JP7135334B2 - Xylanase and its use - Google Patents

Description

This specification relates to xylanases and uses thereof.

Effective use of biomass containing hemicellulose is an important issue for efficient use of biomass. Various modified xylanases and novel xylanases that degrade hemicellulose have been provided (Patent Documents 1 and 2).

JP 2016-49035 A Japanese Patent Application Laid-Open No. 2017-35001

According to studies by the present inventors, it may be efficient to subject biomass containing hemicellulose to continuous treatment at about 50° C. for several days, for example. In addition, according to the present inventors, the xylanase that has been provided so far has a high optimum temperature, but the durability at high temperature has hardly been studied, and the xylanase also has that function. Do not mean.

The present specification provides a xylanase with high temperature tolerance and its use.

The present inventors constructed a xylanase variant library, screened it, and were able to identify useful mutations for exhibiting high temperature tolerance. This specification provides the following means based on such knowledge.

(1) a xylanase,
selected from the group consisting of positions 6, 46, 47, 55, 80, 94, 99, 125 and 164 when aligned with the amino acid sequence represented by SEQ ID NO: 1 A xylanase having mutations at positions corresponding to one or more positions.
(2) the mutation is
at the position corresponding to position 6, any of E, I, V, S, C, M and Q;
I at the position corresponding to position 46,
At the position corresponding to position 47, either C or A,
At the position corresponding to position 55, it is either S, D or E,
At the position corresponding to position 80, it is either M, A or I,
at the position corresponding to position 94, any of H, C, Y and A;
at the 99-equivalent position, either G or A;
At the position corresponding to position 125, it is either A or S,
The xylanase according to (1), wherein the position corresponding to position 164 is M.
(3) the mutation is
At the position corresponding to position 6, it is either E, I, V or S,
I at the position corresponding to position 46,
At the position corresponding to position 47, either C or A,
At the position corresponding to the 55th place, it is S,
At the position corresponding to position 80, it is either M, A or I,
at the 94-equivalent position, either H, C or Y;
at the 99-equivalent position, either G or A;
At the position corresponding to the 125th place, it is A,
The xylanase according to (2), wherein the position corresponding to position 164 is M.
(4) further consists of positions 37, 56, 71, 119, 120, 137, 140, 154 and 161 when aligned with the amino acid sequence represented by SEQ ID NO: 1 The xylanase according to any one of (1) to (3), which has mutations at positions corresponding to one or more positions selected from the group.
(5) the mutation is
At the position corresponding to position 37, it is I,
At the position corresponding to the 56th position, it is T,
At the position corresponding to the 71st place, it is A,
at the 119-equivalent position, either V or C,
At the position corresponding to position 120, it is either K or A,
at the 137-equivalent position, any of W, A, L and V;
I at the position corresponding to 140th place,
At the position corresponding to 154 phases, it is A,
The xylanase according to (4), wherein the position corresponding to position 161 is either A or E.
(6) The xylanase according to any one of (1) to (5), which has mutations at 4 or more positions.
(7) The xylanase according to any one of (1) to (6), wherein the xylanase has a half-life of xylanase activity at 50° C. of 30 hours or longer.
(8) The xylanase according to any one of (1) to (7), wherein the xylanase has a half-life of xylanase activity at 50° C. of 100 hours or longer.
(9) The xylanase according to any one of (1) to (8), wherein the xylanase has 80% or more identity with the amino acid sequence represented by SEQ ID NO:2.
(10) A polynucleotide encoding the amino acid sequence of the xylanase according to any one of (1) to (9).
(11) An expression vector comprising the polynucleotide according to (10).
(12) A transformant expressing the xylanase according to any one of (1) to (9).
(13) A method for decomposing xylan, comprising a step of decomposing xylan using the xylanase according to any one of (1) to (9).
(14) a step of degrading xylan using the xylanase according to any one of (1) to (9);
a step of converting the xylan into a useful substance;
A method of producing a useful substance, comprising:
(15) A composition comprising the xylanase according to any one of (1) to (9).
(16) A composition comprising a transformant that produces the xylanase according to any one of (1) to (9).
(17) A method for screening a xylanase, comprising:
When aligned with the amino acid sequence represented by SEQ ID NO: 1, positions 6, 37, 46, 47, 55, 56, 71, 80, 94, 99, 119, A step of preparing a xylanase candidate having mutations at positions corresponding to one or more positions selected from the group consisting of positions 120, 125, 137, 140, 154, 161 and 164. When,
For the xylanase candidate,
(a) the predetermined temperature when the xylanase activity becomes half the activity when stored at 25° C. for 2 hours when stored at a predetermined temperature for 2 hours, and (b) the xylanase activity when stored at 50° C. evaluating for at least one of the half-lives;
A method.
(18) The method according to (17), wherein the evaluation step is a step of screening for a xylanase having a half-life of xylanase activity of 30 hours or longer when stored at 50°C.

FIG. 10 is a diagram showing an increase in heat-resistant temperature (Δ heat-resistant temperature) of a variant having a single mutation. FIG. 10 is a diagram showing an increase in heat-resistant temperature (Δ heat-resistant temperature) for some variants having a single mutation. FIG. 10 is a diagram showing the Δ heat resistance temperature of additive mutants. FIG. 10 is a diagram showing a comparison result between an additive predicted value and an experimental value of the Δ heat resistance temperature of an additive mutant. FIG. 4 shows the results of alignment of partial amino acid sequences of 81 xylanases belonging to GHF11. FIG. 10 shows the results of alignment of other partial amino acid sequences of 81 xylanases belonging to GHF11. The number represents the UniProtKB registration number. FIG. 10 shows the results of alignment of other partial amino acid sequences of 81 xylanases belonging to GHF11. The number represents the UniProtKB registration number. FIG. 10 shows the results of alignment of other partial amino acid sequences of 81 xylanases belonging to GHF11. The number represents the UniProtKB registration number.

The present disclosure relates to xylanases and uses thereof.

The present inventor searched for a Trichoderma reesei-derived xylanase from the viewpoint of high-temperature durability, which is an unprecedented viewpoint for practical and/or efficient biomass processing. In addition to obtaining mutation sites for high temperature tolerance, the inventors confirmed the additive effect of these mutations.

As used herein, the term "xylanase" refers to endo-1,4-β-xylanase, which is an enzyme of EC No. 3.2.1.8. Xylanase is an enzyme that breaks down xylan into xylose. Xylan is a major component of hemicellulose present in the cell walls of plant cells. The xylanases disclosed herein and their uses are described in detail below.

