Xylanase mutant and application thereof

Abstract

The invention discloses a xylanase mutant and application thereof, and relates to the technical field of gene engineering and enzyme engineering. The invention provides a xylanase mutant XYN77, the amino acid sequence of the xylanase mutant XYN77 is as shown in SEQ ID NO: 2, the optimal pH value of the xylanase XYN77 is 7.0, and the catalytic activity of the xylanase XYN77 is maintained to be 60% or above within the pH range of 5.0-8.0; the optimum temperature is 50 DEG C. The enzyme activity is 70% or above after the enzyme is treated for 1 h under the condition that the pH is 5.0-9.0, the enzyme activity is 60% or above after the enzyme is treated for 3 min at the temperature of 75 DEG C, and good thermal stability is achieved. The xylanase mutant disclosed by the invention has a wide application prospect in the fields of feed, food processing and papermaking.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and enzyme engineering, and in particular to a xylanase mutant and application thereof. Background Art

[0002] Xylanase is a general term for a series of enzymes that degrade xylan into xylo-oligosaccharides. It is mainly composed of β-1,4-endo-xylanase, β-xylosidase, α-D-glucuronidase, α-L-arabinofuranosidase, acetyl-xylanase and phenolic acid esterase, which can degrade most xylans in nature. It is widely used in many industrial fields such as food, feed, papermaking, textiles and biofuels. By genetically engineering the natural xylanase, mutants with better performance can be obtained, such as enhanced thermal stability, wider pH adaptability range or higher catalytic efficiency.

[0003] These improvements are critical to improving the efficiency and economy of industrial processes. As an extension of cellulase, xylanase is gradually being explored for its potential in industrial applications. Xylan is closely related to cellulose in structure and is also found in plant cell walls. Through its catalytic action, xylanase can effectively break down xylan into smaller sugar units, providing raw materials for the production of biofuels and other industrial processes.

[0004] In modern industrial applications, the application scope of xylanase is constantly expanding. It not only plays a role in the papermaking and textile industries, but also shows its unique value in food processing, feed improvement and bioenergy production. In view of the functional similarity between xylanase and cellulase, as well as the synergistic effect in application, xylanase is also classified as a cellulase enzyme preparation.

[0005] Chinese patent CN105087525A discloses a xylanase mutant, the optimal pH value of which is 5.5, which is consistent with the wild-type xylanase XynPF, but the optimal temperature is 60°C, and it has stronger heat resistance than the wild-type xylanase. Among them, the xylanase mutant XynC1 can maintain about 45% of the enzyme activity after being treated at 65°C for 5 minutes, but its enzyme activity is 35% and 27% after being treated at 70°C and 75°C for 5 minutes, and its stability at high temperature is poor.

[0006] The prior art discloses a recombinant xylanase xynP (Effect of the N-terminus of recombinant xylanase xynP on acid stability and its application in apple juice clarification, Qin Chi, Journal of Chinese Institute of Food Science and Technology). The optimum temperature of xylanase xyn P-1 is 40°C and the optimum pH is 5.0, but its thermal stability is poor. When the temperature is higher than 50°C, its enzyme activity is less than 20%.

[0007] In-depth research and improvement of xylanase will help improve its efficiency and stability in the process of cellulose decomposition. This will not only promote the optimization of existing industrial processes, but also provide possibilities for the development of new bioconversion technologies. Therefore, it is urgent to provide a xylanase with high thermal stability and high activity under neutral conditions. Summary of the invention

[0008] The purpose of the present invention is to provide a xylanase mutant and application thereof.

[0009] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides a xylanase mutant XYN77, wherein the amino acid sequence of the xylanase mutant is shown in SEQ ID NO:2.

[0010] SEQ ID NO:2: AEFQSFCSSASHSGQSVKVTGNKVGTIGGVGYELWADSGNNSATFYSDGSSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYAWTRSPLVEYY IVLNWLSPFPGDWVGNKKHGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGAIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD Specifically, the xylanase mutant XYN77 is derived from anaerobic fungi ( Neocallimastix patriciarum. ) by subjecting the xylanase XYN to point mutations G104A, D117L, and T177A, the amino acid sequence of XYN being shown in SEQ ID NO.1.

