CN113943723A

CN113943723A - Xylanase mutant with improved thermostability, preparation and application thereof

Info

Publication number: CN113943723A
Application number: CN202010693613.3A
Authority: CN
Inventors: 周志华; 邢宏观; 邹根; 刘春燕; 柴顺星; 刘睿; 严兴; 李新良
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2020-07-17
Filing date: 2020-07-17
Publication date: 2022-01-18
Anticipated expiration: 2040-07-17
Also published as: CN113943723B

Abstract

The invention provides a xylanase mutant with improved thermostability, and preparation and application thereof. The invention determines the amino acid site related to the thermal stability, and obtains the mutant with obviously improved thermal stability by carrying out site-specific modification. The invention also provides nucleic acid for encoding the mutant, a construction body for expressing the mutant, a xylanase mutant library covering a plurality of mutants, a method for degrading xylan by using the mutant and the like.

Description

Xylanase mutant with improved thermostability, preparation and application thereof

Technical Field

The invention belongs to the field of protein engineering and genetic engineering, and relates to a xylanase mutant with improved heat stability and better specific activity, and preparation and application thereof.

Background

Hemicellulose is the most abundant non-cellulosic heteropolysaccharide present on earth and is a major constituent of plant cells and cell walls. The hemicellulose is efficiently converted into other valuable byproducts, so that the method not only has good economic benefit, but also plays a positive role in environmental protection.

The main component of hemicellulose in plant cells and cell walls is xylan. Xylan is the major structural polysaccharide in plant cells, the second most abundant polysaccharide in nature, accounting for about one third of all renewable organic carbons on earth.

Xylan, a main chain of Xylose molecules (D Xylose) linked by β -1, 4-glycosidic bonds; an arabinofuranosyl group, a glucuronyl group, an acetyl group, or the like is linked to form a branched chain. The raw materials rich in xylan in nature are widely available, and include agricultural, forest and industrial wastes such as hardwood, softwood, straw, bran and the like, municipal solid wastes and the like. The amount of xylan contained in different plants is different, the hard wood contains more xylan than the soft wood, the hard wood can account for 15-30% of the dry weight, and the soft wood generally accounts for 7-12% of the dry weight. In some annual plants such as wheat and cotton seed hulls, the xylan content is as high as over 30%.

Xylanases are glycosidases (O-glycoside hydrolases, EC3.2.1. x) that catalyze the endo-hydrolysis of 1,4- β D-xylosidic bonds (β -1, 4-glycosidic bonds) in xylan. Xylanases can be classified mainly into the GH10 family and the GH11 family, according to their structure and catalytic domain. The family of xylanases, GH10, includes endo-1, 4-beta-xylanases (EC3.2.1.8), endo-1, 3-beta-xylanases (EC 3.2.1.32) and cellobiohydrolases (EC 3.2.1.91), with endo-1, 4-beta-xylanases being the predominant. Compared with GH10 family, GH11 family can only act on D-xylose specifically, can be called xylanase in the true sense, and has the characteristics of low molecular weight, high activity and the like.

In industry, xylanases have a wide range of applications. In the food industry, xylanases are used in the processing of fruits, vegetables and plants etc. to facilitate the impregnation process; in the paper industry, xylanases are used to facilitate the pulping process; in the textile industry, xylanase is used in the enzymolysis of textiles to reduce or replace chemical hemp mixing methods so as to reduce environmental pollution; in the feed industry, xylanases are added to the feed of monogastric and ruminant animals to improve the digestibility and nutritional value of the feed; in the field of biological energy, xylanase can be applied to the industrial production of converting lignocellulose into fuel ethanol together with other cellulase and hemicellulase, and in recent years, with the research and development of pentose fermentation ways and strains, the process for producing fuel ethanol by fermenting xylan hydrolysate xylose by using bacteria, yeast and filamentous fungi is mature day by day, and the role played by xylanase in the field of biological energy is more and more important.

Most xylanases used in industrial applications are derived from bacteria or fungi, and most of them are mesophilic. However, high temperature treatment is often required in the production process, which results in great loss of xylanase performance. The xylanase mutant strain with excellent thermal stability can be obtained, so that the xylanase mutant strain is wider in application and higher in efficiency. Therefore, there is a need in the art to develop a method for maintaining a desired enzyme activity under various high temperature conditions

Disclosure of Invention

The present invention aims to provide xylanase mutants with improved thermostability, their preparation and their use.

In a first aspect of the invention, there is provided a method of increasing the thermostability of a xylanase, comprising: mutating the amino acid sequence of the xylanase, corresponding to the amino acid sequence shown by the wild-type xylanase (SEQ ID NO:2), at a site selected from the group consisting of: 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 133 th, 192 th, 203 th bits.

In a preferred embodiment, the 14 th mutation is his (h); mutation at position 28 to Val (V); mutation at position 29 to Ser (S) or Ala (A); mutation at position 30 to Ser (S) or Pro (P); mutation at position 31 to His (H); the 41 th mutation is Arg (R); the 52 th mutation is Leu (L); mutation at 131 th site to Cys (C); mutation at position 133 to Glu (E); mutation at position 192 to Asp (D); and/or, a mutation at position 203 to Val (V).

In another aspect of the invention, there is provided a xylanase mutant which is: (a) a protein having an amino acid sequence corresponding to a wild-type xylanase, mutated at a site or combination of sites selected from the group consisting of: 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 133 th, 192 th, 203 th bits; (b) a protein derived from (a) and having the function of (a) a protein, which is obtained by substituting, deleting or adding one or more (e.g., 1 to 20; preferably 1 to 15; more preferably 1 to 10; e.g., 5, 3) amino acid residues in the amino acid sequence of the protein (a), but has the same amino acid as the mutated amino acid at the corresponding position of the protein (a) at the 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 133 th, 192 th or 203 th positions corresponding to the wild-type xylanase; (c) and (b) a protein derived from (a) which has more than 80% homology (preferably more than 85%, more preferably more than 90%, more preferably more than 95%, e.g., 98%, 99%) with the amino acid sequence of the protein (a) and which has the function of the protein (a), but the amino acid corresponding to the 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 133 th, 192 th or 203 th position of the wild-type xylanase is the same as the mutated amino acid at the corresponding position of the protein (a).