(xylanase)
The xylanase disclosed in the present specification (hereinafter also referred to as the present xylanase) has a mutation introduced at a predetermined site when aligned with the amino acid sequence of the xylanase consisting of the amino acid sequence represented by SEQ ID NO: 1. It can be defined as a xylanase.

Here, the xylanase consisting of the amino acid sequence represented by SEQ ID NO: 1 is a Trichoderma reesei-derived xylanase, and is a xylanase mature protein specified by UniProtKB accession number P36217 (amino acid sequence defined by accession number P36217 ( Positions 34 to 223 of SEQ ID NO: 3), consisting of an amino acid sequence consisting of 190 amino acid residues, a signal peptide (positions 1 to 19) (SEQ ID NO: 4) and a propeptide (positions 20 to 33rd) (excluding SEQ ID NO: 5)). (Hereinafter, this xylanase is referred to as the first xylanase. The first xylanase is classified into 11 of the Glycoside Hydrolase Family (http://www.cazy.org/Glycoside-Hydrolases.html). In, unless otherwise specified, the position in the amino acid sequence is represented as the position in the amino acid sequence of the first xylanase consisting of 190 amino acid residues.In addition, positions 10 to 188 of the first xylanase ( corresponding to positions 43 to 221 of the amino acid sequence defined by accession number P36217 and represented by SEQ ID NO: 2) is the catalytic domain.

(Heatproof temperature)
The present xylanase preferably has a higher heat resistance temperature than the first xylanase. The heat resistance temperature of the present xylanase can be evaluated as, for example, Δ heat resistance temperature (°C). As used herein, the Δ heat-resistant temperature is the temperature differential (°C) of the heat-resistant temperature (°C) of the present xylanase with respect to the heat-resistant temperature (°C) of the first xylanase. For example, the storage temperature at which the activity when stored at a certain storage temperature for 2 hours becomes half the activity after storage at 25° C. for 2 hours can be defined as the heat resistant temperature of the xylanase.

More specifically, for example, the xylanase is stored at 25° C., 48° C. and 52° C. for 2 hours each, and then its activity is measured by a predetermined method. As a result, when the storage temperature is plotted on the horizontal axis and the xylanase activity is plotted on the vertical axis, an activity decreasing curve can be obtained. In this activity decrease curve, the storage temperature at which the auxiliary line corresponding to half the activity when stored at 25° C. for 2 hours intersects the activity decrease curve can be defined as the heat resistant temperature (° C.). A value obtained by subtracting the heat resistant temperature of the first xylanase from the heat resistant temperature of the present xylanase thus obtained is defined as the Δ heat resistant temperature. Specifically, the Δ heat-resistant temperature can be measured by adopting the conditions described in Examples described later.

The present xylanase has, for example, a Δ heat resistant temperature of 1° C. or more. Further, for example, the Δ heat resistance temperature is 1.5° C. or higher, for example, 2° C. or higher, for example, 3° C. or higher, for example, 4° C. or higher, or for example, 5° C. or higher. An increase in the Δ thermotolerant temperature increases the half-life of xylanase activity at 50° C., for example. In particular, from the viewpoint of the activity half-life of the present xylanase, the Δ heat resistant temperature is 4° C. or higher, preferably 5° C. or higher, more preferably 6° C. or higher, still more preferably 7° C. or higher, still more preferably 8° C. or higher. Preferably, it is 9°C or higher. The upper limit is also not particularly limited, but from the viewpoint of activity half-life, for example, it is 12° C. or lower, for example, 11° C. or lower, or for example, 10° C. or lower.

(Activity half-life)
The present xylanase preferably has a longer half-life of xylanase activity than the first xylanase, for example, when stored at a predetermined storage temperature. As used herein, the activity half-life of xylanase is the time (hr) at which the residual activity of xylanase is halved when stored at a certain temperature. The activity half-life of the present xylanase can be evaluated, for example, using 50° C., which is the temperature at which biomass containing hemicellulose is degraded, as the storage temperature.

For more specific storage conditions and xylanase activity, the method disclosed in the Examples below can be adopted. Note that the storage temperature is not limited to 50°C, and a predetermined temperature can be set in the range of 50°C or higher and 65°C or lower, for example. For example, it may be residual activity at 55°C or residual activity at 60°C. Alternatively, it may be residual activity at 65°C.

The first xylanase has a half-life of about 3 hours at 50°C, but the present xylanase has a half-life of, for example, 30 hours or more at 50°C, or 40 hours or more, and For example, it is 50 hours or more, for example 60 hours or more, for example 70 hours or more, for example 80 hours or more, for example 90 hours or more, or for example 100 hours or more. Further, for example, 120 hours or more, for example, 140 hours or more, for example, 160 hours or more, for example, 180 hours or more, for example, 200 hours or more, or for example, 220 hours or more hours or more, and for example, 240 hours or more. If the half-life at 50 ° C. is 100 hours or more, the activity can be generally maintained during the treatment time (about 60 to 200 hours) for decomposition (saccharification) of biomass containing hemicellulose, and biomass can be efficiently converted. can be decomposed.

The present xylanase having a Δ heat resistant temperature of 4°C or higher also has an activity half-life at 50°C of 30 hours or longer, more preferably 40 hours or longer, still more preferably 50 hours or longer, and still more preferably 60 hours or longer. Many, typically 80 hours or more. In addition, the xylanase having a Δ heat resistant temperature of 6° C. or higher, preferably 7° C. or higher, more preferably 8° C. or higher has an activity half-life at 50° C. of 100 hours or longer, more preferably 120 hours or longer. It is often preferably 140 hours or longer, more preferably 160 hours, and still more preferably 180 hours or longer.

(mutation)
Mutations possessed by this xylanase are defined with reference to the first xylanase. This is because these mutations were obtained by trying various modifications to the first xylanase to identify mutations that contribute to high temperature tolerance. On the other hand, the mutations disclosed herein apply not only to the amino acid sequence of the first xylanase, but also to other xylanases. That is, the first xylanase is classified as GHF11, and other xylanases belonging to GHF11 have similar amino acid sequences and three-dimensional structures. From these facts, it is understood by those skilled in the art that the present xylanase can be obtained by applying the mutations disclosed herein to at least a xylanase belonging to the GH11 family.

The xylanase of the present invention includes naturally-occurring xylanases that are inherently equipped with the mutations disclosed herein, as well as those in which mutations are introduced into the first xylanase.

