Mutant polypeptides having xylanase activity

Abstract

The present application relates to a mutant polypeptide having xylanase activity and uses thereof.

Description

[Technical Field]

[0001] The present application relates to a mutant polypeptide having xylanase activity and uses thereof. [Background technology]

[0002] Xylanase (EC 3.2.1.8) is a hydrolase that randomly degrades the β-1,4 backbone of xylan, a component of plant cell walls. Xylanases are primarily used to degrade biomass in areas such as animal feed, bread making, and pulp bleaching (Beg QK, Kapoor M, Mahajan L, Hoondal GS. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol. 2001 Aug;56(3-4):326-38. doi: 10.1007 / s002530100704. PMID: 11548999.). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Beg QK, Kapoor M, Mahajan L, Hoondal GS. Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol. 2001 Aug;56(3-4):326-38. doi: 10.1007 / s002530100704. PMID: 11548999 [Non-patent document 2] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]:2444 [Non-patent document 3] Rice et al., 2000, Trends Genet. 16: 276-277

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[0004] For the convenience of xylanases used in various fields, they are required to be tolerant and active under harsh conditions (high temperature, basic conditions). However, most xylanases have problems in being difficult to apply to various fields due to their low pH (4.0-6.0) and low thermostability. [Means for solving the problem]

[0005] One object of the present application is to provide mutant polypeptides that have xylanase activity.

[0006] Another object of the present application is to provide a composition comprising said mutant polypeptide.

[0007] Another object of the present application is to provide a use of said mutant polypeptide or said composition for reacting with a xylan-containing substance.

[0008] Another object of the present application is to provide a method for decomposing a xylan-containing substance, and / or a method for producing xylooligosaccharides and / or xylose, which comprises contacting the mutant polypeptide, a host cell expressing the mutant polypeptide, and / or a composition containing the mutant polypeptide with a xylan-containing substance.

[0009] Another object of the present application is to provide a polynucleotide encoding said mutant polypeptide; a nucleic acid construct comprising said polynucleotide; a vector comprising said polynucleotide or nucleic acid construct; and / or a host cell comprising said polynucleotide, nucleic acid construct or vector.

[0010] Another object of the present application is to provide a method for producing the mutant polypeptide. [Effects of the Invention]

[0011] The mutant polypeptides having xylanase activity of the present application are useful in a variety of industrial fields. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the results of confirming the thermal stability of the mutant polypeptide of the present application. DETAILED DESCRIPTION OF THE INVENTION

[0013] One aspect of the present application is a mutant polypeptide having xylanase activity.

[0014] In one embodiment, i) the variant polypeptide is a polypeptide having a sequence identity of 70% or more but less than 100% with SEQ ID NO: 1; and / or ii) the variant polypeptide is a polypeptide encoded by a polynucleotide having 70% or more but less than 100% sequence identity with a sequence encoding the mature polypeptide of SEQ ID NO: 1; and / or iii) the variant polypeptide is a polypeptide encoded by a polynucleotide that hybridizes to (a) the mature polypeptide coding sequence of SEQ ID NO: 1, b) a cDNA thereof, or (c) a full-length complement of (a) or (b) under low stringency, medium stringency, medium-high stringency, high stringency, or very high stringency conditions; and / or iv) the variant polypeptide is a functional fragment of the polypeptide of i), ii) or iii) that has xylanase activity; and The mutant polypeptide comprises any one of the following modifications: Substitution of amino acids at one or more of positions 3, 5, 20, 34, 36, and 41 with other amino acids, disulfide bond formation, and combinations thereof; Here, the position numbers are positions corresponding to the positions in the polypeptide of SEQ ID NO:1.

[0015] In one specific example of any of the above specific examples, the amino acid at position 3 before the modification may be arginine (R), the amino acid at position 5 may be serine (S), the amino acid at position 20 may be phenylalanine (F), the amino acid at position 34 may be serine (S), the amino acid at position 36 may be threonine (T), and / or the amino acid at position 41 may be alanine (A).

[0016] In one embodiment of any of the above embodiments, the variant polypeptide may include an amino acid variation at a position selected from the following:

[0017] i)3+36; ii) 5+34; iii) 20+41; iv) 3+36+5+34; v)3+36+20+41; vi) 5+34+20+41; and vii)3+36+5+34+20+41; Here, the position numbers are positions corresponding to the positions in the polypeptide of SEQ ID NO:1.

[0018] In one embodiment of the above, the modification at each position of the mutant polypeptide may include one or more of the following modifications i) to vi):

[0019] i) substitution of amino acid 3 with cysteine; ii) substitution of amino acid 5 with cysteine; iii) substitution of amino acid 20 with cysteine; iv) substitution of amino acid 34 with cysteine; v) substitution of amino acid 36 with cysteine; and vi) substitution of amino acid 41 with cysteine; Here, the position numbers are positions corresponding to the positions in the polypeptide of SEQ ID NO:1.

[0020] In one embodiment of any of the above embodiments, the variant polypeptide may include any one or more modifications selected from the following:

[0021] R3C+T36C; S5C+S34C; F20C+A41C; R3C+T36C+S5C+S34C; R3C+T36C+F20C+A41C; S5C+S34C+F20C+A41C; and R3C+T36C+S5C+S34C+F20C+A41C.

[0022] In one embodiment of any of the above examples, the mutant polypeptide may comprise a substitution of amino acids at two or more of positions 3, 5, 20, 34, 36, and 41 with cysteine, forming a disulfide bridge between the two substituted amino acids.

[0023] In one embodiment of any of the above examples, the mutant polypeptide may include substitutions of the amino acid pair at positions 3 and 36; the amino acid pair at positions 5 and 34; and / or the amino acid pair at positions 20 and 41 with cysteine, and modifications such that the amino acid pairs form disulfide bridges.

[0024] As a specific example of any one of the above-mentioned specific examples, the mutant polypeptide may have increased heat resistance and / or thermal stability compared to the polypeptide consisting of the amino acid sequence of SEQ ID NO:1.

[0025] Another aspect of the present application is a composition for reaction with a variant polypeptide of the present application and / or a xylan-containing material comprising a variant polypeptide of the present application.

[0026] Another aspect of the present application is the use of said mutant polypeptide and / or a composition comprising said mutant polypeptide for reaction with a xylan-containing substance.

[0027] Another aspect of the present application is a method for producing xylose and / or xylo-oligosaccharides, comprising contacting a xylan-containing substance with the mutant polypeptide, a host cell expressing the mutant polypeptide, and / or a composition containing the mutant polypeptide.

[0028] Another aspect of the present application is a method for degrading a xylan-containing substance, comprising treating the xylan-containing substance with the mutant polypeptide, a host cell expressing the mutant polypeptide, and / or a composition containing the mutant polypeptide.

[0029] Another aspect of the present application is a polynucleotide encoding said mutant polypeptide.

[0030] Another aspect of the present application is a nucleic acid construct comprising said polynucleotide.

[0031] Another aspect of the present application is a vector comprising said polynucleotide or said nucleic acid construct.

[0032] Another aspect of the present application is a host cell comprising said mutant polypeptide, said polynucleotide, said nucleic acid construct, and / or said vector.

[0033] Another aspect of the present application is a method for producing a mutant polypeptide, comprising culturing the host cell described above.

[0034] Specific details for implementing the invention are as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to different descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the following specific description is not intended to limit the scope of this application.

[0035] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the present application described herein, and such equivalents are intended to be encompassed by this application.

[0036] As used in this specification and the appended claims, the singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless the context dictates otherwise, singular terms include plurals and plural terms include the singular. In this specification and the appended claims, the use of "or" may be used to mean "and / or" unless specifically stated otherwise.

[0037] In this application, the term "about" may be used before a specific numerical value. As used in this application, the term "about" includes not only the exact number listed after the term, but also approximately that number or a range close to that number. Whether a number is close to or approximately the specific number mentioned can be determined by considering the context in which the number is presented. As an example, the term "about" can refer to a range of -10% to +10% of the numerical value. As another example, the term "about" can refer to a range of -5% to +5% of the given numerical value. However, the present invention is not limited to this.

