum pH, respectively. The buffer used was KCl-HCl buffer at pH 0.5-2.2, citric acid-disodium hydrogen phosphate series buffer at pH 2.2-8.0, and Tris-HCl series buffer at pH 8.0-10.0.

[0095] The results of the determination of the pH suitability of the β-mannanase Man27-4s in different pH buffer systems at 37°C (Figure 2) showed that the optimum pH value of the recombinant mannanase Man27-4s was 6.0. Within the pH range of 5.0-9.0, the enzyme could maintain more than 65% of the enzyme activity. Compared with the control, the pH optimum range was significantly improved, making it more suitable for neutral to alkaline conditions and widening its application range.

[0096] The enzyme solution was treated in different pH buffers at 37°C for 60 min, and the enzyme activity was determined to study the pH stability of the enzyme. The results (Figure 3) showed that the recombinant mannanase Man27-4s was very stable between pH 6.0-10.0, and could still maintain more than 80% of the enzyme activity after 60 min of treatment within this pH range, indicating that the enzyme had good pH stability. Compared with the control group of mannanase Man27 (CK group), the pH stability of the recombinant mannanase Man27-4s under the conditions of pH 6.0-10.0 was improved to a certain extent.

[0097] 2. The determination method of the optimum temperature and thermal stability of mannanase is as follows:

[0098] The determination of the optimum temperature of mannanase was carried out by measuring the enzymatic reaction activity of the recombinant mannanase Man27-4s and the control group of mannanase Man27 at different temperatures (20-85°C) under the condition of pH 6.0. The optimum reaction temperature of the recombinant mannanase Man27-4s was 60°C (Figure 4).

[0099] The determination of temperature resistance was carried out by treating the mannanase at 85°C for 5 min and then measuring the enzyme activity at 37°C. The temperature resistance experiment showed that the residual enzyme activity of the recombinant mannanase Man27-4s was still more than 70% after treatment at 85°C for 5 min (Figure 5), while the control group of mannanase Man27 (CK group) lost most of the enzyme activity after treatment at 85°C for 5 min, indicating that the thermal stability of the recombinant mannanase Man27-4s was significantly improved compared with the control group, and it could adapt to high-temperature granulation and other processing processes.

[0100] 3. The determination of the anti-pepsin and trypsin ability of the recombinant mannanase Man27-4s is as follows:

[0101] 0.1 mg / mL pepsin was prepared with pH 2.0 KCl-HCl buffer, and 0.1 mg / mL trypsin was prepared with pH 7.0 Tris-HCl buffer. 0.5 mL of the purified enzyme solution diluted with pH 2.0 KCl-HCl buffer was added to 0.5 mL of pepsin, and 0.5 mL of the purified enzyme solution diluted with pH 7.0 Tris-HCl buffer was added to 0.5 mL of trypsin, and the mixture was mixed, the ratio of protease / mannase (w / w) was 0.1, and the mixture was incubated at 37°C for 60 min and 120 min, and samples were taken, and the enzyme activity was determined at pH 7.0 and 60°C.

[0102] The experimental results show that after treatment with pepsin and trypsin for 120 min, the enzyme activity of Man27-4S is increased by about 10% compared with that before treatment. The enzyme activity of the control group of mannase after treatment with trypsin is 95% of that before treatment, and the enzyme activity after treatment with pepsin is 98% of that before treatment. The above results show that the recombinant mannase Man27-4S after mutation has improved resistance to pepsin and trypsin, and has good resistance to pepsin and trypsin.

[0103] Comparative Example 1

[0104] The present application provides a recombinant mannase Man27-3S, the amino acid sequence of which is based on SEQ ID NO: 2 with V86Q, A248Y, and H317D mutations, and the preparation method of the mutant Man27-3S and the recombinant enzyme Man27-3S is as described above. The thermal stability of the recombinant mannase Man27-3S was tested according to the stability determination method of the present application. The results show that after incubation at 85°C for 5 min, the enzyme activity of Man27-3S is only 40%.

[0105] 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 β-mannanase mutant, characterized in that, The beta-mannanase mutant contains the following mutations compared with the amino acid sequence of SEQ ID NO: 2: V86Y, A248P, H317N; the amino acid sequence of the beta-mannanase mutant is shown in SEQ ID NO:

4.

2. A β-mannanase mutant gene, characterized in that, The beta-mannanase mutant gene encodes the beta-mannanase mutant of claim 1; the nucleotide sequence of the beta-mannanase mutant gene is shown in SEQ ID NO:

5.

3. A recombinant vector comprising the beta-mannanase mutant gene of claim 2.

4. A recombinant strain comprising the beta-mannanase mutant gene of claim 2.

5. A method for producing the β-mannanase mutant according to claim 1, characterized by, The method comprises the following steps: (1) fusing the beta-mannanase mutant gene with an expression vector to construct a recombinant vector, transforming a host cell with the recombinant vector to obtain a recombinant strain; (2) culturing the recombinant strain to induce expression of the recombinant beta-mannanase; (3) recovering and purifying the expressed beta-mannanase to obtain the beta-mannanase mutant.

6. The production method according to claim 5, wherein The expression vector in step (1) is selected from one or more of pPIC9K, pET-28a, pEZZ18, pTA1529, pINIII-ompA, pUB110, pE194, pUCX05-bgaB, pHT304, pMK3, pPIC9, pHIL-S1, pPICZ alpha, pYAM75P, PNZ8149-usp45.

7. The production method according to claim 6, characterized by, The expression vector in step (1) is pPIC9K.

8. The preparation method according to claim 5, characterized in that The host cell in step (1) is selected from any one or more of an Escherichia coli cell, a Bacillus subtilis cell, a Pichia pastoris cell, a Saccharomyces cerevisiae cell, a Saccharomyces pombe cell, and a mammalian cell.

9. The beta-mannanase mutant of claim 1, the beta-mannanase mutant gene of claim 2, the recombinant vector of claim 3, the recombinant strain of claim 4, or the beta-mannanase mutant prepared by the method of any one of claims 5-8, for use in food, feed, papermaking, or textile.

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