Xylanase mutant with synchronously improved thermal stability and pH tolerance and application of xylanase mutant

Abstract

The invention discloses a xylanase mutant with synchronously improved thermal stability and pH tolerance and application of the xylanase mutant, and belongs to the field of gene engineering and protein engineering. According to the invention, xylanase AbXyL10A from Agaricus bisporus var burnettii is taken as a female parent, and two amino acid sites of Thr234 and Arg268 are subjected to mutation, so that two types of xylanase mutants T234N and R268L are obtained. Compared with wild type xylanase, the thermal stability and pH tolerance of the two xylanase mutants are obviously improved. In the aspect of specific activity, when the beech xylan is used as a substrate, the specific activity of the two xylanase mutants is obviously improved compared with that of wild type xylanase. The invention provides a theoretical reference for the improvement of xylanase, and the obtained xylanase mutant with synchronously improved thermal stability and pH tolerance has a huge application prospect in the aspects of animal feed and biomass degradation sugar production.

Description

Technical Field

[0001] The 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 are connected by hydrogen bonds, covalent bonds, etc., forming the overall structure of plant cell walls, and are abundant renewable biomass resources in nature. Cellulose accounts for the largest proportion, and hemicellulose is the second. Xylan is the main component of hemicellulose, accounting for about 15%-40% in the cell wall, and is the second polysaccharide after cellulose. Xylan is a complex linear polymer composed of β-(1,4)-linked xylose residues. These xylose residues can be replaced by different functional groups on their molecular chains, including arabinose residues, acetyl groups, glucuronic acid (GlcA) and 4-O-methyl glucuronic 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, which can randomly cut the β-1,4 glycosidic bonds in the main chain to produce xylo-oligosaccharides. Xylanases are widely used in the energy, feed, food and other industries and have received high attention. However, most of the xylanases used in industry today are mesophilic enzymes, which are difficult to adapt to the high temperature requirements in production. Currently, the most reported are GH10 family xylanases and GH11 family xylanases. Among them, GH10 family xylanases have good thermal stability, but low catalytic efficiency, which limits their application in industrial production. Therefore, it is very important to develop new high-temperature resistant high-specific activity xylanases or to modify the thermal stability and catalytic activity of existing enzymes to promote the development of the industry.

[0004] Traditional modification methods are mainly based on the spatial structure and amino acid sequence of xylanase, and a high-throughput mutant library is established through directed evolution. Then, mutants with higher thermal stability or catalytic activity are screened out from the mutant library. This method generally takes a lot of time. As protein structure and function identification technology becomes more and more mature, people's research on protein structure is becoming more and more in-depth, and rational strategies for molecular modification of proteins based on rational design have developed. At present, a series of reasonable strategies have been developed to improve the thermal stability and catalytic activity of enzymes, such as using computers to predict "hot spots" for enzyme stability or activity enhancement; using the physicochemical properties of amino acid residue sites in protein structure and virtual mutagenesis to predict changes in protein-ligand binding energy, etc. Summary of the invention

[0005] The purpose of the present invention is to provide a xylanase mutant with improved thermal stability and pH tolerance and application thereof, so as to solve the problems existing in the above-mentioned prior art. By taking the xylanase AbXyL10A from Agaricus bisporus var burnettii as the parent, respectively mutating the 234th The in the amino acid sequence to Asn and the 268th Arg to Leu, two xylanase mutants T234N and R268L with improved thermal stability and pH tolerance are obtained, 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 thermal stability and pH tolerance. The xylanase mutant is prepared by using xylanase AbXyL10A from Agaricus bisporus var burnettii as a parent, and mutating two amino acid sites T234 and R268 to obtain a xylanase mutant T234N and a xylanase mutant 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 invention also provides a recombinant vector comprising the gene.

[0012] The present invention also provides a recombinant bacterium comprising the recombinant vector. In the embodiment 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 the degradation of xylan;

[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 the use of the recombinant bacteria in preparing xylanase with 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 invention also provides a catalytic preparation for xylan degradation, the active component of which is the xylanase mutant.