[0038] In this application, terms such as "first, second, third...," "i), ii), iii)...," or "(a), (b), (c), (d)..." are used to distinguish between similar components and do not imply sequential or order. For example, when the terms are used in connection with steps of a method, use, or analysis, the steps may be performed without any time interval between them, simultaneously, or separated by a few seconds, minutes, hours, days, or months.

[0039] In this application, the term "consisting essentially of" means that unspecified components may be present if the characteristics of the subject matter claimed in this application are not substantially affected by the presence of the unspecified components.

[0040] In this application, the term "consisting of" means that the proportions of the specified component(s) total 100%. The components or features following the term "consisting of" may be essential or mandatory. In some embodiments, other optional or non-essential components may be excluded, other than the components or features following "consisting of."

[0041] In this application, the term "comprising" means the presence of the feature, step or component described below that term, and does not exclude the presence or addition of one or more features, steps or components. In this application, the components or features described below as "comprising" may be essential or mandatory, but some embodiments may further include other optional or non-essential components or features.

[0042] In this application, the term "comprising" may in some embodiments be modified to refer to "consisting essentially of" or "consisting of."

[0043] In the present application, in relation to an amino acid sequence, a polypeptide "comprising" an amino acid sequence set forth in a particular SEQ ID NO, a polypeptide "consisting of" an amino acid sequence set forth in a particular SEQ ID NO, or a polypeptide or protein "having" an amino acid sequence set forth in a particular SEQ ID NO, is clearly understood to be used in the present application as well, provided that it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the SEQ ID NO. For example, a protein having an amino acid sequence with partial deletion, modification, substitution, conservative substitution, or addition may have, but is not limited to, an addition of a sequence at the N-terminus and / or C-terminus of the amino acid sequence that does not alter the function of the protein, a naturally occurring mutation, a silent mutation, or a conservative substitution thereof.

[0044] As used herein, the term "protein" or "polypeptide" refers to a polymer or oligomer of consecutive amino acid residues. As used herein, "polypeptide," "protein," and "peptide" are used interchangeably with "amino acid sequence."

[0045] In some cases, amino acid sequences that exhibit activity can be referred to as “enzymes.” In this application, amino acid sequences are written in the N-terminal to C-terminal orientation unless otherwise indicated.

[0046] As used herein, the term "recombinant" in reference to a cell, nucleic acid, polypeptide, or vector means that the cell, nucleic acid, polypeptide, or vector has been altered by the introduction of a heterologous nucleic acid or polypeptide or the alteration of a naturally occurring nucleic acid or polypeptide, or that the cell is derived from a cell so altered. Thus, for example, a recombinant cell may express genes that are not found within the native (non-recombinant) form of the cell, or may express naturally occurring genes that are expressed, not expressed at all, or abnormally expressed.

[0047] In this application, the term "isolated" refers to a substance that is in an environment in which it does not naturally occur or in a form that does not exist in nature, including that the substance (e.g., a sequence, enzyme, or nucleic acid) is at least substantially free from at least one other component with which it is naturally associated and with which it is found in nature, e.g., the sequence, enzyme, or nucleic acid.

[0048] For example, the isolated sequences, enzymes, or nucleic acids provided herein can be provided in a form that is substantially free of one or more contaminants.

[0049] Examples of isolated substances include, but are not limited to, i) any substance that is not naturally occurring; ii) any substance from which one, more, or all naturally occurring components associated with it have been removed (e.g., an enzyme, mutant, nucleic acid, protein, peptide, or cofactor); iii) any substance found in nature that has been artificially modified; or iv) a substance that has been modified to alter the amount of the substance relative to other components with which it is naturally associated (e.g., increasing the copy number of a gene encoding a particular substance; modifying a promoter naturally linked to a gene encoding a particular substance with a more active promoter, etc.).

[0050] In this application, the term "wild-type" means a naturally occurring polypeptide that does not have any artificial modifications. When the term "wild-type" is used in reference to a polypeptide, it means a naturally occurring polypeptide that does not have any artificial mutations (substitutions, insertions, deletions, etc.) at one or more amino acid positions. Similarly, when the term "wild-type" is used in reference to a polynucleotide, it means that it does not have any artificial modifications (substitutions, insertions, deletions) at one or more nucleotides. However, a polynucleotide encoding a wild-type polypeptide is not limited to naturally occurring polynucleotides, and also includes a sequence that encodes any wild-type polypeptide.

[0051] In this application, the term "parent sequence" or "backbone" refers to a reference sequence into which modifications are introduced to form a mutant polypeptide. That is, the parent sequence may be used as a starting sequence to introduce mutations such as substitutions, insertions, and / or deletions. The parent sequence may be a naturally occurring or wild-type sequence, or may be a variant of the naturally occurring or wild-type sequence in which one or more substitutions, insertions, or deletions have occurred, or may be an artificially synthesized sequence. When the parent sequence is an amino acid sequence that exhibits activity, i.e., the amino acid sequence of an enzyme, it may be referred to as a "parent enzyme."

[0052] In this application, the term "reference sequence" refers to a sequence used to determine the position of an amino acid in a given amino acid sequence. By aligning the given amino acid sequence with the reference sequence, the position of an amino acid in the given amino acid sequence that corresponds to a particular position in the reference sequence can be determined.

[0053] As used herein, the term "fragment" in connection with an amino acid or nucleic acid sequence refers to a portion of the parent sequence, such as a polypeptide in which one or more amino acids have been removed from the C- or N-terminus of the parent sequence.

[0054] In this application, the term "fragment" of an enzyme may refer to a "functional fragment." A "functional fragment," also known as an active fragment, refers to a polypeptide that is a part of the parent enzyme and has the enzymatic activity of the parent enzyme. For example, a functional fragment of an enzyme may include the catalytic site of the enzyme.

[0055] A fragment of an enzyme may comprise a portion of the full length of the parent enzyme, for example, but not limited to, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more, but less than 100%, of the amino acids of the full length of the parent enzyme.

[0056] In this application, "mutating / modifying" means changing or altering. This may be a change from what occurs naturally. As an example, an enzyme may be altered in such a way that it changes from a parent or reference sequence.

[0057] In the present application, the modified enzyme may be one that does not itself occur in nature, ie, is a non-naturally occurring enzyme.

[0058] As used herein, the term "modified" means, for example, altered from its naturally occurring form. Modified enzymes of the present application include non-naturally occurring enzymes or naturally occurring variants. By way of example, modified enzymes of the present application are modified enzymes that are not found in nature. By way of example, but not limitation, modified enzymes of the present application may be those that are not spontaneously occurring.

[0059] In this application, the term "modification", when used in connection with an amino acid / nucleic acid sequence, may include substitution of an amino acid / nucleic acid residue of a parent sequence for a different amino acid / nucleic acid residue at one or more positions in the amino acid sequence, deletion of an amino acid / nucleic acid residue (or a series of amino acid / nucleic acid residues) of the parent sequence at one or more positions, insertion of an amino acid / nucleic acid residue (or a series of amino acid / nucleic acid residues) of the parent sequence at one or more positions, truncation of the N-terminal and / or C-terminal amino acid sequence or 5' and / or 3' nucleic acid sequence, and any combination thereof.

[0060] As used herein, a "variant" or "modified polypeptide" of an enzyme refers to a protein that differs from a parent enzyme in one or more amino acids by conservative substitutions and / or modifications. The terms "variant" and "mutant polypeptide" may be used interchangeably. The variant or mutant polypeptide may be, but is not limited to, non-naturally occurring.

[0061] Such variants differ from the sequence of the parent enzyme by one or more modifications, such as amino acid substitutions, deletions and / or insertions.

[0062] Such variants may generally be identified by modifying one or more amino acids in the parent enzyme and evaluating the properties of the modified protein, i.e., the performance of the variant may be increased, unchanged, or decreased compared to the parent enzyme.

[0063] Some variants may also include mutant polypeptides in which one or more portions have been removed, such as the N-terminal leader sequence or the transmembrane domain.

[0064] Other variants may include variants in which portions have been removed from the N- and / or C-terminus of the mature protein.

[0065] The term "mutant" or "mutant polypeptide" may be used interchangeably with terms such as mutation, deformation, mutated protein, and mutation (in English, modification, modified protein, mutant, mutein, divergent, variant, etc.), and is not limited to these terms as long as they are used in the sense of mutation.

