Xylanase mutant with simultaneous improvement of thermal stability and pH tolerance and application thereof

Abstract

The application discloses a xylanase mutant with simultaneously improved thermal stability and pH tolerance and application thereof, and belongs to the field of genetic engineering and protein engineering. The xylanase AbXyL10A from Agaricus bisporus var burnettii is used as a parent to obtain two xylanase mutants T234N and R268L after mutation of two amino acid sites Thr234 and Arg268. Compared with the wild-type xylanase, the thermal stability and pH tolerance of the two xylanase mutants are obviously improved. In terms of specific activity, when beech xylan is used as a substrate, the specific activity of the two xylanase mutants is obviously improved compared with the wild-type xylanase. The application provides a theoretical reference for improvement of xylanase, and the obtained xylanase mutant with simultaneously improved thermal stability and pH tolerance has great application prospect in animal feed and biomass degradation sugar.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and protein engineering, and in particular to a xylanase mutant with synchronously improved thermal stability and pH tolerance and application thereof. Background Art

[0002] Cellulose, hemicellulose, and lignin, linked by hydrogen and covalent bonds, form the overall structure of plant cell walls and are abundant renewable biomass resources in nature. Cellulose accounts for the largest proportion, followed by hemicellulose. Xylan is the primary component of hemicellulose, comprising approximately 15%-40% of the cell wall, making it the second most abundant polysaccharide after cellulose. Xylan is a complex linear polymer composed of β-(1,4)-linked xylose residues. These xylose residues can be replaced by various functional groups within their molecular chains, including arabinose residues, acetyl groups, glucuronic acid (GlcA), and 4-O-methylglucuronic acid (Me-GlcA).

[0003] The degradation of xylan requires the synergistic action of multiple enzymes, among which xylanase is the most critical enzyme in the degradation process, randomly cleaving β-1,4 glycosidic bonds in the backbone to produce xylo-oligosaccharides. Xylanases are widely used in the energy, feed, and food industries and have received considerable attention. However, most xylanases used in industry today are mesophilic enzymes, making them difficult to adapt to the high temperatures required for production. Currently, the GH10 and GH11 family xylanases have been the most widely reported. GH10 family xylanases have good thermal stability but low catalytic efficiency, limiting their application in industrial production. Therefore, developing new, high-specific-activity, heat-resistant xylanases or modifying existing enzymes for thermal stability and catalytic activity is crucial for promoting industry development.

[0004] Traditional modification methods primarily rely on the spatial structure and amino acid sequence of xylanase, using directed evolution to create a high-throughput mutant library. These libraries are then screened for mutants with enhanced thermal stability or catalytic activity. This approach is generally time-consuming. However, as protein structure and function identification technologies mature, research on protein structure is deepening, and rational strategies for molecular modification of proteins based on rational design have emerged. A range of rational strategies have been developed to enhance enzyme thermal stability and catalytic activity, including computer-generated prediction of "hotspots" for enhanced enzyme stability or activity, and prediction of changes in protein-ligand binding energy using the physicochemical properties of amino acid residues within protein structure and virtual mutagenesis. Summary of the Invention

[0005] The present invention aims to provide a xylanase mutant with simultaneously improved thermal stability and pH tolerance and its application, so as to solve the problems existing in the above-mentioned prior art. By using the xylanase AbXyL10A from Agaricus bisporus var burnettii as the parent, The at position 234 in the amino acid sequence is mutated to Asn, and Arg at position 268 in the amino acid sequence is mutated to Leu, respectively, to obtain two xylanase mutants T234N and R268L with simultaneously improved thermal stability and pH tolerance, thereby providing new xylanases for animal feed and biomass degradation and sugar production.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a xylanase mutant with improved thermostability and pH tolerance. The xylanase mutant is prepared by using xylanase AbXyL10A from Agaricus bisporus var burnettii as a parent, and mutations are made at amino acid sites T234 and R268 to obtain xylanase mutants T234N and R268L.

[0008] The amino acid sequence of the xylanase mutant T234N is shown in SEQ ID NO.2; the amino acid sequence of the xylanase mutant R268L is shown in SEQ ID NO.3.

[0009] The present invention also provides a gene encoding the xylanase mutant.