In another preferred embodiment, the xylanase mutant comprises a protein selected from the group consisting of: corresponding to wild-type xylanase:

(1) the 14 th position is mutated into His, and the 28 th position is mutated into Val; mutation at the 29 th position to Ser, mutation at the 30 th position to Pro, mutation at the 31 st position to His, mutation at the 52 th position to Leu, mutation at the 133 th position to Glu, mutation at the 192 th position to Asp (D), and mutation at the 203 th position to Val (L28V/K133E/G192D/Q14H/D29S/A203V/S30P/T31H/A52L);

(2) the 14 th mutation is His, the 28 th mutation is Val, the 29 th mutation is Ser, the 30 th mutation is Pro, the 31 th mutation is His, the 52 th mutation is Leu, the 133 th mutation is Glu, the 192 th mutation is Asp, the 203 th mutation is Val, and the 41 th mutation is Arg (L28V/K133E/G192D/Q14H/D29S/A203V/S30P/T31H/A52L/K41R);

(3) the 14 th position is mutated into His, the 28 th position is mutated into Val, the 29 th position is mutated into Ser, the 203 th position is mutated into Val, the 30 th position is mutated into Pro, the 31 th position is mutated into His, the 52 th position is mutated into Leu, the 131 th position is mutated into Cys, the 133 th position is mutated into Glu, and the 192 th position is mutated into Asp (L28V/K133E/G192D/Q14H/D29S/A203V/S30P/T31H/A52L/Q131C);

(4) mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 133 to Glu, and mutation at position 192 to Asp (L28V/K133E/G192D/Q14H/D29S/A203V/S30P/T31H);

(5) mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 133 to Glu, and mutation at position 192 to Asp (L28V/K133E/G192D/Q14H/D29S/A203V/S30P)

(6) Mutation at position 29 to Ser, mutation at position 28 to Val, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 52 to Leu, mutation at position 133 to Glu, and mutation at position 192 to Asp (L28V/K133E/G192D/Q14/D29S/A203V/S30P/T31H/A52L);

(7) mutation at position 14 to His, mutation at position 28 to Val, mutation at position 203 to Val, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 52 to Leu, mutation at position 133 to Glu, and mutation at position 192 to Asp (L28V/K133E/G192D/Q14H/D29/A203V/S30P/T31H/A52L);

(8) mutation at position 14 to His, mutation at position 28 to Val, mutation at position 29 to Ser, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 52 to Leu, mutation at position 133 to Glu, and mutation at position 192 to Asp (L28V/K133E/G192D/Q14H/D29S/A203/S30P/T31H/A52L);

(9) mutation at position 29 to Ser, mutation at position 28 to Val, mutation at position 30 to Pro, mutation at position 31 to His, mutation at position 52 to Leu, mutation at position 133 to Glu, and mutation at position 192 to Asp (L28V/K133E/G192D/Q14/D29S/A203/S30P/T31H/A52L);

(10) the 14 th position is mutated into His, the 28 th position is mutated into Val, the 29 th position is mutated into Ser, the 30 th position is mutated into Ser, the 203 th position is mutated into Val, the 133 th position is mutated into Glu, and the 192 th position is mutated into Asp (L28V/N30S/K133E/G192D/Q14H/D29S/A203V); or

(11) The 14 th mutation is His, the 28 th mutation is Val, the 29 th mutation is Ala, the 30 th mutation is Ser, the 203 th mutation is Val, the 133 th mutation is Glu, and the 192 th mutation is Asp (L28V/N30S/K133E/G192D/Q14H/D29A/A203V); or

(12) Mutation at position 28 to Val, mutation at position 30 to Ser, mutation at position 133 to Glu, and mutation at position 192 to Asp (L28V/N30S/K133E/G192D (Xyn 370)).

In a preferred embodiment, the xylanase mutant comprises a protein selected from the group consisting of: 14, 16, 18, 12, 10, 20, 22, 24, 26, 8, 6 or 4.

In another aspect of the invention, there is provided an isolated polynucleotide, wherein said nucleic acid encodes any of the xylanase mutants described above; preferably, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 11, SEQ ID NO. 9, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 7, SEQ ID NO. 5 or SEQ ID NO. 4.

In another aspect of the invention, there is provided a vector comprising said polynucleotide.

In another aspect of the invention, there is provided a genetically engineered host cell comprising said vector, or having said polynucleotide integrated into its genome.

In a preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cells include Escherichia coli cells, Bacillus subtilis cells and the like; preferably, the eukaryotic cell comprises a mold cell, a yeast cell, an insect cell, a plant cell, a fungal cell, or a mammalian cell, and the like.

In another aspect of the invention, there is provided a method for preparing a xylanase mutant as defined in any preceding claim, the method comprising:

(i) culturing said host cell;

(ii) collecting a culture containing the xylanase mutant;

(iii) isolating the xylanase mutant from the culture.

In another aspect of the present invention, there is provided a composition for degrading xylan, comprising effective amounts of: a xylanase mutant as described in any preceding claim; or said host cell or a culture or lysate thereof; and a dietetically or industrially acceptable carrier.

In another aspect of the invention there is provided the use of a xylanase mutant as defined in any preceding claim, or a composition as defined, for the degradation of xylan; preferably, the xylanase mutant cleaves beta-1, 4-glycosidic bonds, thereby hydrolyzing xylan.

In another aspect of the present invention, there is provided a method for degrading xylan, the method comprising: degrading xylan with a xylanase mutant or a composition as described in any of the preceding; preferably, the xylanase mutant cleaves beta-1, 4-glycosidic bonds, thereby hydrolyzing xylan.

In a preferred embodiment, the degradation or enzyme digestion is degradation or enzyme digestion at a high temperature; preferably, the high temperature is 45-95 ℃; preferably 50-90 deg.C (such as 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C or 90 deg.C).