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

[0021] The invention uses the 10th family xylanase AbXyL10A (NCBI Reference Sequence: XP_007330618.1) derived 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 constructs a recombinant strain containing the mutant with the assistance of machine learning, and screens out two xylanase mutants T234N and R268L with greatly improved thermal stability and pH tolerance after induction culture. The optimum pH of xylanase mutants T234N and R268L was 5.0, and the optimum pH of wild-type xylanase was 6.0. In terms of pH tolerance, wild-type xylanase and xylanase mutants were treated at different pH values ​​(1.0-12.0) for 1 h. The pH tolerance of the two mutants in strong acid (pH 1-3) and strong alkali (pH 10-12) was significantly improved compared with wild-type xylanase, and they had excellent acid-base tolerance. The optimum temperature of wild-type xylanase was between 50-60℃, and the optimum temperature of xylanase mutants T234N and R268L was between 80-90℃. In terms of thermal stability, the T values ​​of xylanase mutants T234N and R268L were 0. 50The values ​​were 28.5℃ and 30.5℃ higher than those of the wild-type xylanase (53℃), respectively, and the half-lives at 80℃ were 5.1 times and 8.3 times higher than those of the wild-type xylanase (7.5min), 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 850.6U / mg and 684.0U / mg, respectively, which were 233% and 167% higher than those of the wild-type AbXyL10A (255.8U / mg), respectively. When sugarcane xylan was used as substrate, the specific activities of the xylanase mutants T234N and R268L were 455.2U / mg and 475.3U / mg, respectively, which were 174% and 186% higher than those of the wild-type AbXyL10A (166.2U / mg), respectively. It can be seen that the present invention obtains two xylanase mutants with improved high thermal stability and pH tolerance, and has great potential for application in animal feed and biomass degradation and sugar production. 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0023] Figure 1 The 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 enzyme xylanase AbXyL10A, xylanase mutant T234N and xylanase mutant R268L, respectively;

[0024] Figure 2 The optimum pH results of xylanase mutants and wild-type xylanase are shown in Figure 2.

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

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

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

[0028] Figure 6 is the half-life of the xylanase mutant and wild-type xylanase at 80°C 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 test materials involved in the embodiments of the present invention are:

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

[0036] (2) Enzymes and other biochemical reagents: Taq enzyme was purchased from Quanshijin Company, endonuclease was purchased from Quanshijin Company, and beech wood xylan was purchased from Sigma Company. Other reagents were domestic analytical grade reagents (all available from ordinary 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 of the xylanase gene AbXyL10A from Agaricus bisporus var burnettii (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.6) as a template, the Thr234 and Arg268 sites were mutated by site-directed mutagenesis. The primer design is shown in Table 1. The mutation method and cloning method refer 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 xylanase mutants 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 colony PCR was used for verification 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 a 10 mL test tube containing 2 mL of BMGY medium, and placed in a shaking incubator at 30°C and 220 rpm for 48 h. The culture solution was centrifuged at 3000 g for 5 min, the supernatant was discarded, the precipitate was resuspended in 2 mL of BMMY medium containing 0.5% methanol, and the precipitate was again placed in an induction incubation at 30°C and 220 rpm for 48 h. The supernatant was used for enzyme activity detection, 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) with improved enzyme activity compared with the wild enzyme were screened.

[0051] The wild-type GS115 / AbXyL10A and two mutants GS115 / T234N and GS115 / R268L were enlarged into a fermentation system. First, the seed culture was inoculated into YPD medium, and then inoculated into a 1L Erlenmeyer flask containing 300mL 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 BMMY medium containing 0.5% methanol, and then placed at 30°C and 220rpm for induction culture again. 0.5mL of methanol was added every 12h to keep 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 anion exchange method was used to purify the protein. 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 about 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)

[0054] The specific method is as follows: Under the optimal pH and temperature conditions, 1 mL of the reaction system includes 100 μL of diluted enzyme solution, 900 μL of substrate, and the reaction is carried out for 10 minutes. 1.5 mL of DNS is added to terminate the reaction and the reaction is boiled in water for 5 minutes. After cooling, the OD value is measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose xylan to produce 1 μmoL reducing sugar per minute under 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 improved thermostability and pH tolerance