Mutation positions that this xylanase can have are positions 6, 37, 46, 47, 55, 56, 71 and 80 when aligned with the amino acid sequence represented by SEQ ID NO: 1. , 94th, 99th, 119th, 120th, 125th, 137th, 140th, 154th, 161st and 164th positions corresponding to one or more positions selected from the group consisting of be. Such a mutation position in this xylanase is obtained by BLAST (https://blast.ncbi.nlm. nih.gov/Blast.cgi) or Pfam (http://pfam.xfam.org/), for example, by aligning with default parameters.

All of these mutation positions are positions where it has been confirmed that the Δ heat resistant temperature (°C) can be positive. The possible replacement amino acid residues at each mutation position are shown below. The substituted amino acid residues were obtained based on evaluation of a saturated gene library of single mutants substituted with the remaining 19 amino acids other than the native amino acid residues at the following positions in the first xylanase: there is The numbers in parentheses are the positions in the first xylanase (positions 1 to 190).

Among the substituted amino acid residues at the mutation positions, the amino acid residues marked with an asterisk (*) are mutations with a Δ heat resistant temperature of about 1° C. or higher.

Glycine (position 6): glutamic acid*, isoleucine, valine, serine, cysteine, methionine, glutamine Valine (position 37): isoleucine Valine (position 46): isoleucine*
Glycine (47th position): cysteine*, alanine*
Threonine (position 55): serine*, aspartic acid, lysine glutamate (position 56): threonine asparagine (position 71): alanine serine (position 80): methionine*, alanine*, isoleucine,
Glycine (position 94): histidine*, cysteine*, alanine, tyrosine* (position 99): glycine*, alanine Arginine (position 119): valine, cysteine Threonine (position 120): lysine, alanine Glutamine (position 125): alanine *, Serine Tyrosine (position 137): tryptophan, alanine, leucine, valine valine (position 140): isoleucine asparagine (position 154): alanine glutamine (position 161): alanine, glutamic acid leucine (position 164): methionine

6 (6E), 46 (46I), 47 (47C, 47A), 55 (55S), 80 (80M, 80A), 94 from the viewpoint that the Δ heat resistance temperature is about 1 ° C. or higher (94H, 94C), 99 (99G), 125 (125A) and 164 (164M) are useful mutation positions and mutations. Mutations having a Δ heat resistant temperature of about 1.5° C. or more are 6E, 47C, 47A, 55S, 80M, 80A, 94H, 99G, and 125A. Mutations having a temperature of about 2.0° C. or more are 47C, 80M, and 99G. Furthermore, the mutations with the same temperature of 2.5°C or higher are 47C and 99G. Furthermore, the mutation with the same 3.0° C. or higher is 47C.

In addition, from the viewpoint that there are multiple mutations that contribute to obtaining an effective Δ heat resistant temperature, preferable mutation positions are positions 6, 47, 55, 90, 94, 99 and 119. 120th, 125th, 137th, and 161st. Among them, 6th, 55th, 80th, 94th, and 137th are listed.

In addition, for example, position 47 is a mutation position that contributes to an increase in the Δ heat resistant temperature and an increase in the activity half-life at, for example, 50°C. A mutation is 47C. Position 99 is also a useful mutation position as well. The mutation is 99G. Additionally, position 80 is also a useful mutation. Mutations are 80M or 80A. Position 125 is also a useful mutation position. A mutation is 125A. Furthermore, position 55 is also a useful mutation position. A mutation is 55S.

Such mutation positions are position 120 in addition to these. The mutation is 120K. Position 94 is also a useful mutation position. Mutations are 94C and 94H. Position 164 is also a useful mutation position. The mutation is 164M.

Also, such a mutation position is position 6 in addition to these. The mutation is 6E. Position 71 is also a useful mutation position. The mutation is 71A. Position 137 is also a useful mutation position. The mutation is 137W.

Furthermore, such a mutation position is position 46 in addition to these. A mutation is 46I. Position 47 is also a useful mutation position. A mutation is 47A.

The xylanase can have one or more mutations at these mutation positions. By combining (additively) two or more mutations, the present xylanase can obtain a Δ heat resistant temperature (° C.) that is equal to or greater than the Δ heat resistant temperature based on each mutation. This is because the mutations found by the present inventor independently contribute to the Δ heat resistant temperature (°C) and/or activity half-life.

When combining two or more mutations, there is no particular limitation, but three or more mutations are preferably combined, more preferably four or more. When four or more mutations are combined, the Δ heat resistance temperature is greatly improved compared to the case of three or less mutations. More preferably, it is 5 or more. Also, the number of mutations is preferably about 8 or less. When the number of mutations is large, the Δ heat resistance temperature tends to be added, but this is because, for example, the activity half-life at 50° C. does not change. The number is preferably 7 or less, more preferably 6 or less. Typically, 4 or more and 5 or more and 6 or less are preferable from the viewpoint of improving the Δ heat resistance temperature and increasing the half-life at 50°C or the like.

Although the optimum temperature for the xylanase activity of the present xylanase is not particularly limited, it is preferably 50°C or higher and 65°C or lower. Within this range, both activity and durability at high temperatures can be achieved. The optimum temperature is also, for example, between 55°C and 65°C. The optimum temperature and xylanase activity can be evaluated and defined by the methods disclosed in the examples below.

The combination of two or more mutations possessed by the present xylanase can be appropriately determined by selecting from the aforementioned mutations. Among others, it can be selected from the viewpoint of the aforementioned Δ heat resistant temperature and half-life of activity. . Also, for example, the xylanase is selected from mutations disclosed below, for example, 2 or more and 10 or less, such as 3 or more and 9 or less, or such as 4 or more and 8 or less, or such as 5 1 or more and 7 or less mutations, or, for example, 5 or more and 5 or less mutations can be appropriately selected and combined. The individual mutations in this xylanase and combinations of suitable ranges are exemplified below.

The xylanase preferably has 80% or more identity and/or similarity with the amino acid sequence (SEQ ID NO: 2) of the catalytic domain of the first xylanase. Also, for example, 85% or more, for example, 90% or more, for example, 95% or more, for example, 96% or more, for example, 97% or more, or for example, 98% or more Yes, for example 99% or more, or for example 99.5% or more. Also for example, the xylanase has less than 100% identity with the amino acid sequence represented by SEQ ID NO:2.