[0010] Optionally, the gene sequence encoding the xylanase mutant T234N is shown as SEQ ID NO.4, and the gene sequence encoding the xylanase mutant R268L is shown as SEQ ID NO.5.

[0011] The present invention also provides a recombinant vector comprising the gene.

[0012] The present invention also provides a recombinant bacterium comprising the recombinant vector. In the embodiments of the present invention, Pichia pastoris GS115 is used as an example for illustration.

[0013] The present invention also provides the use of the xylanase mutant in any of the following:

[0014] (1) Application in xylan degradation;

[0015] (2) Application in the preparation of catalytic preparations for xylan degradation;

[0016] (3) Application in the preparation of feed additives.

[0017] The present invention also provides use of the recombinant bacteria in preparing xylanase with simultaneously improved thermal stability and pH tolerance.

[0018] The present invention also provides a method for producing xylanase mutants by fermenting the recombinant bacteria, comprising fermenting the recombinant bacteria, separating the fermentation broth and collecting the precipitate, then inducing the culture using a fermentation medium containing methanol, and obtaining the xylanase mutants from the supernatant of the culture broth.

[0019] The present invention also provides a catalytic preparation for xylan degradation, the active ingredient of which is the xylanase mutant.

[0020] The present invention discloses the following technical effects:

[0021] The present invention uses family 10 xylanase AbXyL10A (NCBI Reference Sequence: XP_007330618.1) from Agaricus bisporus var burnettii as the parent, mutates the Thr234 and Arg268 sites (mutates The at position 234 to Asn, and mutates Arg at position 268 to Leu) to obtain two mutants. Specifically, recombinant strains containing the mutants are constructed with the assistance of machine learning, and two xylanase mutants T234N and R268L with greatly improved thermal stability and pH tolerance are screened out after induction culture. The optimal pH for xylanase mutants T234N and R268L is 5.0, while that for wild-type xylanase is 6.0. In terms of pH tolerance, wild-type xylanase and xylanase mutants were treated at different pH levels (1.0-12.0) for 1 h. The pH tolerance of the two mutants in strong acid (pH 1-3) and strong base (pH 10-12) was significantly improved compared with that of wild-type xylanase, indicating that they have excellent acid-base tolerance. The optimal temperature for wild-type xylanase is between 50-60°C, while that for xylanase mutants T234N and R268L is between 80-90°C. In terms of thermal stability, the T values ​​of xylanase mutants T234N and R268L are significantly higher than those of wild-type xylanase. 50The values ​​were 28.5°C and 30.5°C higher than those of the wild-type xylanase (53°C), respectively, and the half-lives at 80°C were 5.1-fold and 8.3-fold higher than those of the wild-type xylanase (7.5 min), respectively. In terms of catalytic activity, when beech wood xylan was used as substrate, the specific activities of the xylanase mutants T234N and R268L were 850.6 U / mg and 684.0 U / mg, respectively, which were 233% and 167% higher than those of the wild-type AbXyL10A (255.8 U / mg), respectively. When sugarcane xylan was used as substrate, the specific activities of the xylanase mutants T234N and R268L were 455.2 U / mg and 475.3 U / mg, respectively, which were 174% and 186% higher than those of the wild-type AbXyL10A (166.2 U / mg), respectively. It can be seen that the present invention has obtained two xylanase mutants with improved high thermal stability and pH tolerance, and they have great potential for application in animal feed and biomass degradation to produce sugar. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Protein purification diagrams of wild-type xylanase AbXyL10A (A) and xylanase mutants T234N (B) and R268L (C); M is a marker, and AC are wild-type enzyme xylanase AbXyL10A, xylanase mutant T234N, and xylanase mutant R268L, respectively;

[0024] Figure 2 The optimum pH values ​​of xylanase mutants and wild-type xylanase are determined;

[0025] Figure 3 The pH stability test results of xylanase mutants and wild-type xylanase are shown;

[0026] Figure 4 The results of the optimal temperature determination of xylanase mutants and wild-type xylanase;

[0027] Figure 5 T is the difference between the xylanase mutant and the wild-type xylanase 50 Measurement results;

[0028] Figure 6 is the half-life of the xylanase mutant and wild-type xylanase at 80°C, t 1 / 2 Measurement results. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The main experimental materials involved in the embodiments of the present invention are:

[0035] (1) Strains and vectors: The expression host Pichiapastoris GS115 was maintained in our laboratory, and the expression plasmid vector pPIC9r was prepared in our laboratory;

[0036] (2) Enzymes and other biochemical reagents: Taq enzyme was purchased from Quanshijin Company, endonuclease was purchased from Quanshijin Company, and beechwood xylan was purchased from Sigma Company. Other reagents were domestic analytical grade (all available from general biochemical reagent companies).

