WO2023075496A1 - Polypeptide variant possédant une activité xylanasique - Google Patents

Polypeptide variant possédant une activité xylanasique Download PDF

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WO2023075496A1
WO2023075496A1 PCT/KR2022/016685 KR2022016685W WO2023075496A1 WO 2023075496 A1 WO2023075496 A1 WO 2023075496A1 KR 2022016685 W KR2022016685 W KR 2022016685W WO 2023075496 A1 WO2023075496 A1 WO 2023075496A1
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polypeptide
variant polypeptide
amino acid
variant
seq
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PCT/KR2022/016685
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English (en)
Korean (ko)
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양태주
양성재
최은정
심지현
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씨제이제일제당 (주)
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Publication of WO2023075496A1 publication Critical patent/WO2023075496A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present application relates to variant polypeptides having xylanase activity and uses thereof.
  • Xylanase (EC 3.2.1.8) is a hydrolase that randomly degrades the ⁇ -1,4 backbone of xylan, a plant cell wall component. Xylanase is mainly used to break down biomass in fields such as animal feed, bakery, 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.)
  • One object of the present application is to provide a variant polypeptide having xylanase activity.
  • Another object of the present application is to provide a composition comprising the variant polypeptide.
  • Another object of the present application is to provide a use of the variant polypeptide or the composition for reaction with a xylan-containing material.
  • Another object of the present application is a xylan-containing material comprising contacting the variant polypeptide, a host cell expressing the variant polypeptide, and/or a composition comprising the variant polypeptide with the xylan-containing material. disassembly method; And / or to provide a method for producing xylooligosaccharide and / or xylose.
  • Another object of the present application is a polynucleotide encoding the variant polypeptide; a nucleic acid construct comprising the polynucleotide; a vector containing the polynucleotide or nucleic acid construct; and/or a host cell containing the polynucleotide, nucleic acid construct or vector.
  • Another object of the present application is to provide a method for producing the variant polypeptide.
  • the variant polypeptide having xylanase activity of the present application can be usefully used in various industrial fields.
  • One aspect of the present application is a variant polypeptide having xylanase activity.
  • the variant polypeptide is a polypeptide having a sequence identity of 70% or more and less than 100% with SEQ ID NO: 1; and/or
  • the variant polypeptide is a polypeptide encoded by a polynucleotide having 70% or more and less than 100% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1; and/or
  • the variant polypeptide comprises (a) the mature polypeptide coding sequence of SEQ ID NO: 1, (b) a cDNA thereof, or (c) the full-length complement of (a) or (b) above and low stringency a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions, medium stringency conditions, medium high stringency conditions, high stringency conditions, or very high stringency conditions; and/or
  • the variant polypeptide is a functional fragment of i), ii) or iii) polypeptide having xylanase activity;
  • the variant polypeptide comprises a substitution of another amino acid in an amino acid at one or more of positions 46 and 93:
  • the position number is a position corresponding to the position of the polypeptide of SEQ ID NO: 1.
  • amino acid number 46 before the modification is glutamic acid (E); And/or amino acid number 93 may be asparagine (N).
  • the variant polypeptide may include any one or more of the following modifications:
  • the variant polypeptide may include the following substitutions:
  • the position number is a position corresponding to the position of the polypeptide of SEQ ID NO: 1.
  • any one of the above embodiments it may include substitution of any one or more of amino acids corresponding to positions 28 and 52 of SEQ ID NO: 1.
  • amino acid number 28 or amino acid number 52 before the modification may be asparagine (N).
  • the variant polypeptide may include any one or more of the following modifications:
  • the variant polypeptide may include the following substitutions:
  • the position number is a position corresponding to the position of the polypeptide of SEQ ID NO: 1.
  • the variant polypeptide may include an amino acid pair forming a disulfide bridge.
  • the pair of amino acids forming the disulfide bridge may include substitution of amino acids corresponding to positions 3 and 36 of SEQ ID NO: 1 with cysteine.
  • the variant polypeptide may include a modification in which amino acid pairs at positions 3 and 36 form a disulfide bridge.
  • the mutant polypeptide may have increased heat resistance and/or heat stability compared to a polypeptide composed of the amino acid sequence of SEQ ID NO: 1.
  • compositions for reaction with a variant polypeptide of the present application and/or a xylan-containing material comprising the variant polypeptide of the present application.
  • variant polypeptide and/or a composition comprising the variant polypeptide for reaction with a xylan-containing material.
  • xylose and / or xylose comprising contacting a xylan-containing material with the variant polypeptide, a host cell expressing the variant polypeptide, and / or a composition comprising the variant polypeptide. or a method for producing xylo-oligosaccharide.
  • Another aspect of the present application is a xylan-containing material comprising treating the variant polypeptide, a host cell expressing the variant polypeptide, and/or a composition comprising the variant polypeptide to the xylan-containing substance how to decompose
  • Another aspect of the present application is a polynucleotide encoding the variant polypeptide.
  • nucleic acid construct comprising the polynucleotide.
  • Another aspect of the present application is a vector comprising the polynucleotide or the nucleic acid construct.
  • Another aspect of the present application is a host cell comprising the variant polypeptide, the polynucleotide, the nucleic acid construct, and/or the vector.
  • Another aspect of the present application is a method for producing a variant polypeptide comprising culturing the host cell.
  • the term “about” may be presented before a specific numeric value.
  • the term “about” includes not only the exact number that follows the term, but also a range that is or is close to that number. It can be determined whether the number is close to or nearly the specific number mentioned, given the context in which it is presented.
  • the term “about” can refer to a range of -10% to +10% of a numerical value.
  • the term “about” can refer to a range of -5% to +5% of a given numerical value. However, it is not limited thereto.
  • the term "consisting essentially of” means that a non-specified component may be present if the characteristics of the subject matter claimed in this application are not substantially affected by the presence of the non-specified component.
  • the term “consisting of” means that the proportions of the specified component(s) total 100%. Ingredients or features that come after the term “consisting of” may be essential or mandatory. In some embodiments, in addition to the ingredients or features that come under “consisting of”, any other ingredients or non-essential ingredients may be excluded.
  • the amino acid sequence has an addition of a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation thereof, or a conservative substitution to the N-terminus and/or C-terminus of the amino acid sequence. It may, but is not limited thereto.
  • protein or “polypeptide” refers to a polymer or oligomer of contiguous amino acid residues.
  • polypeptide polypeptide
  • protein and “peptide” may be used interchangeably with “amino acid sequence”.
  • amino acid sequence that exhibits an activity may be referred to as an "enzyme.”
  • amino acid sequences are written in N-terminal to C-terminal orientation unless otherwise indicated.
  • a recombinant cell in this application means that a cell, nucleic acid, polypeptide or vector has been modified by introduction of a heterologous nucleic acid or polypeptide or alteration of a natural nucleic acid or polypeptide; or that the cell is derived from a cell so modified.
  • a recombinant cell may express a gene that is not found in the native (non-recombinant) form of the cell, or may express a native gene that is expressed or not expressed at all, or otherwise abnormally expressed.
  • isolated refers to a substance in a form that does not exist or exists in a non-naturally occurring environment. This includes at least substantially the separation of a substance (sequence, enzyme or nucleic acid) from at least one other component with which it is naturally associated in nature and which has the same substance, eg, a sequence, enzyme or nucleic acid, as found in nature. .