In another preferred embodiment, the substrate for degradation of the xylanase mutant or composition is xylan or a xylan-containing material.

In another preferred embodiment, the xylan includes (but is not limited to): beech xylan, corncob xylan, oat bran xylan.

In another preferred embodiment, the xylan-containing material includes (but is not limited to): industrial products, agricultural, forestry, industrial waste and municipal solid waste; preferably, including (but not limited to): pulp, feed, food, straw, hardwood, softwood, straw, bran.

In another aspect of the invention there is provided a use of a xylanase mutant as defined in any preceding claim, or a composition as defined, comprising: as a dietary supplement; as a plant (including, e.g., fruit or vegetable) processing additive; as a paper making process additive; as a textile processing additive; as a feed supplement; and/or for converting lignocellulose to fuel ethanol.

In another aspect of the invention, there is provided a library of xylanase mutants or polynucleotides encoding the same, the library comprising: at least 5, preferably at least 10, of the xylanase mutants or polynucleotides encoding same described above; more preferably it comprises said xylanase mutant or a polynucleotide encoding same.

In another aspect of the invention there is provided the use of a library of said xylanase mutants or polynucleotides encoding same to provide an appropriate xylanase mutant to degrade xylan depending on the temperature of the reaction system in which the xylan to be degraded is located.

In another aspect of the present invention, there is provided a kit for degrading xylan, comprising: a xylanase mutant or combination of mutants as described in any preceding claim; the host cell of (a); the composition as described; or a library of said xylanase mutants or polynucleotides encoding same.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1-1 to FIG. 1-13, summary of sequence information for wild type and mutant.

Detailed Description

The inventor starts from a wild xylanase from a GH11 family, determines amino acid sites related to thermostability by an error-prone PCR (polymerase chain reaction) technology of directed evolution and long-term research experience of the inventor aiming at the xylanase, and obtains a mutant with remarkably improved thermostability by performing site-specific modification.

As used herein, the term "xylose" refers to a monosaccharide containing five carbon atoms. The molecular formula is C4H9O4 CHO. The "xylan" is a polymer of "xylose".

As used herein, unless otherwise indicated, the terms "mutant xylanase", "mutant xylanase" and "mutants thereof are used interchangeably and refer to enzymes (polypeptides/proteins) that are constituted after mutation at some site identified by the present inventors to have a correlation with the thermostability of the enzyme, preferably corresponding to the amino acid sequence shown in SEQ ID NO:2, at a site selected from the group consisting of: 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 203 th bits.

If desired, the xylanase (wild-type) before mutation may be a "protein having an amino acid sequence as set forth in SEQ ID NO: 2". Unless otherwise stated, the mutation sites of the mutants of the present invention are based on the sequence shown in SEQ ID NO. 2.

In the present invention, unless otherwise indicated, the designation of xylanase mutants uses the expression "amino acid substituted at the original amino acid position" to denote the mutated amino acid in the xylanase mutant, e.g., Q14H, which denotes the substitution of the amino acid at position 14 from Q to H of the parent xylanase.

As used herein, "isolated xylanase" means a xylanase mutant that is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Those skilled in the art are able to purify xylanase mutants using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.

As used herein, "increased thermostability" refers to a statistically significant increase, or a significant increase, in the thermostability of a mutated xylanase compared to the xylanase starting polypeptide prior to alteration. For example, after a certain period of heat treatment at the same treatment temperature, the residual enzyme activity of the mutant xylanase with improved heat stability is significantly improved by 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 70% or more, 80% or more, 100% or more, 150% or more, etc. compared with the enzyme before modification.

As used herein, the term "xylanase mutant or a library of polynucleotides encoding the same" refers to a collection of polypeptides or polynucleotides comprising a series of mutant xylanases provided by the present invention. A plurality of xylanase mutants with different stability or different temperature adaptability or polynucleotides encoding the xylanase mutants are assembled in a library, and the appropriate xylanase or a nucleic acid encoding the xylanase is selected by a person skilled in the art according to the reaction conditions required by the person.

The mutant proteins of the invention can be chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, higher plant, insect, and mammalian cells).

The invention also includes fragments, derivatives and analogues of the xylanase mutants. As used herein, the terms "fragment," "derivative," and "analog" refer to a protein that retains substantially the same biological function or activity as a native xylanase mutant of the invention. A protein fragment, derivative or analog of the invention may be (i) a protein in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein having a substituent group in one or more amino acid residues, or (iii) a protein in which an additional amino acid sequence is fused to the protein sequence (e.g., a leader or secretory sequence or a sequence used to purify the protein or a pro-protein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art, as defined herein. However, the xylanase mutant and the amino acid sequences of the fragments, derivatives and analogues thereof must have at least one mutation as described above, preferably the mutation is a mutation corresponding to the amino acid sequence shown in SEQ ID NO. 2, and is selected from the group consisting of amino acid 14, 28, 29, 30, 31, 41, 52, 131, 133, 192 and/or 203.

In the present invention, the term "xylanase mutant" also includes (but is not limited to): deletion, insertion and/or substitution of several (usually 1 to 20, more preferably 1 to 10, still more preferably 1 to 8, 1 to 5, 1 to 3, or 1 to 2) amino acids, and addition or deletion of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition or deletion of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of xylanase mutants. However, in these variants, there must be at least one mutation according to the invention as described above, preferably a mutation corresponding to the amino acid sequence shown in SEQ ID No. 2, selected from the group consisting of the amino acids at position 14, 28, 29, 30, 31, 41, 52, 131, 133, 192 and/or 203.

In the present invention, the term "xylanase mutant" also includes (but is not limited to): derived proteins having more than 80%, preferably more than 85%, more preferably more than 90%, even more preferably more than 95%, such as more than 98% or more than 99% sequence identity with the amino acid sequence of the xylanase mutant and retaining the protein activity. Likewise, in these derived proteins, there must be at least one mutation according to the invention as described above, preferably a mutation corresponding to the amino acid sequence shown in SEQ ID No. 2, selected from the group consisting of amino acids 14, 28, 29, 30, 31, 41, 52, 131, 133, 192 and/or 203.

