Novel polypeptides having DON degrading activity

Abstract

The present application relates to variant polypeptides having DON degrading activity.

Description

Technical Field

[0001] This disclosure relates to modified polypeptides having deoxynivalenol (DON) degradation activity. Background Technology

[0002] Mycotoxins are a collective term for metabolites derived from fungi, and they are toxic to animals. These mycotoxins are produced when crops or stored agricultural products are infected or contaminated by mycotoxin-producing fungi, posing health risks to animals or humans. Trichothecenes, commonly found in grains, are sesquiterpenoid mycotoxins with a 12,13-epoxy-trichothecene-9-ene structure, primarily produced by species in the genera *Fusarium*, *Myrothecium*, *Stachybotrys*, and *Trichothecium*. From an economic perspective, *Fusarium* is the most important species producing trichothecenes. Among the toxins, the most frequently detected in grains are deoxynivalenol (DON), nivalenol (NIV), and their acetylated derivatives.

[0003] Trichothecene toxins act on eukaryotic cells, exhibiting various harmful effects such as inhibition of protein synthesis, cytotoxicity, and cell death. When humans consume feed or food contaminated with trichothecene (DON), they may experience symptoms such as immunosuppression, anemia, headache, nausea, and abdominal pain, while animals may exhibit symptoms such as loss of appetite, vomiting, growth retardation, and reproductive disorders. Due to the effects of global warming, annual exposure levels to trichothecene toxins in humans and animals are projected to exceed tolerable levels.

[0004] Most countries have laws regulating permissible levels of DON in feed, food, and harvested grains. Despite efforts to reduce DON contamination through pesticides targeting Fusarium spp. or through breeding, DON still contaminates grains. Various chemicals have been used to detoxify DON, such as ozone, ammonia, chlorine, hydrogen peroxide, and sodium bisulfite, but these have not been scaled up due to cost, safety concerns, and negative impacts on grain quality. The most effective method is treatment with sodium metabisulfite. However, this is prohibited in Europe for use in edible grains; therefore, a safe method for DON detoxification is needed.

[0005] [Existing technical documents]

[0006] [Non-patent literature]

[0007] (Non-patent document 1) S Chakraborty et al., Virulence. 2015 Jan; 6(1): 50-65(2014.12.17), Lactoylglutathione lyase, a critical enzyme in methylglyoxaldetoxification, contributes to survival of Salmonella in the nutrient richenvironment. Summary of the Invention

[0008] Technical issues

[0009] The inventors have developed a modified polypeptide with deoxynivalenol (DON) degradation activity.

[0010] Technical solution

[0011] One object of this disclosure is to provide a modified polypeptide having deoxynivalenol (DON) degradation activity, wherein an amino acid at a position corresponding to any one or more positions selected from positions 28, 29, 87, 91, 101 and 152 of SEQ ID NO: 1 is replaced by another amino acid.

[0012] Another object of this disclosure is to provide a polynucleotide encoding the modified polypeptide of this disclosure.

[0013] Another object of this disclosure is to provide a microorganism comprising one or more of the following: the modified polypeptide; and a polynucleotide encoding the modified polypeptide.

[0014] Another object of this disclosure is to provide a composition for degrading deoxynivalenol (DON), said composition comprising one or more of the following: the modified polypeptide; and a microorganism expressing said modified polypeptide.

[0015] Another object of this disclosure is to provide a feed additive composition comprising one or more of the following: the modified polypeptide; and a microorganism expressing the modified polypeptide.

[0016] Another object of this disclosure is to provide a method for degrading deoxynivalenol (DON), the method comprising the steps of reacting DON with any one or more of the following: the modified polypeptide; and a microorganism expressing the modified polypeptide.

[0017] Another object of this disclosure is to provide a method for preparing a feed product, the method comprising the step of mixing feed components with a feed additive composition, the feed additive composition comprising one or more of the following: the modified polypeptide; and microorganisms expressing the modified polypeptide.

[0018] Another object of this disclosure is to provide a method for preparing modified peptides with DON degradation activity, the method comprising the following steps:

[0019] Cultivating microorganisms, said microorganisms comprising any one or more of the following: the modified polypeptide; and a polynucleotide encoding said modified polypeptide; and

[0020] The modified polypeptides of this disclosure with DON degradation activity expressed during the culture step are recovered.

[0021] Beneficial effects

[0022] The modified polypeptides disclosed herein exhibit improved DON degradation activity, thermal stability, heat resistance, and acid resistance, and can be effectively applied in various industrial fields. Attached Figure Description

[0023] Figure 1 The examination of the DON degradation activity of the parental DON-degrading enzyme (GhSPG-216M2; SEQ ID NO: 1) and the modified polypeptide (GhSPG-216M3-8) of this disclosure having DON degradation activity is shown.