[0066] Variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a protein N-terminal signal (or leader) sequence involved in co- or post-translational protein transfer. The polypeptide may also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0067] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid of similar structural and / or chemical properties. Such amino acid substitutions are generally made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0068] Throughout this application, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, and amino acids referred to by abbreviations in this application are written according to the IUPAC-IUB nomenclature system.

[0069] Alanine Ala, A Arginine Arg, R Asparagine Asn, N Aspartic acid Asp, D Cysteine ​​Cys, C Glutamic acid Glu, E Glutamine Gln, Q Glycine Gly, G Histidine His, H Isoleucine Ile, I Leucine (Leu), L Lysine (Lys), K Methionine Met, M Phenylalanine Phe, F Proline Pro, P Serine Ser, S Threonine Thr, T Tryptophan Trp, W Tyrosine Tyr, Y Valine Val, V

[0070] On the other hand, any amino acid can be represented by Xaa, X.

[0071] In addition, commonly accepted three-letter codes are used for naturally occurring amino acids as well as other amino acids such as Aib (2-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, etc.

[0072] Amino acids can generally be grouped based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues. In this regard, amino acid substitutions can generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0073] For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include glutamic acid and aspartic acid; among amino acids having uncharged side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and among the nonpolar amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0074] As used herein, the term "gene" refers to a polynucleotide that encodes a polypeptide and includes regions preceding and following the coding region. In some embodiments, a gene has sequences (introns) inserted between each coding region (exon).

[0075] As used herein, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid or nucleotide sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0076] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences generally hybridize under moderately or highly stringent conditions over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or over the entire length of the sequence. Hybridization obviously also includes polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0077] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity may be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it may be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), as well as the GCG program package (Devereux, J., et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [Ed.] Academic Press, San Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48: 1073. For example, but not limited to, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0078] Homology, similarity, or identity of polynucleotides or polypeptides may be determined by comparing sequence information using a GAP computer program, such as that of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0079] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. Suitable defined hybridization conditions are within the skill of the art and may be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0080] As used herein, the term "mature polypeptide" refers to a polypeptide in a form that lacks a signal sequence or propeptide sequence. A mature protein / polypeptide / peptide may be a functional form of a protein / polypeptide / peptide. A mature polypeptide may be a final form that has undergone post-translational or post-translational modifications. Examples of post-translational modifications include, but are not limited to, modifications of the N- or C-terminus, glycosylation, phosphorylation, removal of a leader sequence, etc.

[0081] As used herein, the term "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule that contains one or more regulatory sequences and that has been artificially synthesized, engineered to contain a specific sequence in a manner that does not occur in nature, or isolated from nature.

[0082] In the present application, the term "expression" includes any step involved in the production of a polypeptide, such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0083] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule that contains a coding sequence and regulatory sequences operably linked thereto for the expression thereof.

[0084] As used herein, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned appropriately so that it directs the expression of a coding sequence. Thus, "operably linked" includes a regulatory region, such as a promoter, terminator, signal sequence, or enhancer region, which is a functional domain having a known or desired activity, attached or linked to a target (gene or polypeptide) in such a way that it can regulate the expression, secretion, or function of the target in accordance with the known or desired activity.

[0085] As used herein, the term "cDNA" refers to a DNA sequence that can be produced through reverse transcription from a mature, spliced ​​mRNA molecule that can be obtained from a eukaryotic or prokaryotic cell. A cDNA sequence does not include intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before being expressed as a mature, spliced ​​mRNA.

[0086] In this application, the term "regulatory sequence" refers to a polynucleotide sequence required for the expression of a coding sequence. Each regulatory sequence may be native to the coding sequence (having the same origin) or foreign (derived from another gene). Examples of the regulatory sequence include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination. The minimum unit of the regulatory sequence may include a promoter, a transcription and translation termination sequence.

[0087] The following nomenclature is used to describe the variants provided in this application.

[0088] In this application, a reference to a specific position in an amino acid sequence may include a reference to the amino acid present or substituted at that position. A reference to an amino acid at a specific position can be described in various ways. For example, "position 003" can be described as "position 3," "amino acid 3," or "the third amino acid." For example, if the amino acid at position 3 is arginine (R), it can be described as "R3" or "Arg3."

[0089] Amino acid substitutions can be expressed by listing the original amino acid, its position, and the substituted amino acid in that order. The amino acids can be represented using standard one-letter and three-letter codes. For example, if the amino acid serine at position 5 of a particular sequence is substituted with cysteine, it can be represented as "S5C" or "Ser5Cys."

[0090] Any amino acid at a particular position can be designated as "X." For example, X6 refers to any amino acid at position 6. Also, when a substituted amino acid is designated as X, it means that the amino acid present before substitution is replaced with another amino acid. For example, "V6X" indicates that V at position 6 is replaced with any amino acid other than V.

[0091] Different alternations can be expressed by simultaneously listing various amino acids using the " / " or "," symbols. For example, the substitution of amino acid (F) at position 20 with S or C can be expressed as F20S / C or F20S,C. In another example, F / S20C means that the amino acid F or S at position 20 before substitution is substituted with C.

[0092] Multiple mutations can be indicated using "+", for example, "R3C+T36C" means that the amino acid at position 3, arginine, is replaced by cysteine, and the amino acid at position 8, threonine, is replaced by cysteine, respectively.

[0093] As used herein, the term "corresponding to" refers to an amino acid residue at a recited position in a protein or polypeptide, or an amino acid residue that is similar, identical, or homologous to a recited residue in a protein or polypeptide. Identifying an amino acid at a corresponding position may be determining the specific amino acid of a sequence that references a particular sequence. As used herein, a "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0094] SEQ ID NO: 1 may be used as a reference sequence to determine the position of an amino acid within any amino acid sequence in this application.

[0095] That is, SEQ ID NO: 1 disclosed in this application is used to determine the corresponding amino acid residues in any polypeptide having xylanase activity, and unless otherwise specified in this application, the residues of a particular amino acid sequence are numbered based on SEQ ID NO: 1.

[0096] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in this application can identify the position of an amino acid relative to a query sequence (also referred to as a "reference sequence"), or the position where a variation such as a substitution, insertion, or deletion occurs.

[0097] For such alignment, for example, but not limited to, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277), etc. can be used.

[0098] Additionally, corresponding amino acid residues in other xylanases can be identified through multiple sequence alignment. Examples of multiple sequence alignment programs known in the art include MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: Examples of such programs include EMBOSS EMMA (1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680) using ClustalW, and basic parameters of each of the above programs can be used without limitation.

[0099] Alternatively, when enzymes diverging from the mature polypeptide of SEQ ID NO: 1 cannot be detected by conventional sequence-based comparison, other pairwise sequence comparison algorithms are used (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Higher sensitivity can be achieved from sequence-based searches by using search programs that use probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program calculates profiles through an iterative database search process and can detect remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Much greater sensitivity can be achieved when a polypeptide family or superfamily has more than one representation in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from a variety of sources, such as PSI-BLAST, secondary structure predictions, structural alignment profiles, and solvation potentials, as input to neural networks that predict structural folds for a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919, is used to align sequences with unknown structures to superfamily models present in the SCOP database. These alignments are in turn used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.

[0100] Several tools and resources are available for searching and creating structural alignments for proteins of known structure. For example, the SCOP superfamily of proteins has been structurally aligned, and the alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms, such as distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (CE) (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747). Implementations of these algorithms can additionally be used to query structural databases with the target structure to find possible structural homologs (Holm and Park, 2000, Bioinformatics 16: 566-567).

[0101] The above method is an example and is not limiting.

[0102] A specific example of the present application will be described in more detail below.

[0103] In the present application, xylanase refers to an enzyme that catalyzes the endohydrolysis of 1,4-beta-D-xylosidic bonds in xylan, including, but not limited to, an enzyme with the EC number 3.2.1.8.

[0104] For purposes of this application, xylanase activity can be measured and assessed using methods known in the art, including the embodiments described herein.