  • an isolated sequence, enzyme or nucleic acid provided herein may be provided in a form that is substantially free of one or more contaminants.
  • an isolated material examples include i) any material that is not naturally occurring, ii) any material from which one, more, or all naturally occurring constituents associated with it in nature have been removed. (e.g., enzymes, variants, nucleic acids, proteins, peptides or cofactors), iii) any substance that has been artificially modified from a substance found in nature, or iv) other constituents with which it is naturally associated. (e.g., increased copy number of a gene encoding a specific substance; modification of a promoter naturally linked to a gene encoding a specific substance into a highly active promoter, etc.) It may, but is not limited thereto.
  • wild type means a naturally-occurring one without artificial modification.
  • wild type when used in reference to a polypeptide, it is meant to be a naturally occurring polypeptide, which does not have artificial variance (substitutions, insertions, deletions, etc.) at one or more amino acid positions.
  • wild type when used in reference to a polynucleotide, it means not having artificial modifications (substitutions, insertions, deletions) of one or more nucleotides.
  • polynucleotides encoding wild-type polypeptides are not limited to wild-type polynucleotides, and include sequences encoding any wild-type polypeptides.
  • a parent sequence or backbone refers to a reference sequence that becomes a mutant polypeptide by introducing modification. That is, the parental sequence may be a target for introducing mutations such as substitution, insertion, and/or deletion as a starting sequence.
  • the parental sequence may be a naturally occurring or wild type, or a variant in which one or more substitutions, insertions or deletions have occurred in the natural or wild type, or may be an artificially synthesized sequence. there is.
  • the parent sequence is an amino acid sequence showing activity, that is, an amino acid sequence of an enzyme, it may be referred to as a parent enzyme.
  • reference sequence refers to a sequence used to determine the position of amino acids within any amino acid sequence. Any amino acid sequence can be aligned with a reference sequence to determine the position of an amino acid within any amino acid sequence that corresponds to a particular position in the reference sequence.
  • fragment in relation to an amino acid or nucleic acid sequence in this application means a portion of a parent sequence.
  • it may be a polypeptide in which one or more amino acids in the parental sequence are removed from the C or N terminus.
  • a "fragment” of an enzyme may refer to a "functional fragment”.
  • “Functional fragment” may also be referred to as an active fragment, and refers to a polypeptide that is part of a parent enzyme and has the enzymatic activity of the parent enzyme.
  • a functional fragment of an enzyme may include a catalytic site of an enzyme.
  • a fragment of an enzyme may include a portion of the full length of the parent enzyme. For example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more, but less than 100% amino acids of the full length of the parent enzyme. It may include, but is not limited thereto.
  • modifying means changing or altering. This may be a change from naturally occurring.
  • an enzyme can be altered in such a way that the enzyme is altered from a parental or reference sequence.
  • a modified enzyme may be an enzyme that does not itself exist in nature, i.e., is non-naturally occurring.
  • modified means altered from, for example, its naturally occurring form.
  • Modified enzymes of this application include non-naturally occurring enzymes or naturally occurring variants.
  • the modified enzyme of the present application is a modified enzyme not found in nature.
  • the modified enzyme of the present application may not occur spontaneously (spontaneously), but is not limited thereto.
  • modification when used in reference to an amino acid/nucleic acid sequence, it is a substitution of an amino acid/nucleic acid residue of a parent sequence for a different amino acid/nucleic acid residue at one or more sites in an amino acid sequence, one Deletion of a parental amino acid/nucleic acid residue (or set of amino acid/nucleic acid residues) at an abnormal site, insertion of a parental amino acid/nucleic acid residue (or set of amino acid/nucleic acid residues) at one or more sites , truncation of N-terminal and/or C-terminal amino acid sequences or 5' and/or 3' nucleic acid sequences, and any combination thereof.
  • a “variant” or “modified polypeptide” of an enzyme refers to a protein that differs from the parent enzyme in one or more amino acids by conservative substitution and/or modification. do. “Variant” or “variant polypeptide” may be used interchangeably. The variant or variant polypeptide may be non-naturally occurring, but is not limited thereto.
  • Such variants differ from the sequence of the parent enzyme by one or more modifications, eg, amino acid substitutions, deletions and/or insertions.
  • Such variants can generally be identified by modifying one or more amino acids in the parent enzyme and evaluating the properties of the modified protein. That is, the ability of the variant may be increased, unchanged, or reduced compared to the parent enzyme.
  • variants may include variant polypeptides in which one or more portions such as an N-terminal leader sequence or a transmembrane domain are removed.
  • variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein.
  • mutant or “mutant polypeptide” may be used interchangeably with terms such as variant, modification, mutated protein, mutation (in English, modification, modified protein, mutant, mutein, divergent, variant, etc.), If the term is used in a modified sense, it is not limited thereto.
  • Variants may include deletions or additions of amino acids that have minimal effect on the secondary structure and properties of the polypeptide.
  • a polypeptide may be conjugated with a signal (or leader) sequence at the N-terminus of a protein that is involved in protein transfer either co-translationally or post-translationally.
  • the polypeptide may also be conjugated with other sequences or linkers to allow identification, purification, or synthesis of the polypeptide.
  • conservative substitution means the substitution of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions can generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
  • any amino acid can be described as Xaa, X.
  • Amino acids can generally be classified based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
  • amino acid substitutions can generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
  • amino acids with electrically charged side chains positively charged (basic) amino acids are arginine, lysine, and histidine, and negatively charged (acidic) amino acids are glutamic acid and aspartic acid.
  • Nonpolar amino acids among amino acids with uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and are polar or hydrophilic ( hydrophilic) amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and aromatic amino acids among the non-polar amino acids include phenylalanine, tryptophan, and tyrosine.
  • the term "gene” refers to a polynucleotide that encodes a polypeptide and a polynucleotide that includes regions preceding and following the coding region.
  • a gene may have sequences (introns) inserted between each coding region (exon).
  • homology refers to the degree of relatedness between two given amino acid sequences or nucleotide sequences and can be expressed as a percentage.
  • homology and identity are often used interchangeably.
  • Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, together with default gap penalties established by the program used. Substantially homologous or identical sequences are generally under moderate or high stringency conditions along at least about 50%, 60%, 70%, 80% or 90% of the entire or full-length sequence. It can hybridize under stringent conditions. It is obvious that hybridization also includes polynucleotides containing common codons or codons considering codon degeneracy in polynucleotides.
  • GAP program can define the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids).
  • the default parameters for the GAP program are (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or 10 gap opening penalty, 0.5 gap extension penalty); and (3) no penalty for end gaps.
  • mature polypeptide refers to a polypeptide in its form without a signal sequence or propeptide sequence.
  • a mature protein/polypeptide/peptide may be a functional form of the protein/polypeptide/peptide.
  • a mature polypeptide may be in its final form post-translational or subjected to post-translational modifications. Examples of post-translational modifications include, but are not limited to, N- or C-terminal modifications, glycosylation, phosphorylation, leader sequence removal, and the like.
  • nucleic acid construct includes one or more regulatory sequences, is artificially synthesized, engineered to include a specific sequence by a method that does not exist in nature, or derived from nature. Refers to an isolated single or double-stranded nucleic acid molecule.