[0037] (3) Culture medium:

[0038] YPD medium: 2% glucose, 2% peptone, 1% yeast extract;

[0039] LB medium: 1% peptone, 0.5% yeast extract, 1% NaCl, 1% agar powder (solid);

[0040] MD medium: 1.5% agarose, 2% glucose, 0.00004% Biotin, 1.34% YNB;

[0041] BMGY medium: 2% peptone, 1% yeast extract, 1% glycerol (V / V), 0.00004% Biotin, 1.34% YNB;

[0042] BMMY medium: 2% peptone, 1% yeast extract, 1.34% YNB, 0.5% methanol (V / V), 0.00004% Biotin.

[0043] Example 1 Obtaining a gene encoding a xylanase mutant with improved thermostability and pH tolerance

[0044] Using the recombinant expression vector pic9r-AbXyL10A containing the xylanase gene AbXyL10A from Agaricus bisporus var burnettii (amino acid sequence shown in SEQ ID NO. 1, nucleotide sequence shown in SEQ ID NO. 6) as a template, site-directed mutagenesis was performed at Thr234 and Arg268. The primer designs are shown in Table 1. The mutagenesis and cloning methods were referred to the literature (Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase; You et al., 2019).

[0045] Table 1 Primer sequences

[0046]

[0047]

[0048] Example 2 Preparation of a xylanase mutant with improved thermostability and pH tolerance

[0049] The linear recombinant expression vector obtained by PCR in Example 1 was directly transformed into DMT competent cells and verified by colony PCR to obtain the nucleic acid sequence of the target site mutant. The recombinant plasmid was linearized and transformed into Pichia pastoris GS115 to obtain recombinant yeast strains GS115 / T234N and GS115 / R268L.

[0050] The GS115 strain containing the recombinant plasmid was inoculated into 2 mL of BMGY medium in a 10 mL test tube and incubated at 30°C, 220 rpm, and shaker for 48 hours. The culture was then centrifuged at 3000 g for 5 minutes, the supernatant discarded, and the pellet resuspended in 2 mL of BMMY medium containing 0.5% methanol. The culture was again incubated at 30°C, 220 rpm, for 48 hours. The supernatant was used for enzyme activity assays, and mutants T234N (amino acid sequence shown in SEQ ID NO. 2, nucleotide sequence shown in SEQ ID NO. 4) and R268L (amino acid sequence shown in SEQ ID NO. 3, nucleotide sequence shown in SEQ ID NO. 5) were screened for enzyme activity compared to the wild-type enzyme.

[0051] The wild-type GS115 / AbXyL10A and two mutants GS115 / T234N and GS115 / R268L were scaled up to obtain a fermentation system. First, the seed culture was inoculated into YPD medium, and then inoculated into a 1L Erlenmeyer flask containing 300mL of BMGY medium at a 1% inoculum. The flask was placed at 30°C and shaken at 220rpm for 48h. The culture was then centrifuged at 4000g for 5min, the supernatant was discarded, and the precipitate was resuspended in 100mL of BMMY medium containing 0.5% methanol and induced again at 30°C and 220rpm. 0.5mL of methanol was added every 12h to maintain the methanol concentration in the bacterial solution at 0.5%, and the supernatant was taken for enzyme activity detection. Finally, the supernatant was concentrated to 30mL for enzyme property determination and comparison. The protein was purified using anion exchange method. After the expressed xylanase was purified, polyacrylamide gel electrophoresis ( Figure 1 ) showed that the molecular weight of the xylanase mutant and wild-type xylanase in the fermentation supernatant was approximately 80-90 kDa.