[0105] As used herein, the term "parent xylanase" refers to a xylanase that is modified to produce a mutant or variant polypeptide of the present application. Specifically, a parent xylanase, parent enzyme, or parent sequence may be a naturally occurring or wild-type polypeptide, a mature polypeptide thereof, or a variant or functional fragment thereof, but is not limited thereto, as long as it has xylanase activity and can serve as a parent for a variant.

[0106] The parent xylanase provided in the present application may be, but is not limited to, the polypeptide of SEQ ID NO: 1. Furthermore, the parent xylanase may be a polypeptide having about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the polypeptide of SEQ ID NO: 1, as long as it has xylanase activity. Any polypeptide having the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 may be included within the scope of the parent xylanase without limitation.

[0107] The parent xylanase of the variants provided in the present application may be derived from the genus Orpinomyces sp., Neocallimastix sp., Piromyces sp., or Ruminococcus sp. In particular, it may be derived from Orpinomyces sp.

[0108] Meanwhile, the above-mentioned microorganisms are examples of microorganisms from which the xylanase provided in the present application is derived, and include those derived from taxonomically homologous microorganisms, regardless of the name of the microorganism.

[0109] The above-mentioned microorganisms can be obtained from well-known microorganism depositories such as ATCC, DSMZ, CBS, NRRL, KCTC, and KCCM.

[0110] In this application, a sequence "derived from" a particular microorganism is not limited to sequences that are naturally produced or producible in that microorganism, but also includes sequences encoded by a gene that is produced or isolated from a microorganism that contains that gene.

[0111] For example, xylanases derived from Orpinomyces sp. include not only enzymes having xylanase activity that are naturally produced in Orpinomyces microorganisms, but also those produced by Orpinomyces microorganisms and those produced in other host cells through genetic modifications known in the art (e.g., transformation with a sequence encoding the enzyme).

[0112] In the present application, a "variant polypeptide having xylanase activity" may be a variant of a parent xylanase.

[0113] As used herein, the term "variant of a parent xylanase" or "xylanase variant" refers to a protein that differs from the amino acid sequence of the parent xylanase by one or more amino acids and that retains the activity of a xylanase.

[0114] The terms "mutant polypeptide having xylanase activity," "mutant of parent xylanase," and "mutant xylanase" can be used interchangeably.

[0115] The variants provided herein may contain one or more amino acid modifications in the parent xylanase sequence while retaining xylanase activity, which may be amino acid substitutions and / or disulfide bond formation.

[0116] Furthermore, i) the variant may be a polypeptide having 70% or more but less than 100% sequence identity with SEQ ID NO: 1; and / or ii) the variant is a polypeptide encoded by a polynucleotide having 70% or more but less than 100% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1; and / or iii) the variant is a polypeptide encoded by a polynucleotide that hybridizes to (a) the mature polypeptide coding sequence of SEQ ID NO: 1, (b) its cDNA, or (c) the full-length complement of (a) or (b) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions; and / or iv) the variant may be a functional fragment of i), ii), or ii) polypeptide that has xylanase activity.

[0117] Specifically, the variants provided herein may have xylanase activity while also having one or more altered functions or properties compared to the parent xylanase, including modifications of one or more amino acids in the parent xylanase sequence.

[0118] In one embodiment, the variants provided herein retain xylanase activity while having one or more altered functions or properties compared to the parent xylanase, including one or more amino acid modifications in the parent xylanase sequence, and may have one or more conservative substitutions.

[0119] The variants provided in this application are variants of a parent xylanase and may be polypeptides that have xylanase activity.

[0120] In one embodiment, the variants provided herein may include modifications at one or more positions corresponding to positions 3, 5, 20, 34, 36 and 41 of SEQ ID NO:1.

[0121] In one embodiment, the variants provided herein may include an amino acid modification at a position selected from the following i) to vii):

[0122] i)3+36; ii) 5+34; iii) 20+41; iv) 3+36+5+34; v)3+36+20+41; vi) 5+34+20+41; and vii)3+36+5+34+20+41.

[0123] In this application, position numbers correspond to positions in the polypeptide of SEQ ID NO: 1, where "corresponding" is as explained above.

[0124] In one specific example, the variants provided herein may include modifications of amino acids corresponding to one or more of R3, S5, F20, S34, T36 and A41 of SEQ ID NO:1.

[0125] As a specific example, the amino acid corresponding to position 3 in SEQ ID NO: 1 before modification provided in the present application may be arginine (R); the amino acid corresponding to position 5 may be serine (S); the amino acid corresponding to position 20 may be phenylalanine (F); the amino acid corresponding to position 34 may be serine (S); the amino acid corresponding to position 36 may be threonine (T); and / or the amino acid corresponding to position 41 may be alanine (A).

[0126] In one specific example, the variants provided herein may include a substitution of the amino acid corresponding to position 3 of SEQ ID NO: 1 with G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K or H, specifically with S, T, C, Y, N or Q, and more specifically with C.

[0127] In one specific example, the variants provided herein may include a substitution of the amino acid corresponding to position 5 of SEQ ID NO: 1 with G, A, V, L, I, M, F, W, P, T, C, Y, N, Q, D, E, K, R or H, specifically with T, C, Y, N or Q, and more specifically with C.

[0128] In one specific example, the variants provided herein may include a substitution of the amino acid corresponding to position 20 of SEQ ID NO: 1 with G, A, V, L, I, M, W, P, S, T, C, Y, N, Q, D, E, K, R or H, specifically with S, T, C, Y, N or Q, and more specifically with C.

[0129] In one specific example, the variants provided herein may include a substitution of the amino acid corresponding to position 34 of SEQ ID NO: 1 with G, A, V, L, I, M, F, W, P, T, C, Y, N, Q, D, E, K, R or H, specifically with T, C, Y, N or Q, and more specifically with C.

[0130] In one specific example, the variant provided in the present application may include a substitution of the amino acid corresponding to position 36 of SEQ ID NO: 1 with G, A, V, L, I, M, F, W, P, S, C, Y, N, Q, D, E, K, R or H, specifically with S, C, Y, N or Q, and more specifically with C.

[0131] In one specific example, the variants provided herein may include a substitution of the amino acid corresponding to position 41 of SEQ ID NO: 1 with G, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R or H, specifically with S, T, C, Y, N or Q, and more specifically with C.

[0132] In one embodiment, the variants provided herein may comprise a substitution of the amino acid corresponding to position 3 of SEQ ID NO:1 with a polar amino acid.

[0133] In one embodiment, the variants provided herein may comprise a substitution of the amino acid corresponding to position 5 of SEQ ID NO:1 with a polar amino acid.

[0134] In one embodiment, the variants provided herein may comprise a substitution of the amino acid corresponding to position 20 of SEQ ID NO:1 with a polar amino acid.

[0135] In one specific example, the variants provided herein may comprise a substitution of the amino acid corresponding to position 34 of SEQ ID NO:1 with a polar amino acid.

[0136] In one embodiment, the variants provided herein may comprise a substitution of the amino acid corresponding to position 36 of SEQ ID NO:1 with a polar amino acid.

[0137] In one embodiment, the variants provided herein may comprise a substitution of the amino acid corresponding to position 41 of SEQ ID NO:1 with a polar amino acid.

[0138] In one specific example, the variants provided herein may include one or more of the following substitutions of SEQ ID NO:1: R3C, S5C, F20C, S34C, T36C, and A41C.

[0139] In one specific example, the variants provided herein may include two or more of the following substitutions of SEQ ID NO:1: R3C, S5C, F20C, S34C, T36C, and A41C.

[0140] In one embodiment, the variants provided herein may contain any one or more substitutions selected from the following:

[0141] R3C+T36C; S5C+S34C; F20C+A41C; R3C+T36C+S5C+S34C; R3C+T36C+F20C+A41C; S5C+S34C+F20C+A41C; R3C+T36C+S5C+S34C+F20C+A41C.

[0142] Specifically, a mutant comprising an R3C+T36C substitution in SEQ ID NO: 1 can be represented by SEQ ID NO: 3, a mutant comprising an S5C+S34C substitution in SEQ ID NO: 1 can be represented by SEQ ID NO: 5, and a mutant comprising an F20C+A41C substitution in SEQ ID NO: 1 can be represented by SEQ ID NO: 7.