The invention also provides analogues of the xylanase mutants. These analogs can differ from the xylanase mutant by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

As a preferred mode of the present invention, in the preferred stability-related site, the 14 th mutation is his (h); mutation at position 28 to Val (V); mutation at position 29 to Ser (S) or Ala (A); mutation at position 30 to Ser (S) or Pro (P); mutation at position 31 to His (H); the 41 th mutation is Arg (R); the 52 th mutation is Leu (L); mutation at 131 th site to Cys (C); mutation at position 133 to Glu (E); mutation at position 192 to Asp (D); and/or, a mutation at position 203 to Val (V).

In the specific embodiment of the invention, the inventor utilizes an error-prone PCR technology to carry out a first round of random mutation on wild type Xyn11 (the nucleic acid sequence is shown as SEQ ID NO:1, and the protein sequence is shown as SEQ ID NO:2) from a xylanase 11 family, wherein the capacity of a random mutation library is 10000, and a preferred mutant Xyn68 is obtained. The optimized Xyn68 is used as a template, error-prone PCR is used for carrying out second round random mutation on Xyn68, the capacity of a mutation library is 20000, and the optimized mutant Xyn370 is obtained (the nucleic acid sequence is shown as SEQ ID NO:3, and the protein sequence is shown as SEQ ID NO: 4). Compared to the wild type, Xyn370 includes mutations: L28V/N30S/K133E/G192D.

Sequencing the preferred mutants obtained in the two rounds (including Xyn68 and Xyn370, but not limited to Xyn68 and Xyn370) shows that most of mutation sites are concentrated in Q14H, L28V, D29A, N30S, S93A, R97L, V99L, K133E, E159G, A187D, G192D and A203V.

The mutant sites of Q14H, L28V, D29A, N30S, S93A, R97L, V99L, K133E, E159G, A187D, G192D and A203V are singly and one by one introduced into wild type Xyn11, and the results show that Q14H, D29A and A203V have the function of improving the thermal stability of wild type Xyn11

The preferred mutant Xyn370 is introduced into the Q14H, the D29A and the V203 in a single, two-combination or three-combination mode to obtain the preferred mutant Xyn370-Q14H/D29A/A203V (the nucleic acid sequence is shown as SEQ ID NO:5, and the protein sequence is shown as SEQ ID NO: 6)

Carrying out saturation mutation on the 14 th amino acid residue of the preferred Xyn370-Q14H/D29A/A203V mutant, and replacing the residue Q with 18 amino acid residues except H; carrying out saturation mutation on the 29 th amino acid residue, and replacing the residue D with 18 other amino acids except A; saturation mutation is carried out on the 203 nd amino acid residue, and the amino acid residue is replaced by other 18 amino acids except A, so as to obtain the preferred mutant Xyn370-Q14H/D29S/A203V (the nucleic acid sequence is shown as SEQ ID NO:7, and the protein sequence is shown as SEQ ID NO: 8)

And carrying out homologous modeling on the preferred mutant Xyn370-Q14H/D29S/A203V by using an online server to obtain a structure diagram of the preferred mutant Xyn370-Q14H/D29S/A203V 3D.

And (3) predicting the B-factor value of the 3D stereo structure of the preferred mutant Xyn370-Q14H/D29S/A203V by using an online server to obtain the B-factor value information of each residue of the preferred mutant Xyn 370-Q14H/D29S/A203V.

Selecting residues with higher B-factor value in the preferred mutant Xyn370-Q14H/D29S/A203V, wherein the residues are respectively as follows: 30S, 31T, 41K, 52A, 53V, 108G, 109V, 110Q, 131Q, 170Q, 171K.

The preferred mutants Xyn370-Q14H/D29S/A203V are subjected to saturation mutation on 30S, 31T, 41K, 52A, 53V, 108G, 109V, 110Q, 131Q, 170Q and 171K respectively to construct a saturation mutation library, and S30P, T31H, K41R, A52L and Q131C mutation residues are screened from the saturation mutation library, so that the thermal stability of the preferred mutants Xyn370-Q14H/D29S/A203V is improved.

Respectively introducing S30P, T31H, K41R, A52L and Q131C into the preferred mutant Xyn370-Q14H/D29S/A203V in an S30P, S30P/T31H, S30P/T31H/A52L, S30P/T31H/A52L/K41R and S30P/T31H/A52L/Q131C iterative combination mode to obtain the preferred mutant Xyn 370-Q14H/D29S/A203V/S203 30P (the nucleic acid sequence is shown as SEQ ID NO:9, and the protein sequence is shown as SEQ ID NO: 10); preferably, the mutant Xyn370-Q14H/D29S/A203V/S30P/T31H (the nucleic acid sequence is shown as SEQ ID NO:11, and the protein sequence is shown as SEQ ID NO: 12); preferably, the mutant Xyn370-Q14H/D29S/A203V/S30P/T31H/A52L (the nucleic acid sequence is shown as SEQ ID NO:13, and the protein sequence is shown as SEQ ID NO: 14); xyn370-Q14H/D29S/A203V-S30P/T31H/A52L/K41R (the nucleic acid sequence is shown as SEQ ID NO:15, and the protein sequence is shown as SEQ ID NO: 16); preferably, the mutant Xyn370-Q14H/D29S/A203V/S30P/T31H/Q131C (the nucleic acid sequence is shown as SEQ ID NO:17, and the protein sequence is shown as SEQ ID NO: 18).