As used herein, identity or similarity is a relationship between two or more proteins or two or more polynucleotides determined by comparing sequences, as known in the art. As used in the art, "identity" refers to an identity between protein or polynucleotide sequences, as determined by an alignment between protein or polynucleotide sequences, or optionally between stretches of such sequences. means the degree of sequence invariance of Similarity also refers to the relationship between protein or polynucleotide sequences as determined by alignments between protein or polynucleotide sequences, or in some cases by alignments between stretches of partial sequences. means the degree of More specifically, it is determined by sequence identity and conservation (substitutions that maintain specific amino acids in the sequence or physicochemical properties in the sequence). The similarity is referred to as similarity in the sequence homology search results of BLAST, which will be described later. Methods to determine identity and similarity are preferably those designed to give the longest alignment between the sequences being compared. Methods to determine identity and similarity are provided as publicly available programs. For example, the BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (see, for example, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)). Conditions when using software such as BLAST are not particularly limited, but default values are preferably used.

The present xylanase can have mutations at the specific mutation positions described below, but may have mutations such as substitutions and deletions at positions other than the specific mutation positions as long as the effects of the mutations are not impaired. That is, such additional mutations can be at sites other than the catalytic domain (positions 10-188) in the amino acid sequence of the first xylanase, and the like. As such mutations, examples of amino acid substitutions are preferably conservative substitutions, specifically substitutions within the following bracketed groups. For example, (glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

A person skilled in the art can prepare and evaluate various additive forms based on the mutation positions and suitable substitution examples of the present xylanase disclosed herein to obtain a xylanase having the intended high-temperature durability. can.

In addition, those skilled in the art can add either or both regions of the signal peptide and propeptide of the first xylanase to the N-terminal side of the xylanase disclosed herein, or A suitable protein for the production of the present xylanase can be obtained by appropriately adding a peptide having the same function as these.

For example, this xylanase is obtained by molecularly evolving DNA (SEQ ID NO: 6) (GenBank: EU532196) encoding the amino acid sequence of the first xylanase using conventional mutagenesis, site-directed mutagenesis, and error-prone PCR. A modified DNA can be obtained by modification by a method or the like, and can be obtained based on the modified DNA or the like. Techniques for obtaining the modified DNA include known techniques such as the Kunkel method and the gapped duplex method, and methods based thereon; for example, mutagenesis using various commercially available site-directed mutagenesis methods. Mutations are introduced using a kit for

For example, the present xylanase is obtained by transforming a suitable host such as eukaryotic cells with the DNA construct containing the modified DNA thus obtained, culturing the transformed cells according to a conventional method known to those skilled in the art, and culturing the transformed cells. It can be obtained by recovering the present xylanase from cells or culture medium. A soluble fraction is obtained from the cultured cells by a separation operation such as centrifugation after disrupting the cells, and the polypeptide can be recovered from this fraction. For example, the xylanase can be isolated using a combination of conventional purification techniques. Such techniques include ammonium sulfate fractionation, organic solvent treatment, centrifugation, ultrafiltration, various types of chromatography (e.g., gel filtration chromatography, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, etc.). , high performance liquid chromatography (HPLC), electrophoresis, and the like.

In addition, those skilled in the art should refer to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)). Thus, for example, DNAs of various aspects can be obtained based on known sequences such as SEQ ID NO: 1 or 6, and the present xylanases can be obtained using the DNAs.

This xylanase can be used at 50°C, which is a practical temperature for decomposition (saccharification) and decomposition of biomass containing hemicellulose by a cellulase complex or a transformant expressing cellulase, and fermentation production of useful substances such as ethanol, compared to conventional xylanases. high durability. Therefore, this xylanase is useful for producing useful substances from biomass.

The xylanase is also useful for purposes other than producing useful substances from biomass. Xylanase is used for bleaching pulp, improving the digestibility of silage, and as a food additive, as well as being used in various food fields. From the viewpoint of industrial use and storage stability, high temperature durability is an important factor. The xylanases are also useful in food applications and food manufacturing applications.

The xylanase is used as a purified or unpurified protein depending on the application, and as described later, the xylanase is transformed into a microorganism such as a transformant that expresses it intracellularly, displays it on its surface, or secretly expresses it. It can also take a form. When the present xylanase is used as a protein, it may be configured as a composition containing the present xylanase, other cellulases, and additives. Moreover, when the present xylanase is used as a microbial form, it may be constructed as a cytoplasmic body that co-expresses other cellulases.

(Polynucleotide)
The polynucleotides disclosed herein (hereinafter also simply referred to as the polynucleotides) can encode the amino acid sequences of the xylanases. Such polynucleotides can take a variety of forms, but the coding region for the amino acid sequence is DNA or RNA, typically DNA.

This polynucleotide can take any known form suitable for transformation, such as a DNA fragment or an RNA fragment, as well as a plasmid or vector.

The present polynucleotide can be obtained as DNA for obtaining the present xylanase. A nucleic acid fragment can be obtained by performing PCR amplification using a nucleic acid derived from a cDNA library, a genomic DNA library, or the like as a template. Alternatively, a nucleic acid fragment can be obtained by hybridization using a nucleic acid derived from the library or the like as a template and a DNA fragment that is part of the xylanase gene as a probe. Alternatively, it may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.

(expression vector)
The expression vector disclosed herein (hereinafter also referred to as the present vector) can comprise the present polynucleotide. The purpose of this vector is to express the present xylanase in host cells. Expression vectors can take various forms depending on the type of cells to be transformed, the purpose, and the like. Examples of this vector include, for example, a vector for obtaining a microorganism that industrially produces this protein, a microorganism that displays or secretively expresses this protein on the cell surface and, if necessary, produces useful substances such as ethanol by fermentation. vectors for obtaining A person skilled in the art can easily construct such a vector based on the well-known technique at the time of filing the present application.

Elements of a vector for obtaining a microorganism capable of degrading biomass with this xylanase will be described below.

The vectors can include suitable promoters, terminators, markers and other elements. Such vector elements are appropriately selected according to the host cell to be transformed and the expression form of the present xylanase.