[0024] Figure 2 The thermal stability of the parental DON-degrading enzyme and the modified polypeptides of this disclosure with DON-degrading activity were examined.

[0025] Figure 3 The acid resistance of the parental DON-degrading enzyme and the DON-degrading modified polypeptide of this disclosure is shown.

[0026] Figure 4 A comparative assessment of the activity of the variants is shown, in which the amino acid at position 101 of the parental DON degrading enzyme in each variant is replaced by another amino acid.

[0027] Figure 5 A comparative assessment of the heat resistance of the variants is shown, in which the amino acid at position 101 of the parental DON degrading enzyme in each variant is replaced by another amino acid.

[0028] Figure 6 A comparative assessment of the acid resistance of the variants is shown, wherein the amino acid at position 101 of the parental DON degrading enzyme in each variant is replaced by another amino acid; and

[0029] Figure 7 The examination of the DON degradation activity of the parental DON-degrading enzyme and the DON-degrading modified peptides of this disclosure is shown. Detailed Implementation

[0030] This disclosure will be described in detail below. Furthermore, each description and embodiment disclosed herein can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific descriptions below.

[0031] Furthermore, those skilled in the art can recognize or confirm many equivalent substitutions for specific aspects of this disclosure using only conventional experiments. Moreover, it is intended that these equivalent substitutions be included in this disclosure.

[0032] Furthermore, numerous academic papers and patent documents are referenced and cited throughout the specification. The disclosures of these cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter to which this disclosure pertains.

[0033] One aspect of this disclosure provides a modified polypeptide having deoxynivalenol (DON) degradation activity, wherein an amino acid at a position corresponding to any one or more positions selected from positions 28, 29, 87, 91, 101, and 152 of SEQ ID NO: 1 is replaced by another amino acid.

[0034] As used in this article, "deoxynivalenol (DON) degradation activity" refers to the catalytic degradation of deoxynivalenol (DON) by converting it into the isodon form.

[0035] In this disclosure, the chemical formula of deoxynivalenol (DON) is C 15 H 20 O6, with a molecular weight of 296.3 g / mol (CAS No.: 51481-10-8), is a mycotoxin belonging to the trichothecene type B group. Trichothecene type B toxins are a type of epoxysesquiterpenoid and are also known as vomitoxins. Deoxynivalenol can be produced by fungi that cause plant diseases, with *Fusarium* species being known producers. Ingestion can cause symptoms such as immunosuppression, anemia, headache, nausea, and abdominal pain. In animals, it can cause symptoms such as loss of appetite, vomiting, growth inhibition, and reproductive disorders.

[0036] Meanwhile, the degradation of deoxynivalenol (DON) can be used interchangeably with the detoxification, inactivation, and removal of DON contamination (decontamination).

[0037] Although the modified polypeptides with DON degradation activity provided in this disclosure are defined as polypeptides having amino acid substitutions at positions corresponding to any one or more of positions selected from SEQ ID NO: 1 at positions 28, 29, 87, 91, 101, and 152, this does not exclude the addition of meaningless sequences upstream or downstream of the amino acid sequence of SEQ ID NO: 1, the presence of naturally occurring mutations or their silenced mutations, and it will be apparent to those skilled in the art that any protein having the same or corresponding activity as a protein composed of the amino acid sequence of SEQ ID NO: 1 can belong to the polypeptides with DON degradation activity provided in this disclosure.

[0038] In other words, although described in this disclosure as "a protein or polypeptide having the amino acid sequence shown in a specific SEQ ID NO" or "a protein or polypeptide containing the amino acid sequence shown in a specific SEQ ID NO", it is obvious that any protein with a missing, modified, substituted or added amino acid sequence in a portion of the sequence may also be used in this disclosure, as long as the protein can have the same or corresponding activity as the polypeptide composed of the amino acid sequence of the corresponding SEQ ID NO.

[0039] As used herein, the term "wild-type" refers to a naturally occurring state without artificial modifications. When the term "wild-type" is used to refer to a polypeptide, it refers to a naturally occurring polypeptide without artificial mutations (substitutions, insertions, deletions, etc.) at one or more amino acid positions. Similarly, when the term "wild-type" is used to refer to a polynucleotide, it means without artificial modifications (substitutions, insertions, deletions) in one or more nucleotides. However, polynucleotides encoding wild-type polypeptides are not limited to wild-type polynucleotides, but include sequences encoding any wild-type polypeptide.