Through a series of transformation, the inventor obtains a plurality of preferable mutant strains with greatly improved thermal stability, but finds that the specific enzyme activity of the preferable mutant strains is also inevitably reduced while the thermal stability is improved, and in order to balance the relationship between the thermal stability and the specific enzyme activity, three sites which are possibly influenced by the enzyme activity of the preferable mutant strains Xyn370-Q14H/D29S/A203V/S P/T31H/A52L are respectively subjected to reversion mutation on the basis of the preferable mutant strains Xyn370-Q14/D S/A203V/S30P/T31H/A52L (the nucleic acid sequence is shown as SEQ ID NO:19, and the protein sequence is shown as SEQ ID NO: 20); xyn370-Q14H/D29/A203V/S30P/T31H/A52L (the nucleic acid sequence is shown as SEQ ID NO:21, and the protein sequence is shown as SEQ ID NO: 22); xyn370-Q14H/D29S/A203/S30P/T31H/A52L (the nucleic acid sequence is shown as SEQ ID NO:23, and the protein sequence is shown as SEQ ID NO: 24); xyn370-Q14/D29S/A203/S30P/T31H/A52L (the nucleic acid sequence is shown as SEQ ID NO:25, and the protein sequence is shown as SEQ ID NO: 26).

The invention also provides a polynucleotide sequence for encoding the xylanase mutant or conservative variant protein thereof.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The polynucleotides encoding the mature proteins of the mutants include: a coding sequence that encodes only the mature protein; the coding sequence for the mature protein and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature protein.

A "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, and may also include additional coding and/or non-coding sequences.

The invention also relates to a vector comprising the polynucleotide of the invention, a genetically engineered host cell transformed with the vector or xylanase mutant coding sequence of the invention, and a method for producing the protein of the invention by recombinant techniques.

The polynucleotide sequences of the invention can be used to express or produce recombinant xylanase mutants by conventional recombinant DNA techniques. Generally, the following steps are performed:

(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a xylanase mutant, or with a recombinant expression vector comprising the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) isolating and purifying the protein from the culture medium or the cells.

In the present invention, the xylanase mutant polynucleotide sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences encoding xylanase mutants and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

In the present invention, the host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as mold cells, yeast cells; or higher eukaryotic cells, such as plant cells. Representative examples are: escherichia coli, Bacillus subtilis, Streptomyces, and Agrobacterium; eukaryotic cells such as yeast, plant cells, and the like. In a specific embodiment of the present invention, Escherichia coli is used as the host cell.

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells. In a preferred form of the invention, the expression vector used is pET28 a; the microbial host cells transformed by the expression vector are all escherichia coli.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The xylanase can act on the inside of a long-chain molecule of xylan, and acts on a beta-1, 4-xyloside bond inside a main chain of the xylan in an internal mode to hydrolyze macromolecular polyxylose into simple sugars (such as xylo-oligosaccharide).

As used herein, the term "simple sugar" broadly refers to a generic term for a group of sugars formed after the xylan chain is cleaved, and the chain length is shorter than that before cleavage. For example, the simple sugar contains 1-50 xylose, preferably 1-30 xylose; more preferably, it contains 1-15 xylose; more preferably 1-10 xylose, such as 2, 3, 4, 5, 6, 7, 8, 9 xylose. The simple sugar comprises: monosaccharides, xylobiose, xylotriose, xylotetraose, and the like. In the present invention, the simple sugar refers to: xylo-oligosaccharide, small amount of xylose, and xylooligosaccharide.

After obtaining the xylanase mutant enzyme of the invention, the skilled person can conveniently apply the enzyme to act to hydrolyse a substrate, in particular xylan, according to the teachings of the present invention. As a preferred mode of the present invention, there is also provided a method for degrading xylan, the method comprising: the xylanase mutant enzyme of the invention is used to treat a substrate to be hydrolyzed, including but not limited to beech xylan, corncob xylan, oat bran xylan, and the like. In a selection mode of the invention, the xylanase degrades or enzyme-cuts a substrate at high temperature; preferably, the high temperature is 45-95 ℃; preferably 50-90 deg.C, such as 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C or 90 deg.C.

The invention also provides a composition comprising an effective amount of a xylanase mutant of the invention together with a dietetic or industrial acceptable carrier or excipient. Such vectors include (but are not limited to): water, buffer, glucose, water, glycerol, ethanol, and combinations thereof. One skilled in the art can determine the effective amount of xylanase mutant in the composition based on the actual use of the composition.

The composition can also be added with substances for regulating the enzymatic activity of the xylanase mutant of the invention. Any substance having a function of enhancing the enzymatic activity is usable. For example, some substances that may increase the enzymatic activity of the xylanase mutants of the invention are selected and validated from the group consisting of: k⁺、Mg²⁺、Cu²⁺、Co²⁺、K⁺、Mn²⁺、Cu²⁺、Co²⁺、Ni²⁺、Zn²⁺、Fe³⁺EDTA, etc.

The xylanase mutant, the composition containing the same, the cell expressing the same and the like can be contained in a kit so as to be convenient for enlarged application or commercial application. Preferably, the kit further comprises instructions for use to instruct a person to use the kit.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

Culture medium and culture medium formula

LB liquid medium: 5.0g/L yeast extract, 10.0g/L peptone and 10.0g/L NaCl.

LB solid medium: 5.0g/L yeast extract, 10.0g/L peptone, 10.0g/L NaCl and 2g/L agar.

Protein expression purification

The host BL21 containing xylanase wild type or preferred mutant expression vector is picked up to LB test tube, shaken at 37 deg.C and 180r/min overnight, transferred to LB triangular flask, shaken at 37 deg.C and 180r/min to OD600 to 0.4-0.6, placed on ice for 10min, and IPTG is added to final concentration of 40uM, 16 deg.C, 110r/min for induction for 16 h. After the induction, the cells were collected by centrifugation and lysed with lysis buffer (50mM NaH)₂PO₄300mM NaCl, 10mM imidazole, pH 8.0) and pressed. The crude enzyme solution was purified by using a Ni column purchased from Shanghai assist in san, washed with 20mM imidazole, eluted with 200mM imidazole, and the eluate was collected and subjected to protein SDS-PAGE electrophoresis.