[0052] Example 3 Comparative analysis of enzymatic properties of recombinant xylanase mutants with improved thermostability and pH tolerance and wild-type xylanase

[0053] 1. Dinitrosalicylic acid colorimetric method (DNS) determination

[0054] The specific method is as follows: At the respective optimal pH and temperature conditions, a 1 mL reaction system consists of 100 μL of diluted enzyme solution and 900 μL of substrate. The reaction is allowed to proceed for 10 minutes. 1.5 mL of DNS is added to terminate the reaction, and the reaction is then boiled in water for 5 minutes. After cooling, the OD value is measured at 540 nm. One unit (U) of enzyme activity is defined as the amount of enzyme required to degrade xylan to produce 1 μmol / L of reducing sugars per minute under the given conditions.

[0055] 2. Determination of properties of recombinant xylanase mutants and wild-type xylanase with improved thermostability and pH tolerance

[0056] 2.1 Method for determining the optimal pH of recombinant xylanase mutants and wild-type xylanase with simultaneously improved thermostability and pH tolerance

[0057] The xylanase mutants and wild-type xylanase obtained in Example 2 were subjected to enzymatic reactions at different pH values ​​(1.0-7.5) to determine their optimal pH values. Xylanase activity was determined at 55°C using the substrate xylan in 0.1 mol / L citric acid-sodium dihydrogen phosphate buffer at different pH values ​​(1.0, 1.5, 2.0, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7.0).

[0058] The results are as follows Figure 2 As shown, the optimal pH of mutants T234N and R268L is 5.0. Compared with the wild type (pH 6.0), the optimal pH shifts 1 pH unit toward the acidic environment. In the acidic environment, taking pH 4.0 as an example, the relative enzyme activities of mutants T234N and R268L are increased by 71.7% and 60.1% respectively compared with the wild type.

[0059] 2.2 pH Stability Determination Method for Recombinant Xylanase Mutants with Simultaneously Improved Thermal Stability and pH Tolerance and Wild-Type Xylanase

[0060] The xylanase mutants and wild-type xylanase were diluted with 0.1 mol / L citric acid-disodium hydrogen phosphate buffer at different pH values ​​(1.0-12.0) and placed in a 37°C constant temperature water bath for 1 h. The relative residual enzyme activity was then determined under standard conditions, with the untreated enzyme activity serving as the 100% control.

[0061] The results are as follows Figure 3 As shown, the pH tolerance of the two xylanase mutants under strong acid (pH 1-3) and strong base (pH 10-12) was significantly improved compared with the wild type, showing good acid-base tolerance.

[0062] 2.3 Method for determining the optimal temperature of wild-type xylanase and xylanase mutants

[0063] The method for determining the optimal temperature of the recombinant xylanase mutant and wild-type xylanase with simultaneously improved high thermal stability and pH tolerance is: performing enzymatic reactions in a 0.1 mol / L citric acid-disodium hydrogen phosphate buffer (pH 4.5) buffer system and at different temperatures (30-100°C).

[0064] The results are as follows Figure 4 As shown, the optimum temperature of the wild-type xylanase is between 50-60°C, and the optimum temperature of the xylanase mutants T234N and R268L is between 80-90°C. The relative enzyme activity at high temperature (80-90°C) is significantly improved compared with the wild-type xylanase.

[0065] 2.4 Determination of the thermal stability of wild-type xylanase and xylanase mutants

[0066] T was measured at 40-90°C. 50 Value: After the xylanase mutants and wild-type xylanase were treated at different temperatures of 40-90℃ for 30 min, the residual enzyme activities of each were detected.

[0067] The half-life (t 1 / 2 ):The xylanase mutants and wild-type xylanase were treated at 85°C for different time periods, up to 90 min, and their residual enzyme activities were detected.

[0068] T at 40-90℃ 50 The results of the value determination are as follows Figure 5 As shown, the T of xylanase mutant T234N 50 The T value of xylanase mutant R268L was 81.5℃, which was 28.5℃ higher than that of wild-type xylanase (53℃). 50 The half-life of 85℃ was 83.5℃, which was 30.5℃ higher than that of wild type (53℃). Figure 6 As shown, the half-life of the xylanase mutant T234N at 85°C was 46 minutes, a 5.1-fold increase compared to the wild-type xylanase (7.5 minutes). The half-life of the mutant R268L at 85°C was 70 minutes, an 8.3-fold increase compared to the wild-type xylanase (7.5 minutes). Both xylanase mutants showed significantly improved thermal stability compared to the wild-type xylanase.