  • expression includes, but is not limited to, any step involved in the production of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, secretion, and the like.
  • expression vector refers to a linear or circular nucleic acid molecule comprising a coding sequence and operably linked regulatory sequences for its expression.
  • operably linked refers to a configuration in which regulatory sequences are positioned in appropriate positions such that the regulatory sequences direct expression of the coding sequence.
  • operably linked means that a regulatory region of a functional domain having a known or desired activity, such as a promoter, terminator, signal sequence, or enhancer region, functions to express, secrete, or function a target (gene or polypeptide). It includes those attached to or linked to their targets so that they can be modulated according to the desired activity.
  • cDNA refers to a DNA sequence that can be prepared by reverse transcription from mature, spliced mRNA molecules obtainable from eukaryotic or prokaryotic cells.
  • a cDNA sequence does not include intronic sequences that may be present in the corresponding genomic DNA.
  • Early primary RNA transcripts are precursors of mRNA before they are processed through a series of steps including splicing to appear as mature spliced mRNA.
  • regulatory sequence refers to a polynucleotide sequence necessary for the expression of a coding sequence.
  • Each regulatory sequence may be native (of the same origin) or foreign (from another gene) to the coding sequence.
  • 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 that regulates transcription and translation termination. contains sequence.
  • the minimum unit of the regulatory sequence may include a promoter, transcriptional and translational termination sequences.
  • reference to a specific position in an amino acid sequence may include reference to an amino acid present or substituted at that position.
  • Referencing an amino acid at a specific position can be described in various ways. For example, “position 003” can be written as “position 3", “amino acid 3", “3 amino acid”. Also, for example, when the amino acid at position 3 is arginine (R), it may be described as “R3” or "Arg3".
  • Amino acid substitution can be expressed by describing the amino acid before substitution, the position, and the amino acid to be substituted in the order.
  • the amino acids can be expressed using conventional one-letter and three-letter codes. For example, when serine, an amino acid at position 5 of a specific sequence, is substituted with cysteine, it may be described as "S5C” or "Ser5Cys".
  • Any amino acid at a particular position may be referred to as "X”.
  • X6 refers to any amino acid at position 6.
  • the amino acid to be substituted when expressed as X, it means that it is substituted with an amino acid different from the amino acid present before substitution.
  • V6X indicates that V at position 6 is substituted with any amino acid other than V.
  • 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. Identification of the amino acid at the corresponding position may be determining the specific amino acid in the sequence that references the specific sequence.
  • corresponding region generally refers to a similar or corresponding position in a related or reference protein.
  • SEQ ID NO: 1 may be used as a reference sequence to determine the position of amino acids in any amino acid sequence in this application.
  • SEQ ID NO: 1 disclosed in this application can be used to determine the corresponding amino acid residue in any polypeptide having xylanase activity, and unless otherwise indicated in this application, the residue of a specific amino acid sequence is SEQ ID NO: They are numbered based on 1.
  • any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 1.
  • sequence alignment algorithms such as those described herein, can identify the location of amino acids, or locations where modifications such as substitutions, insertions, or deletions occur, compared to a query sequence (also referred to as a “reference sequence”).
  • proteins of known structure For proteins of known structure, several tools and resources are available to search for and create structural alignments.
  • the SCOP superfamily of proteins has been structurally aligned, and these alignments are accessible and downloadable.
  • the structure of two or more proteins can be structured in a variety of ways, such as a distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or a combinational extension (CE) Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747). It can be sorted using an algorithm. Implementations of these algorithms can additionally be used to query structural databases with the structure of interest, to discover possible structural homologs (Holm and Park, 2000, Bioinformatics 16: 566-567).
  • xylanase refers to an enzyme that catalyzes the endohydrolysis of 1,4-beta-D-xylosidic bonds in xylan.
  • it may be an enzyme having an EC number of 3.2.1.8, but is not limited thereto.
  • Xylanase activity in this application can be measured and evaluated using methods known in the art, including the embodiments described in this application.
  • Parent xylanase refers to a xylanase to which a modification is applied to produce a variant or variant polypeptide of the present application.
  • the parent xylanase, parent enzyme, or parent sequence may be a naturally occurring polypeptide or wild-type polypeptide, may be a mature polypeptide thereof, may include a variant or functional fragment thereof, but may not inhibit xylanase activity. It is not limited thereto as long as it has a polypeptide that can be the parent of a variant.
  • the moxylanase provided in the present application may be the polypeptide of SEQ ID NO: 1, but is not limited thereto. In addition, as long as it has xylanase activity, about 60%, 70%, 75% with the polypeptide of SEQ ID NO: 1. 80%. It may be a polypeptide having a sequence identity of 85%, 90%, 95%, 96%, 97%, 98% or 99% or more, and if it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, all It may be included without limitation in the scope of xylanase.
  • the parent xylanases of the variants provided in this application are Orpinomyces sp., Neocallimastix sp., Piromyces sp., Ruminococcus sp. may be of origin. Specifically Orpinomyces sp. may be of origin.
  • microorganism is an example of a microorganism from which the xylanase provided in the present application can be derived, and includes those derived from microorganisms taxonomically homologous to the microorganism regardless of the name of the microorganism.
  • microorganisms may be distributed from known microorganism depository institutions such as ATCC, DSMZ, CBS, NRRL, KCTC, and KCCM.
  • a sequence "derived from” a specific microorganism is not limited to those naturally produced or producible in that microorganism, but also includes sequences encoded by genes produced and isolated from the microorganism containing the gene. do.
  • Orpinomyces sp. Derived xylanase is an enzyme having xylanase activity naturally produced in microorganisms of the genus Orpinomyces as well as those produced in microorganisms of the genus Orpinomyces, and genetic modifications known in the art (eg, sequences encoding the enzymes). It also includes those produced in other host cells through transformation into).
  • mutant polypeptide having xylanase activity may be a variant of a parent xylanase.
  • parent xylanase variant or “xylanase variant” refers to a protein having at least one amino acid different from the amino acid sequence of parent xylanase and having xylanase activity.
  • mutant polypeptide having xylanase activity can be used interchangeably.
  • the variant provided in the present application may have xylanase activity and include one or more amino acid modifications in the parent xylanase sequence.
  • the modification may be amino acid substitution and/or disulfide bond formation.
  • the variant is a polypeptide having a sequence identity of 70% or more and less than 100% with SEQ ID NO: 1; and/or ii) the variant is a polypeptide encoded by a polynucleotide having at least 70% and less than 100% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1; and/or iii) the variant is low in sequence with (a) the mature polypeptide coding sequence of SEQ ID NO: 1, (b) the cDNA thereof, or (c) the full-length complement of (a) or (b) above.
  • the variant provided in the present application may have xylanase activity and at least one modified function or characteristic compared to parent xylanase, including modification of one or more amino acids in the parent xylanase sequence. there is.
  • the variant provided in the present application has xylanase activity, and has one or more altered functions or properties compared to the parent xylanase, including modification of one or more amino acids in the parent xylanase sequence. , can have one or more conservative substitutions.
  • the mutant provided in the present application is a mutant of a parent xylanase, and may be a polypeptide having xylanase activity.
  • the variant provided in the present application may include modifications at one or more positions corresponding to positions 46 and 93 of SEQ ID NO: 1.