[0011] SEQ ID NO: 1: AEFQSFCSSASHSGQSVKVTGNKVGTIGGVGYELWADSGNNSATFYSDGSSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYY IVDNWLSPFPGDWVGNKKHGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIGD Specifically, the theoretical molecular weight of xylanase XYN is 24.75 kDa.

[0012] Specifically, the xylanase mutant XYN77 is a neutral xylanase.

[0013] According to some embodiments of the invention, the amino acid sequence of the xylanase mutant has at least 75% homology to the sequence shown in SEQ ID NO:2.

[0014] According to some embodiments of the invention, the amino acid sequence of the xylanase mutant has at least 80% homology to the sequence shown in SEQ ID NO:2.

[0015] According to some embodiments of the invention, the amino acid sequence of the xylanase mutant has at least 85% homology to the sequence shown in SEQ ID NO:2.

[0016] According to some embodiments of the invention, the amino acid sequence of the xylanase mutant has at least 90% homology to the sequence shown in SEQ ID NO:2.

[0017] According to some embodiments of the invention, the amino acid sequence of the xylanase mutant has at least 95% homology to the sequence shown in SEQ ID NO:2.

[0018] In another aspect, the present invention provides a xylanase gene xyn77 , the xylanase gene xyn77 Encodes the above-mentioned xylanase mutant XYN77.

[0019] Specifically, the xylanase gene xyn77 The nucleotide sequence is shown in SEQ ID NO:3.

[0020] SEQ ID NO:3: GCTGAATTTCAATCCTTCTGTAGTAGTGCATCACATTCTGGTCAATCAGTTAAAGTTACTGGTAATAAGGTTGGTACCATTGGTGGAGTTGGCTATGAGTTATGGGCTGACTCCGGTAACAATTCTGCTACGTTTTATTCAGACGGAAGTTTTAGTTGTACGTTCCAAAAC GCTGGTGATTACTTGTGCAGATCTGGATTAAGTTTCGATTCTACCAAGACGCCTAGTCAAATTGGTAGAATGAAAGCCGACTTCAAGTTAGTCAAACAAAATTCCTCTAATGTCGGTTACAGTTATGTCGGAGTGTATGCATGGACTAGGTCCCATTAGTTGAGTACTAC ATAGTGCTTACTGGTTGTCACCATTCCCACCTGGTGATTGGGTTGGTAACAAAAAACACGGTAGTTTTACGATTGACGGTGCTCAATACACTGTCTACGAAAATACTAGGACAGGACCATCAATTGACGGTGACACAACTTTCAATCAATACTTCTCCATAAGACAACAA GCAAGGGACTGCGGTGCTATTGATATTTCTGCTCACTTTGATCAATGGGAAAAGTTAGGAATGACCATGGGAAAGCTGCATGAAGCAAAAGTTTTAGGTGAGGCCGGTCCAGTTAATGGCGGAGCTTCTGGAACTGCTGACTTTCCTTATGCCAAGGTGTATATCGGTGAC The invention synthesizes the xylanase gene xyn77 by a gene synthesis method, and the result of DNA full sequence analysis shows that the structural gene xyn77 of the xylanase mutant XYN77 has a full length of 684 bp.

[0021] In another aspect, the present invention provides a method comprising the above-mentioned xylanase gene xyn77 of the recombinant vector.

[0022] Furthermore, the vector is selected from pPIC9K 、 One or more of pET-28a, pE194, pHT304, pMK3, and pPICZα.

[0023] Furthermore, the vector is pPIC9K.

[0024] According to some embodiments of the present invention, the recombinant vector is pPIC9K- xyn77 .

[0025] According to some embodiments of the present invention, the pPIC9K- xyn77 The preparation method is: inserting the xylanase gene of the present invention into the appropriate restriction enzyme cutting site of the expression vector, so that the nucleotide sequence can be operably connected with the expression control sequence. As a most preferred embodiment of the present invention, it is preferred to insert the xylanase gene of the present invention into the plasmid pPIC9K. EcoR I and Not I The restriction enzyme cutting sites were separated so that the nucleotide sequence was located downstream of the AOX1 promoter and regulated by it, and the recombinant yeast expression plasmid pPIC9K- xyn77 .