[0069] 2.5 Kinetic parameter determination method of xylanase mutants and wild-type xylanase

[0070] The detection method was referred to the literature (Athermophilic and acid stable family-10xylanase from the acidophilic fungus Bispora sp MEY-1. Extremophiles. 2009; 13: 849-57. Luo, et al., 2009) to determine the first-order reaction time of the reaction. m Value and V max The reaction time was 5 min. Different concentrations of beechwood xylan and sugarcane xylan (10, 8, 5, 2.5, 2, 1.25, 1 and 0.75 mg / mL) were used as substrates. The enzyme activity was measured under the optimal conditions (temperature, pH), and the corresponding reaction rate was calculated. K was calculated using GraFit7 software. m Value and V max .

[0071] The results are shown in Table 2. Under their respective optimal conditions, when beech wood xylan was used as the substrate, the specific activities of the xylanase mutants T234N and R268L were 850.6 U / mg and 684.0 U / mg, respectively, which were 233% and 167% higher than those of the wild-type AbXyL10A (255.8 U / mg); the catalytic efficiencies were 502.9 mL / s·mg and 326.7 mL / s·mg, respectively, which were 363% and 201% higher than those of the wild-type AbXyL10A (108.7 mL / s·mg). Under their respective optimal conditions, when using sugarcane xylan as substrate, the specific activities of mutants T234N and R268L were 455.2 U / mg and 475.3 U / mg, respectively, which were 174% and 186% higher than those of wild-type AbXyL10A (166.2 U / mg); the catalytic efficiencies were 106.8 mL / s·mg and 61.1 mL / s·mg, respectively, which were 507% and 247% higher than those of wild-type GtXyn10 (17.6 mL / s·mg).

[0072] Table 2 Comparison of specific activity and catalytic efficiency of wild-type enzyme and mutant

[0073]

[0074] The present invention relates to the sequence:

[0075] SEQ ID NO.1:

[0076] QLNTLAVRAGKKYFGTATDNPELGDAPYVAQLGNTADFNQITAGNSMKWDATEPSRGTFTFANGDTVANMARNRGQLLRGHTCVWHSQLPNWVTSGNFDNSTLLSIVQNHCSTLVSHYRGQMYSWDVVNEPFNEDGSFRQSVFFQKTGTAYIATALRAARNADPNTKLYINDFNIEGTGAKSTGMINLVRSLQQQNVPIDGIGVQAHLIVGQIPSSIQQNLQNFANLGVEVAITELDIRMTLPVTQQKLEQQQEDYRTVIRACKAVSRCVGVTVWDWTDRYSWVPGVFTGEGAACPWDENLAKKPAYQGIVDGWSQ。

[0077] SEQ ID NO.2:

[0078] QLNTLAVRAGKKYFGTATDNPELGDAPYVAQLGNTADFNQITAGNSMKWDATEPSRGTFTFANGDTVANMARNRGQLLRGHTCVWHSQLPNWVTSGNFDNSTLLSIVQNHCSTLVSHYRGQMYSWDVVNEPFNEDGSFRQSVFFQKTGTAYIATALRAARNADPNTKLYINDFNIEGTGAKSTGMINLVRSLQQQNVPIDGIGVQAHLIVGQIPSSIQQNLQNFANLGVEVAI N ELDIRMTLPVTQQKLEQQQEDYRTVIRACKAVSRCVGVTVWDWTDRYSWVPGVFTGEGAACPWDENLAKKPAYQGIVDGWSQ。

[0079] SEQ ID NO.3:

[0080] QLNTLAVRAGKKYFGTATDNPELGDAPYVAQLGNTADFNQITAGNSMKWDATEPSRGTFTFANGDTVANMARNRGQLLRGHTCVWHSQLPNWVTSGNFDNSTLLSIVQNHCSTLVSHYRGQMYSWDVVNEPFNEDGSFRQSVFFQKTGTAYIATALRAARNADPNTKLYINDFNIEGTGAKSTGMINLVRSLQQQNVPIDGIGVQAHLIVGQIPSSIQQNLQNFANLGVEVAITELDIRMTLPVTQQKLEQQQEDYRTVIRACKAVS L CVGVTVWDWTDRYSWVPGVFTGEGAACPWDENLAKKPAYQGIVDGWSQ。

