Novel polypeptide having don-degrading activity

Abstract

The present application relates to a variant polypeptide having deoxynivalenol (DON) degrading activity, a microorganism comprising the variant polypeptide, a DON degrading composition and a feed additive composition comprising the variant polypeptide or the microorganism, and a DON degrading method using the variant polypeptide or the microorganism. The variant polypeptide has improved DON degrading activity, and thus can be usefully applied to various industrial fields.

Description

Novel polypeptides having DON decomposition activity

[0001] The present application relates to a mutant polypeptide having DON decomposition activity.

[0002]

[0003] Mycotoxin is a general term for fungal metabolites that are toxic to animals. When crops or stored agricultural products are infected or contaminated with mycotoxin-producing fungi, the mycotoxins produced can pose a health risk to animals and humans. Trichothecenes, which are often found in grains, are sesquiterpenoid mycotoxins with the structure 12,13-epoxi-trichothec-9-ene, and are mainly produced by Fusarium, Myrothercium, Stachybotrys, and Trichothecium. From an economic point of view, the most important species producing trichothecenes is Fusarium, and the most commonly detected toxins in cereals are deoxynivalenol (DON), nivalenol (NIV), and their acetylated derivatives.

[0004] Trichothecenes exert a variety of toxic effects on eukaryotic cells, including inhibition of protein synthesis, cytotoxicity, and cell death. Ingestion of feed or food contaminated with DON can cause immunosuppression, anemia, headaches, nausea, and abdominal pain in humans, while animals can experience symptoms such as food refusal, vomiting, growth retardation, and reproductive disorders. Due to global warming, annual exposure to trichothecenes in humans and animals is expected to exceed tolerable levels.

[0005] Most countries have laws regulating the acceptable levels of DON in feed, food, and harvested grain. Despite efforts to reduce DON contamination through pesticides targeting Fusarium spp. and through breeding, DON continues to contaminate grain. Various chemicals, including ozone, ammonia, chlorine, hydrogen peroxide, and sodium bisulfite, have been used to detoxify grains. However, these methods have not been scaled up due to cost, safety concerns, and negative impacts on grain quality. The most effective method is sodium metabisulfite, but its application to food grains is prohibited in Europe. Therefore, a safe and effective method for detoxifying DON is urgently needed.

[0006]

[0007] [Prior Art Literature]

[0008] [Non-patent literature]

[0009] (Non-patent Document 1) S Chakraborty et al, Virulence. Jan 2015; 6(1): 50-65 (2014.12.17), Lactoylglutathione lyase, a critical enzyme in methylglyoxal detoxification, contributes to survival of Salmonella in the nutrient rich environment.

[0010]

[0011] The problem to be solved by the present application is to provide a mutant polypeptide having DON decomposition activity.

[0012]

[0013] The purpose of the present application is to provide a mutant polypeptide having DON degradation (Deoxynivalenol degradation) activity, wherein the mutant polypeptide has an amino acid at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83, and 133 of SEQ ID NO: 1 replaced with a different amino acid.

[0014] Another object of the present application is to provide a polynucleotide encoding the mutant polypeptide of the present application.

[0015] Another object of the present application is to provide a microorganism comprising at least one of the above mutant polypeptides and a polynucleotide encoding the above mutant polypeptides.

[0016] Another object of the present application is to provide a composition for decomposing DON (Deoxynivalenol), comprising the mutant polypeptide and at least one microorganism expressing the mutant polypeptide.

[0017] Another object of the present application is to provide a feed additive composition comprising at least one of the mutant polypeptide and a microorganism expressing the mutant polypeptide.

[0018] Another object of the present application is to provide a method for decomposing DON, comprising the step of reacting at least one of the mutant polypeptide and a microorganism expressing the mutant polypeptide with DON.

[0019] Another object of the present application is to provide a method for producing a feed product, comprising the step of mixing a feed ingredient with a feed additive composition comprising at least one of the mutant polypeptide and a microorganism expressing the mutant polypeptide.

[0020] Another object of the present application is to provide a method for producing a mutant polypeptide comprising: culturing a microorganism comprising at least one of the mutant polypeptide and a polynucleotide encoding the mutant polypeptide; and

[0021] The present invention provides a method for producing a mutant polypeptide having DON decomposition activity, comprising a step of recovering a mutant polypeptide having DON decomposition activity of the present application expressed in the above culturing step.

[0022]

[0023] The mutant polypeptide having DON decomposition activity of the present application has improved DON decomposition activity and can be usefully applied to various industrial fields.

[0024]

[0025] Figure 1 confirms the DON decomposition activity of a parent DON decomposition enzyme (GhM1: SEQ ID NO: 1) and mutant polypeptides (GhM2 to GhM10) having DON decomposition activity of the present application.

[0026]

[0027] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0028] Furthermore, those skilled in the art will recognize or be able to ascertain, using only routine experimentation, numerous equivalents to the specific embodiments described in this application. Furthermore, such equivalents are intended to be encompassed by this application.

[0029] Additionally, numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0030]

[0031] One aspect of the present application provides a mutant polypeptide having DON (Deoxynivalenol) decomposition activity, wherein the mutant polypeptide has an amino acid at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83, and 133 of SEQ ID NO: 1 replaced with another amino acid.