  • the position number is a position corresponding to the position of the polypeptide of SEQ ID NO: 1, and "corresponding" is as described above.
  • the variant provided in the present application may include modifications of amino acids corresponding to one or more of E46 and N93 of SEQ ID NO: 1.
  • amino acid 46 before modification of the variant provided in the present application is glutamic acid (E); and/or amino acid number 93 may be asparagine (N).
  • the variant provided in this application is G, A, V, L, I, M, F, W, P, S, T, C, Y of the amino acid corresponding to position 46 of SEQ ID NO: 1 , may include substitution with N, Q, D, K, R or H, specifically may include substitution with S, T, C, Y, N or Q, more specifically with Q or C can include
  • the variant provided in this application is G, A, V, L, I, M, F, W, P, S, T, C, Y of the amino acid corresponding to position 93 of SEQ ID NO: 1 , Q, D, E, K, R or H, specifically may include substitution with G, A, V, L, I, M, F, W, Y or H, More specifically, it may include substitution with W.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 46 of SEQ ID NO: 1 with a polar amino acid.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 93 of SEQ ID NO: 1 with a nonpolar or aromatic amino acid.
  • the variant provided in the present application may include substitution of one or more of E46C and N93W of SEQ ID NO: 1.
  • the variants provided herein include all possible combinations of the foregoing variants.
  • the variant may include a modification of an amino acid at a position selected from the following in a combination of the above modifications:
  • the position number is a position corresponding to the position of the polypeptide of SEQ ID NO: 1, and "corresponding" is as described above.
  • the variant provided in this application may further include modifications at one or more positions corresponding to positions 28 and 52 of SEQ ID NO: 1.
  • the variant provided in the present application is G, A, V, L, I, M, F, W, P, S, T, C, Y of the amino acid corresponding to position 28 of SEQ ID NO: 1 , Q, D, E, K, R or H, specifically G, A, V, L, I, M, F, W, P, S, T, C, Y or Q It may include substitution with, and more specifically, it may include substitution with A, G or S.
  • the variant provided in the present application is G, A, V, L, I, M, F, W, P, S, T, C, Y of the amino acid corresponding to position 52 of SEQ ID NO: 1 , Q, D, E, K, R or H, specifically G, A, V, L, I, M, F, W, P, D, E, K, R or H It may include substitution with, and more specifically may include substitution with P, A or E.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 28 of SEQ ID NO: 1 with a hydrophobic or polar amino acid.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 52 of SEQ ID NO: 1 with a hydrophobic or acidic amino acid.
  • the variant provided in the present application may include substitution of one or more of N28A/G/S and N52P/A/E of SEQ ID NO: 1.
  • variants provided in this application may include the following substitutions:
  • variants provided in this application may include the following substitutions:
  • the variant provided in the present application may include an amino acid pair that forms a disulfide bridge.
  • the amino acid pair forming the disulfide bridge is located at two or more positions among positions 3, 5, 20, 34, 36, and 41 of SEQ ID NO: 1. substitution of the corresponding amino acid with a cysteine.
  • the variant provided in this application may include substitution of two or more of R3C, S5C, F20C, S34C, T36C and A41C of SEQ ID NO: 1.
  • the variant provided in this application may include any one or more substitutions selected from the following:
  • the variant may include, but is not limited to, substitution of any one or more of the following i) to vi): i) substitution of amino acid 3 with cysteine; ii) substitution of amino acid 5 with a cysteine; iii) substitution of amino acid 20 with a cysteine; iv) substitution of amino acid 34 with a cysteine; v) substitution of amino acid 36 with a cysteine; and vi) substitution of amino acid 41 with a cysteine.
  • the variant provided in the present application is a pair of amino acids 3 and 36; amino acid pairs at positions 5 and 34; and/or amino acid pairs at positions 20 and 41 may be substituted with cysteine to form a disulfide bridge.
  • the variant provided in the present application may include modifications at positions 3 and 36 of SEQ ID NO: 1.
  • the variant provided in the present application may include modifications of amino acids corresponding to R3 and T36 of SEQ ID NO: 1.
  • the amino acid corresponding to position 3 of SEQ ID NO: 1 before modification provided in the present application is arginine (R); and/or the amino acid corresponding to position 36 may be threonine (T).
  • the variant provided in the present application is G, A, V, L, I, M, F, W, P, S of the amino acid corresponding to position 3 of SEQ ID NO: 1 , T, C, Y, N, Q, D, E, K, or H, and may specifically include substitution with S, T, C, Y, N, or Q, and more Specifically, it may include substitution with C.
  • the variant provided in the present application is G, A, V, L, I, M, F, W, P, S of the amino acid corresponding to position 36 of SEQ ID NO: 1 , C, Y, N, Q, D, E, K, R, or H, and may specifically include substitution with S, C, Y, N, or Q, more specifically may include substitution with C.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 3 of SEQ ID NO: 1 with a polar amino acid.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 36 of SEQ ID NO: 1 with a polar amino acid.
  • the variant provided in this application may include R3C and T36C substitutions of SEQ ID NO: 1.
  • the variant provided in the present application comprises substitution of amino acids corresponding to positions 3 and 36 of SEQ ID NO: 1 with cysteine, and between the two amino acids substituted It may be to form a disulfide bridge.
  • the variant provided in the present application may include modifications at positions 5 and 34 of SEQ ID NO: 1.
  • the variant provided in the present application may include modifications of amino acids corresponding to S5 and S34 of SEQ ID NO: 1.
  • the amino acid corresponding to position 5 of SEQ ID NO: 1 before modification provided in the present application is serine (S); and/or the amino acid corresponding to position 34 may be serine (S).
  • the variant provided in the present application is G, A, V, L, I, M, F, W, P, T of the amino acid corresponding to position 5 of SEQ ID NO: 1 , C, Y, N, Q, D, E, K, R, or H, specifically may include substitution with T, C, Y, N, or Q, more specifically may include substitution with C.
  • the variant provided in the present application is G, A, V, L, I, M, F, W, P, T of the amino acid corresponding to position 34 of SEQ ID NO: 1 , C, Y, N, Q, D, E, K, R, or H, specifically may include substitution with T, C, Y, N, or Q, more specifically may include substitution with C.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 5 of SEQ ID NO: 1 with a polar amino acid.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 34 of SEQ ID NO: 1 with a polar amino acid.
  • the variant provided in the present application may include S5C and S34C substitutions of SEQ ID NO: 1.
  • the variant provided in the present application comprises substitution of amino acids corresponding to positions 5 and 34 of SEQ ID NO: 1 with cysteine, and between the two amino acids substituted It may be to form a disulfide bridge.
  • the variant provided in the present application may include modifications at positions 20 and 41 of SEQ ID NO: 1.
  • the variant provided in the present application may include amino acid modifications corresponding to F20 and A41 of SEQ ID NO: 1.
  • the amino acid corresponding to position 20 of SEQ ID NO: 1 before modification provided in the present application is phenylalanine (F); and/or the amino acid corresponding to position 41 may be alanine (A).
  • the variant provided in this application is G, A, V, L, I, M, W, P, S, T of the amino acid corresponding to position 20 of SEQ ID NO: 1 , C, Y, N, Q, D, E, K, R, or H, and may specifically include substitution with S, T, C, Y, N, or Q, and more Specifically, it may include substitution with C.