[0040] As used herein, the parental sequence or backbone refers to the reference sequence in which modifications are introduced to serve as a reference for the modified polypeptide. That is, the parental sequence can serve as a starting sequence in which mutations, such as substitutions, insertions, and / or deletions, can be introduced. The parental sequence can be naturally occurring or wild-type, or a variant of the natural or wild-type sequence with one or more substitutions, insertions, or deletions, or it can be a synthetically produced sequence. When the parental sequence is an active amino acid sequence, i.e., the amino acid sequence of an enzyme, it can be called a parent enzyme.

[0041] Regarding the amino acid or nucleic acid sequences in this disclosure, the term "fragment" refers to a portion of the parental sequence. For example, it can be a polypeptide in the form of one or more amino acids removed from the C-terminus or N-terminus of the parental sequence.

[0042] As used herein, the term "fraction" of an enzyme can refer to a "functional fragment." A "functional fragment" can also be called an active fragment and refers to a polypeptide that is part of the parent enzyme and has the enzymatic activity of the parent enzyme. For example, the functional fragment of an enzyme can contain the enzyme's catalytic site.

[0043] A fragment of an enzyme may contain a portion of the full length of the parent enzyme. For example, a fragment of an enzyme may contain at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more or less than 100% of the full length of the parent enzyme, but is not limited thereto.

[0044] As used in this article, the term "modification" means change or alteration. It can be a change from a naturally occurring state. For example, an enzyme can be modified in such a way that the enzyme is altered from a parental sequence or a reference sequence.

[0045] In this disclosure, the modified enzyme can be an enzyme that does not exist in nature, i.e., an enzyme that does not exist naturally.

[0046] As used herein, the term "modified" refers to, for example, a change from its naturally occurring form. Modified enzymes of this disclosure include enzymes that are not naturally occurring or naturally occurring variants. For example, the modified enzymes of this disclosure are modified enzymes that have not yet been found in nature. For example, the modified enzymes of this disclosure can be non-spontaneously occurring enzymes, but are not limited thereto.

[0047] As used herein, the term "modification" when applied to an amino acid / nucleic acid sequence may include: replacing an amino acid / nucleic acid residue of a parent sequence with another amino acid / nucleic acid residue at one or more positions in the amino acid sequence; deleting an amino acid / nucleic acid residue (or a series of amino acid / nucleic acid residues) at one or more positions in the parent sequence; inserting an amino acid / nucleic acid residue (or a series of amino acid / nucleic acid residues) at one or more positions in the parent sequence; truncating an N-terminal and / or C-terminal amino acid sequence or a 5' and / or 3' nucleic acid sequence, and any combination thereof.

[0048] As used herein, the term "variant" or "modified polypeptide" of an enzyme refers to a protein that has one or more amino acids different from the parent enzyme through conserved substitution and / or modification. "Variant" and "modified polypeptide" are used interchangeably. Variants or modified polypeptides may be non-natural, but are not limited to this.

[0049] Variants differ from the parent enzyme sequence through one or more modifications (e.g., amino acid substitution, deletion, and / or insertion).

[0050] These variants can typically be identified by modifying one or more amino acids of the parent enzyme and assessing the properties of the modified protein. That is, the ability of the variant may be enhanced, unchanged, or reduced compared to the ability of the parent enzyme.

[0051] In addition, some variants may include one or more of these regions, such as the N-terminal leader sequence or the modified polypeptide with the transmembrane domain removed.

[0052] Other variants may include those in which a portion has been removed from the N-terminus and / or C-terminus of the mature protein.

[0053] The term “variant” or “modified polypeptide” may be used interchangeably with, and is not limited to, terms such as modification, modified protein, mutant, mutated protein, diverter, variant, etc., as long as the term is used to refer to mutation.

[0054] Variants can also contain the deletion or addition of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, a polypeptide can be conjugated at the N-terminus with a signal (or leader) sequence that participates in protein translocation in a co-translational or post-translational manner. Furthermore, polypeptides can be conjugated with another sequence or linker to identify, purify, or synthesize polypeptides.

[0055] As used herein, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions can typically occur based on the similarity of the residues' polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties.

[0056] As used herein, “parental DON-degrading enzyme” refers to a DON-degrading enzyme modified for the production of the variant or modified polypeptide of this disclosure. Specifically, the parental DON-degrading enzyme, parental enzyme, or parental sequence may be a naturally occurring polypeptide or a wild-type polypeptide, or a mature polypeptide thereof, and may contain, but is not limited to, a variant or a functional fragment thereof, as long as the polypeptide has DON-degrading activity and can be a parent of the variant.

[0057] The parental DON-degrading enzyme disclosed herein may be, but is not limited to, the polypeptide of SEQ ID NO: 1. Furthermore, it may be a polypeptide having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the polypeptide of SEQ ID NO: 1, as long as it has DON-degrading activity, and any polypeptide may be included within the scope of parental DON-degrading enzymes, provided it has the same or corresponding activity as the polypeptide composed of the amino acid sequence of SEQ ID NO: 1.