Host cells to be transformed with this vector are not particularly limited, but prokaryotic cells and eukaryotic cells can be used. Prokaryotic cells include, for example, E. coli such as Escherichia coli. Eukaryotic cells include fungi such as Aspergillus oryzae and yeasts in consideration of substance production and the like. Aspergillus genus, such as Aspergillus aculeatus (Aspergillus aculeatus) and Aspergillus oryzae (Aspergillus orizae), is mentioned as koji mold. As the yeast, various known yeasts can be used, and yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts of the genus Schizosaccharomyces such as Schizosaccharomyces pombe, Candida Candida yeast such as Candida shehatae, Pichia yeast such as Pichia stipitis, Hansenula yeast, Klockckera yeast, Schwanniomyces yeast and yeast of the genus Yarrowia, yeast of the genus Trichosporon, yeast of the genus Brettanomyces, yeast of the genus Pachysolen, yeast of the genus Yamadazyma, yeast of the genus Kluyveromyces marxianus ), yeast of the genus Kluyveromyces such as Kluveromyces lactis, and yeast of the genus Isatokenkia such as Issatchenkia orientalis. Among them, yeast of the genus Saccharomyces is preferable from the viewpoint of industrial usability. Among them, Saccharomyces cerevisiae is preferable.

Any promoter can be applied to this vector as long as it can operate in the host and achieve the intended expression mode. Promoters may also be inducible or constitutive. For example, constitutive promoters in yeast include 3-phosphoglycerate kinase (PGK) promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, alcohol dehydrogenase 1 (ADH1) promoter, histidine trophic function gene (HIS3). Promoters include cytochrome bc1 complex (CYC1) promoter and hyperosmotic response 7 gene (HOR7) promoter and variants thereof.

A terminator to be applied to this vector may also be one that can operate in the host and achieve the intended mode of expression. Furthermore, this vector can also be provided with enhancers, origins of replication (ori), markers and the like.

The vector may be maintained extrachromosomally in the host cell, but is preferably configured to be maintained chromosomally.

General manipulations necessary for constructing such expression vectors and handling yeast as a recombinant host are routinely performed by those skilled in the art. Maniatis, J. Sambrook, et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, 1989, 2001) and the like can be appropriately referred to by those skilled in the art.

(transformant)
The transformant disclosed herein (hereinafter also simply referred to as the present transformant) can express the present xylanase. Since this transformant can express this xylanase, this transformant can be used not only for industrial production of this xylanase but also for decomposition of biomass and the like and saccharification and fermentation.

This transformant can be obtained by introducing the above-described present vector into a host. Methods for introduction into a host include conventionally known methods such as calcium phosphate method, transformation method, transfection method, conjugation method, protoplast method, electroporation method, lipofection method, lithium acetate method and other methods. mentioned. Such a method is described in the above-mentioned experiment manual and the like. This transformant can be obtained by selection using a marker gene and selection by expression of activity from the microorganism into which the vector has been introduced.

This transformant preferably expresses xylose isomerase (XI). That is, it is preferred that the ability to convert xylose to xylulose is imparted. By imparting XI activity, it can grow and ferment using xylose as a carbon source via the pentose phosphate pathway.

When the present transformant is a eukaryotic cell, the DNA encoding the present xylanase may be retained extrachromosomally, but is preferably retained on the chromosome. Also, in order to exhibit high xylose resolution, it is preferable to retain, for example, multiple copies.

The transformant may also secrete and express cellulase or other hemicellulase to the cell surface or extracellularly. Examples thereof include various cellulases such as endoglucanase, cellobiohydrolase, and β-glucosidase, as well as other biomass-degrading enzymes such as other hemicellulases. By expressing such proteins, it becomes possible to effectively utilize sugars other than lignin derived from lignocellulose. In addition, the transformant disclosed herein may be genetically engineered, such as introduction of foreign genes or disruption of endogenous genes, if necessary.

Genes encoding such enzymes may be endogenous or exogenous in eukaryotic cells such as yeast. In addition, known genes encoding these enzymes can be used as appropriate. Any gene of any origin can be used as long as it can enhance glycolysis. That is, the gene may be derived from another species of yeast other than the host yeast, or from another genus of yeast. may be derived from Information on such genes can be obtained by those skilled in the art as appropriate by accessing HP such as NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov). The gene may be cDNA or the like in addition to genomic DNA.

(Method for decomposing xylan and method for producing xylose)
The method for decomposing xylan disclosed in the present specification (hereinafter also simply referred to as the present decomposition method) can comprise a step of decomposing xylan using the present xylanase. According to this decomposition method, a high decomposition temperature, for example, about 50° C. can be set to decompose xylan. Moreover, according to the decomposition method of the present invention, xylan can be decomposed at once without adding xylanase, which is advantageous even when the high decomposition temperature is set. This decomposition method can also be implemented as a method for producing xylose, which is a decomposition product of xylan.

According to this decomposition method, if xylan is contained as a carbon source, it can be effectively used. Xylan-containing carbon sources are generally plants or their parts, and include general biomass resources as well as food wastes such as coffee lees, sugar beet lees, and the like.

By degrading xylan with xylanase, xylose can be obtained as a degradation product. Xylan is a general term for polysaccharides such as arabinoxylan and glucuronoxylan, and xylose is a constituent monosaccharide. Xylan naturally constitutes one component of hemicellulose and is present in biomass such as lignocellulose.

The present degradation method can be a step of degrading xylan using the present xylanase as a purified or unpurified protein, or a step of degrading xylan using the present transformant expressing the present xylanase extracellularly. may be Using this xylanase as a protein may be useful in the food industry, and using this transformant may be useful from the viewpoint of utilization of biomass resources and saccharification/fermentation.

When the present xylanase is used as a protein, the present xylanase directly degrades the xylan. As for the conditions in this case, the decomposition temperature, time, etc. can be appropriately set in consideration of the Δ heat resistant temperature and half-life of activity of the present xylanase. Typically, the temperature can be from 45° C. to 65° C. for several hours to several tens of hours.

In addition, in the step of degrading xylan using the xylanase expressed by the present transformant, a culture step is performed in which a microorganism that extracellularly expresses xylan is added to xylan and cultured under culture conditions suitable for fermentation of the microorganism. Thus, xylan can be decomposed. The culture conditions can be appropriately set according to the type of microorganism. Aerobic conditions and aerobic conditions can be selected as appropriate, and the culture temperature is also not particularly limited, but can be in the range of 25°C to 55°C. In addition, the culture time is also set according to need, but it can be about several hours to 150 hours. Moreover, the pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During cultivation, antibiotics such as ampicillin and tetracycline can be added to the medium as necessary.