Preparation method of reagent DNS (Domain name System) used in xylanase activity determination

Xylanase enzyme activity assays include wild-type and mutant assays. 10g of NaOH are weighed out and dissolved in about 400ml of ddH₂In O, 10g of dinitrosalicylic acid, 2g of phenol, 0.5g of anhydrous sodium sulfite, and 200g of potassium sodium tartrate tetrahydrate were weighed and dissolved in about 300ml of ddH₂And in O, mixing the two solutions, metering the volume to 1 liter, and storing in a dark place.

Preparation of substrate used in xylanase activity

Substrate selection: beech xylan (from Sigma), beech xylan (from Megazyme, corncob xylan, oat bran xylan.

The same applies to beech xylan from Sigma.

0.5g beech xylan (from Sigma) was weighed into 20mL 0.1% NaOH, stirred in a water bath at 70 ℃ for 1.5h, then 0.2M acetate buffer (pH 6.0) was added to 45mL, adjusted to pH 6.0 with glacial acetic acid, and finally made up to 50mL with 0.2M acetate buffer to a final concentration of 1% (W/V).

Xylanase enzyme activity determination method

Substrate selection: beech xylan (from Sigma), beech xylan (from Megazyme), corncob xylan, oat skin xylan.

The same applies to beech xylan from Sigma.

50 mu L of 1% beech xylan (purchased from Sigma) +50 mu L of enzyme solution diluted by a proper amount, uniformly mixing, reacting at 37 ℃ for 10min, then adding 100 mu L of DNS to terminate the reaction, adding the enzyme solution after adding 100 mu L of DNS into the reaction system, performing color reaction at 95 ℃ for 10min, and finally measuring the light absorption value at the wavelength of 540nm by using a microplate reader and converting into the corresponding enzyme activity unit.

Definition of enzyme Activity Unit (U)

1U is the amount of enzyme required to catalytically hydrolyze xylan to 1. mu. mol xylose per minute.

Example 1 first round mutation library construction and screening

Random mutation library construction: random Mutagenesis was performed on xylanase wild-type coding gene Xyn11(SEQ ID NO:1) using GeneMorph II Random Mutagenesis Kit error-prone PCR Kit (PCR protocol refer to GeneMorph II Random Mutagenesis Kit), the mutated target gene fragment was recovered in gel, recombined into pET28a vector using Vazyme Clon express II One Step Cloning Kit, electrotransformed into E.coli BL21 competent cells and plated with LB solid plate containing Kana antibiotics, and incubated overnight at 37 ℃. The overnight incubated transformant was picked up with a toothpick into a 96-well LB liquid medium plate containing Kana antibiotic, incubated at 37 ℃ for 12 hours, transferred to a new 96-well LB liquid plate at an inoculum size of 1%, IPTG was added to a final concentration of 2mM, and induced at 37 ℃ for 12 hours.

Screening of random mutation library: the 96-well plate induced at 37 ℃ for 12h was centrifuged, the medium was removed, and then 50ul of PBS buffer was added per well for resuspension, followed by treatment at 65 ℃ for 2.5 h. After the heat treatment, 50ul of 1% beech xylan was added, the reaction was carried out at 37 ℃ for 10 minutes, 100ul of DNS was added to terminate the reaction, and the reaction was developed at 95 ℃ for 10 minutes and then transferred to a 96-well microplate to read OD 540. And marking the positive transformant with the OD540 higher than the wild type, and picking out and preserving the strain for further verification. The round covered 10000 transformants.

Verification of thermostability of preferred mutants: from 10000 transformants, the inventor selects 5 preferable mutants with greatly improved thermal stability compared with the wild Xyn11, and the 5 preferable mutants are expressed and purified to determine the thermal stability. The residual enzyme activity (%) of the preferred mutants in the first round of random mutation library after heat treatment at 65 ℃ for various periods of time is shown in table 1, and it can be seen that the preferred mutant Xyn68 is the most stable to heat, and is the most preferred mutant in this round.

TABLE 1

Example 2 second round construction and screening of random mutation library

Random mutation is carried out on the Xyn68 encoding gene by using the first round of preferred mutant Xyn68 as a template and a GeneMorph II Random Mutagenesis Kit easy-error PCR Kit, and the subsequent construction method is not different from the first round of Random mutation library construction method. The screening procedure was identical to the first round of screening procedure except that the first round of treatment at 65 ℃ for 2.5h was changed to 80 ℃ for 30min, the library covering 20000 transformants. The mutant is preferably purified and then subjected to thermal stability verification, the results of the residual enzyme activity (%) of the mutant after heat treatment at 70 ℃ for different time periods are shown in Table 2, and Xyn370 is the preferred mutant in this round.

TABLE 2

Example 3 site-directed mutagenesis

Sequencing and analyzing preferred mutants (including Xyn68 and Xyn370, but not limited to Xyn68 and Xyn370) in the two rounds of random mutation, the inventor finds that the frequency of amino acid sites such as Q14H, L28V, D29A, N30S, S93A, R97L, V99L, K133E, E159G, A187D, G192D, A203V and the like is more frequent, and the inventors speculate that the thermal stability of xylan is improved, and then the sites are subjected to site-specific mutation one by oneMeans (site-directed mutagenesis program reference to Hieff Mut^TMsite-directed mutagenesis kit) was introduced into xylanase wild type Xyn 11. The residual enzyme activity (%) after introducing 14H, 29A, 203V mutant sites into the xylanase wild type and heat-treating at 70 ℃ for various times is shown in Table 3.

TABLE 3

As can be seen from Table 3, Q14H, D29A and A203V have an effect of improving the thermal stability of the wild type Xyn 11.

To further determine whether the three amino acid positions have an important effect on the xylanase thermostability, the inventors introduced the three positions into the preferred mutant Xyn370 in the second round of random mutation in a combination of single mutation as well as double mutation and triple mutation. The residual enzyme activity (%) of the xylanase mutant Xyn370 after introducing the 14H, 29A and 203V mutation sites and carrying out heat treatment at 75 ℃ for different times is shown in Table 4.