[0143] In one embodiment, the variants provided in this application include all possible combinations of the aforementioned variations.

[0144] For example, the variant may include an amino acid variation at a position selected from the following, with a combination of the above variations:

[0145] 3 5 20 34 36 41 3 + 5 3 + 20 3 + 34 3 + 36 3 + 41 5 + 20 5 + 34 5 + 36 5 + 41 20 + 34 20 + 36 20 + 41 34 + 36 34 + 41 36 + 41 3 + 5 + 20 3 + 5 + 34 3 + 5 + 36 3 + 5 + 41 3 + 20 + 34 3 + 20 + 36 3 + 20 + 41 3 + 34 + 36 3 + 34 + 41 3 + 36 + 41 5 + 20 + 34 5 + 20 + 36 5 + 20 + 41 5 + 34 + 36 5 + 34 + 41 5 + 36 + 41 20 + 34 + 36 20 + 34 + 41 20 + 36 + 41 34 + 36 + 41 3 + 5 + 20 + 34 3 + 5 + 20 + 36 3 + 5 + 20 + 41 3 + 5 + 34 + 36 3 + 5 + 34 + 41 3 + 5 + 36 + 41 3 + 20 + 34 + 36 3 + 20 + 34 + 41 3 + 20 + 36 + 41 3 + 34 + 36 + 41 5 + 20 + 34 + 36 5 + 20 + 34 + 41 5 + 20 + 36 + 41 5 + 34 + 36 + 41 20 + 34 + 36 + 41 3 + 5 + 20 + 34 + 36 3 + 5 + 20 + 34 + 41 3 + 5 + 20 + 36 + 41 3 + 5 + 34 + 36 + 41 3 + 20 + 34 + 36 + 41 5 + 20 + 34 + 36 + 41 3 + 5 + 20 + 34 + 36 + 41

[0146] As another example, the variant may include, but is not limited to, any one or more of the following substitutions: i) substitution of amino acid 3 with cysteine; ii) substitution of amino acid 5 with cysteine; iii) substitution of amino acid 20 with cysteine; iv) substitution of amino acid 34 with cysteine; v) substitution of amino acid 36 with cysteine; and vi) substitution of amino acid 41 with cysteine.

[0147] As another example, the variant may include, but is not limited to, any one or more variations selected from the following:

[0148] R3C S5C F20C S34C T36C A41C R3C + S5C R3C + F20C R3C + S34C R3C + T36C R3C + A41C S5C + F20C S5C + S34C S5C + T36C S5C + A41C F20C + S34C F20C + T36C F20C + A41C S34C + T36C S34C + A41C T36C + A41C R3C + S5C + F20C R3C + S5C + S34C R3C + S5C + T36C R3C + S5C + A41C R3C + F20C + S34C R3C + F20C + T36C R3C + F20C + A41C R3C + S34C + T36C R3C + S34C + A41C R3C + T36C + A41C S5C + F20C + S34C S5C + F20C + T36C S5C + F20C + A41C S5C + S34C + T36C S5C + S34C + A41C S5C + T36C + A41C F20C + S34C + T36C F20C + S34C + A41C F20C + T36C + A41C S34C + T36C + A41C R3C + S5C + F20C + S34C R3C + S5C + F20C + T36C R3C + S5C + F20C + A41C R3C + S5C + S34C + T36C R3C + S5C + S34C + A41C R3C + S5C + T36C + A41C R3C + F20C + S34C + T36C R3C + F20C + S34C + A41C R3C + F20C + T36C + A41C R3C + S34C + T36C + A41C S5C + F20C + S34C + T36C S5C + F20C + S34C + A41C S5C + F20C + T36C + A41C S5C + S34C + T36C + A41C F20C + S34C + T36C + A41C R3C + S5C + F20C + S34C + T36C R3C + S5C + F20C + S34C + A41C R3C + S5C + F20C + T36C + A41C R3C + S5C + S34C + T36C + A41C R3C + F20C + S34C + T36C + A41C S5C + F20C + S34C + T36C + A41C R3C + S5C + F20C + S34C + T36C + A41C

[0149] In one specific example, the variants provided herein may comprise a substitution of amino acids corresponding to two or more of positions 3, 5, 20, 34, 36 and 41 of SEQ ID NO: 1 with cysteine, forming a disulfide bridge between the two substituted amino acids.

[0150] In one specific example, the variants provided herein may have the amino acid pair at positions 3 and 36; the amino acid pair at positions 5 and 34; and / or the amino acid pair at positions 20 and 41 substituted with cysteines, such that the amino acid pairs form disulfide bridges.

[0151] In one embodiment, the variants provided herein may have about 60% or more, e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, but less than 100% sequence identity with the parent xylanase; its mature polypeptide or a functional fragment thereof.

[0152] In one specific example, the variants provided herein may have about 60% or more, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more and less than 100% sequence identity with SEQ ID NO: 1.

[0153] In one specific example, the variant provided herein may be a polypeptide encoded by a polynucleotide having about 60% or more, e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more but less than 100% nucleobase sequence identity to the nucleobase sequence encoding the mature polypeptide of SEQ ID NO:1.

[0154] In one specific example, the variant provided in the present application may be a polypeptide encoded by a polynucleotide that hybridizes to (a) a sequence encoding the mature polypeptide of SEQ ID NO: 1, (b) its cDNA, or (c) a full-length complement of (a) or (b) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions.

[0155] In one specific example, the variants provided herein may have about 60% or more, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more and less than 100% sequence identity to a functional fragment of SEQ ID NO: 1.

[0156] In one specific example, the variant provided herein may be a polypeptide encoded by a polynucleotide having a nucleic acid sequence identity of about 60% or more, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more and less than 100%, to the nucleic acid sequence set forth in SEQ ID NO:2.

[0157] In one specific example, the variant provided herein may be a polypeptide in which the amino acids at one or more of amino acid positions 3, 5, 20, 34, 36, and 41 in the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 are fixed, and the variant has about 60% or more, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity to the amino acid sequence of said SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, its mature polypeptide, or a functional fragment thereof.

[0158] In one embodiment, the variants provided herein may be encoded by the polynucleotide of SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.

[0159] As one specific example, a variant containing the R3C+T36C substitution of SEQ ID NO: 1 of the present application may be encoded by the polynucleotide represented by SEQ ID NO:4.

[0160] As one specific example, a variant containing the S5C+S34C substitution of SEQ ID NO: 1 of the present application may be encoded by the polynucleotide represented by SEQ ID NO: 6.

[0161] As one specific example, a variant containing the F20C+A41C substitution of SEQ ID NO: 1 of the present application may be encoded by the polynucleotide represented by SEQ ID NO:8.

[0162] The variants provided herein may have one or more altered properties or attributes of the polypeptide that may be selected or detected compared to other xylanases, such as wild-type xylanases, parent xylanases, other xylanase variants, etc.

[0163] Such properties or attributes include oxidative stability, substrate specificity, catalytic activity, thermostability, alkaline stability, pH activity profile, resistance to proteolysis, Km, k cat , k catThese include, but are not limited to, the ability to bind to a ligand, the ability to bind to a receptor, the ability to be secreted, the ability to be displayed on the surface of a cell, the ability to form oligomers, the ability to transmit a signal, the ability to promote cell proliferation, the ability to inhibit cell proliferation, the ability to induce apoptosis, the ability to be modified by phosphorylation or glycosylation, and / or the ability to treat a disease.

[0164] In one embodiment, the variants provided herein may have increased thermotolerance and / or thermostability compared to the parent sequence.

[0165] In the present application, "enzymatic activity" refers to at least one catalytic activity. cat The conversion efficiency of the enzyme may be expressed primarily in terms of / Km, but is not limited thereto.

[0166] k cat Km is the catalytic constant of the rate at which an enzyme converts a substrate to a product per unit time when the enzyme is completely saturated with the substrate, and is also called the turnover number. Km is the substrate concentration at which the reaction velocity is half of its maximum value (Vmax).

[0167] An example of a method for expressing enzyme activity is specific activity (µmol of converted substrate x mg). -1 x min -1 ) or volumetric activity (umol of converted substrate x mL -1 x min -1 ) etc.