In the culturing step, glucose can be included as a carbon source. This is because glucose is a carbon source that can be metabolized by microorganisms. In addition, when the transformant has the ability to decompose cellulose, the medium may contain cellulose or a partial decomposition product thereof as a carbon source, or biomass such as lignocellulose containing hemicellulose may be used as a carbon source. good too. The present transformant is capable of efficiently degrading cellulose by loosening the lignocellulose structure by degrading the xylan with the present xylanase, and is also able to metabolically proliferate using the glucose resulting from degrading cellulose as a carbon source. Therefore, the expression level of the xylanase can be easily increased, and xylan can be efficiently decomposed. Furthermore, by decomposing biomass such as lignocellulose containing hemicellulose as a carbon source with this transformant, simultaneous saccharification and fermentation can be performed by this transformant, and useful substances can also be produced by this transformant. .

In the culture step, culture conditions generally applied to the transformant host can be appropriately selected and used. Typically, culture for fermentation may be static culture, shaking culture, aeration agitation culture, or the like. Aeration conditions can be appropriately selected from anaerobic conditions, microaerobic conditions, aerobic conditions, and the like. The culture temperature is also not particularly limited, but can be in the range of 25°C to 55°C. In addition, the culture time is also set according to need, but it can be about several hours to 150 hours. Moreover, the pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During cultivation, antibiotics such as ampicillin and tetracycline can be added to the medium as necessary.

(Method for producing useful substances)
The method for producing a useful substance disclosed in the present specification (hereinafter also simply referred to as the present production method) comprises the steps of degrading xylan using the present xylanase, and converting the xylose obtained in the above step into a useful substance. and a step of performing. According to this production method, various useful substances derived from xylose can be obtained by efficiently decomposing xylan and converting the resulting xylose into useful substances.

Useful substances to be produced by this production method are not particularly limited. Ethanol, lactic acid, acetic acid, 1,3-propane-diol, propanol, butanol, succinic acid, ethylene and glycerol produced by microorganisms using sugars are included. Furfural, hydroxymethylfurfural, furfuryl alcohol derived from these compounds, tetrahydrofuran, furandicarboxylic acid and xylose obtained by dehydrating hexoses such as glucose and pentoses such as xylulose in the presence of an acid catalyst. xylitol obtained by reduction, and the like.

The decomposition step in this production method can be carried out in the same manner as the xylan decomposition step. In addition, the conversion step in this production method is a step of converting the xylose obtained in the decomposition step into useful substances by further enzymatic treatment, industrial treatment, fermentation treatment with microorganisms, etc. to obtain various useful substances from xylose. The method for this is well known to those skilled in the art at the time of filing of the present application, and those skilled in the art can use this production method and add steps as appropriate, depending on the type of the desired useful substance.

In this production method, although not particularly limited, it is preferable to use this transformant in the step of decomposing xylan. Due to the xylose assimilation ability of this transformant and/or by using other carbon sources, the decomposition step and the conversion step can be carried out simultaneously, and substances that can be produced by the metabolic ability of this transformant are produced as useful substances. can. In this case, when the present transformant is yeast, one or more enzymes in the metabolic system from glucose can be replaced or added by gene recombination to produce compounds that are not original metabolites as useful substances. In this case, specifically, in addition to lower alcohols such as ethanol, organic acids such as lactic acid and acetic acid, 1,3-propane-diol, propanol, butanol, succinic acid, ethylene and glycerol, addition of isoprenod synthesis route terpenoid farnesol, geranylgeraniol, squalene, fine chemicals (coenzyme Q10, vitamins and their raw materials, etc.), glycerin by modification of glycolysis, raw materials for plastics and chemical products, and the like. Since yeast has a high alcoholic fermentation ability, this transformant can efficiently produce ethanol using a medium containing biomass containing cellulose as a carbon source in addition to xylan. Yeast having a high alcoholic fermentation ability also has a high ability to produce other useful substances such as organic acids even when the glycolytic system is modified to produce such useful substances.

A useful substance is produced according to the ability of the transformant of the present invention to produce a useful substance by carrying out the conversion step such as the culture step. For example, if the transformant of the present invention has the ability to produce ethanol, it will produce ethanol, and if it has been modified to produce lactic acid, it will produce lactic acid and the like. After completion of fermentation, a step of recovering a useful substance-containing fraction from the culture solution, and a step of purifying or concentrating the same can also be carried out. The recovery step, purification step, and the like are appropriately selected according to the type of the useful substance.

(Screening method)
The xylanase screening method disclosed in the present specification (hereinafter also referred to as the present screening method) is performed at positions 6, 37, 46 and 47 when aligned with the amino acid sequence represented by SEQ ID NO: 1. 55th, 56th, 71st, 80th, 94th, 99th, 119th, 120th, 125th, 137th, 140th, 154th, 161st and 164th A step of preparing a xylanase candidate having mutations at positions corresponding to one or more positions; and (b) evaluating at least one of the half-life of the xylanase activity when stored at 50° C. and the predetermined temperature when the xylanase activity reaches half the activity at the time of storage. The above mutation positions have been found to have good Δ thermostability in single mutants. Therefore, by combining mutations at these mutation positions, it is possible to efficiently screen this xylanase that is excellent in high-temperature resistance.

In this screening method, either evaluation of (a) or (b) described above may be performed. It may be selected as appropriate, or both may be performed. The evaluation of (a) above is effective for primary screening.

In this screening method, the evaluation step can be a step of screening for a xylanase having a half-life of xylanase activity of 30 hours or longer when stored at 50°C. A xylanase with such a half-life would be useful for the xylan decomposition process.

Various aspects of the present xylanase already described can be applied to the mutation positions applied to screening candidates, the manner of mutation at the mutation positions, the number of mutations, and the like. In addition, acquisition of screening candidates can also be carried out in the same manner as acquisition of the present xylanase. In addition, in this screening method, efficient screening becomes possible when screening candidates are obtained by a cell-free protein synthesis system.

In this screening method, screening candidates with additional mutations in addition to the mutation positions described above may be used.

Examples are given below as specific examples in order to more specifically describe the disclosure of the present specification. The following examples are intended to illustrate the disclosure herein and are not intended to dictate its scope.