TABLE 4

As can be seen from Table 4, the thermal stability of the triple mutant Xyn370-Q14H/D29A/A203V is remarkably improved compared with that of the wild Xyn11 and the starting strain Xyn 370. Compared with the wild type and the single mutant, the thermal stability of the Xyn370-A203V/Q14H double mutant is also improved to a certain extent.

Example 4 preferred mutant Xyn370-Q14H/D29A/A203V saturation mutagenesis

With Hieff Mut^TMsite-directed mutagenesis kit saturation mutagenesis was performed on the 14 th, 29 th and 203 th positions of the preferred mutant Xyn370-Q14H/D29A/A203V in example 4: substitution of amino acid residue Q at position 14 with another 18 amino acid residues other than H; substitution of amino acid residue D at position 29 with another 18 amino acids other than a; the substitution of amino acid residue V at position 203 with another 18 amino acids other than A. Wood polymerThe residual enzyme activity (%) of the carbohydrase mutant Xyn370-Q14H/D29A/A203V after 29 th saturation mutation and treatment at 80 ℃ for different time is shown in Table 5.

TABLE 5

As can be seen from Table 5, after residue D at position 29 is replaced by S, the thermostability of the mutant Xyn370-Q14H/D29S/A203V is further remarkably improved, so that the preferred mutant Xyn370-Q14H/D29S/A203V is obtained in example 4.

Example 5 rational design of the preferred mutant Xyn370-Q14H/D29S/A203V

And logging in a protein database protein databank (https:// www.rcsb.org /), selecting a crystal structure with high homology with the three-dimensional structure of the preferred mutant Xyn370-Q14H/D29S/A203V as a template after BLAST comparison, and then performing homology modeling by using an online server to obtain the structural diagram of the preferred mutant Xyn370-Q14H/D29S/A203V 3D. B-factor value prediction is carried out on the three-dimensional structure model of the optimized mutant Xyn370-Q14H/D29S/A203V by using an online server, and the B-factor value of each residue is obtained. Preferably, the high B-factor amino acid residues in the mutant Xyn370-Q14H/D29S/A203V are shown in Table 6.

TABLE 6

Residue number	30	31	41	52	53	108	109	110	130	170
											B-factor value	29.21	30.22	32.17	30.87	30.883	33.92	35.29	33.34	30.71	30.36

According to Table 6, residues with higher B-factor values were selected for saturation mutagenesis to construct a mutation library.

The saturation mutagenesis method was the same as in example 4 above, and the library screening method was the same as in example 1 or example 2 above. The inventor further screens out mutation libraries in which the mutation of sites S30P, T31H, K41R, A52L and Q131C has an effect of improving the thermal stability of the preferred mutant Xyn370-Q14H/D29S/A203V, and then introduces the preferred mutant Xyn370-Q14H/D29S/A203V in an S30P, S30P/T31H, S30P/T31H/A52L, S30P/T31H/A52L/K41R and S30P/T31H/A52L/Q131C iterative combination mode respectively, and measures the thermal stability after expression and purification. Preferably, the residual enzyme activity (%) of the xylanase mutant Xyn370-Q14H/D29S/A203V after different mutation sites are introduced and heat treatment is carried out for different time is shown in Table 7 (in the table, Xyn370-Q14H/D29S/A203V is abbreviated as Xyn 370-A29S).

TABLE 7

As can be seen from Table 7, the mutants Xyn370-Q14H/D29S/A203V/S30P, Xyn370-Q14H/D29S/A203V-S30P/T31H, Xyn 370-Q14H/D29/A203H-S30H/T31H/A52H, Xyn 370-Q14/D29H/A203H/S30H/T31H/A52H/K41H, Xyn 370-Q14H/D29/A203H/S30/T31H/A52H/Q131H have improved thermal stability compared with the mutants Xyn370-Q14/D H/A203/S30/S72/T31/A52/Q H/Q131/Q H/A H/X H/D31/A203/D H/D31/Q131H/X31/X72/X31/X72/X3/X31/X3/X72/X3/X72/X3/X72/X3/X72/X3/, the residual enzyme activity is 85.90% +/-5.24% and 80.27% +/-1.33% respectively, and the Xyn370-Q14H/D29S/A203V is only left about 4%, so that the thermal stability is obviously improved.

Example 6 determination of specific enzyme Activity of xylanase mutants and Back-mutation of Xyn370-Q14H/D29S/A203V/S30P/T31H/A52L

The specific enzyme activities of the wild type and the preferred mutant strain are determined by taking beech (purchased from Sigma) xylan as a substrate at 37 ℃, the unit is U/mg, and the specific enzyme activities of the mutant strain of the xylanase are shown in a table 8 (Xyn 370-Q14H/D29S/A203V is abbreviated as Xyn370-A29S in the table).

TABLE 8

As can be seen from Table 8, the specific enzyme activities of the preferred mutants are all reduced, and in the modification of protein thermostability, the improvement of thermostability is inevitably at the expense of enzyme activity. In order to balance the relationship between the enzyme activity and the thermal stability, the inventor carries out reversion mutation on the optimal mutant strains Xyn370-Q14H/D29S/A203V/S30P/T31H/A52L, namely Q14H, D29S and A203V to obtain Q14, D29 and A203 respectively, and measures the residual enzyme activity change and the specific enzyme activity after the mutant strains are treated for different times at 85 ℃. The residual enzyme activity (%) and the specific enzyme activity (U/mg) of the Xyn370-Q14H/D29S/A203V/S30P/T31H/A52L back-mutation after heat treatment at 85 ℃ for different times are determined as shown in Table 9.

TABLE 9

As can be seen from Table 9, after Q14H, D29S and A203V are respectively restored and mutated into Q14, D29 and A203, compared with the original strain Xyn370-Q14H/D29S/A203V/S30P/T31H/A52L, the thermal stability is reduced, the thermal stability of D29 is reduced most obviously, and the influence of A203 is minimal. Compared with the original strain, the specific enzyme activity of the xylanase is increased, wherein the Q14 is increased to the maximum from 593.2 +/-2.7 to 1158.8 +/-24.4 of the original strain, and the fact that three sites of Q14, D29 and A203 have great influence on the enzyme activity of the xylanase is proved.