[0058] The parental DON-degrading enzyme of the variants disclosed herein can be derived from the genera *Gossypium*, *Handroanthus*, or *Hibiscus*. Specifically, it can be derived from the genus *Gossypium*.

[0059] In this disclosure, the "modified polypeptide with DON degradation activity" may be a variant of the parental DON-degrading enzyme.

[0060] As used herein, the terms “variant of parental DON-degrading enzyme” or “modified polypeptide with DON-degrading activity” refer to proteins with DON-degrading activity in which one or more amino acids have an amino acid sequence different from that of the parental DON-degrading enzyme.

[0061] "Modified polypeptide with DON degradation activity", "variant of parental DON degrading enzyme" and "DON degrading enzyme variant" can be used interchangeably.

[0062] The modified polypeptides disclosed herein may contain one or more amino acids modified in the parental DON-degrading enzyme sequence, thereby possessing DON-degrading activity. This modification may be an amino acid substitution and / or the formation of disulfide bonds.

[0063] Specifically, by modifying the sequence of the parental DON degrading enzyme by including one or more amino acids, the variants provided in this disclosure may have one or more altered functions or properties compared to the parental DON degrading enzyme, while still possessing DON degrading activity.

[0064] In one specific embodiment, by including one or more amino acid modifications in the sequence of the parental DON degrading enzyme, the variants provided in this disclosure may have one or more altered functions or properties compared to the parental DON degrading enzyme, and may have one or more conserved substitutions while possessing DON degrading activity.

[0065] The variants provided in this disclosure are variants of the parental DON-degrading enzyme and may be polypeptides having DON-degrading activity. In one specific embodiment, the variants provided in this disclosure may contain modifications at positions corresponding to one or more of positions selected from SEQ ID NO: 1, specifically positions 28, 29, 87, 91, 101, and 152.

[0066] In this disclosure, a position number refers to the position corresponding to the polypeptide position of SEQ ID NO: 1, and the term "corresponding" is used as described above.

[0067] In one specific embodiment, compared with the amino acid sequence of SEQ ID NO: 1, the modified polypeptide of this disclosure may contain amino acid substitutions at positions corresponding to one or more of positions selected from positions 28, 29, 87, 91, 101 and 152 of SEQ ID NO: 1, but is not limited thereto.

[0068] In specific instances, the modified polypeptides provided in this disclosure may include substitutions at 2, 3, 4, 5, or 6 positions selected from the positions of other amino acids.

[0069] In any of the above specific embodiments, the modified polypeptide may include amino acid modifications at positions selected from i) to iii):

[0070] i) 28 bits; ii) 29 bits; iii) 87 bits; iv) 91 bits; v) 101 bits; vi) 152 bits; vii) 28+29 bits; viiii) 28+87 bits; ix) 28+91 bits; x) 28+101 bits; xi) 28+152 bits; xii) 29+87 bits; xiii) 29+91 bits; xiv) 29+101 bits; xv) 29+152 bits; xvi) 29+152 bits; xvii) 87+91 bits; xviii) 87+101 bits; xix) 87+152 bits; xx) 91+101 bits; xxi) 91+152 bits; xxii) 101+152 bits; xxiii) 28+ 29+87 bits; xxiv) 28+29+101 bits; xxv) 28+29+152 bits; xxvi) 28+87+91 bits; xxvii) 28+87+101 bits; xxviii) 28+87+152 bits; xxix) 28+91+101 bits; xxx) 28+91+152 bits; xxxi) 28+101+152 bits; xxxii) 29+87+91 bits; xxxiii) 29+87+101 bits; xxxiv) 29+87+152 bits; xxxv) 28+91+101 bits; xxxvi) 29+91+152 bits; xxxvii) 29+101+ 152-bit; xxxviii) 87+91+ 101-bit; xxxix) 87+ 91+ 152-bit; xl) 87+ 101+ 152-bit; xli) 91+ 101+ 152-bit; xlii) 28+ 29+ 87+ 91-bit; xliii) 28+ 29+ 87+ 101-bit; xliv) 28+ 29+ 87+ 152-bit; xlv) 28+ 29+ 91+ 101-bit; xlvi) 28+ 29+ 91+ 152-bit; xlvii) 28+ 29+ 101+ 152-bit; xlviii) 28+ 87+91+ 101-bit; xlix) 28+ 87+ 91+ 152 bits; l) 28+ 87+ 101+ 152 bits; li) 28+ 91+ 101+ 152 bits; lii) 29+ 87+ 91+ 101 bits; liii) 29+ 87+ 91+ 152 bits; liv) 29+ 87+ 101+ 152 bits; lv) 29+ 91+ 101+ 152 bits; lvi) 87+ 91+ 101+ 152 bits; lvii) 28+ 29+ 87+ 91+ 101 bits;lviii) 28+ 29+ 87+ 91+ 152 bits; lix) 28+ 29+ 87+ 101+ 152 bits; lx) 28+ 29+ 91+ 101+ 152 bits; lxi) 28+ 87+ 91+ 101+ 152 bits; lxii) 29+ 87+ 91+ 101+ 152 bits; and lxiii) 28+ 29+ 87+91+ 101+ 152.