[0032] In addition, the mutant polypeptide may have an amino acid substitution at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83, and 133 of SEQ ID NO: 1, and an amino acid substitution at a position corresponding to any one or more positions selected from positions 75 and 156 of SEQ ID NO: 1, in addition to a substitution of another amino acid.

[0033]

[0034] In this application, “DON decomposition (Deoxynivalenol degradation) activity” means catalyzing the decomposition of deoxynivalenol (DON) by converting it into the iso-DON form.

[0035] In this application, DON (Deoxynivaleno) has the chemical formula C 15 H 20O6, molecular weight 296.3 g / mol (CAS NO.: 51481-10-8), may be a mycotoxin belonging to Type B trichothecene, an epoxy-sesquiterpenoid, and is also called Vomitoxin. It can be produced by fungi that cause diseases in plants, of which Fusarium is a known producer, and when ingested, it can cause symptoms such as immunosuppression, anemia, headache, nausea, and abdominal pain, and in animals, it can cause symptoms such as refusal to eat, vomiting, growth retardation, and reproductive disorders.

[0036] Meanwhile, the degradation of DON (Deoxynivalenol, DON) can be used in the same sense as detoxification of DON, inactivation of DON, and decontaminating of contamination caused by DON.

[0037] The mutant polypeptide having DON decomposition activity provided in the present application is defined as a polypeptide in which an amino acid at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83, and 133 in SEQ ID NO: 1 is substituted; or, additionally, a polypeptide in which an amino acid at a position corresponding to any one or more positions selected from positions 75 and 156 in SEQ ID NO: 1 is substituted with another amino acid. However, this does not exclude meaningless sequence additions before or after the amino acid sequence of SEQ ID NO: 1, mutations that may occur naturally, or silent mutations thereof, and it is obvious to those skilled in the art that if it has the same or corresponding activity as a protein consisting of the amino acid sequence of SEQ ID NO: 1, it corresponds to a polypeptide having DON decomposition activity provided in the present application.

[0038] That is, even if the present application describes a “protein or polypeptide having an amino acid sequence described by a specific sequence number” or a “protein or polypeptide comprising an amino acid sequence described by a specific sequence number,” it is clear that a protein having an amino acid sequence in which some of the sequences are deleted, modified, substituted or added can also be used in the present application if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number.

[0039]

[0040] In this application, the term "wild type" means a naturally occurring polypeptide without any artificial modifications. When the term "wild type" is used in relation 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 relation to a polynucleotide, it means a polypeptide that 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 a naturally occurring polynucleotide, and also includes a sequence encoding any wild type polypeptide.

[0041]

[0042] In the present application, the parent sequence or backbone refers to a reference sequence into which a modification is introduced to become a mutant polypeptide. That is, the parent sequence may be a starting sequence into which mutations such as substitutions, insertions, and / or deletions are introduced. The parent 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. If the parent sequence is an amino acid sequence that exhibits activity, i.e., an amino acid sequence of an enzyme, it may be referred to as a parent enzyme.

[0043]

[0044] In the present application, with respect to an amino acid or nucleic acid sequence, the term "fragment" means a portion of a parent sequence. For example, it may be a polypeptide in which one or more amino acids from the parent sequence are removed from the C or N terminus.

[0045] In the present application, a "fragment" of an enzyme may refer to a "functional fragment." A "functional fragment," also referred to as an "active fragment," refers to a polypeptide that is part of a parent enzyme and possesses the enzymatic activity of the parent enzyme. For example, a functional fragment of an enzyme may include the catalytic site of the enzyme.

[0046] The enzyme fragment 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 less than 100% of the amino acids of the full length of the parent enzyme.

[0047]

[0048] In this application, "mutating / modifying" means changing or altering. This may be a change from a naturally occurring sequence. For example, an enzyme may be modified in such a way that it is altered from its parent sequence or reference sequence.

[0049] In the present application, the modified enzyme may be a non-naturally occurring enzyme, i.e., an enzyme that does not exist in nature itself.

[0050] The term "modified" in this application means, for example, something that has been altered from its naturally occurring form. The modified enzymes of this application include enzymes that do not occur naturally or naturally occurring variants. For example, the modified enzymes of this application are enzymes that are not found in nature. For example, the modified enzymes of this application may be, but are not limited to, enzymes that do not occur spontaneously.

[0051] When the term "modification" is used in relation to an amino acid / nucleic acid sequence in this application, it may include substitution of an amino acid / nucleic acid residue of the parent sequence for a different amino acid / nucleic acid residue at one or more sites 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 sites, 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 sites, truncation of the N-terminal and / or C-terminal amino acid sequence or 5' and / or 3' nucleic acid sequence, and any combination thereof.

[0052]

[0053] In the present application, 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. The terms "variant" and "modified polypeptide" may be used interchangeably. The variant or modified polypeptide may be, but is not limited to, a non-naturally occurring one.

[0054] The variant differs from the sequence of the parent enzyme by one or more modifications, e.g., amino acid substitutions, deletions and / or insertions.

[0055] Such variants can generally be identified by altering one or more amino acids in the parent enzyme and evaluating the properties of the altered protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the parent enzyme.

[0056] Additionally, some variants may comprise mutant polypeptides in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted.

[0057] Other variants may include variants in which portions are removed from the N- and / or C-terminus of the mature protein.