Subsequently, the inventor carries out reversion mutation on Q14 and A203 at the same time to construct a mutant strain Xyn370-Q14/D29S/A203/S30P/T31H/A52L, and the specific enzyme activity of the mutant strain is further improved (1554.7 +/-156.8, Table 9).

In summary, the sequence information of the wild type and the mutant are summarized in FIGS. 1-1 to 1-13.

The mutant strain can be reasonably selected in practical application, the relationship between the thermal stability and the enzyme activity can be better balanced, and a proper mutant strain is selected according to the practical production requirement to achieve the aim.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A method of increasing the thermostability of a xylanase, comprising: mutating the amino acid sequence of the xylanase, corresponding to the wild-type xylanase, at a site selected from the group consisting of: 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 133 th, 192 th, 203 th bits.

2. The method of claim 1, wherein the 14 th mutation is His; mutation at position 28 to Val; mutation at position 29 to Ser or Ala; the 30 th mutation is Ser or Pro; mutation at position 31 to His;

the 41 th mutation is Arg; the 52 th mutation is Leu; mutation at position 131 to Cys; mutation at position 133 to Glu; mutation at position 192 to Asp; and/or, the 203 rd mutation is Val.

3. A xylanase mutant which is:

(a) a protein having an amino acid sequence corresponding to a wild-type xylanase, mutated at a site or combination of sites selected from the group consisting of: 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 133 th, 192 th, 203 th bits;

(b) a protein derived from (a) by substituting, deleting or adding one or more amino acid residues to the amino acid sequence of the protein (a), wherein the protein has the function of the protein (a), and the amino acid corresponding to the 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 133 th, 192 th or 203 th position of the wild-type xylanase is the same as the mutated amino acid at the corresponding position of the protein (a);

(c) and (b) a protein derived from (a) and having more than 80% homology with the amino acid sequence of the protein (a) and having the function of the protein (a), wherein the amino acid corresponding to the 14 th, 28 th, 29 th, 30 th, 31 th, 41 th, 52 th, 131 th, 133 th, 192 th or 203 th position of the wild-type xylanase is the same as the mutated amino acid at the corresponding position of the protein (a).

4. The xylanase mutant according to claim 3, wherein the 14 th mutation is His; mutation at position 28 to Val; mutation at position 29 to Ser or Ala; the 30 th mutation is Ser or Pro; mutation at position 31 to His; the 41 th mutation is Arg; the 52 th mutation is Leu; mutation at position 131 to Cys; mutation at position 133 to Glu; mutation at position 192 to Asp; and/or, the 203 rd mutation is Val.

5. The xylanase mutant according to claim 3 or 4, comprising a protein selected from the group consisting of: corresponding to the wild-type xylanase, and the xylanase,

6. The xylanase mutant according to claim 5, comprising a protein selected from the group consisting of: 14, 16, 18, 12, 10, 20, 22, 24, 26, 8, 6 or 4.

7. An isolated polynucleotide encoding a xylanase mutant according to any one of claims 3 to 6; preferably, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 11, SEQ ID NO. 9, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 7, SEQ ID NO. 5 or SEQ ID NO. 4.

8. A vector comprising the polynucleotide of claim 7.

9. A genetically engineered host cell comprising the vector of claim 8, or having the polynucleotide of claim 7 integrated into its genome.

10. A method for preparing a xylanase mutant according to any one of claims 3 to 6, comprising:

(i) culturing the host cell of claim 9;

(ii) collecting a culture containing a xylanase mutant according to any one of claims 3-6;

(iii) isolating the xylanase mutant from the culture.

11. A composition for degrading xylan comprising an effective amount of: a xylanase mutant according to any one of claims 3 to 6; or, the host cell of claim 9 or a culture or lysate thereof; and

a dietetic or industrially acceptable carrier.

12. Use of a xylanase mutant according to any one of claims 3 to 6 or a composition according to claim 11 for degrading xylan; preferably, the xylanase mutant cleaves beta-1, 4-glycosidic bonds, thereby hydrolyzing xylan.

13. A method of degrading xylan, said method comprising: degrading xylan with a xylanase mutant according to any one of claims 3 to 6 or a composition according to claim 11; preferably, the xylanase mutant cleaves beta-1, 4-glycosidic bonds, thereby hydrolyzing xylan.

14. The use according to claim 12 or the method according to claim 13, wherein the degradation or cleavage is a degradation or cleavage at elevated temperature; preferably, the high temperature is 45-95 ℃; preferably 50 to 90 ℃.

15. The use according to claim 12 or the method according to claim 13, wherein the substrate for degradation by the xylanase mutant or composition is a xylan or xylan-containing material.

16. The use as claimed in claim 12 or method as claimed in claim 13, wherein the xylan-containing material comprises: industrial products, agricultural, forestry, industrial waste and municipal solid waste; preferably, it comprises: pulp, feed, food, straw, hardwood, softwood, straw, bran.

17. Use of a xylanase mutant according to any one of claims 3 to 6 or a composition according to claim 11, comprising:

as a dietary supplement;

as a plant processing additive;

as a paper making process additive;

as a textile processing additive;

as a feed supplement; and/or

The method is applied to converting lignocellulose into fuel ethanol.

18. A library of xylanase mutants or polynucleotides encoding the same, the library comprising: at least 5, preferably at least 10, of the xylanase mutants of any one of claims 3-6 or polynucleotides encoding same; more preferably it comprises a xylanase mutant according to claim 5 or 6 or a polynucleotide encoding same.

19. Use of a library of xylanase mutants or polynucleotides encoding thereof according to claim 18, for providing an appropriate xylanase mutant to degrade xylan depending on the temperature of the reaction system in which the xylan to be degraded is located.

20. A kit for degrading xylan comprising:

a xylanase mutant or combination of mutants according to any one of claims 3 to 6;

the host cell of claim 9;

the composition of claim 11; or

A library of xylanase mutants or polynucleotides encoding the same according to claim 18.