[0071] In this document, the position number refers to the position corresponding to the polypeptide position of SEQ ID NO: 1.

[0072] In any of the above specific embodiments, the modified polypeptide provided in this disclosure may contain amino acids at positions corresponding to any one or more positions selected from SEQ ID NO: 1, such that the amino acid is replaced by G, A, V, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R or H.

[0073] In any of the above specific embodiments, the modified polypeptide provided in this disclosure may contain amino acids at positions corresponding to any one or more positions selected from SEQ ID NO: 1, such that the amino acids are replaced by polar, nonpolar, hydrophobic or acidic amino acids.

[0074] Furthermore, the modified polypeptides provided in this disclosure may contain an amino acid at position 28 corresponding to SEQ ID NO: 1 that has been replaced by isoleucine.

[0075] Furthermore, the modified polypeptides provided in this disclosure may contain an amino acid at position 29 corresponding to SEQ ID NO: 1 that has been replaced by leucine.

[0076] Furthermore, the modified polypeptides provided in this disclosure may contain an amino acid corresponding to position 87 of SEQ ID NO: 1 replaced by isoleucine.

[0077] Furthermore, the modified polypeptides provided in this disclosure may contain an amino acid at position 91 corresponding to SEQ ID NO: 1 that has been replaced by serine.

[0078] Furthermore, the modified polypeptides provided in this disclosure may contain an amino acid corresponding to position 101 of SEQ ID NO: 1 replaced by phenylalanine.

[0079] Furthermore, the modified polypeptides provided in this disclosure may contain an amino acid corresponding to position 152 of SEQ ID NO: 1 replaced by glycine.

[0080] Furthermore, the modified polypeptides provided in this disclosure may include the amino acid at position 28 of SEQ ID NO: 1 being replaced by isoleucine; and the amino acid at position 101 of SEQ ID NO: 1 being replaced by phenylalanine.

[0081] Furthermore, the modified polypeptides provided in this disclosure may include the amino acid at position 28 of SEQ ID NO: 1 being replaced by isoleucine; and the amino acid at position 152 of SEQ ID NO: 1 being replaced by glycine.

[0082] Furthermore, the modified polypeptide provided in this disclosure may include the amino acid corresponding to position 28 of SEQ ID NO: 1 being replaced by isoleucine; the amino acid corresponding to position 101 of SEQ ID NO: 1 being replaced by phenylalanine; and the amino acid corresponding to position 152 of SEQ ID NO: 1 being replaced by glycine.

[0083] In any of the above specific embodiments, the modified polypeptide may include one or more substitutions selected from the following substitutions:

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] In any of the above specific embodiments, the modified polypeptide provided in this disclosure may include 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: 31, SEQ ID NO: 33 or SEQ ID NO: 35.

[0091] 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 to, identical to, or homologous to a residue listed in a protein or polypeptide. Identifying an amino acid at a corresponding position can be used to determine a specific amino acid in a sequence of a particular sequence. As used herein, "corresponding region" typically refers to a similar or corresponding position in a related or reference protein.

[0092] In this disclosure, SEQ ID NO: 1 can be used as a reference sequence to determine the position of an amino acid in any amino acid sequence.

[0093] As used herein, the term "reference sequence" refers to a sequence used to determine the position of an amino acid within an arbitrary amino acid sequence. An arbitrary amino acid sequence can be aligned with a reference sequence to determine the position of an amino acid in the arbitrary amino acid sequence that corresponds to a specific position in the reference sequence.

[0094] In other words, SEQ ID NO: 1 disclosed herein can be used to identify the corresponding amino acid residues in any polypeptide exhibiting DON degradation activity. The residues in a specific amino acid sequence are based on the SEQ ID NO: 1 number, unless otherwise stated in this disclosure.

[0095] For example, by comparing any amino acid sequence with SEQ ID NO: 1, each amino acid residue in the sequence can be numbered based on the numerical position of the amino acid residues corresponding to the amino acid residues in SEQ ID NO: 1. For example, the sequence alignment algorithm described herein can identify the position of an amino acid or the position of a modification such as substitution, insertion, or deletion compared to the query sequence (also known as the “reference sequence”).