[0058] The term "variant" or "variant polypeptide" may be used interchangeably with terms such as variant, modification, mutated protein, and mutation (in English, modification, modified protein, mutant, mutein, divergent, variant, etc.), and is not limited thereto if the term is used in the meaning of variant.

[0059] Variants may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of the protein that is involved in co-translational or post-translational protein transfer. Furthermore, the polypeptide may be conjugated to other sequences or linkers to facilitate identification, purification, or synthesis of the polypeptide.

[0060]

[0061] In this application, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0062]

[0063] In this application, the term "parent DON degrading enzyme" refers to a DON degrading enzyme that is modified to produce a variant or mutant polypeptide of the present application. Specifically, the parent DON degrading enzyme, parent enzyme, or parent sequence may be a naturally occurring polypeptide or a wild-type polypeptide, may be a mature polypeptide thereof, may include a variant or functional fragment thereof, but is not limited to any polypeptide that has DON degrading activity and can be a parent of a variant.

[0064] The DON degrading enzyme provided in the present application may be, but is not limited to, a polypeptide having SEQ ID NO: 1. In addition, as long as it has DON degrading activity, it may be a polypeptide having a sequence identity of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the polypeptide having SEQ ID NO: 1, and as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, it may be included in the scope of the DON degrading enzyme without limitation.

[0065] The parent DON degrading enzyme of the variant provided in the present application may be derived from the genus Gossypium, the genus Handroanthus, or the genus Hibiscus. Specifically, it may be derived from the genus Gossypium.

[0066]

[0067] In the present application, the “variant polypeptide having DON decomposition activity” may be a variant of the parent DON decomposition enzyme.

[0068] In the present application, the term "variant of a parent DON degrading enzyme" or "variant polypeptide having DON degrading activity" refers to a protein having one or more amino acids different from the amino acid sequence of the parent DON degrading enzyme and having DON degrading activity.

[0069] The above “mutant polypeptide having DON decomposition activity”, “mutant of parent DON decomposition enzyme” and “mutant DON decomposition enzyme” can be used interchangeably.

[0070] The mutant polypeptide provided in the present application may have DON degrading activity and may include modifications of one or more amino acids in the parent DON degrading enzyme sequence. The modifications may be amino acid substitutions and / or disulfide bond formation.

[0071] Specifically, the variants provided in the present application may have DON decomposition activity and may have one or more altered functions or properties compared to the parent DON decomposition enzyme, including modification of one or more amino acids in the parent DON decomposition enzyme sequence.

[0072] In one specific example, the variant provided in the present application has DON degrading activity, and may have one or more altered functions or properties compared to the parent DON degrading enzyme, including modification of one or more amino acids in the parent DON degrading enzyme sequence, and may have one or more conservative substitutions.

[0073]

[0074] The variant provided in the present application is a variant of a parent DON degrading enzyme and may be a polypeptide having DON degrading activity. In one specific example, the variant provided in the present application may include a modification at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83, and 133 of SEQ ID NO: 1.

[0075] In another specific example, the variant provided in the present application may include an amino acid substitution at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83 and 133 of SEQ ID NO: 1, and a modification at a position corresponding to any one or more positions selected from positions 75 and 156 of SEQ ID NO: 1.

[0076] In this application, the position number is a position corresponding to the position of the polypeptide of sequence number 1, and “corresponding” is as described above.

[0077]

[0078] In one specific example, the variant polypeptide of the present application may include, but is not limited to, a substitution at an amino acid at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83, and 133 of SEQ ID NO: 1 compared to the amino acid sequence of SEQ ID NO: 1. In addition, the variant polypeptide may include a substitution at a position corresponding to any one or more positions selected from positions 75 and 156 of SEQ ID NO: 1.

[0079] In a specific example, the variant polypeptide provided in the present application may have a combination of 2, 3, 4, 5, 6 or 7 positions selected from the above positions substituted with different amino acids.

[0080] In any one of the specific examples described above, the variant polypeptide provided in the present application may include a substitution of an amino acid at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83 and 133 of SEQ ID NO: 1 with G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R or H.

[0081] In any one of the specific examples described above, the variant polypeptide provided in the present application may include a substitution of an amino acid at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83 and 133 of SEQ ID NO: 1 with a polar, nonpolar, hydrophobic or acidic amino acid.

[0082] In any one of the specific examples described above, the mutant polypeptide provided in the present application may be one in which the amino acid corresponding to position 48 of SEQ ID NO: 1 is substituted with glutamic acid; the amino acid corresponding to position 60 of SEQ ID NO: 1 is substituted with isoleucine; the amino acid corresponding to position 62 of SEQ ID NO: 1 is substituted with glutamic acid or glutamine; the amino acid corresponding to position 83 of SEQ ID NO: 1 is substituted with valine; or the amino acid corresponding to position 133 of SEQ ID NO: 1 is substituted with valine, but is not limited thereto.

[0083]

[0084] In addition, as another specific example among the specific examples described above, the mutant polypeptide provided in the present application may be one in which an amino acid at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83, and 133 of SEQ ID NO: 1 is substituted with another amino acid, in addition to an amino acid substitution at a position corresponding to any one or more positions selected from positions 75 and 156 of SEQ ID NO: 1.

[0085] In one specific example, the variant polypeptide provided in the present application may include a substitution of an amino acid at a position corresponding to any one or more positions selected from positions 75 and 156 of SEQ ID NO: 1 with a polar, nonpolar, hydrophobic or acidic amino acid as an additional substitution.