[0081] SEQ ID NO.4:

[0082] CAATTGAACACCTTGGCTGTTAGAGCTGGTAAAAAGTATTTTGGTACTGCTACTGATAACCCAGAATTGGGTGATGCTCCATATGTTGCTCAATTGGGTAATACTGCTGATTTTAATCAAATCACTGCTGGTAATTCTATGAAATGGGATGCTACTGAACCATCTAGAGGAACTTTCACTTTTGCTAACGGTGATACTGTTGCTAACATGGCTAGAAACAGAGGTCAATTGTTGAGAGGTCACACTTGTGTTTGGCACTCTCAATTGCCAAACTGGGTTACTTCCGGTAACTTTGATAACTCTACTTTGTTGTCTATCGTTCAAAACCATTGTTCTACTTTGGTTTCTC ATTACAGAGGTCAAATGTACTCTTGGGATGTTGTTAACGAACCTTTTAACGAAGATGGATCTTTTAGACAATCCGTTTTTTTTCAAAAGACCGGTACTGCTTATATTGCTACCGCTTTGAGAGCTGCTAGAAACGCTGATCCTAACACTAAGTTGTACATTAACGACTTTAACATCGAAGGTACTGGTGCTAAGTCTACTGGTATGATTAACTTGGTTAGATCTTTGCAACAACAGAACGTTCCAATTGATGGTATTGGTGTTCAAGCTCATTTGATTGTTGGACAGATTCCTTCTTCTATTCAGCAAAACTTGCAAAACTTTGCTAACTTGGGTGTTGAAGTTGCTATC AAC GAATTGGATATTAGAATGACTTTGCCAGTTACTCAACAGAAGTTGGAGCAACAACAAGAAGATTACAGAACTGTTATTAGAGCTTGTAAGGCTGTTTCTAGATGTGTTGGTGTTACAGTTTGGGATTGGACCGACAGATACTCCTGGGTTCCAGGTGTTTTTACTGGTGAAGGTGCTGCTTGTCCATGGGATGAGAACTTGGCTAAGAAGCCAGCTTACCAAGGTATTGTTGACGGTTGGTCTCAA。

[0083] SEQ ID NO.5:

[0084] CAATTGAACACCTTGGCTGTTAGAGCTGGTAAAAAGTATTTTGGTACTGCTACTGATAACCCAGAATTGGGTGATGCTCCATATGTTGCTCAATTGGGTAATACTGCTGATTTTAATCAAATCACTGCTGGTAATTCTATGAAATGGGATGCTACTGAACCATCTAGAGGAACTTTCACTTTTGCTAACGGTGATACTGTTGCTAACATGGCTAGAAACAGAGGTCAATTGTTGAGAGGTCACACTTGTGTTTGGCACTCTCAATTGCCAAACTGGGTTACTTCCGGTAACTTTGATAACTCTACTTTGTTGTCTATCGTTCAAAACCATTGTTCTACTTTGGTTTCTCATTACAGAGGTCAAATGTACTCTTGGGATGTTGTTAACGAACCTTTTAACG AAGATGGATCTTTTAGACAATCCGTTTTTTTTCAAAAGACCGGTACTGCTTATATTGCTACCGCTTTGAGAGCTGCTAGAAACGCTGATCCTAACACTAAGTTGTACATTAACGACTTTAACATCGAAGGTACTGGTGCTAAGTCTACTGGTATGATTAACTTGGTTAGATCTTTGCAACAACAGAACGTTCCAATTGAT GGTATTGGTGTTCAAGCTCATTTGATTGTTGGACAGATTCCTTCTTCTATTCAGCAAAACTTGCAAAACTTTGCTAACTTGGGTGTTGAAGTTGCTATCACCGAATTGGATATTAGAATGACTTTGCCAGTTACTCAACAGAAGTTGGAGCAACAACAAGAAGATTACAGAACTGTTATTAGAGCTTGTAAGGCTGTTTCT TTA TGTGTTGGTGTTACAGTTTGGGATTGGACCGACAGATACTCCTGGGTTCCAGGTGTTTTTACTGGTGAAGGTGCTGCTTGTCCATGGGATGAGAACTTGGCTAAGAAGCCAGCTTACCAAGGTATTGTTGACGGTTGGTCTCAA。