[0096] In such comparisons, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol. Biol. 48: 443-453), the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), etc., can be used, but are not limited to these.

[0097] In addition, the corresponding amino acid residues in another polypeptide with DON degradation activity can be identified by multiple sequence alignment. Examples of known multiple sequence alignments in this field include MUSCLE (multiple sequence alignment by logarithmic 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: ). The program can be used with ClustalW (1899-1900) and EMBOSSEMMA (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), and can use their respective default parameters, but is not limited to them.

[0098] Furthermore, when enzymes derived from the mature polypeptide of SEQ ID NO: 1 cannot be correlated using conventional sequence-based comparisons, alternative pairwise sequence comparison algorithms can be used (Lindahl and Elofsson, 2000, J.Mol. Biol. 295: 613-615). Higher sensitivity for sequence-based searches can be achieved using search procedures that search a database using probabilistic representations (profiles) of polypeptide families. For example, the PSI BLAST procedure generates profiles through iterative database searches and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even higher sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives 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) utilize information from various sources, such as PSI BLAST, secondary structure prediction, structural alignment maps, and solvation potential, as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919 can be used to align sequences with unknown structures to superfamily models existing in the SCOP database. These alignments can be used to sequence sequences to generate homology models of peptides, and the accuracy of these models can be evaluated using various tools developed for this purpose.

[0099] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins33: 88-96) or CE (Combinatorial extension) (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747). Implementations of these algorithms can also be used to query structure databases containing structures of interest to discover potential structural homologs (Holm and Park, 2000, Bioinformatics 16: 566-567).

[0100] The methods described above are illustrative and not limited thereto.

[0101] Throughout this disclosure, standard single-letter and three-letter codes for naturally occurring amino acids are used. Furthermore, the abbreviations for amino acids used herein are in accordance with the IUPAC-IUB nomenclature rules.

[0102] Alanine (Ala), A; Arginine (Arg), R

[0103] Asparagine Asn, N-aspartic acid Asp, D

[0104] Cysteine ​​(Cys), C; Glutamate (Glu), E

[0105] Glutamine (Gln, Q) and glycine (Gly, G)

[0106] Histidine (His, H) and Isoleucine (Ile, I)

[0107] Leucine (Leu), L-lysine (Lys), K

[0108] Methionine (Met, M) and Phenylalanine (Phe, F)

[0109] Proline (Pro, P) and Serine (S)

[0110] Threonine (Thr), T-tryptophan (Trp), W

[0111] Tyrosine (Tyr, Y) and Valine (V)

[0112] At the same time, any amino acid can be described as Xaa or X.

[0113] In addition, three-letter codes can usually be used not only for naturally occurring amino acids, but also for other amino acids, such as 2-aminoisobutyric acid (Aib), Sar (N-methylglycine), α-methyl-glutamic acid, etc.

[0114] Amino acids can generally be classified based on the similarity of their residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. Therefore, amino acid substitutions can typically occur based on the similarity of these residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity.

[0115] For example, among amino acids with charged side chains (charged amino acids), positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; among amino acids with uncharged side chains (uncharged amino acids), nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and among nonpolar amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0116] In one embodiment, the modified polypeptide with DON degradation activity provided in this disclosure may include a modified polypeptide wherein an amino acid corresponding to one or more positions selected from SEQ ID NO: 1, such as positions 28, 29, 87, 91, 101, and 152, is replaced, 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, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 33, or SEQ ID NO: 35. In another embodiment, the novel modified polypeptide with DON degradation activity provided in this disclosure may have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, 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, SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 33 or SEQ ID NO: 35, or may contain sequences having said homology or identity, or may be substantially composed of or may be composed of.

[0117] As used herein, the terms “homology” or “identity” refer to the degree of correlation between two given amino acid sequences or nucleotide sequences and can be expressed as a percentage. The terms “homology” and “identity” are generally used interchangeably.

[0118] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, and can utilize default gap penalties established by the program used. Essentially, homologous or identical sequences can typically hybridize with at least approximately 50%, 60%, 70%, 80%, or 90% of the full-length sequence under moderately or highly stringent conditions. Clearly, hybridization also involves polynucleotides hybridizing with polynucleotides containing universal codons or codons that take codon degeneracy into account.

[0119] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined, for example, using known computer algorithms, such as the “FASTA” program with default parameters as described in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), such as using the Needleman program (version 5.0.0 or later) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (GCG 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.]). The method described in (1988) SIAMJ Applied Math 48: 1073) can be used, for example, to determine homology, similarity, or identity using BLAST or ClustalW from the National Center for Biotechnology Information, but is not limited thereto.