[0086] In another specific embodiment of the above-described specific embodiments, the variant polypeptide provided in the present application may include a substitution in which the amino acid corresponding to position 75 of SEQ ID NO: 1 is substituted with leucine; an amino acid corresponding to position 156 of SEQ ID NO: 1 is substituted with valine; or a combination thereof.

[0087]

[0088] In another specific example, the mutant polypeptide provided in the present application may be one in which the amino acid corresponding to position 60 of SEQ ID NO: 1 is substituted with isoleucine; and the amino acid corresponding to position 75 of SEQ ID NO: 1 is substituted with leucine.

[0089] In another specific example, the mutant polypeptide provided in the present application may be one in which the amino acid corresponding to position 60 of SEQ ID NO: 1 is substituted with isoleucine; and the amino acid corresponding to position 156 of SEQ ID NO: 1 is substituted with valine.

[0090] In another specific example, the mutant polypeptide provided in the present application may be one in which the amino acid corresponding to position 60 of SEQ ID NO: 1 is substituted with isoleucine; the amino acid corresponding to position 75 of SEQ ID NO: 1 is substituted with leucine; and the amino acid corresponding to position 156 of SEQ ID NO: 1 is substituted with valine.

[0091]

[0092] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a protein or polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the protein or polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0093] In the present application, SEQ ID NO. 1 can be used as a reference sequence to determine the position of an amino acid in any amino acid sequence.

[0094] In this application, the term "reference sequence" refers to a sequence used to determine the position of an amino acid within an arbitrary amino acid sequence. By aligning an arbitrary amino acid sequence with a reference sequence, the position of an amino acid corresponding to a specific position of the reference sequence within the arbitrary amino acid sequence can be determined.

[0095]

[0096] That is, SEQ ID NO: 1 disclosed in the present application can be used to determine the corresponding amino acid residue in a polypeptide having any DON decomposition activity, and unless otherwise indicated in the present application, residues of a specific amino acid sequence are numbered based on SEQ ID NO: 1.

[0097] For example, 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 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 the present application can identify the position of an amino acid, or the position at which a modification such as a substitution, insertion, or deletion occurs, by comparing it to a query sequence (also referred to as a “reference sequence”).

[0098] These alignments can be performed using, but are not limited to, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), and Trends Genet. 16: 276-277).

[0099] Additionally, multiple sequence alignment can be used to identify corresponding amino acid residues in polypeptides with different DON degrading activities. 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: 1899-1900), and EMBOSS using ClustalW. EMMA (1.83 or higher; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), etc., and the default parameters of each of the above programs can be used, but are not limited thereto.

[0100] Additionally, if enzymes diverged from the mature polypeptide of SEQ ID NO: 1 and their relationships cannot be detected by conventional sequence-based comparison, other pairwise sequence comparison algorithms can be used (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Higher sensitivity can be achieved in sequence-based searches by using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and can detect remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide 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 a neural network that predicts the structural folding of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919 can be used to align an unknown sequence with superfamily models available in the SCOP database. These alignments can in turn be used to build homology models for the polypeptide, and these models can be evaluated for accuracy using a variety of tools developed for this purpose.

[0101] For proteins with known structures, several tools and resources are available for searching and generating structural alignments. For example, the SCOP superfamily of proteins is structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms, such as distance alignment matrix alignment (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 containing the target structure to discover possible structural homologues (Holm and Park, 2000, Bioinformatics 16: 566-567).

[0102] The above methods are examples and are not limiting.

[0103]

[0104] Throughout this application, the conventional one-letter and three-letter codes for naturally occurring amino acids are used. Furthermore, amino acids referred to by abbreviations in this application are described according to the IUPAC-IUB nomenclature.

[0105]

[0106] Alanine Ala, A Arginine Arg, R

[0107] Asparagine Asn, N Aspartic acid Asp, D

[0108] Cysteine ​​Cys, C Glutamic acid Glu, E

[0109] Glutamine Gln, Q Glycine Gly, G

[0110] Histidine His, H Isoleucine Ile, I

[0111] Leucine Leu, L Lysine Lys, K

[0112] Methionine Met, M Phenylalanine Phe, F

[0113] Proline Pro, P Serine Ser, S

[0114] Threonine Thr, T Tryptophan Trp, W

[0115] Tyrosine Tyr, Y Valine Val, V

[0116]

[0117] Meanwhile, any amino acid can be written as Xaa, X.

[0118] Additionally, the generally accepted three-letter codes for other amino acids, such as Aib (2-Aminoisobutyric acid), Sar (N-methylglycine), and α-methyl-glutamic acid, may be used, as well as for naturally occurring amino acids.

[0119] Amino acids can generally be classified based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Accordingly, amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0120] For example, among the 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 the amino acids having uncharged side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and 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.

[0121]

[0122] In one embodiment, the mutant polypeptide having DON decomposition activity provided in the present application may include a mutant polypeptide in which an amino acid at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83, and 133 in SEQ ID NO: 1 is substituted, or may be composed of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13. In another embodiment, the novel polypeptide provided in the present application may be a variant polypeptide having DON degrading activity that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13. Or, it may comprise, consist essentially of, or consist of a sequence having the homology or identity.