  • the variant provided in this application is G, V, L, I, M, F, W, P, S, T of the amino acid corresponding to position 41 of SEQ ID NO: 1 , C, Y, N, Q, D, E, K, R, or H, specifically with S, T, C, Y, N, or Q, and more specifically with C can do.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 20 of SEQ ID NO: 1 with a polar amino acid.
  • the variant provided in the present application may include substitution of an amino acid corresponding to position 41 of SEQ ID NO: 1 with a polar amino acid.
  • the variant provided in this application may include F20C and A41C substitutions of SEQ ID NO: 1.
  • the variant provided in the present application comprises substitution of amino acids corresponding to positions 20 and 41 of SEQ ID NO: 1 with cysteine, and between the two amino acids substituted It may be to form a disulfide bridge.
  • the variants provided herein include all possible combinations of the foregoing variants.
  • the variant may include a modification of an amino acid at a position selected from the following in a combination of the above modifications:
  • the variant may include one or more modifications selected from the following, but is not limited thereto:
  • the variant comprising the E46C substitution of SEQ ID NO: 1 is SEQ ID NO: 3
  • the variant comprising the N93W substitution of SEQ ID NO: 1 is SEQ ID NO: 5
  • the variant comprising the N93W+R3C+T36C substitution of SEQ ID NO: 1 It can be represented by SEQ ID NO: 7.
  • the variant provided in the present application is a mo xylanase; At least about 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, 93 with a mature polypeptide or functional fragment thereof. % or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater and less than 100% sequence identity.
  • the variant provided in the present application is about 60% or more, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of SEQ ID NO: 1 , at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100% sequence identity. .
  • the variant provided in the present application is about 60% or more, for example, 65% or more, 70% or more, 75% or more, 80% or more, At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100% sequence It may be a polypeptide encoded by a polynucleotide having identity.
  • the variant provided in the present application is (a) the mature polypeptide coding sequence of SEQ ID NO: 1, (b) its cDNA, or (c) the full-length complementary sequence of (a) or (b) (full -length complement) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions.
  • the variant provided in the present application is a functional fragment of SEQ ID NO: 1 and about 60% or more, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, have a sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100% can
  • the variant provided in the present application is about 60% or more, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more of the nucleotide sequence represented by SEQ ID NO: 2 % 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% Nucleobases It may be a polypeptide encoded by a polynucleotide having sequence identity.
  • the variant provided in the present application may be a polypeptide of any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.
  • the variant provided in this application is SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 in any one of the amino acid sequences, 46, 93, At least one amino acid at position 28, 52, 3 and 36 is fixed, and about 60% or more, for example, 65% or more, with the amino acid sequence of the above sequence number, its mature polypeptide or its functional fragment, 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% It may be a polypeptide having a sequence identity of at least 99%, or at least 100%.
  • Variants provided in this application have one or more selected or detectable polypeptides of any characteristic or attribute compared to other xylanases, e.g., wild-type xylanase, parent xylanase, other xylanase variants, and the like. may have been changed.
  • the above properties or attributes include oxidative stability, substrate specificity, catalytic activity, thermal stability, alkali stability, pH activity profile, resistance to proteolysis, Km, k cat , k cat /Km ratio, protein folding, induction of an immune response, Ability to bind to ligands, to bind to receptors, to be secreted, to be displayed on the surface of cells, to form oligomers, to signal, to promote cell proliferation, to inhibit cell proliferation, to inhibit cell proliferation ability to induce apoptosis, ability to be modified by phosphorylation or glycosylation, and/or ability to treat disease.
  • the variant provided in the present application may have increased heat resistance and/or thermal stability compared to the parental sequence.
  • enzyme activity refers to at least one catalytic activity. Specifically, it may be the conversion efficiency of an enzyme mainly expressed as k cat /Km, but is not limited thereto.
  • k cat means a rate constant (catalytic constant) of conversion to a product in unit time by one enzyme when the enzyme is completely saturated with a substrate, and is also called a turnover number.
  • Km is the substrate concentration when the reaction rate is half of the maximum value (Vmax).
  • Examples of methods of expressing enzyme activity include specific activity (umol of converted substrate x mg -1 x min -1 ) or volumetric activity (umol of converted substrate x mL -1 x min -1 ). .
  • enzyme activity is not limited to the above, and 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.
  • the variant provided in the present application is about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180% compared to the parent enzyme %, about 190%, or about 200% or more increased enzymatic activity.
  • the variant provided in the present application is about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, or It may have a reduced enzymatic activity of about 20% or less.
  • specific activity is the activity of an enzyme per unit weight of protein, and can be expressed as unit/mg. Quantification of the protein can be performed using, for example, SDS-PAGE or Bradford assay.
  • Enzyme stability means that enzyme activity is preserved during storage or reaction time. In order to measure this change in stability, the initial enzyme activity was measured and compared at time zero (100%) and after a certain amount of time (x%) under predetermined conditions to determine the level at which enzyme activity is lost or enzyme stability.
  • Factors that affect enzyme activity include, for example, pH, heat, the presence of other substances (eg, oxidizing agents, chelating agents), and the like.
  • pH stability means the ability of a protein to function in a specific pH range.
  • the variant provided in the present application may have activity at about pH 4.0 to about pH 12.0, but is not limited thereto.
  • a protein When a protein maintains its function in a specific pH range, it can be defined as having “pH stability”, and according to the pH range, it can be defined as having "acid resistance”, “alkali resistance”, and the like.
  • thermal stability means the ability of a protein to function in a specific temperature range.
  • the variant provided in the present application may have activity in the range of about 20 ° C to about 120 ° C, and specifically may have activity in the range of about 60 ° C to about 100 ° C, but is not limited thereto. .
  • thermo tolerance refers to the ability of a protein to function after exposure to a specific temperature, eg, high heat or cryogenic temperature.
  • proteins that are thermotolerant may not function at the temperature they are exposed to, but may become functional again when returned to an optimal temperature environment.
  • the increase in stability may be compared to other enzymes, eg, wild-type enzyme, parent enzyme and/or other variants, maintaining high enzyme activity; This includes increasing the range of pH, temperature and/or time, etc., over which the protein remains functional.
  • other enzymes eg, wild-type enzyme, parent enzyme and/or other variants, maintaining high enzyme activity; This includes increasing the range of pH, temperature and/or time, etc., over which the protein remains functional.
  • Reduction in stability is associated with maintenance of lower enzyme activity compared to other enzymes, e.g., wild-type enzyme, parent enzyme, and/or other variants; This includes reducing the range of pH, temperature and/or time, etc., over which the protein remains functional.
  • the term “substrate specificity” refers to the ability of an enzyme to discriminate between a substrate and molecules that compete with the substrate. Substrate specificity can be determined by measuring the activity of an enzyme on different substrates.
  • the change in substrate specificity may be a change in the direction of increasing specificity for a substrate capable of producing a desired product. In another embodiment, the change in substrate specificity may be a change in a direction in which specificity for a substrate capable of producing a desired product is reduced.
  • modified properties of the variants provided in this application may be suitable for application in various industrial fields, including feed, bakery, pulp bleaching, and the like, and improved activity.