[0168] However, the definition of enzymatic activity is not limited to the above-mentioned content, and can be defined and evaluated based on known content such as Irwin H. Segel, Enzyme kinetics, John Wiley & Sons, 1979; A. G. Marangoni, Enzyme kinetics, Wiley-Interscience, 2003; A. Fersht, Enzyme structure and mechanisms, John Wiley & Sons, 1981; Structure and Mechanism in Protein Science: A guide to enzyme catalysis and protein folding, Alan Fersht, W. H. Freeman, 1999; Fundamentals of Enzyme Kinetics, Athel Cornish-Bowden, Wiley-Blackwell 2012 and Voet et al., "Biochemie" [Biochemistry], 1992, VCH-Verlag, Chapter 13, pages 331-332 with respect to enzymatic activity.

[0169] In one embodiment, the variants provided herein may have an increased enzymatic activity of about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200% or more compared to the parent enzyme.

[0170] In another embodiment, the variants provided herein may have an enzymatic activity that is reduced by about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, or about 20% or less compared to the parent enzyme.

[0171] In this application, the term "specific activity" refers to the activity of an enzyme per unit weight of protein, and can be expressed in units / mg. Protein quantification can be performed using, for example, SDS-PAGE or Bradford assay.

[0172] Enzyme stability refers to the preservation of enzyme activity during storage or reaction. To measure this change in stability, the initial enzyme activity is measured under specified conditions at time zero (100%) and after a certain time (x%), and then compared. This can be used to express the level at which enzyme activity is lost, or enzyme stability.

[0173] Factors that affect enzyme activity include, for example, pH, heat, and the presence of other substances (eg, oxidizing agents, chelating agents).

[0174] As used herein, the term "pH stability" refers to the ability of a protein to function within a specific pH range. In one specific example, the variants provided herein may be active at about pH 4.0 to about pH 12.0, but are not limited thereto.

[0175] When a protein maintains its function within a specific pH range, it can be defined as having "pH stability," and depending on the pH range, it can be defined as having "acid resistance," "alkali resistance," etc.

[0176] As used herein, the term "thermal stability" refers to the ability of a protein to function within a specific temperature range. As a specific example, the mutant provided herein may be active within a temperature range of about 20°C to about 120°C, and more specifically, may be active within a temperature range of about 60°C to about 100°C, but is not limited thereto.

[0177] As used herein, the term "thermal tolerance" refers to the ability of a protein to function after being exposed to a particular temperature, e.g., high heat or cryogenic temperatures. For example, a protein that is thermotolerant may not function at the temperature to which it is exposed, but can regain its functionality when returned to an optimal temperature environment.

[0178] Increased stability includes maintaining higher enzymatic activity compared to other enzymes, e.g., the wild-type enzyme, the parent enzyme, and / or other variants; increasing the range of pH, temperature, and / or time over which the protein remains functional, etc.

[0179] Decreased stability includes reduced maintenance of enzymatic activity compared to other enzymes, e.g., wild-type enzymes, parent enzymes, and / or other variants; a reduced range of pH, temperature, and / or time over which the protein remains functional, etc.

[0180] As used herein, the term "substrate specificity" refers to the ability of an enzyme to distinguish between a substrate and a molecule that competes with the substrate. Substrate specificity can be determined by measuring the activity of an enzyme toward different substrates. In one embodiment, the change in substrate specificity may be a change in the direction of increasing specificity for a substrate that can produce a desired product. In another embodiment, the change in substrate specificity may be a change in the direction of decreasing specificity for a substrate that can produce a desired product.

[0181] The altered properties of the variants provided herein may be suitable or improved activity for applications in a variety of industrial sectors, including feed, baking, pulp bleaching, etc.

[0182] A polynucleotide encoding a variant of the present application may contain the coding sequence of the variant described above. The polynucleotide may have various modifications in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the polypeptide is to be expressed, as long as the amino acid sequence of the polypeptide is not changed.

[0183] Furthermore, the polynucleotide of the present application may include, without limitation, a sequence that hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the base sequence, and encodes the variant of the present application.

[0184] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0185] Examples of conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more, hybridize with each other, while polynucleotides with lower homology or identity do not hybridize with each other; and conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, specifically 60°C, 1xSSC, and 0.1% SDS, specifically 60°C, 0.1xSSC, and 0.1% SDS, and more specifically 68°C, 1xSSC, and 0.1% SDS.

[0186] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0187] Specifically, polynucleotides having homology or identity can be detected using the hybridization conditions described above, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0188] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0189] For example, "high stringency" may occur at about 5-10°C below the Tm of the probe; "intermediate stringency" may occur at about 10-20°C below the Tm of the probe; and "low stringency" may occur at about 20-25°C below the Tm, but is not limited thereto.

[0190] As an example, "low stringency conditions" may be prehybridization and hybridization for 12-24 hours for a probe at least 100 nucleotides long in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 25% formamide at 42°C according to standard Southern blotting procedures. The carrier material may finally be washed two to three times for 15 minutes each in 2X SSC, 0.1-0.2% SDS at 50°C.

[0191] As an example, "medium stringency conditions" may be prehybridization and hybridization for 12-24 hours for a probe at least 100 nucleotides long in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide at 42°C according to standard Southern blotting procedures. The carrier material may be finally washed two to three times for 15 minutes each in 2X SSC, 0.1-0.2% SDS at 55°C. As an example, "medium-high stringency conditions" may be prehybridization and hybridization for a probe at least 100 nucleotides long in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide at 42°C for 12-24 hours according to standard Southern blotting procedures. The carrier material may be finally washed two to three times for 15 minutes each with 1-2X SSC, 0.1-0.2% SDS at 60°C.

[0192] By way of example, "high stringency conditions" may be prehybridization and hybridization for 12-24 hours for a probe at least 100 nucleotides long in 5×SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide at 42° C., according to standard Southern blotting procedures. The carrier material may be finally washed two to three times for 15 minutes each in 2×SSC, 0.1-0.2% SDS at 65° C.

[0193] The nucleic acid constructs provided herein comprise a polynucleotide encoding a variant provided herein operably linked to one or more regulatory sequences that direct the expression of the coding sequence in a suitable host cell under conditions appropriate for the regulatory sequences.

[0194] Polynucleotides can be manipulated in a variety of ways to allow for expression of variants. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide before inserting it into the vector. Such manipulations can be performed using methods known in the art.

[0195] The term "vector" as used herein refers to a DNA construct containing a polynucleotide sequence encoding a variant of the present application operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the variant in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence controlling the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or may be integrated into the genome itself.

[0196] The vectors that can be used in the present application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0197] For example, a polynucleotide encoding a variant provided herein can be inserted into a chromosome via a vector for chromosomal insertion in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of insertion of the target nucleic acid molecule. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, may be used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0198] The host cells of the present application may include, without limitation, any cells that can express the variants of the present application.

[0199] The host cells of the present application may comprise the variants described above, polynucleotides encoding said variants, nucleic acid constructs and / or vectors comprising same.

[0200] The nucleic acid construct or vector may be chromosomally integrated or maintained as an autonomously replicating extrachromosomal vector, as described above.

[0201] A host cell of the present application includes any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0202] The host cell may be any cell useful for the recombinant production of variants, for example, a prokaryotic or eukaryotic cell.

[0203] The prokaryotic host cell may be any gram-positive or gram-negative bacterium.

[0204] Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.

[0205] Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Iriobacter, Neisseria, Pseudomonas, Salmonella, Vibrio (e.g., Vibrio natriegens), and Ureaplasma.

[0206] In one embodiment, the bacterial host cell may be a Bacillus host cell, including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lotus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0207] In one embodiment, the bacterial host cell may be a Streptococcus host cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subspecies Zooepidemicus cells.

[0208] In one embodiment, the bacterial host cell may be a Streptomyces host cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0209] In one embodiment, the bacterial host cell may be a Corynebacterium host cell, such as Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The bacterium may be, but is not limited to, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens.

[0210] In one specific example, the host cell may be a microorganism of the genus Escherichia, such as, but not limited to, Escherichia coli, Escherichia albertii, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, or Escherichia blattae.

[0211] The host cell may be a eukaryotic cell, such as a mammalian, insect, plant or fungal cell.