[0026] In another aspect, the present invention provides a method comprising the above-mentioned xylanase gene xyn77 of the recombinant strains.

[0027] Specifically, the strain is selected from one or more of Escherichia coli, yeast, Bacillus, and Lactobacillus; Further, the strain is yeast; According to some embodiments of the present invention, the strain is Pichia pastoris GS115.

[0028] In another aspect, the present invention provides a method for preparing the above-mentioned xylanase mutant XYN77, comprising the following steps: (1) Transforming host cells with the recombinant vector to obtain a recombinant strain; (2) Cultivating the recombinant strain and inducing the expression of recombinant xylanase; (3) Recovering and purifying the expressed xylanase mutant XYN77.

[0029] Specifically, the vector described in step (1) is selected from one or more of pPIC9K, pET-28a, pE194, pHT304, pMK3, and pPICZα.

[0030] Furthermore, the vector is pPIC9K.

[0031] Specifically, the preparation of the recombinant vector includes the step of enzymatically cutting the expression vector.

[0032] Furthermore, the expression vector is used EcoRI and NotI Perform double enzyme digestion.

[0033] Specifically, the host cell in step (1) is selected from one or more of fungal cells, bacterial cells or plant cells.

[0034] Furthermore, the host cell in step (1) is a yeast cell.

[0035] Furthermore, the host cell in step (1) is a Pichia pastoris cell.

[0036] According to some embodiments of the present invention, the recombinant yeast expression plasmid is transformed into Pichia pastoris cells ( Pichia pastoris )GS115, and obtained the recombinant strain GS115 / XYN77.

[0037] In another aspect, the present invention provides the above-mentioned xylanase mutant XYN77, xylanase gene xyn77 , recombinant vectors, recombinant strains or the xylanase mutant XYN77 prepared by the above preparation method in feed, food processing and papermaking.

[0038] Specifically, when the xylanase mutant XYN77 is used in the feed industry, it can degrade xylan, reduce the viscosity of the feed, reduce the anti-nutritional effects caused by the increased viscosity, and enable animals to digest and absorb nutrients more efficiently, thereby promoting animal growth and improving breeding efficiency.

[0039] Specifically, when the xylanase mutant XYN77 is used in food processing, it can improve the rheological properties of the dough, enhance the stability and extensibility of the dough, produce baked foods such as bread with better taste and larger volume, and extend the shelf life of the food.

[0040] Specifically, when the xylanase mutant XYN77 is used in papermaking, it can decompose xylan in papermaking raw materials, reduce pulp viscosity, and improve the strength and smoothness of paper. At the same time, it can reduce the use of chemical bleaching agents and reduce environmental pollution, helping the papermaking industry to develop in a green and environmentally friendly direction, and has broad application prospects.

[0041] The beneficial effects of the present invention are: The xylanase XYN77 of the present invention has the characteristics of high activity under neutral conditions and high enzyme stability. The xylanase of the present invention has an optimum pH value of 7.0, maintains more than 60% catalytic activity in the pH range of 5.0-8.0; the optimum temperature is 50°C, and has more than 30% activity at 20°C. When treated for 1 hour under pH 5.0-9.0 conditions, the enzyme activity is more than 70%, and when treated for 3 minutes at 75°C, the enzyme activity is more than 60%, and has good thermal stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the optimal pH of xylanase mutant XYN77.

[0043] Figure 2 pH stability of xylanase mutant XYN77.

[0044] Figure 3 is the optimum temperature of xylanase mutant XYN77.

[0045] Figure 4 The thermal stability of xylanase mutant XYN77 at 75°C.

[0046] Figure 1-Figure 4 In the invention, XYN is an unmutated xylanase, and its amino acid sequence is shown in SEQ ID NO:1; the amino acid sequence of XYN77 is shown in SEQ ID NO:2; and the amino acid sequence of XYN77M is mutated to G66A, D84E, and N210P based on SEQ ID NO.1. DETAILED DESCRIPTION

[0047] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0048] Experimental materials and reagents 1. Strains and vectors: Xylanase gene of the present invention xyn77 It was synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd., and the Pichia pastoris expression vector pPIC9K and strain GS115 were purchased from Invitrogen.