  • a polynucleotide encoding a variant of the present application may include the coding sequence of the above-described variant.
  • various modifications may be made to the coding region within a range that does not change the amino acid sequence of the polypeptide due to codon degeneracy or in consideration of codons preferred in organisms in which the polypeptide is to be expressed. .
  • polynucleotide of the present application is a probe that can be prepared from a known gene sequence, for example, a sequence that hybridizes with a complementary sequence to all or part of the nucleotide sequence under stringent conditions to encode the variant of the present application. can be included without limitation.
  • stringent condition means a condition that allows specific hybridization between polynucleotides. These conditions are 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).
  • polynucleotides with high homology or identity 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, more specifically 99% or more 60°C, 1 ⁇ SSC, 0.1% SDS, which is a condition in which polynucleotides of the same identity or identity do not hybridize and polynucleotides having less homology or identity do not hybridize, or washing conditions of conventional southern hybridization.
  • 60° C. 0.1 ⁇ SSC, 0.1% SDS, more specifically at a salt concentration and temperature corresponding to 68 ° C., 0.1 ⁇ SSC, 0.1% SDS, conditions for washing once, specifically 2 to 3 times are listed.
  • Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization.
  • complementary is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.
  • the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the entire sequence.
  • polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions.
  • the Tm value may be 60 °C, 63 °C or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
  • Appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotide and the degree of complementarity, parameters well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
  • “high stringency” occurs about 5 to 10° C. below the Tm of the probe; “Medium stringency” occurs between about 10 and 20° C. below the Tm of the probe; “Low stringency” may occur at, but is not limited to, about 20 to 25° C. below the Tm.
  • low stringency condition is 5X SSPE, 0.3% SDS, sheared and denatured salmon, for probes of at least 100 nucleotides in length, for 12-24 hours according to Southern blotting standard procedures. Prehybridization and hybridization at 42° C. at 200 micrograms/ml of sperm DNA and 25% formamide. The carrier material can be finally washed 2 to 3 times each for 15 minutes using 2 X SSC, 0.1 to 0.2% SDS at 50 °C.
  • “medium stringency condition” is 5X SSPE, 0.3% SDS, sheared and denatured salmon, for probes of at least 100 nucleotides in length, for 12-24 hours according to standard Southern blotting procedures. Prehybridization and hybridization at 42°C at 200 micrograms/ml of sperm DNA and 35% formamide. The carrier material can be finally washed 2-3 times each for 15 minutes using 2 X SSC, 0.1-0.2% SDS at 55 °C.
  • “medium-high stringency condition” is 5X SSPE, 0.3% SDS, shear and denaturation, for probes of at least 100 nucleotides in length, for 12-24 hours according to standard Southern blotting procedures.
  • prehybridization and hybridization at 42° C. in 200 micrograms/ml of prepared salmon sperm DNA and 35% formamide.
  • the carrier material can be finally washed 2 to 3 times at 60° C. with 1 to 2 X SSC, 0.1 to 0.2% SDS for 15 minutes each.
  • high stringency conditions are 5 X SSPE, 0.3% SDS, sheared and denatured, for probes of at least 100 nucleotides in length, for 12-24 hours according to Southern blotting standard procedures. Prehybridization and hybridization at 42° C. in 200 micrograms/ml of salmon sperm DNA and 35% formamide. The carrier material can be finally washed 2 to 3 times each for 15 minutes using 2 X SSC, 0.1 to 0.2% SDS at 65 °C.
  • a nucleic acid construct provided herein comprises a polynucleotide encoding a variant provided herein, operably linked to one or more regulatory sequences that direct expression of the coding sequence in an appropriate host cell under conditions suitable for the regulatory sequence. do.
  • Polynucleotides can be engineered in a variety of ways to allow expression of variants. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to insertion into the vector. Such manipulations may use methods known in the art.
  • the "vector" provided in the present application contains a nucleotide sequence of a polynucleotide encoding the variant operably linked to a suitable expression control region (or expression control sequence) so as to express the variant of the present application in a suitable host.
  • a suitable expression control region or expression control sequence
  • the expression control region may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation.
  • the vector After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can integrate into the genome itself.
  • Vectors that can be used in the present application are not particularly limited, and any vectors known in the art may be used.
  • Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
  • pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors
  • pBR-based, pUC-based, and pBluescriptII-based plasmid vectors pGEM-based, pTZ-based, pCL-based, pET-based, etc.
  • pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.
  • a polynucleotide encoding a variant provided in the present application may be inserted into a chromosome through a vector for chromosomal insertion into a cell. Insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.
  • a selection marker for determining whether the chromosome is inserted may be further included. Selectable markers are used to select cells transformed with a vector, that is, to determine whether a target nucleic acid molecule has been inserted, and to give selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. markers may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive or exhibit other expression traits, so transformed cells can be selected.
  • Host cells of the present application may be included without limitation as long as they can express the variants of the present application.
  • the host cell of the present application may include the above-described variant, a polynucleotide encoding the variant, a nucleic acid construct containing the same, and/or a vector.
  • the nucleic acid construct or vector may be maintained as a chromosomally integrated or self-replicating extrachromosomal vector as described above.
  • a host cell of the present application includes any progeny of the parent cell that are not identical to the parent cell due to mutations occurring during replication.
  • a host cell may be any cell useful for recombinant production of variants, such as a prokaryotic or eukaryotic cell.
  • a prokaryotic host cell can be any gram-positive or gram-negative bacterium.
  • Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.
  • Gram-negative bacteria include Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Iliobacter, Neisseria, Pseudomonas, Salmonella, Vibrio (e.g. Vibrio natriegens) ) and Ureaplasma.
  • the bacterial host cell may be a host cell of the genus Bacillus, specifically, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumillus, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis cells. don't
  • the bacterial host cell may be a host cell of the genus Streptococcus, specifically Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis and Streptococcus equi subspecies Zooepidemicus cells.
  • the bacterial host cell may be a host cell of the genus Streptomyces, specifically Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces Coelicola, Streptomyces griseus and Streptomyces lividans cells.
  • the bacterial host cell may be a host cell of the genus Corynebacterium, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare ( Corynebacterium singulare), Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli , Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, but is not limited thereto.
  • the host cell may be a microorganism of the genus Escherichia, Escherichia coli, (Escherichia coli), Escherichia albertii, Escherichia fergusonii ), Escherichia hermannii, Escherichia vulneris, or Escherichia blattae, but is not limited thereto.
  • the host cell may be a eukaryote such as a mammalian, insect, plant or fungal cell.
  • the host cell may be a fungal cell.
  • fungi includes ascomycetes, basidiomycota, phylum phylum and zygomycota, as well as phylum oomycetes and all imperfect fungi.
  • Fungal host cells may be yeast cells.
  • yeast in this application refers to ascosporogenous yeast (Endomycetales), basidiosporogenous yeast and yeast belonging to Fungi imperfecti (Blastomycetes). includes However, these classifications are subject to change, and classifications may be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980). .
  • Yeast host cells are Candida, Hansenula, Kluyveromyces, Pichia, Komagataella, Saccharomyces, Schizosaccharomyces ( Schizosaccharomyces or Yarrowia cells, for example Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, saccharomyces Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces It may be Saccharomyces oviformis, Komagataella phaffii or Yarrowia lipolytica cells.