[0123] In another embodiment, the variant polypeptide having DON decomposition activity provided in the present application may comprise a variant polypeptide in which an amino acid is substituted at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83 and 133 of SEQ ID NO: 1, and an amino acid is substituted at a position corresponding to any one or more positions selected from positions 75 and 156 of SEQ ID NO: 1, or may be composed of SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19. In another embodiment, the novel polypeptide provided in the present application may be a variant polypeptide having DON degrading activity that has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity to SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 19. Or, it may comprise, consist essentially of, or consist of a sequence having said homology or identity.

[0124]

[0125] In this application, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or base sequences are related, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0126] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences are generally capable of hybridizing under moderate or high stringency conditions, typically along at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or its entire length. It should be appreciated that hybridization also encompasses polynucleotides containing common codons or codons considered codon degeneracy.

[0127] Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can 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, 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) can be determined using 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, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information database.

[0128] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the GAP program can be defined as 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). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0129] Additionally, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and appropriate hybridization conditions are within the skill of the art and can 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).

[0130]

[0131] In the present application, "enzymatic activity" or "DON decomposition activity" refers to at least one catalytic activity. Specifically, it may be, but is not limited to, the conversion efficiency of the enzyme, which is mainly expressed as kcat / Km.

[0132] kcat is the catalytic constant for the conversion of a single enzyme into a product per unit time when the enzyme is completely saturated with substrate, and is also called the turnover number. Km is the substrate concentration when the reaction rate is half of the maximum value (Vmax).

[0133] As an example of how to express enzyme activity, 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 ) etc. However, defining enzyme activity is not limited to the above-mentioned content, and can be defined and evaluated based on content known in the art.

[0134] In this application, enzyme stability refers to the preservation of enzyme activity during storage or reaction time. To measure changes in this stability, the initial enzyme activity is measured at time zero (100%) and after a specified time (x%) under specified conditions, and compared. This allows for the expression of the level of enzyme activity loss or enzyme stability.

[0135]

[0136] The term "specific activity" in this application refers to the activity of an enzyme per unit weight of protein, expressed as unit / mg. Protein quantification can be performed using, for example, SDS-PAGE or the Bradford assay.

[0137]

[0138] Factors that affect enzyme activity include, for example, pH, heat, and the presence of other substances (e.g., oxidizing agents, chelating agents).

[0139]

[0140] In one specific example, the mutant polypeptide provided in the present application has an activity of about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about It may have an increased enzyme activity of at least 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138%, about 139%, about 140%, about 141%, about 142%, about 145%, or about 150%.

[0141]

[0142] In any one of the embodiments described above, the mutant polypeptide having increased enzyme activity (improved DON decomposition activity) compared to the polypeptide of SEQ ID NO: 1 may include one or more substitutions selected from the following.

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] In any one of the embodiments described above, the properties of the mutant polypeptide having DON decomposition activity provided in the present application may be an activity or improved activity suitable for application in various industrial fields including feed, baking, pulp bleaching, etc.

[0152]

[0153] Another aspect of the present application is a polynucleotide encoding a variant polypeptide having DON degrading activity of the present application.

[0154] In this application, the term "polynucleotide" means a DNA or RNA strand of a certain length or longer, which is a polymer of nucleotides in which nucleotide units (monomers) are linked in a long chain shape by covalent bonds.

[0155] The polynucleotide encoding the variant polypeptide having DON decomposition activity of the present application may include, without limitation, any polynucleotide encoding the variant polypeptide; or a polypeptide of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 and a polypeptide having an activity corresponding thereto.

[0156] The polynucleotide encoding the mutant polypeptide having the DON decomposition activity of the present application can have various modifications made to the coding region within a range that does not change the amino acid sequence of the polypeptide due to the degeneracy of the codon or in consideration of the codon preferred in the organism that is to express the mutant polypeptide.

[0157] For example, the polynucleotide encoding the variant polypeptide having DON decomposition activity of the present application may be a polynucleotide sequence encoding an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19, or a polypeptide having homology or identity therewith.

[0158] In one embodiment, a polynucleotide encoding a variant polypeptide having DON degrading activity of the present application may have or include a base sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, or 90% homologous or identical, but less than 100%, to a polynucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20. In another embodiment, the polynucleotide encoding the variant polypeptide having DON degrading activity of the present application may be composed of or consist essentially of a base sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with a sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, but is not limited thereto. In addition, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known gene sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application.

[0159]

[0160] The above "stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions in which polynucleotides having high homology or identity hybridize with each other, polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS, which are washing conditions of typical southern hybridization, are performed.

[0161] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

[0162] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0163] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

[0164]

[0165] Another aspect of the present application provides a vector comprising a polynucleotide encoding a variant polypeptide having DON degrading activity of the present application. The variant polypeptide and polynucleotide are as described in the other aspect above.

[0166] The term "vector" as used herein refers to a DNA construct containing a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide 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 ribosome binding site, and sequences regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or may be integrated into the genome itself.

[0167] For example, a polynucleotide encoding a target protein within a chromosome can be replaced with a mutated polynucleotide through a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule. Markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface protein, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, thereby allowing the selection of transformed cells.

[0168] The vector used in this application is not particularly limited, and any vector known in the art can be used.

[0169] Examples of vectors commonly used in prokaryotes include pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, which can be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series, which can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pDCM2 vectors, etc. can be used.