[0057] The xylanase mutant and wild-type xylanase obtained in Example 2 were subjected to enzymatic reaction at different pH values ​​(1.0-7.5) to determine their optimum pH values. The xylanase activity of the substrate xylan was determined at 55° C. 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, 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, and in an 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 of recombinant xylanase mutants and wild-type xylanase with improved thermostability and pH tolerance

[0060] The xylanase mutants and wild-type xylanase were diluted with 0.1 mol / L citric acid-disodium hydrogen phosphate buffer of different pH (1.0-12.0) and placed in a 37°C constant temperature water bath for 1 h. Then, their relative residual enzyme activities were determined under standard conditions, with the untreated enzyme activity as 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 reaction 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 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 30min, 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 value was 81.5℃, which was 28.5℃ higher than that of the wild-type xylanase (53℃). The T 50 The half-life at 85°C was 83.5°C, which was 30.5°C higher than that of the wild type (53°C). Figure 6 As shown, the half-life of the xylanase mutant T234N at 85°C was 46 min, 5.1 times higher than that of the wild-type xylanase (7.5 min), and the half-life of the mutant R268L at 85°C was 70 min, 8.3 times higher than that of the wild-type xylanase (7.5 min). The thermal stability of the two xylanase mutants was significantly improved compared with the wild-type xylanase.

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

[0070] The detection method was based on 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, and the enzyme activity was measured under the optimal conditions (temperature, pH). The corresponding reaction rate was calculated and 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 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 sugarcane xylan was used 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] CAATTGAACACCTTGGCTGTTAGAGCTGGTAAAAAGTATTTTGGTACTGCTACTGATAACCCAGAATTGGGTGATGCTCCAATATGTTGCTCAATTGGGTAATACTGCTGATTTTAATCAAATCACTGCTGGTAATTCTATGAAATGGGATGCTACTGAACCATCTAGAGGAACTTTCACTTTTGCTAACGGTGATACTGTTGCTAACATGGCTAGAAACAGAGGTCAATTGTTGAGAGGTCACACTTGTGTTTGGCACTCTCAATTGCCAAACTGGGTTACTTCCGGTAACTTTGATAACTCTACTTTGTTGTCTATCGTTCAAAACCATTGTTCTACTTTGGTTTCTC ATTACAGAGGTCAAATGTACTCTTGGGATGTTGTTAACGAACCTTTTAACGAAGATGGATCTTTTAGACAATCCGTTTTTTTTCAAAAGACCGGTACTGCTTATATTGCTACCGCTTTGAGAGCTGCTAGAAACGCTGATCCTAAACACTAAGTTGTACATTAACGACTTTAACATCGAAGGTACTGGTGCTAAGTCTACTGGTATGATTAACTTGGTTAGATCTTTGCAACAACAGAACGTTCCAATTGATGGTATTGGTGTTCAAGCTCATTTGATTGTTGGACAGATTCCTTCTTCTATTCAGCAAAACTTGCAAAACTTTGCTAACTTGGGTGTTGAAGTTGCTATC AAC GAATTGGATATTAGAATGACTTTGCCAGTTACTCAACAGAAGTTGGAGCAACAACAAGAAGATTACAGAACTGTTATTAGAGCTTGTAAGGCTGTTTCTAGATGTGTTGGTGTTACAGTTTGGGATTGGACCGACAGATACTCCTGGGTTCCAGGTGTTTTTACTGGTGAAGGTGCTGCTTGTCCATGGGATGAGAACTTGGCTAAGAAGCCAGCTTACCAAGGTATTGTTGACGGTTGGTCTCAA。

[0083] SEQ ID NO.5:

[0084] CAATTGAACACCTTGGCTGTTAGAGCTGGTAAAAAGTATTTTGGTACTGCTACTGATAACCCAGAATGGGTGATGCTCCATATGTTGCTCAATTGGGTAATACTGCTGATTTTAATCAAATCACTGCTGGTAATTCTATGAAATGGGATGCTACTGAACCATCTAGAGGAACTTTCACTTTTGCTAACGGTGATACTGTTGCTAACATGGCTAGAAACAGAGGTCAATTGTTGAGAGGTCACACTTGTGTTTGGCACTCTCAATTGCCAAACTGGGTTACTTCCGGTAACTTTGATAACTCTACTTTGTTGTCTATCGTTCAAAACCATTGTTCTACTTTGGTTTCTCATTACAGAGGTCAAATGTACTCTTGGGATGTTGTTAACGAACCTTTTAACG AAGATGGATCTTTTAGACAATCCGTTTTTTTTCAAAAGACCGGTACTGCTTATATTGCTACCGCTTTGAGAGCTGCTAGAAACGCTGATCCTAACACTAAGTTGTACATTAACGACTTTAACATCGAAGGTACTGGTGCTAAGTCTACTGGTATGATTAACTTGGTTAGATCTTTGCAACAACAGAACGTTCCAATTGAT GGTATTGGTGTTCAAGCTCATTTGATTGTTGGACAGATTCCTTCTTCTATTCAGCAAAACTTGCAAAACTTTGCTAACTTGGGTGTTGAAGTTGCTATCACCGAATTGGATATTAGAATGACTTTGCCAGTTACTCAACAGAAGTTGGAGCAACAACAAGAAGATTACAGAACTGTTATTAGAGCTTGTAAGGCTGTTTCT TTA TGTGTTGGTGTTACAGTTTGGGATTGGACCGACAGATACTCCTGGGTTCCAGGTGTTTTTACTGGTGAAGGTGCTGCTTGTCCATGGGATGAGAACTTGGCTAAGAAGCCAGCTTACCAAGGTATTGTTGACGGTTGGTCTCAA。

[0085] SEQ ID NO.6:

[0086] CAATTGAACACCTTGGCTGTTAGAGCTGGTAAAAAGTATTTTGGTACTGCTACTGATAACCCAGAATTGGGTGATGCTCCAATATGTTGCTCAATTGGGTAATACTGCTGATTTTAATCAAATCACTGCTGGTAATTCTATGAAATGGGATGCTACTGAACCATCTAGAGGAACTTTCACTTTTGCTAACGGTGATACTGTTGCTAACATGGCTAGAAACAGAGGTCAATTGTTGAGAGGTCACACTTGTGTTTGGCACTCTCAATTGCCAAACTGGGTTACTTCCGGTAACTTTGATAACTCTACTTTGTTGTCTATCGTTCAAAACCATTGTTCTACTTTGGTTTCTC ATTACAGAGGTCAAATGTACTCTTGGGATGTTGTTAACGAACCTTTTAACGAAGATGGATCTTTTAGACAATCCGTTTTTTTTCAAAAGACCGGTACTGCTTATATTGCTACCGCTTTGAGAGCTGCTAGAAACGCTGATCCTAAACACTAAGTTGTACATTAACGACTTTAACATCGAAGGTACTGGTGCTAAGTCTACTGGTATGATTAACTTGGTTAGATCTTTGCAACAACAGAACGTTCCAATTGATGGTATTGGTGTTCAAGCTCATTTGATTGTTGGACAGATTCCTTCTTCTATTCAGCAAAACTTGCAAAACTTTGCTAACTTGGGTGTTGAAGTTGCTATC ACC GAATTGGATATTAGAATGACTTTGCCAGTTACTCAACAGAAGTTGGAGCAACAACAAGAAGATTACAGAACTGTTATTAGAGCTTGTAAGGCTGTTTCT AGATGTGTTGGTGTTACAGTTTGGGATTGGACCGACAGATACTCCTGGGTTCCAGGTGTTTTTACTGGTGAAGGTGCTGCTTGTCCATGGGATGAGAACTTGGCTAAGAAGCCAGCTTACCAAGGTATTGTTGACGGTTGGTCTCAA.

[0087] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A xylanase mutant with improved thermostability and pH tolerance, characterized in that: The xylanase mutant is based on the xylanase AbXyL10A from Agaricus bisporus var burnettii as the parent, and the two amino acid sites T234 and R268 are mutated to obtain the xylanase mutant T234N and the xylanase mutant 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, characterized in that 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 the degradation of xylan; (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 xylanase 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 with a fermentation medium containing methanol, and obtaining the xylan 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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