[0120] Homology, similarity, or identity of polynucleotides or polypeptides can be determined, for example, by comparing sequence information using computer programs such as GAP, as disclosed in Needleman et al. (1970), J Mol Biol. 48: 443, and Smith and Waterman, Adv. Appl. Math (1981) 2:482. In short, the GAP program defines homology, similarity, or identity as a value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP procedure may include: (1) a binary comparison matrix (with a value of 1 for identity and a value of 0 for non-identity), and a weighted comparison matrix as disclosed in Schwartz and Dayhoff, ed., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or the EDNAFULL permutation matrix (EMBOSS version of NCBI NUC4.4)); (2) a penalty of 3.0 for each empty space, and an additional penalty of 0.10 for each symbol in each empty space (or a penalty of 10 for an open empty space and a penalty of 0.5 for an extended empty space); and (3) no penalty for terminal empty spaces.

[0121] Furthermore, whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical to each other can be determined by comparing the sequences under specific and stringent Southern hybridization conditions, and suitable hybridization conditions are within the scope 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; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0122] In any of the above specific embodiments, the modified polypeptide may have one or more altered properties among the following i) to vii) compared to the polypeptide composed of the amino acid sequence of SEQ ID NO: 1:

[0123] i) Increased enzyme activity;

[0124] ii) Increased specific activity;

[0125] iii) Increased pH stability;

[0126] iv) Increased storage stability;

[0127] v) Increased acid resistance;

[0128] vi) Increased thermal stability; and

[0129] vii) Modified substrate specificity.

[0130] As used herein, “enzyme activity” or “DON degradation activity” refers to at least one catalytic activity. Specifically, it can be the enzyme’s conversion efficiency, primarily expressed as kcat / Km, but is not limited thereto.

[0131] When an enzyme is completely saturated with substrate, kcat refers to the rate constant (catalytic constant) of an enzyme in converting substrate into product per unit time, also known as the turnover number. Km is the substrate concentration at which the reaction rate is half of its maximum value (Vmax).

[0132] Examples of ways to express enzyme activity include specific activity (µmol of substrate converted to mg). -1 x min -1 ) or volumetric activity (substrate converted in μmol x mL) -1 x min -1 However, the definition of enzyme activity is not limited to the above description, and enzyme activity can be defined and evaluated based on information known in the art.

[0133] As used herein, “enzyme stability” refers to the maintenance of enzyme activity during storage or reaction time. To measure changes in this stability, initial enzyme activity can be measured and compared under defined conditions at zero time (100%) and after a predetermined time period (x%), thus representing the level of enzyme activity loss or enzyme stability.

[0134] Factors affecting enzyme activity include, for example, pH, heat, and the presence of other substances (e.g., oxidants, chelating agents).

[0135] In one specific embodiment, the modified polypeptide provided in this disclosure may have an enzyme activity that is about 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200% or more, about 210% or more, about 220% or more, or about 230% or more, compared to the parent enzyme.

[0136] As used herein, the term "specific activity" refers to the enzyme activity per unit weight of protein, and can be expressed in units per mg. Protein quantification can be performed using, for example, SDS-PAGE or the Bradford assay.

[0137] As used herein, “enzyme stability” refers to the maintenance of enzyme activity during storage or reaction time. To measure changes in this stability, initial enzyme activity can be measured and compared under defined conditions at zero time (100%) and after a predetermined time (x%), thus representing the level of enzyme activity loss or enzyme stability.

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

[0139] As used herein, the term "thermal stability" refers to the ability of a protein to function within a specific temperature range. In one embodiment, the polypeptides provided in this disclosure may be active in the range of about 20°C to about 120°C, specifically, in the range of about 60°C to about 100°C. In any of the above embodiments, the polypeptides provided in this disclosure having DON degradation activity may be active in the range of 50°C to 80°C, but are not limited thereto.

[0140] As used herein, the term "thermosity" refers to the ability of a protein to function after exposure to a specific temperature, such as high heat or low temperature. For example, a thermostable protein may not function at the temperature it is exposed to, but may become functional when it returns to its optimal temperature environment.

[0141] As used herein, the term "pH stability" refers to the ability of a protein to function within a specific pH range. In one specific embodiment, the variants provided in this disclosure may be active at about pH 2.0 to about pH 12.0, but are not limited thereto.

[0142] When a protein maintains its function within a specific pH range, it can be defined as having "pH stability," or it can be defined as having "acid resistance," "alkali resistance," etc., depending on the pH range.

[0143] In one embodiment of this disclosure, the variants provided herein may have “acid resistance” at about pH 3.0 or lower, but are not limited thereto.

[0144] Increased stability may include maintaining high enzyme activity compared to other enzymes (e.g., wild-type enzymes, parental enzymes, and / or other modified peptides); and increasing the range of pH, temperature, and / or time at which the protein maintains its function.