[0085] SEQ ID NO. 6:

[0086] CAATTGAACACCTTGGCTGTTAGAGCTGGTAAAAAGTATTTTGGTACTGCTACTGATAACCCAGAATTGGGTGATGCTCCATATGTTGCTCAATTGGGTAATACTGCTGATTTTAATCAAATCACTGCTGGTAATTCTATGAAATGGGATGCTACTGAACCATCTAGAGGAACTTTCACTTTTGCTAACGGTGATACTGTTGCTAACATGGCTAGAAACAGAGGTCAATTGTTGAGAGGTCACACTTGTGTTTGGCACTCTCAATTGCCAAACTGGGTTACTTCCGGTAACTTTGATAACTCTACTTTGTTGTCTATCGTTCAAAACCATTGTTCTACTTTGGTTTCTC ATTACAGAGGTCAAATGTACTCTTGGGATGTTGTTAACGAACCTTTTAACGAAGATGGATCTTTTAGACAATCCGTTTTTTTTCAAAAGACCGGTACTGCTTATATTGCTACCGCTTTGAGAGCTGCTAGAAACGCTGATCCTAACACTAAGTTGTACATTAACGACTTTAACATCGAAGGTACTGGTGCTAAGTCTACTGGTATGATTAACTTGGTTAGATCTTTGCAACAACAGAACGTTCCAATTGATGGTATTGGTGTTCAAGCTCATTTGATTGTTGGACAGATTCCTTCTTCTATTCAGCAAAACTTGCAAAACTTTGCTAACTTGGGTGTTGAAGTTGCTATC ACC GAATTGGATATTAGAATGACTTTGCCAGTTACTCAACAGAAGTTGGAGCAACAACAAGAAGATTACAGAACTGTTATTAGAGCTTGTAAGGCTGTTTCT AGATGTGTTGGTGTTACAGTTTGGGATTGGACCGACAGATACTCCTGGGTTCCAGGTGTTTTTACTGGTGAAGGTGCTGCTTGTCCATGGGATGAGAACTTGGCTAAGAAGCCAGCTTACCAAGGTATTGTTGACGGTTGGTCTCAA.

[0087] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A xylanase mutant with improved thermostability and pH tolerance, characterized in that: The xylanase mutants are based on the xylanase AbXyL10A from Agaricus bisporus var burnettii as the parent, and the amino acid sites T234 and R268 are mutated to obtain the xylanase mutants T234N and R268L. The amino acid sequence of the xylanase mutant T234N is shown in SEQ ID NO.2; the amino acid sequence of the xylanase mutant R268L is shown in SEQ ID NO.

3.

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

3. The gene according to claim 2, wherein The gene sequence encoding the xylanase mutant T234N is shown in SEQ ID NO. 4, and the gene sequence encoding the xylanase mutant R268L is shown in SEQ ID NO.

5.

4. A recombinant vector comprising the gene according to claim 2 or 3.

5. A recombinant bacterium comprising the recombinant vector according to claim 4.

6. Use of the xylanase mutant according to claim 1 in any of the following: (1) Application in xylan degradation; (2) Application in the preparation of catalytic preparations for xylan degradation; (3) Application in the preparation of feed additives.

7. Use of the recombinant bacterium according to claim 5 in preparing a xylanase mutant with improved thermostability and pH tolerance.

8. A method for producing a xylanase mutant by fermentation using the recombinant bacteria according to claim 5, characterized in that: The method comprises fermenting the recombinant bacteria, separating the fermentation liquid and collecting the precipitate, then inducing the culture using a fermentation medium containing methanol, and obtaining the xylanase mutant from the supernatant of the culture liquid.

9. A catalytic preparation for xylan degradation, characterized in that: The active ingredient is the xylanase mutant according to claim 1.

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