[0049] 2. Enzymes and other biochemical reagents: Endonucleases were purchased from TaKaRa, ligases were purchased from Invitrogen, mannan was purchased from Sigma, and the others were domestic reagents (all available from common biochemical reagent companies).

[0050] 3. Culture medium: (1) Yeast medium YPD: 1% peptone, 0.5% yeast extract, 1% glucose, 2% agar, pH 7.0.

[0051] (2) Escherichia coli culture medium LB: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0052] (3) BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (V / V).

[0053] (4) BMMY medium: except that 0.5% methanol is used instead of glycerol, the rest of the ingredients are the same as BMGY.

[0054] The molecular biology experimental methods not specifically described in the following examples were all performed with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions.

[0055] Example 1 Anaerobic fungi ( Neocallimastix patriciarum ) Xylanase encoding genes xyn77 Synthesis The present invention uses xylanase from anaerobic fungi xyn The amino acid sequence was used as a reference, and the following mutations were made to the sequence (G104A, D117L, T177A), and the 5' and 3' ends of the mutated sequence were added EcoR I and Not I The restriction enzyme cutting site was added and the sequence was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for artificial gene synthesis. The amino acid sequence of the artificially synthesized xylanase mutant is shown in SEQ ID NO: 2. xyn The nucleotide sequence of the gene is shown in SEQ ID NO:3.

[0056] Example 2 Xylanase Gene xyn Clone The synthesized gene vector was stored in the form of puncture bacteria. The puncture bacteria were picked up with a sterile toothpick in the clean bench and placed in an LB shaking tube containing Amp antibiotic (working concentration: 100 μg / ml). They were cultured overnight at 37°C and 220 rpm. The next day, the gene-containing vector was extracted according to the instructions of the PurePlasmid Mini Kit (CW0500).

[0057] According to the xylanase gene sequence, the following primers were designed and synthesized: P1: CCGGAATTCGCTGAATTTCAATCCTTCTGTAGTAGT (SEQ ID NO:4) P2: AAATGCGGCCGCtAAATTAGTCACCGATATACACCTTGGCAT (SEQ ID NO:5) The extracted vector was used as a template for PCR amplification. The PCR reaction parameters were: 94℃ denaturation for 5 min; then 94℃ denaturation for 30 sec, 55℃ annealing for 30 sec, 72℃ extension for 1 min, and 72℃ incubation for 10 min after 30 cycles. A fragment of about 684 bp was obtained, which was recovered and connected to the pMD19 vector and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The predicted protein molecular weight was 24.75 kDa.

[0058] According to the nucleotide sequence obtained by sequencing, the obtained nucleotide sequence was compared with xyn The sequences were compared and confirmed to be correct.

[0059] Example 3 Preparation of recombinant xylanase XYN The expression vector pPIC9K was double-digested ( EcoR I + Not I ), and the gene encoding xylanase xyn Double enzyme digestion ( EcoR I + Not I ), the gene fragment encoding xylanase was cut out and connected with the expression vector pPIC9K to obtain a gene fragment containing xylanase gene xyn The recombinant plasmid pPIC9K- xyn And transformed Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 / XYN.

[0060] Take the GS115 strain containing the recombinant plasmid and the control strain (i.e. the unmutated strain GS115 / xyn ), inoculated into 300 mL BMGY culture medium, cultured at 30℃ 200 rpm for 48h, and then centrifuged to collect the cells. Then resuspended in 150 mL BMMY medium, cultured at 30℃ 200 rpm. After 72h of induction, the supernatant was collected by centrifugation and the activity of xylanase was determined.

[0061] Example 4 Activity analysis of recombinant xylanase XYN 3,5-Dinitrosalicylic acid (DNS) method: The specific method is as follows: at pH 7.0 and 37°C, 1 ml of the reaction system includes 100 ul of diluted enzyme solution and 900 ul of substrate, react for 10 min, add 1.5 ml of DNS to terminate the reaction, boil in boiling water for 5 min, and measure the OD value at 540 nm after cooling. One unit of enzyme activity (U) is defined as the amount of enzyme that releases 1 umol of reducing sugar per minute under given conditions.