[0170] As an example of a vector used in a eukaryotic cell, the yeast expression vector can be both an integrative yeast plasmid (YIp) and an extrachromosomal plasmid vector. The extrachromosomal plasmid vector can include an episomal yeast plasmid (YEp), a replicative yeast plasmid (YRp), and a yeast centromer plasmid (YCp). In addition, artificial yeast chromosomes (YACs) can also be used as the vector of the present application. As specific examples, available vectors include pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPα A, pGAPα B, pGAPα C, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2 / CT, pYD1 Yeast Display Vector, pYES2, pYES2 / CT, pYES2 / NT A, pYES2 / NT B, pYES2 / NT C, pYES2 / CT, pYES2.1, pYES-DEST52, pTEF1 / Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form, pRS403, pRS404, pRS405, pRS406, pYJ403, Including but not limited to pYJ404, pYJ405 and pYJ406.

[0171]

[0172] The term "transformation" in this application refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell or microorganism, thereby enabling expression of the protein encoded by the polynucleotide within the host cell. The transformed polynucleotide may be located within the chromosome of the host cell or located extrachromosomally, as long as it can be expressed within the host cell. Furthermore, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0173]

[0174] Additionally, the term "operably linked" as used herein means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target polypeptide of the present application.

[0175] The method for transforming the vector of the present application includes any method for introducing nucleic acids into cells, and can be performed by selecting an appropriate standard technique known in the art depending on the host cell. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0176]

[0177] Another aspect of the present application is a microorganism comprising a variant polypeptide having DON decomposition activity of the present application; and at least one polynucleotide encoding the variant polypeptide.

[0178] The above microorganism may comprise the above-described mutant polypeptide, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, and / or a vector. The nucleic acid construct or vector may be integrated into a chromosome or maintained as an extrachromosomal vector that replicates autonomously.

[0179] The microorganism of the present application may be included without limitation as long as it can express the variant polypeptide having DON degrading activity of the present application. For example, any cell useful for the recombinant production of the variant polypeptide having DON degrading activity of the present application is included, and for example, the microorganism may be a prokaryotic cell or a eukaryotic cell. As another example, examples of the microorganism include fungi and bacteria.

[0180] In one embodiment, the microorganism of the present application may be a microorganism of the genus Escherichia, such as Escherichia coli, Escherichia albertii, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, or Escherichia blattae. In any one of the aforementioned embodiments, the microorganism may be, but is not limited to, Escherichia coli.

[0181]

[0182] Another aspect of the present application is a composition for decomposing DON (Deoxynivalenol), comprising a mutant polypeptide having DON decomposition activity of the present application; and at least one microorganism expressing the mutant polypeptide.

[0183] Another aspect of the present application is a method for decomposing DON, comprising the step of reacting a variant polypeptide having DON decomposition activity of the present application with DON and at least one microorganism expressing the variant polypeptide.

[0184] The mutant polypeptide having DON decomposition activity of the present application and / or a microorganism expressing the mutant polypeptide can be used to decompose a substance containing DON.

[0185]

[0186] In one specific example, the polypeptide of the present application can be used to induce detoxification of DON, detoxification of DON, conversion of DON (deoxynivalenol) to iso-DON (iso-deoxynivalenol), and / or decomposition through conversion of iso-DON.

[0187] Additionally, in another additional embodiment of the present application, DON can be used to detoxify, decontaminate or detoxify (e.g., decompose) food contaminated with DON.

[0188]

[0189] In one embodiment, the composition for decomposing DON provided in the present application may further include a naturally occurring substance or a non-naturally occurring substance in addition to the mutant polypeptide of the present application.

[0190] In one embodiment, the composition for decomposing DON provided in the present application may further include any component suitable for application in various industrial fields such as animal feed, baking, biomass saccharification, and pulp bleaching.

[0191] Examples of substances that may be added include, but are not limited to, stabilizers, surfactants, builders, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, compounding agents, lubricants, disintegrants, excipients, solubilizers, suspending agents, colorants, flavorings, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, diluents, lubricants, preservatives, and the like.

[0192]

[0193] Another aspect of the present application is a feed additive composition comprising a mutant polypeptide having DON decomposition activity of the present application; and at least one microorganism expressing the mutant polypeptide.

[0194] Another aspect of the present application is a method for producing a feed product, comprising the step of mixing a feed additive composition comprising a mutant polypeptide having DON decomposition activity of the present application and at least one microorganism expressing the mutant polypeptide, and a feed ingredient.

[0195] The mutant polypeptide having DON decomposition activity of the present application and / or the microorganism expressing the mutant polypeptide may be used in any of the following applications:

[0196] a) additives in animal feed ingredients; and / or

[0197] b) animal feed supplements; and / or

[0198] c) Decomposition of grain-based materials (e.g., this may be whole grains or parts of grains).

[0199] The feed product of the present application may mean any natural or artificial diet, meal, etc. or ingredients of the meal for eating, ingesting, and digesting by animals or suitable therefor, and may be manufactured in various forms known in the art.

[0200] In one specific example, the feed product may be a grain-based material (including whole grains or partial grains or malted grains, e.g., wheat, barley, corn, oats, rye, rice, sorghum, etc.).