  • Fungal host cells may be filamentous fungal cells.
  • “Filamentous fungi” includes all filamentous forms of the subphylum Mycobacterium and Oomycota (as defined above by Hawksworth et al., 1995). Filamentous fungi are generally characterized by hyphal walls composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is by mycelial elongation, and carbon catabolism is strictly aerobic. In contrast, vegetative growth by yeasts, such as Saccharomyces cerevisiae, is by germination of unicellular thalluses, and carbon catabolism can be fermentative.
  • Filamentous fungal host cells include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus ( Coprinus), Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycellioptora, Neocalimastics (Neocallimastix), Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus ), Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or Trichoderma cells can be
  • filamentous fungal host cells are Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus ), Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis curry Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrupa subrufa), Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense , Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropic
  • the method for preparing a variant of the present application may include culturing a host cell.
  • culture means growing the host cells under appropriately controlled environmental conditions.
  • the culture process of the present application may be performed according to suitable media and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culture may be batch, continuous and fed-batch, but is not limited thereto.
  • the term "medium” refers to a material in which nutrients necessary for culturing the host cells are mixed as main components, and supplies nutrients and growth factors, including water essential for survival and growth.
  • the medium and other culture conditions used for culturing the host cells of the present application can be any medium without particular limitation as long as it is a medium used for culturing conventional host cells. It can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing phosphorus, inorganic compounds, amino acids, and/or vitamins.
  • Examples of the carbon source in the present application include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included.
  • natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sorghum pomace and corn steep liquor can be used, specifically glucose and sterilized pretreated molasses (i.e. converted to reducing sugar).
  • Carbohydrates such as molasses
  • other carbon sources in an appropriate amount may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.
  • nitrogen source examples include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, etc., organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.
  • inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate
  • Amino acids such as glutamic acid, methionine, glutamine, etc.
  • organic nitrogen sources such as peptone, NZ-amine,
  • the number of persons may include monopotassium phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto.
  • the inorganic compound sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and amino acids, vitamins, and/or appropriate precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, it is not limited thereto.
  • 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 of the host cells.
  • an antifoaming agent such as a fatty acid polyglycol ester.
  • oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without gas injection or nitrogen, hydrogen or carbon dioxide gas may be injected to maintain the anaerobic and non-aerobic state. It is not.
  • the temperature of the medium may be 20 °C to 55 °C, specifically 25 °C to 40 °C, but is not limited thereto.
  • the culturing period may be continued until a desired production amount of a useful substance is obtained, specifically, it may be 24 hours to 196 hours, but is not limited thereto.
  • the method for producing a variant polypeptide having xylanase activity of the present application may further include recovering the variant polypeptide having xylanase activity of the present application expressed in the culturing step. there is.
  • the mutant expressed in the culturing step can be recovered using a method known in the art to which the present invention belongs.
  • variants can be recovered from nutrient media by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation or precipitation.
  • the recovery method may be to collect variants using a suitable method known in the art according to the host cell culture method of the present application, for example, batch, continuous, or fed-batch culture.
  • a suitable method known in the art for example, batch, continuous, or fed-batch culture.
  • centrifugation, filtration, treatment with a precipitating agent for crystallized proteins salting out method
  • extraction sonic disruption
  • ultrafiltration dialysis
  • molecular sieve chromatography gel filtration
  • adsorption chromatography ion exchange chromatography
  • affinity chromatography HPLC
  • variants expressed by the host cell in the culture step may not be recovered.
  • the host cell itself expressing the variant may be used as a source of the variant.
  • composition of the present application can be used to degrade xylan-containing materials.
  • compositions of the present application may be used to convert xylan-containing materials to xylose and/or xylo-oligosaccharides.
  • composition of the present application may further include other components in addition to the variants provided in the present application.
  • a person skilled in the art can appropriately select components added to the composition of the present application.
  • composition of the present application may further include any component suitable for conversion of xylan-containing material to xylose and/or xylo-oligosaccharide.
  • composition of the present application may further include optional components suitable for application in various industrial fields such as animal feed, baking, biomass saccharification, and pulp bleaching.
  • materials that may be added are stabilizers, surfactants, builders, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, formulations, lubricants, disintegrants, excipients, solubilizers, suspending agents, colorant, flavoring agent, buffering agent, preservative, soothing agent, solubilizer, tonicity agent, stabilizer, diluent, lubricant, preservative and the like, but are not limited thereto.
  • composition provided in the present application may further include a naturally occurring material or a non-naturally occurring material, in addition to the variant provided in the present application.
  • composition provided in this application may further include additional enzymes commonly used in various industrial fields, including animal feed, baking, biomass saccharification, pulp bleaching, etc., in addition to the variants provided in this application. can
  • the additional enzymes include beta-amylase, cellulases (beta-glucosidase, cellobiohydrolase and endoglucanase), glucoamylase, hemicellulases (endo-xylanase, ⁇ -xylosidase) , ⁇ -L-arabinofuranosidase, ⁇ -D-glucuronidase, feruloyl esterase, coumaroyl esterase, ⁇ -galactosidase, ⁇ -gal lactosidase, ⁇ -mannanase or ⁇ -mannosidase), isoamylase, isomerase, lipase, phytase, protease, pullulanase and/or alpha-amylase that are useful in commercial processes. Any one or more enzymes selected from the group consisting of external enzymes may be further included.
  • xylanase variants of the present application or compositions comprising the xylanase variants of the present application can be used to degrade any xylanase-containing material.
  • a xylan-containing material is any material that can be degraded by xylanase.
  • the xylan-containing material may be hemicellulose.
  • the xylan-containing material may be a material selected from xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan.
  • the xylan-containing material may be xylan, but is not limited thereto.
  • the present application provides a method for degrading (or disintegrating) a xylan-containing material. This may also be referred to as solubilization of xylan and/or solubilization of pentosans.
  • the method relates to degradation (eg, degradation) of a polymer derived from xylan degradation.
  • Decomposition products e.g., glucose
  • biofuels e.g., bioethanol
  • biochemicals e.g., biobased isoprene
  • Xylan can be degraded using the variant of the present application, a host cell expressing the same, and a composition comprising the variant and/or the host cell.
  • cofactors, coenzymes, etc. may be added together in the hydrolysis step of xylan.
  • the step of hydrolyzing the substrate can be performed under optimal pH, temperature conditions, etc., and appropriate conditions can be selected by those skilled in the art.
  • xylanase variants of the present application may be used in any of the following applications:
  • grain-based material eg, which may be whole grains or parts of grains.
  • the xylanase variant of the present application can be used as a feed ingredient.
  • the xylan-containing material may be a feed ingredient or feed ingredient.
  • the feed composition of the present application may mean any natural or artificial diet, one meal, etc., or a component of the one meal for animals to eat, ingest, and digest, or suitable therefor, in various forms known in the art. can be manufactured
  • the xylanase variant of the present application may be used for a food composition or its preparation.
  • the xylan-containing material may be a grain-based material (including whole or partial grain or malted grains, such as malted barley).
  • the xylan-containing material may be grain flour (eg wheat, oat, rye or barley flour).
  • grain flour eg wheat, oat, rye or barley flour.
  • the xylan-containing material may be barley malt or saccharide, or malted barley or a combination thereof.