[0212] The host cell may be a fungal cell. In this application, "fungi" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all Fungi Imperfecti.

[0213] The fungal host cell may be a yeast cell. As used herein, "yeast" includes ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and yeasts belonging to the Fungi imperfecti (Blastomycetes). However, such classifications may vary and may be defined as explained in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0214] Yeast host cells can be Candida, Hansenula, Kluyveromyces, Pichia, Komagataella, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, The cell may be a Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Komagataella phaffii, or Yarrowia lipolytica cell.

[0215] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the phylum Eumycota and subdivision Oomycota (as defined supra (Hawksworth et al., 1995)). Filamentous fungi are generally characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts, such as Saccharomyces cerevisiae, is by germination of a unicellular thallus, and carbon catabolism may be fermentative.

[0216] Filamentous fungal host cells include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophysola, and Neocallimasticus. ocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cells.

[0217] For example, filamentous fungal host cells include those of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, and Ceriporiopsis ribulosa. rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellensecrookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophysora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatumThe cell may be, but is not limited to, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0218] The method of producing the variant of the present application may include culturing the host cell.

[0219] In the present application, the term "culturing" means growing the host cells under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using appropriate media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, or fed-batch culture.

[0220] In the present application, the term "culture medium" refers to a mixture of nutrients required for culturing the host cells, including water as the main component, and provides nutrients and growth factors essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the host cells of the present application are not particularly limited as long as they are media used for culturing conventional host cells. The host cells of the present application can be cultured under aerobic conditions in a conventional culture medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling the temperature, pH, etc.

[0221] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used. A variety of other suitable carbon sources may also be used without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0222] Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its hydrolyzed products, defatted soybean cake or its hydrolyzed products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0223] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, they are not limited thereto.

[0224] During the culture of the host cells, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. During the culture, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas can be injected into the medium to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection or to maintain an anaerobic or microaerobic state, but this is not limiting.

[0225] The temperature of the medium may be, but is not limited to, 20° C. to 55° C., specifically 25° C. to 40° C. The culture period may be continued until a desired amount of useful substance is produced, specifically 24 hours to 196 hours, but is not limited to this.

[0226] In one specific example, the method for producing the mutant polypeptide having xylanase activity of the present application may further comprise a step of recovering the mutant polypeptide having xylanase activity of the present application expressed in the culturing step.

[0227] In another embodiment, the variant expressed during the culturing step can be recovered using methods known in the art, for example, the variant can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0228] The recovery method may involve collecting the mutant using a suitable method known in the art based on the host cell culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods may be used, and the mutant can be recovered from the medium or host cells using a suitable method known in the art.

[0229] In another embodiment, the variant expressed by the host cells during the culturing step may not be recovered, in which case the host cells expressing the variant may themselves be used as the source of the variant.

[0230] The compositions of the present application are used to degrade xylan-containing materials.

[0231] The compositions of the present application are used to convert xylan-containing materials into xylose and / or xylo-oligosaccharides.

[0232] The composition of the present application may further contain other components in addition to the variants provided in the present application. Those skilled in the art can appropriately select the components to be added to the composition of the present application.

[0233] In one embodiment, the composition of the present application may further comprise any component suitable for application in converting xylan-containing materials into xylose and / or xylooligosaccharides.

[0234] As one specific example, the composition of the present application may further comprise any component suitable for application in various industrial fields such as animal feed, baking, biomass saccharification, and pulp bleaching.

[0235] Examples of substances that may be added include, but are not limited to, stabilizers, surfactants, builders, chelating agents, dispersing agents, enzymes, enzyme stabilizers, catalysts, activators, carriers, compounding agents, lubricants, disintegrants, excipients, solubilizers, suspending agents, dyes, flavorings, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, diluents, lubricants, preservatives, and the like.

[0236] In one embodiment, the compositions provided herein may further comprise naturally occurring or non-naturally occurring substances in addition to the variants provided herein.

[0237] In one embodiment, the compositions provided herein may further contain, in addition to the variants provided herein, additional enzymes commonly used in various industrial fields, including animal feed, bread making, biomass saccharification, pulp bleaching, etc.

[0238] For example, the additional enzymes may further comprise any one or more enzymes selected from the group consisting of β-amylase, cellulase (β-glucosidase, cellobiohydrolase, and endoglucanase), glucoamylase, hemicellulase (endo-xylanase, β-xylosidase, α-L-arabinofuranosidase, α-D-glucuronidase, feruloyl esterase, coumaroyl esterase, α-galactosidase, β-galactosidase, β-mannanase, or β-mannosidase), isoamylase, isomerase, lipase, phytase, protease, pullulanase, and / or other enzymes useful in commercial processes together with α-amylase.

[0239] The xylanase variants of the present application or compositions comprising the xylanase variants of the present application can be used to degrade any xylan-containing material.

[0240] In the present application, a xylan-containing substance is any substance that can be decomposed by xylanase. For example, the xylan-containing substance may be hemicellulose. Specifically, the xylan-containing substance may be a substance selected from the group consisting of xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. For example, the xylan-containing substance may be xylan, but is not limited thereto.

[0241] In one embodiment, the present application provides a method for degrading (or disintegrating) xylan-containing materials, which may also be referred to as xylan solubilization and / or pentosan solubilization.

[0242] In additional embodiments of the application, the methods relate to the degradation (eg, degradation) of polymers derived from the degradation of xylan.

[0243] The degradation products (e.g., glucose) are feedstocks for any fermentation process and can be used, for example, in the production of biofuels (e.g., bioethanol) or in the production of other products, such as biochemicals (e.g., bio-based isoprene).

[0244] Xylan can be decomposed using the mutants of the present application, host cells expressing the mutants, and compositions containing the mutants and / or host cells. In the xylan hydrolysis step, cofactors, coenzymes, etc. may be added in addition to the mutants of the present application. The substrate hydrolysis step is carried out under optimal conditions such as pH and temperature, and appropriate conditions can be selected by those skilled in the art.

[0245] The xylanase variants of the present application may be used in any one of the following applications:

[0246] a) additives in animal feed ingredients; and / or b) animal feed supplements; and / or c) Decomposition of cereal-based materials (for example, this may be whole grains or parts of cereals).

[0247] In one embodiment, the xylanase variants of the present application are used in feed ingredients.

[0248] In one embodiment, the xylan-containing material may be a feedstuff or ingredient.

[0249] The feed composition of the present application means any natural or artificial diet, meal, or component of such a meal, intended to be eaten, ingested, and digested by an animal or suitable for this purpose, and can be prepared in a variety of forms known in the art.

[0250] In one embodiment, the xylanase variants of the present application may be used in food compositions or in the preparation thereof.

[0251] In one embodiment, the xylan-containing material may be a grain-based material (including whole grain or partial grain or malted grain, eg, malted barley).

[0252] In one embodiment, the xylan-containing material may be a cereal flour (eg, wheat, oat, rye or barley flour).

[0253] In one embodiment, the xylan-containing material may be malt or saccharified liquor, or malted barley, or a combination thereof.

[0254] By way of example, the food composition may be a fermented beverage, including beer and wine. By way of another example, the food composition may be a bakery product, including, but not limited to, loaves, rolls, buns, pizza, pretzels, tortillas, cakes, cookies, biscuits, and crackers.

[0255] The xylanase variants of the present application are used for the separation of wheat gluten starch.

[0256] Fractionation of wheat endosperm flour into starch and gluten fractions, after initial separation of the bran and wheat germ from the endosperm, can be used to obtain high quality α-starch and by-products β-starch and active gluten.

[0257] In a method for separating flour (e.g., wheat flour) into starch and gluten fractions, the method comprises mixing flour (e.g., wheat flour), water, and a xylanase variant. The flour, water, and xylanase variant may be mixed simultaneously or sequentially. In some embodiments, the flour (e.g., wheat flour) and water can be mixed before mixing with the xylanase variant.

[0258] The application of the xylanase variants of the present application in wheat gluten starch separation can produce higher pre-starch yields and / or better quality gluten (eg, better quality vital gluten).

[0259] In other embodiments, the xylanase variants of the present application may be used to degrade grain-based materials and may be used as part of the biofuel (eg, bioethanol) production process.