[0201]

[0202] Another aspect of the present application comprises the steps of culturing a microorganism comprising a variant polypeptide having DON decomposition activity of the present application; and at least one polynucleotide encoding the variant polypeptide; and

[0203] A method for producing a mutant polypeptide having DON decomposition activity, comprising a step of recovering a mutant polypeptide having DON decomposition activity of the present application expressed in the above-mentioned culturing step.

[0204]

[0205] The above culturing may include a step of culturing the microorganism in a culture medium.

[0206] In this application, the term "cultivation" refers to growing the host cells under appropriately controlled environmental conditions. The culturing process of this application can be performed using any suitable medium and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, or fed-batch, but is not limited thereto.

[0207] In this application, the term "medium" refers to a material containing nutrients as a main component necessary for culturing the host cells, and supplies nutrients and growth factors, including water, which is essential for survival and development. Specifically, the medium and other culture conditions used for culturing the host cells of the present application may be any medium used for culturing conventional host cells without particular limitation, but the host cells of the present application may be cultured under aerobic conditions while controlling temperature, pH, etc. in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0208] 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.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugar) may be used, and other appropriate amounts of carbon sources 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.

[0209] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0210] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0211] In addition, during the cultivation of the host cells, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or in order to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without injecting gas, but is not limited thereto.

[0212] The temperature of the medium may be, but is not limited to, 20°C to 55°C, specifically 25°C to 40°C. The incubation period may continue until the desired amount of useful material is obtained, and specifically may be, but is not limited to, 24 hours to 196 hours.

[0213] In one embodiment, the method for producing a mutant polypeptide having DON decomposition activity of the present application may further include a step of recovering the mutant polypeptide having DON decomposition activity of the present application expressed in the culturing step.

[0214] In another embodiment, the mutant polypeptide having DON degrading activity expressed in the above-described culturing step can be recovered using methods known in the art. For example, the mutant polypeptide can be recovered from the medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0215] The above recovery method may be to collect the polypeptide using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC and a combination of these methods may be used, and the mutant polypeptide of the present application can be recovered from a medium or host cell using a suitable method known in the art.

[0216] In another embodiment, the mutant polypeptide expressed by the host cell during the culture step may not be recovered. In this embodiment, the host cell itself expressing the mutant polypeptide may be used as a source of the mutant polypeptide.

[0217]

[0218] Another aspect of the present application provides a use of the variant polypeptide of the present application; and a microorganism expressing the variant polypeptide for decomposing DON (Deoxynivalenol).

[0219] The definitions of the above terms are as described above.

[0220]

[0221] Hereinafter, this application will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify this application and the scope of this application is not limited to these examples and experimental examples.

[0222]

[0223] Example 1: Preparation of GhSPG-M1 variants

[0224]

[0225] Example 1-1. Production of GhM1

[0226] A polynucleotide (SEQ ID NO: 2) encoding a mutant (hereinafter referred to as GhM1, SEQ ID NO: 1) with 31 mutations added to the hypothetical protein sequence (SEQ ID NO: 37) derived from Gossypium harknessii was synthesized by Cosmo genetech and cloned into a pET vector (Novagen) to create an expression vector.

[0227]

[0228] Example 1-2. Production of GhM1 point mutations and combination mutants

[0229] To improve the activity of GhM1 of Example 1-1, mutation sites were selected and primers were designed to produce 9 point mutation and combination mutants (hereinafter referred to as GhM2, GhM3, GhM4, GhM5, GhM6, GhM7, GhM8, GhM9, GhM10, SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19). The mutation sites, amino acids after mutation, and primer sequences for producing mutants based on the amino acid sequence of SEQ ID NO: 1 are sequentially described in Table 1 below.

[0230]

[0231]

[0232]

[0233] Specifically, nine GhM1 point mutations and combination mutants were produced by PCR using the polynucleotide encoding GhM1 (SEQ ID NO: 1) produced in Example 1-1 cloned into a pET vector as a template, and primers (SEQ ID NOs: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 in Table 1) and PCR premix (iNtRON, cat no. 25185). PCR was performed using an Eppendorf Mastercycler Nexus GX2, and the reaction conditions were as follows.

[0234]

[0235] Initial denaturation - 94℃, 2 min

[0236] Denaturation - 94℃, 20sec

[0237] Annealing - 50℃, 10sec

[0238] Extension - 72℃, 8 min (15 cycles from denaturation to extension)

[0239] Final Extension - 72℃, 5min

[0240]

[0241] The generated truncated mutants were ligated using the QuickChange Site-Directed Mutagenesis kit (Agilent, Cat# 200518), and then transformed into the E. coli Dh5α strain to confirm sequence mutations through sequencing.

[0242]

[0243] Example 2: Characterization of GhM1 point and combination mutants

[0244]

[0245] Example 2-1. Comparative evaluation of the activity of GhM1 point and combination mutants.

[0246] The expression vectors of GhM1 and 9 mutants produced in Example 1-1 were transformed into E. coli BL21 (DE3), inoculated into sterilized LB medium (BD Difco), and pre-cultured at 37°C and 200 rpm for 16 hours. Afterwards, 1 / 100 of the medium volume was inoculated into a flask containing sterilized LB medium, and then the absorbance (OD) was measured at 37°C and 200 rpm. 600) was cultured until the confluency was between 0.4 and 0.5, then IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 1 mM, and cultured for an additional 16 hours. Then, the cells were harvested by centrifugation. 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole) was added to the harvested cells and redispersed. Then, the crude enzyme solution was secured through sonication and centrifugation. The crude enzyme solution was adsorbed on Ni-NTA resin (Qiagen, Cat no. 30230), and then the enzyme was purified by sequentially flowing the washing buffer (20 mM imidazole concentration in the lysis buffer composition) and the elution buffer (250 mM imidazole concentration in the lysis buffer composition).