[0062] Example 5 Determination of properties of the recombinant xylanase mutant XYN77 The enzymatic properties of the recombinant xylanase mutant XYN77 and the unmutated xylanase XYN were measured and compared. At the same time, a mutant recombinant xylanase mutant XYN77M was added for enzymatic property comparison. The sequence of XYN77M was mutated (G66A, D84E, N210P) based on SEQ ID NO.1. The specific preparation methods of the XYN77 mutant and the XYN77M recombinant enzyme are as described in the above examples.

[0063] 1. The optimum pH and pH stability of the recombinant xylanase mutant XYN77 were determined as follows: The purified xylanase XYN, XYN77, and XYN77M were subjected to enzymatic reactions at different pH values ​​to determine their optimum pH. The substrate xylan was used to determine the xylanase activity at 37°C in 0.1 mol / L citric acid-sodium hydrogen phosphate buffer at different pH values. Figure 1 ) showed that the optimum pH of the three was 7.0.

[0064] The purified xylanases XYN, XYN77, and XYN77M were treated in the above buffers of different pH values ​​at 37°C for 60 min, and then the enzyme activity was measured in a pH 7.0 buffer system at 37°C to study the pH tolerance of the enzyme. Figure 2 ) showed that the recombinant xylanase mutant XYN77 was very stable between pH 5.0 and 9.0, and had higher stability compared with XYN and XYN77M.

[0065] 2. The optimum temperature and thermal stability of the recombinant xylanase mutant XYN77 were determined as follows: The optimum temperature of xylanase was determined by performing enzymatic reactions in a citric acid-sodium hydrogen phosphate buffer (pH 7.0) buffer system at different temperatures. The temperature tolerance was determined by treating xylanase at 75°C for 3 minutes and then measuring enzyme activity at 37°C. The optimum temperature of the recombinant xylanase mutant XYN77 was determined ( Figure 3 ) showed that its optimum temperature was 50°C, and the enzyme activity remained high at 30-60°C.

[0066] The enzyme thermal stability test showed that ( Figure 4 ), XYN77 has good thermal stability and can maintain more than 60% enzyme activity after incubation at 75℃ for 3 minutes. However, the remaining enzyme activity of XYN is only 20%, and that of XYN77M is only 40%. Compared with the two, XYN77 has significantly improved thermal stability and good heat resistance.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A xylanase mutant XYN77, characterized in that The amino acid sequence of the xylanase mutant is shown in SEQ ID NO:

2.

2. The xylanase mutant XYN77 according to claim 1, characterized in that The xylanase mutant XYN77 is a mutant produced by subjecting xylanase XYN derived from anaerobic fungi to point mutations G104A, D117L and T177A. The amino acid sequence of xylanase XYN is shown in SEQ ID NO.

1.

3. A xylanase gene xyn77 , characterized in that, The xylanase gene xyn77 Encoding the xylanase mutant XYN77 according to any one of claims 1-2.

4. The xylanase gene according to claim 3 xyn77 , characterized in that, The xylanase gene xyn77 The nucleotide sequence is shown in SEQ ID NO:

3.

5. Comprising the xylanase gene according to any one of claims 3 to 4 xyn77 The recombinant vector is characterized in that The vector is selected from pPIC9K 、 One or more of pET-28a, pE194, pHT304, pMK3, and pPICZα.

6. The recombinant vector according to claim 5, characterized in that The vector is pPIC9K.

7. A method comprising the xylanase gene according to any one of claims 3 to 4. xyn77 of the recombinant strains.

8. The recombinant strain according to claim 7, characterized in that The strain is selected from one or more of Escherichia coli, yeast, Bacillus, and Lactobacillus.

9. The method for preparing the xylanase mutant XYN77 according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Transforming host cells with the recombinant vector to obtain a recombinant strain; (2) Cultivating the recombinant strain and inducing the expression of recombinant xylanase; (3) Recovering and purifying the expressed xylanase mutant XYN77.

10. The xylanase mutant XYN77 according to any one of claims 1 to 2, and the xylanase gene according to any one of claims 3 to 4 xyn77 , the use of the recombinant vector according to any one of claims 5-6, the recombinant strain according to any one of claims 7-8 or the xylanase mutant XYN77 prepared by the preparation method according to claim 9 in feed, food processing and papermaking.

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