[0260] As an example, the xylanase variants of the present application can improve the production of biofuels (eg, bioethanol) and utilization of grain-based materials in the biofuel industry.

[0261] By way of example, steps including liquefaction, saccharification, fermentation, simultaneous saccharification, and mixing of the biofuel and xylanase variant before or during fermentation, after fermentation, or a combination thereof can be used.

[0262] When the xylanase variants of the present application are applied to biofuel production processes, more dry matter saccharification solution can be used in the process; a higher solids content can be obtained in the final syrup; better heat transfer; lower energy requirements; reduced evaporator fouling; reduced cleaning costs; increased final fuel yield; improved by-product quality; easier separation of the solid and liquid portions of the residue after distillation; or a combination of the above benefits can be obtained.

[0263] The xylanase variants of the present application may be used in pulp bleaching.

[0264] For example, when colored lignin in pulp is linked to crystalline cellulose via xylan, treatment with a xylanase mutant can degrade the xylan and release the colored lignin, thereby facilitating pulp bleaching. [Example]

[0265] Example The present application will be described in more detail below through examples and experimental examples. However, these examples and experimental examples are intended to exemplify the present application and are not intended to limit the scope of the present application.

[0266] Example 1: Production of single xylanase mutants 1-1. Template production The gene (SEQ ID NO: 2) of a xylanase mutant (hereinafter referred to as Op Xyn, SEQ ID NO: 1) was amplified from the genomic DNA of Orpinomyces sp. PC-2 strain and cloned into the pHCE vector (Takara) and used as a template.

[0267] 1-2. Construction of xylanase mutants The amino acid pairing site for forming a disulfide bond in Op Xyn was selected, and primers were designed to mutate the amino acid sequence to cysteine, producing seven mutants as shown in Table 1 below (Table 1). The mutation details in Table 1 below include the amino acid before mutation, the mutation position based on the amino acid sequence of SEQ ID NO: 1, and the amino acid after mutation, in that order.

[0268] [Table 1]

[0269] Specifically, the xylanase single mutant was constructed by PCR using a template, primers, and PCR premix (iNtRON, cat. no. 25185). PCR was performed using an Eppendorf Mastercycler Nexus GX2 under the following reaction conditions:

[0270] Initial denaturation - 94℃ 2 min Denaturation - 94℃ 20sec Annealing - 55℃, 10 seconds Extension - 72℃ 10 min (30 cycles from denaturation to extension) Final Extension - 72℃, 5min

[0271] The seven mutants were ligated using the In-Fusion HD cloning kit (Takara, Cat. No. 639650), transformed into E. coli DH5α, and sequenced to confirm sequence mutations.

[0272] Example 2: Selection of mutants with improved heat tolerance and confirmation of heat tolerance effect E. coli Dh5α strains transformed with the mutants and Op Xyn gene prepared in Example 1 were inoculated into sterilized LB medium (BD Difco) and cultured at 37°C and 180 rpm for 24 hours. The cells were then harvested by centrifugation. The harvested cells were resuspended in 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole) and sonicated and centrifuged to obtain the coenzyme solution. The coenzyme solution was adsorbed onto Ni-NTA resin (Qiagen, Cat. No. 30230). The enzyme was purified by sequentially passing through a washing buffer (lysis buffer, containing 20 mM imidazole only) and an elution buffer (lysis buffer, containing 250 mM imidazole only). The purified enzyme was then used for titer and heat resistance evaluation.

[0273] The enzyme concentration was 4 μl of diluted enzyme solution + Bradford solution (Quick Start TM After mixing with 196 μl of Bradford 1× Dye Reagent (#5000205), the absorbance at 595 nm was measured.

[0274] The enzyme activity was measured by mixing 96 μl of 1% beachwood xylan (Megazyme, P-XYLNBE-10G) with 4 μl of 1M pH 6.5 phosphate buffer, followed by 100 μl of the diluted enzyme solution, and reacting at 37°C for 15 minutes. The reaction was stopped by adding 300 μl of DNS solution, and the mixture was boiled for 7 minutes to develop color, then cooled in ice water. 500 μl of distilled water was added to this mixture, and the absorbance at 550 nm was measured. The activity was measured using a standard curve prepared with xylose (Sigma-Aldrich, X1500).

[0275] The DNS solution was prepared as follows: 6.3 g of 3,5-dinitrosalicylic acid (Samchun, D1267) was added to a beaker containing 500 ml of distilled water, the temperature was adjusted to 50°C, and 21 g of sodium hydroxide (Daejung, 7571-4400) was added. 300 ml of water and 182 g of potassium sodium tartrate tetrahydrate (Daejung, 6618-4400) were added, followed by 5 g of phenol (Sigma-Aldrich, P1037) and 5 g of sodium sulfite anhydrous (Daejung, 7634-4405). The solution was stirred until dissolved and then cooled. Distilled water was added to the resulting solution to bring the total volume to 1000 ml, filtered, and stored in a brown bottle for at least 7 days before use.

[0276] To evaluate the heat resistance, the purified enzyme was diluted to a concentration of 0.5 mg / ml, incubated in a water bath at 70°C for 10 minutes, and then the residual activity was measured.

[0277] [Table 2]

[0278] As a result, when enzyme activity was measured as a ratio, three mutants (DS1, DS2, and DS5) were selected that had enzyme activity relative to Op Xyn in the range of 50-300% and heat resistance (residual activity) improved by more than five times.

[0279] The changes in the residual titers of the selected mutant strains DS1, DS2, and DS5 depending on the incubation time at 70°C were further investigated and are shown in Figure 1. As a result, it was confirmed that there was almost no change in the residual titers of DS1, DS2, and DS5 even with the incubation time being extended.

[0280] From the above description, those skilled in the art to which the present invention pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A mutant polypeptide having xylanase activity, the variant polypeptide has a sequence identity of 90% or more but less than 100% with SEQ ID NO: 1; The mutant polypeptide has increased thermotolerance and / or thermostability compared to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, and includes one or more modifications selected from the following: Substitution of an amino acid at a position selected from the following i) to vii) with cysteine, disulfide bond formation, and combinations thereof: i) 3 + 36; ii) 5+34; iii) 20+41; iv) 3+36+5+34; v) 3+36+20+41; vi) 5 + 34 + 20 + 41; and vii) 3+36+5+34+20+41; Here, the position numbers are positions corresponding to the positions in the polypeptide of SEQ ID NO:

1.

2. The third amino acid of the mutant polypeptide having xylanase activity before modification is arginine (R); The fifth amino acid is serine (S); The 20th amino acid is phenylalanine (F); Amino acid number 34 is serine (S); Amino acid number 36 is threonine (T); and / or the amino acid at position 41 is alanine (A).

3. The variant polypeptide comprises any one or more modifications selected from the following:

1. A mutant polypeptide according to claim 1: R3C+T36C; S5C+S34C; F20C+A41C; R3C+T36C+S5C+S34C; R3C+T36C+F20C+A41C; S5C+S34C+F20C+A41C; and R3C+T36C+S5C+S34C+F20C+A41C.

4. The mutant polypeptide of claim 1, wherein the mutant polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 3, 5, or 7.

5. The mutant polypeptide may have any one of the following substitutions of the amino acid pair at positions 3 and 36 with cysteine; the amino acid pair at positions 5 and 34 with cysteine; or the amino acid pair at positions 20 and 41 with cysteine. and a modification in which the amino acid pair forms a disulfide bridge.

6. A method for producing xylooligosaccharides or xylose, comprising contacting a xylan-containing substance with a mutant polypeptide according to any one of claims 1 to 5, a host cell expressing the mutant polypeptide, and / or a composition comprising the mutant polypeptide.

7. A method for decomposing a xylan-containing substance, comprising treating the xylan-containing substance with a mutant polypeptide according to any one of claims 1 to 5, a host cell expressing the mutant polypeptide, and / or a composition comprising the mutant polypeptide.

8. A polynucleotide encoding the mutant polypeptide of any one of claims 1 to 5.

9. A host cell comprising a mutant polypeptide according to any one of claims 1 to 5 and / or a polynucleotide according to claim 8.

10. A method for producing a mutant polypeptide having xylanase activity, comprising culturing the host cell of claim 9.

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