[0247] Protein concentration was determined by mixing 5 ㎕ of diluted enzyme solution and 250 ㎕ of Bradford solution (Quick Start Bradford 1x Dye Reagent, #5000205) and measuring the absorbance at 595 nm.

[0248] To analyze the activity of purified GhM1 and GhM1 point and combination mutants toward DON, DON (CAS 51481-10-8, Deoxynivalenol) dissolved in distilled water and purified enzyme reaction solution (25 mM Tris HCl, 150 mM NaCl, pH 7.4) were prepared, and after treating with 500 μM NiCl2 as a cofactor, the reaction was carried out at 50°C for 24 hours and then stopped by leaving at 100°C for 5 minutes. The amount of DON remaining in the reaction solution was measured using an HPLC-UV (High performance liquid chromatography-Ultraviolet) detector, and the relative activity of the mutants was measured using the ratio of the total degraded DON, and the results are shown in Table 2 and Fig. 1.

[0249]

[0250] As a result, when the enzyme activity was expressed as a relative ratio, it was confirmed that the activity of 9 GhM1 point mutants and combination mutants (GhM2, GhM3, GhM4, GhM5, GhM6, GhM7, GhM8, GhM9, GhM10) was improved by 5% to 42% or more compared to the template GhM1.

[0251]

[0252] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

[0253]

[0254] [Sequence List]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

Claims

1. A mutant polypeptide having DON (Deoxynivalenol) decomposition activity, wherein the mutant polypeptide is a mutant polypeptide in which an amino acid at a position corresponding to any one or more positions selected from positions 48, 60, 62, 83, and 133 of sequence number 1 is substituted with a different amino acid.

2. In the first paragraph, the mutant polypeptide further comprises a mutant polypeptide in which the amino acid corresponding to position 75 of SEQ ID NO: 1 is substituted with leucine; the amino acid corresponding to position 156 of SEQ ID NO: 1 is substituted with valine; or a combination thereof.

3. In the first paragraph, the mutant polypeptide is a mutant polypeptide in which the amino acid corresponding to position 48 of SEQ ID NO: 1 is substituted with glutamic acid; the amino acid corresponding to position 60 of SEQ ID NO: 1 is substituted with isoleucine; the amino acid corresponding to position 62 of SEQ ID NO: 1 is substituted with glutamic acid or glutamine; the amino acid corresponding to position 83 of SEQ ID NO: 1 is substituted with valine; or the amino acid corresponding to position 133 of SEQ ID NO: 1 is substituted with valine.

4. In the second paragraph, the mutant polypeptide is a mutant polypeptide in which the amino acid corresponding to position 60 of sequence number 1 is substituted with isoleucine; and the amino acid corresponding to position 75 of sequence number 1 is substituted with leucine.

5. In the second paragraph, the mutant polypeptide is a mutant polypeptide in which the amino acid corresponding to position 60 of sequence number 1 is substituted with isoleucine; and the amino acid corresponding to position 156 of sequence number 1 is substituted with valine.

6. In the second paragraph, the mutant polypeptide is a mutant polypeptide in which the amino acid corresponding to position 60 of SEQ ID NO: 1 is substituted with isoleucine; the amino acid corresponding to position 75 of SEQ ID NO: 1 is substituted with leucine; and the amino acid corresponding to position 156 of SEQ ID NO: 1 is substituted with valine.

7. A mutant polypeptide according to claim 1, wherein the mutant polypeptide comprises any one selected from SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO:

13.

8. A mutant polypeptide according to claim 2, wherein the mutant polypeptide comprises any one selected from SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO:

19.

9. In the first paragraph, the mutant polypeptide has improved DON decomposition activity compared to a polypeptide consisting of the amino acid sequence of sequence number 1.

10. A polynucleotide encoding a mutant polypeptide of any one of claims 1 to 9.

11. A microorganism comprising a mutant polypeptide according to any one of claims 1 to 9; and at least one polynucleotide encoding the mutant polypeptide.

12. A composition for decomposing DON (Deoxynivalenol), comprising a mutant polypeptide according to any one of claims 1 to 9; and at least one microorganism expressing the mutant polypeptide.

13. A feed additive composition comprising a mutant polypeptide according to any one of claims 1 to 9; and at least one microorganism expressing the mutant polypeptide.

14. A method for decomposing DON, comprising the step of reacting a mutant polypeptide according to any one of claims 1 to 9; and at least one microorganism expressing the mutant polypeptide with DON.

15. A method for manufacturing a feed product, comprising the step of mixing the feed additive composition of Article 13 and a feed ingredient.

16. A step of cultivating the microorganism of clause 11; and A method for producing a mutant polypeptide having DON decomposition activity, comprising a step of recovering a mutant polypeptide having DON decomposition activity of any one of claims 1 to 9 expressed in the above culturing step.

17. A mutant polypeptide according to any one of claims 1 to 9; and a use for decomposing DON (Deoxynivalenol) of at least one microorganism expressing the mutant polypeptide.

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