(Synthesis of various mutants)
As the xylanase (hereinafter referred to as XYN), one classified into 11 of the Glycoside Hydrolase Family (http://www.cazy.org/Glycoside-Hydrolases.html) of Trichoderma reesei-derived UniProtKB registration number P36217 (XYN2_HYPJR) was used. Mutant gene library in which 183 residues of the 190 residues of the full-length amino acid sequence of mature xylanase, excluding 7 residues whose wild-type sequence is alanine, are replaced with alanine using a 2-step PCR mutagenesis method. built. In the 1st PCR, the forward primer (T7p) of the T7 promoter and rbs added to the 5' end of the wild-type XYN gene, the reverse primer (rp) of the mutation introduction site, the forward primer (fp) of the mutation introduction site and the XYN gene Two fragments were amplified using a reverse primer (T7t) with a T7 terminator (transcription termination region) added to the 3' end. In the 2nd PCR, T7p and T7t primers were used to amplify the full length sequence.

Using the 2nd PCR products of the natural type and each mutant as templates, XYN was synthesized by an E. coli-derived cell-free protein synthesis system, and a mutant protein library was constructed on a 96-well PCR plate. For cell-free protein synthesis, 2.5 μl of the PCR product was added to a transcription/translation reaction solution (S30 E. coli extract, 56.4 mM Tris-acetate, pH 7.4, 1.2 mM ATP, 1 mM GTP, 1 mM CTP, 1 mM UTP, 40 mM creatine phosphate, 0 .7 mM 20 amino acid mix, 4.1% (w/w) polyethylene glycol 6000, 35 μg/ml folinic acid, 0.2 mg/ml E. coli tRNA, 36 mM ammonium acetate, 0.15 mg/ml creatine kinase, 10 mM magnesium acetate, 100 mM acetic acid 12.5 μl of potassium, 10 μg/ml rifampicin, 7.7 μg/ml T7 RNA polymerase) was added, and the mixture was incubated at 26° C. for 3 hours.

1 μl of the cell-free protein synthesis product was added to 29 μl of 25 mM citrate buffer (pH 5) and incubated at 25° C. and at 1° C. intervals between 48-52° C. for 2 hours each. Then, after adding 30 μl of AXB solution as a substrate, the mixture was kept at 40° C. for 15 minutes to react XYN with the substrate. The AXB solution used was obtained by dissolving 1 g of AZO-XYLAN (derived from BIRCHWOOD, manufactured by Megazyme) in 100 ml of sterilized water. 140 μl of ethanol was added to the enzymatic reaction solution, stirred, and allowed to stand at room temperature for 5 minutes to terminate the reaction. After centrifugation at 3000 rpm for 10 minutes with a plate centrifuge, 100 μl of the supernatant was placed in a 96-well assay plate, and blue absorption was measured at 590 nm using a plate reader (SPECTRA MAXPLUS 384 manufactured by Molecular Devices).

As described above, in each mutant incubated at 25°C for 2 hours and at 48°C to 52°C for 2 hours, the temperature at which the residual activity becomes 1/2 when the activity after incubation at 25°C for 2 hours is 1, Defined as heat resistant temperature. That is, when the residual activity at the two temperatures near which the residual activity becomes 0.5 is plotted on the y axis and the temperature is plotted on the x axis, a linear approximation formula is obtained, and Y = 0.5 is inserted into the formula. x was taken as the heat resistance temperature (° C.). When the heat resistant temperature was not within the range of 48 to 52° C., the activity was similarly measured near the assumed heat resistant temperature and plotted to obtain a linear approximation formula to obtain the heat resistant temperature. The heat resistance was evaluated by taking the temperature difference (Δ heat resistance temperature) obtained by subtracting the heat resistance temperature of the wild type from the heat resistance temperature of each mutant.

Based on the above results, 45 sites were selected as candidates for subsequent mutation introduction. A saturated mutant gene library was constructed by substituting the amino acid residues of candidates for mutation introduction with wild-type amino acids and the remaining 18 types of amino acids excluding alanine. Mutations were introduced by 2-step PCR in the same manner as above, and the enzyme was synthesized by a cell-free synthesis system.

(Heat resistance of single mutation)
A total of 1010 mutants of the alanine-substituted library and the 45-place saturation mutation library were evaluated for thermostability. The results are shown in FIG. FIG. 2 shows the result of extracting 40 single mutants having a positive Δ heat resistance temperature.

As shown in FIG. 1, among the 1010 mutants, only a few mutants had a positive Δthermal temperature. Most of the mutants had negative Δ thermostability and had lower thermostability than the parent xylanase. Further, as shown in FIG. 2, 11 mutants had a Δ heat resistant temperature of about 1° C. or more. In addition, at positions 6, 47, 55, 80, 94, 99, 119, 120, 125, 137 and 161 in SEQ ID NO: 1, two or more amino acids are effective. It was thought that this is the part that greatly contributes to heat resistance. The above 40 mutation positions were evaluated by substituting with all 19 kinds of amino acids, and the heat resistance was shown.

(Evaluation of heat resistance of additive mutants)
For mutations that were highly effective at each mutation position confirmed in Example 2, 21 types of additive mutants were prepared according to Example 1, and Δ heat resistant temperature was evaluated in the same manner as in Example 1. The mutations possessed by the additive mutants are shown in the table below, and the results of evaluating the Δ heat resistance temperature are shown in FIG.

As shown in FIG. 3, by adding mutations, the heat resistance temperature is improved at once, and by adding 2 to 3 mutations, the Δ heat resistance temperature exceeds 4 ° C., and 5 to 8 mutations are added. By adding the mutation of , the Δ heat-resistant temperature was about 8°C or more, and the Δ heat-resistant temperature was 10°C or more in 7 kinds of mutants.

FIG. 4 shows the result of comparing the additive predicted value obtained by adding the Δ heat resistant temperature for these additive mutants and the Δ heat resistant temperature obtained by the actual experiment of the additive mutant. As shown in FIG. 4, it was found that there was a correlation between the addition predicted value and the actual measurement value, and the heat resistance was improved as the mutation was added.

(Evaluation of activity half-life of additive mutants)
Using the additive mutants prepared in Example 3, the half-life at 50°C was evaluated. The xylanase activity was evaluated in the same manner as in Example 1, except that the temperature was 50°C.

As shown in the above table, the residual activity of the wild type decreased to about 80% after standing at 50°C for 2 hours, and the half-life of the residual activity when kept at 50°C was 3.1 hours in the wild type. rice field. Mutants with 5 or more mutations had a half-life of 120 hours or more, and mutR89b, which has the longest half-life, had a half-life of 263 hours. The relative ratio of mutR89b to the wild-type half-life of 1 was 84-fold.