  • the food composition may be a fermented beverage including beer and wine.
  • the food composition may be bakery products including lobes, rolls, buns, pizza, pretzels, tortillas, cakes, cookies, biscuits, and crackers. However, it is not limited thereto.
  • the xylanase variants of the present application can be used for wheat gluten-starch separation.
  • fractionation of wheat endosperm flour into starch and gluten fractions can be used to obtain high quality A-starch and by-product B-starch and active gluten.
  • the process includes mixing flour (eg wheat flour), water and a xylanase variant.
  • the flour, water and xylanase variant may be mixed simultaneously or sequentially.
  • flour e.g., wheat flour
  • water may be mixed prior to mixing with the xylanase variant.
  • Higher A-starch yields and/or higher quality gluten can be produced by applying the xylanase variants of the present application to wheat gluten-starch separation.
  • the xylanase variants of the present application are used for the degradation of grain-based materials and can be used as part of a biofuel (eg, bioethanol) production process.
  • a biofuel eg, bioethanol
  • the xylanase variants of the present application can improve the production of biofuels (eg, bioethanol) and the use of grain-based materials in the biofuel industry.
  • biofuels eg, bioethanol
  • the biofuel and the xylanase variant may be used including mixing before or during liquefaction, saccharification, fermentation, simultaneous saccharification and fermentation, and after fermentation, or a combination thereof.
  • the xylanase variants of the present application can be used for pulp bleaching.
  • treatment with a xylanase variant can degrade the xylan and release the colored lignin, thereby promoting pulp bleaching.
  • the gene (SEQ ID NO: 2) of the xylanase mutant (hereinafter referred to as Op Xyn, SEQ ID NO: 1) was amplified from the genomic DNA of the PC-2 strain and cloned into the pHCE vector (Takara). It was used as a template.
  • the xylanase variant was prepared through PCR using a template (Template, Example 1-1), primers (Primer, Table 1), and PCR premix (iNtRON, cat no. 25185). PCR was performed using Eppendorf Mastercycler Nexus GX2, and the reaction conditions were as follows.
  • the resulting variants were ligated using an In-Fusion HD cloning kit (Takara, Cat. No. 639650), and then transformed into an E.coli Dh5 ⁇ strain, followed by sequencing to confirm sequence mutation.
  • E.coli DH5 ⁇ strains each transformed with the genes of variants E46C, N93W and Op Xyn prepared in Example 1 were plated on LB plates containing antibiotics (Kanamycin) and then cultured in a 37 ° C incubator for 24 hours. Colonies obtained here were inoculated into a 96-well plate in which 0.4 ml LB medium was dispensed one by one, cultured for 24 hours at 37°C and 750 rpm, and then centrifuged at 4000 rpm for 15 minutes to recover cells (Eppendorf Centrifuge 5810R).
  • the recovered cells were treated with 100 ⁇ l of B-PER Bacterial Protein Extraction reagent (Thermo, #78248), incubated for 10 minutes, mixed with 300 ⁇ l of distilled water, and centrifuged at 4000 rpm for 15 minutes to obtain a crude enzyme solution. After heat-treating the crude enzyme solution in a water bath at 70° C. for 15 minutes, a xylanase titer assay scaled down to 1/5 in a 96-well plate was used.
  • Enzyme titer was measured by mixing 4 ⁇ l of 1M pH 6.5 phosphate buffer with 96 ⁇ l of 1% xylan from beachwood (Megazyme, P-XYLNBE-10G), then mixing 100 ⁇ l of the diluted enzyme solution, and incubating at 37 ° C. The reaction was carried out for 15 minutes and measured. 300 ⁇ l of the DNS solution was mixed with the reaction solution to stop the reaction, and the color was developed by boiling for 7 minutes and then cooled in ice water. The absorbance at 550 nm of the solution mixed with 500 ⁇ l of distilled water was measured, and the titer was measured using a standard curve made of xylose (Sigma-Aldrich, X1500).
  • the method for preparing the DNS solution is as follows. 6.3 g of 3,5-dinitrosalicylic acid (samchus, D1267) was added to a beaker containing 500 ml of distilled water, and 21 g of sodium hydroxide (Daejung, 7571-4400) was added at 50 ° C. .
  • An amino acid pair site for generating a disulfide bond was selected, and a primer for mutating the amino acid sequence to cysteine was designed.
  • disulfide bond variants were produced by PCR under the same conditions as in Example 1-2 using a template (Template, Example 1-1), primers (Table 3), and PCR premix (iNtRON, cat no. 25185) did
  • Variants N28G, N52P, DS1+N28G, DS1+N52P, and DS1+N93W were prepared by introducing a single mutation through PCR under the same conditions as in Example 1-2 to Op Xyn or disulfide-linked variants using the primers shown in Table 4 below. did In addition, additional mutants were prepared through PCR under the same conditions as in Example 1-2 using the primers in Tables 1 and 4 for the mutants thus prepared.
  • Example 3-2 The variant prepared in Example 3-2 was transformed into E.coli DH5 ⁇ strain using the same method as in Example 2, and then purified enzyme was prepared, and specific activity, 70 ° C heat stability, and 80 ° C heat stability etc. was measured.

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Abstract

La présente invention concerne : un polypeptide variant possédant une activité xylanasique ; et son utilisation.
PCT/KR2022/016685 2021-10-29 2022-10-28 Polypeptide variant possédant une activité xylanasique WO2023075496A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110117663A (ko) * 2008-12-23 2011-10-27 대니스코 에이/에스 자일라나제 활성을 갖는 폴리펩타이드
US8927248B2 (en) * 2006-04-12 2015-01-06 National Research Council Canada Modification of xylanases to increase thermophilicity, thermostability and alkalophilicity
CN105219664A (zh) * 2015-09-24 2016-01-06 新疆农业大学 一种重组基因工程菌构建及高活性β-D-1,4-内切木聚糖酶的制备与应用
US10519432B2 (en) * 2016-07-08 2019-12-31 Novozymes A/S Xylanase variants and polynucleotides encoding same

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Publication number Priority date Publication date Assignee Title
US8927248B2 (en) * 2006-04-12 2015-01-06 National Research Council Canada Modification of xylanases to increase thermophilicity, thermostability and alkalophilicity
KR20110117663A (ko) * 2008-12-23 2011-10-27 대니스코 에이/에스 자일라나제 활성을 갖는 폴리펩타이드
CN105219664A (zh) * 2015-09-24 2016-01-06 新疆农业大学 一种重组基因工程菌构建及高活性β-D-1,4-内切木聚糖酶的制备与应用
US10519432B2 (en) * 2016-07-08 2019-12-31 Novozymes A/S Xylanase variants and polynucleotides encoding same

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LARISSA MATTOS TREVIZANO; RAFAELA ZANDONADE VENTORIM; SEBASTIO TAVARES DE REZENDE; FLORIANO PAES SILVA JUNIOR; VALRIA MONTEZE GUIM: "Thermostability improvement ofsp. xylanase by directed evolution", JOURNAL OF MOLECULAR CATALYSIS B : ENZYMATIC, vol. 81, 28 April 2012 (2012-04-28), pages 12 - 18, XP028495603, ISSN: 1381-1177, DOI: 10.1016/j.molcatb.2012.04.021 *

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