(Secretion expression and heat resistance evaluation of additive mutants by yeast)
The xylanase coding regions of wild type and additive mutant R89 were amplified by PCR and subcloned into a single copy yeast secretion expression vector, pAURGAPSSRG. pAURGAPSSRG has a secretion signal downstream of the TDH3 promoter and is capable of secreting the enzyme outside the cell. Yeast (Saccharomyces cerevisiae strain BJ5465) was transformed with this vector, and SD-URA agar medium (yeast nitrogen base without amino acids without ammonia sulfate 1.7 g, casamino acid 5 g, amino acid mix, glucose 20 g, deionized water 10 g, deionized water 10 g, , pH 6.0) at 30°C for 3 days. The grown colony was inoculated into 2 ml of SD-URA liquid medium (yeast nitrogen base without amino acids without ammonia sulfate 1.7 g, casamino acid 5 g, -URA amino acid mix 0.77 g, glucose 20 g, deionized water 1000 ml, pH 6.0). The bacterial solution was cultured at 30°C for 24 hours and used as a preculture solution. 100 μl of preculture was inoculated into 2 ml of SD-URA liquid medium and main culture was carried out at 30° C. for 24 hours. The culture supernatant obtained by centrifugation was used as a crude enzyme solution.

30 μl of the substrate AXB solution was added to 30 μl of the crude enzyme solution, the activity was measured in the same manner as in Example 1, and the Δ heat-resistant temperature of the crude enzyme solution was measured in the same manner as when evaluating the heat-resistant temperature of the cell-free protein synthesis product. I checked with As a result, the Δ heat resistant temperature of the additive mutant R89 produced by secretion in yeast was 9.7°C, which was almost the same as 10.1°C when the E. coli-derived cell-free synthetic system product was used. From this, it was found that a xylanase with excellent Δ heat-resistant temperature can be stably obtained regardless of whether the transformant is a prokaryote or a eukaryote.

(Xylanase alignment of GH11)
Pham ID; 81 sequences of xylanase of GHF11 registered in Glyco_hydro_11 (PF00457) were aligned by the following method. Alignment results are shown in FIGS. 5A-5D. In FIG. 5, P36217 is a xylanase derived from Trichoderma reesei and represents UniProtKB accession number P36217, and other numbers also represent UniProtKB.
http://pfam.xfam.org/family/PF00457#tabview=tab3
Alignment types: seed (the curated alignment from which the HMM for the family is built
Selex format

As a result, positions 6, 37, 46, 47, 55, 56, 71, 80, 94, 99, 119, 120 and 125 of the amino acid sequence of accession number P36217 (XYN2_HYPJR) Positions 137, 140, 154, 161 and 164 have similar amino acid residues in 81 xylanases, and 174 xylanases are catalytic It had 37%-94% identity and 77%-100% similarity to the domain, positions 10-188 (SEQ ID NO: 2).

Claims (18)

a xylanase,
One or have mutations at positions corresponding to two or more positions;
The mutation at each position is either E, I, V or S at the position corresponding to position 6,
I at the position corresponding to position 46,
At the position corresponding to position 47, either C or A,
At the position corresponding to the 55th place, it is S,
At the position corresponding to position 80, it is either M, A or I,
at the 94-equivalent position, either H, C or Y;
at the 99-equivalent position, either G or A;
A xylanase that is M at the 164-equivalent position. Mutations at each of the above positions are
At the position corresponding to 6th place, it is E,
I at the position corresponding to position 46,
At the position corresponding to position 47, either C or A,
At the position corresponding to the 55th place, it is S,
At the position corresponding to position 80, it is either M or A,
at the position corresponding to position 94, either H or C;
At the position corresponding to the 99th position, it is G,
2. The xylanase according to claim 1, wherein the position corresponding to position 164 is M. Mutations at each of the above positions are
At the position corresponding to 6th place, it is E,
I at the position corresponding to position 46,
At the position corresponding to position 47, it is C,
At the position corresponding to the 55th place, it is S,
At the position corresponding to the 80th place, it is M,
H at the position corresponding to the 94th position,
At the position corresponding to the 99th position, it is G,
3. The xylanase according to claim 1 or 2, wherein the position corresponding to position 164 is M. Furthermore, when aligned with the amino acid sequence represented by SEQ ID NO: 1, one or two selected from the group consisting of positions 37, 56, 71, 120, 125, 137 and 140 having mutations at positions corresponding to one or more positions,
Mutations at each of the above positions are
At the position corresponding to position 37, it is I,
At the position corresponding to the 56th position, it is T,
At the position corresponding to the 71st place, it is A,
K at the position corresponding to 120th place,
At the position corresponding to the 125th place, it is A,
at the 137-equivalent position, any of W, A, L and V;
At the position corresponding to 140th place, it is I,
The xylanase according to any one of claims 1-3 . Mutations at each of the above positions are
At the position corresponding to position 37, it is I,
At the position corresponding to the 56th position, it is T,
At the position corresponding to the 71st place, it is A,
K at the position corresponding to 120th place,
At the position corresponding to the 125th place, it is A,
At the position corresponding to position 137, it is either W or A,
5. The xylanase according to claim 4 , wherein the position corresponding to position 140 is I. The xylanase according to any one of claims 1 to 5 , which has mutations at two or more positions. The xylanase according to any one of claims 1 to 6 , which has mutations at 4 or more positions. When stored at a predetermined temperature for 2 hours, the xylanase having the amino acid sequence represented by SEQ ID NO. 8. The xylanase according to claim 6 or 7 , which is higher than °C. The xylanase according to any one of claims 1 to 8 , wherein the xylanase has a half-life of xylanase activity at 50°C of 30 hours or longer. The xylanase according to any one of claims 1 to 9 , wherein the xylanase has a half-life of xylanase activity at 50°C of 100 hours or longer. 11. The xylanase according to any one of claims 1 to 10 , wherein said xylanase has 90% or more identity with the amino acid sequence represented by SEQ ID NO:2. A polynucleotide encoding the amino acid sequence of the xylanase according to any one of claims 1-11 . An expression vector comprising the polynucleotide of claim 12 . A transformant expressing the xylanase according to any one of claims 1 to 11 . A method for decomposing xylan, comprising a step of decomposing xylan using the xylanase according to any one of claims 1 to 11 . a step of decomposing xylan using the xylanase according to any one of claims 1 to 11 ;
a step of converting the xylan into a useful substance;
A method of producing a useful substance, comprising: A composition comprising the xylanase according to any one of claims 1-11 . A composition comprising a transformant that produces the xylanase according to any one of claims 1 to 11 .

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