[0145] In this disclosure, the increase in stability can be compared based on parental DON degrading enzymes, such as, but not limited to, the DON degrading enzyme based on SEQ ID NO: 1.

[0146] In one embodiment, the modified polypeptide with DON degradation activity provided in this disclosure can be improved in terms of one or more properties selected from heat resistance, thermal stability and acid resistance compared with the polypeptide of SEQ ID NO: 1.

[0147] In any of the above embodiments, the modified polypeptide can maintain a relative activity of at least 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 37% or more, or 40% or more at 50°C to 80°C, but is not limited thereto.

[0148] In any of the above embodiments, the modified peptide may maintain a relative activity of at least 1% or more, 3% or more, 5% or more, 8% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more at pH 3 or lower, but is not limited thereto.

[0149] In any of the above embodiments, the modified polypeptide having increased enzymatic activity (improved DON degradation activity) compared to the polypeptide of SEQ ID NO. 1 may include any one or more substitutions selected from the following substitutions.

[0150]

[0151]

[0152]

[0153] In any of the above embodiments, the modified polypeptide having improved heat resistance compared to the polypeptide of SEQ ID NO. 1 may include any one or more substitutions selected from the following substitutions.

[0154]

[0155]

[0156] In any of the above embodiments, the modified polypeptide having improved acid resistance compared to the polypeptide of SEQ ID NO. 1 may include one or more substitutions selected from the following substitutions.

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] Increased stability may include maintaining high enzyme activity compared to other enzymes (e.g., wild-type enzymes, parental enzymes, and / or other variants); and increasing the range of pH, temperature, and / or time at which the protein maintains its function.

[0163] Decreased stability may include maintaining low enzyme activity compared to other enzymes (e.g., wild-type enzymes, parental enzymes, and / or other variants); or reducing the range of pH, temperature, and / or time at which the protein maintains its function.

[0164] In any of the above embodiments, the properties of the modified polypeptide with DON degradation activity provided in this disclosure may be suitable for or have improved activity for application in various industrial fields, including feed, baking, pulp bleaching, etc.

[0165] Another aspect of this disclosure provides a polynucleotide encoding a modified polypeptide having DON degradation activity as disclosed herein.

[0166] As used herein, the term "polynucleotide" refers to a long-chain polymer of nucleotides formed by covalently linking nucleotide monomers, meaning a DNA or RNA chain of predetermined length or longer.

[0167] The polynucleotide encoding the modified polypeptide with DON degradation activity disclosed herein may include any polynucleotide without limitation, as long as it is a polypeptide or a polynucleotide encoding the modified polypeptide; 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: 31, SEQ ID NO: 33, or SEQ ID NO: 35.

[0168] Due to codon degeneracy or taking into account preferred codons in the organism to which the polypeptide is to be expressed, the polynucleotides encoding modified polypeptides with DON degradation activity disclosed herein can be modified in various ways in the coding region without altering the amino acid sequence of the polypeptide.

[0169] For example, the polynucleotide encoding the modified polypeptide with DON degradation activity disclosed herein may have the 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: 31, SEQ ID NO: 33, or SEQ ID NO: 35, or the polynucleotide encoding a polypeptide that is homologous or identical to it.

[0170] In one embodiment, the polynucleotide encoding the modified polypeptide having DON degradation activity disclosed herein may have or contain a nucleotide sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, or 90% or more and less than 100% homology or identity with the 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: 32, SEQ ID NO: 34, or SEQ ID NO: 36. In another embodiment, the polynucleotide encoding the modified polypeptide having DON degradation activity of this disclosure may consist of, or substantially consist of, nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the sequences 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: 32, SEQ ID NO: 34, or SEQ ID NO: 36, but is not limited thereto. Furthermore, the polynucleotide of this disclosure may include probes that can be prepared from known gene sequences, such as any sequence capable of hybridizing under stringent conditions with all or part of the complementary sequence of the polynucleotide sequence of this disclosure, without limitation.

[0171] “Strict conditions” refer to conditions that enable specific hybridization between polynucleotides. Such conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, stringent conditions may include conditions under which polynucleotides with high homology or identity, 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, while polynucleotides with less homology or identity do not hybridize with each other, or typical Southern hybridization washing conditions, i.e., washing once, particularly twice or three times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0172] Hybridization requires two nucleic acids to contain complementary sequences, although mismatches between bases are possible depending on the strictness 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 this disclosure may comprise separate nucleotide fragments complementary to the whole sequence as well as nucleic acid sequences substantially similar to them.

[0173] Specifically, hybridization conditions including a hybridization step with a Tm value of 55°C can be used to detect polynucleotides homologous to or identical to the polynucleotides disclosed herein under the above conditions. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to their purpose.

[0174] The appropriate stringency of hybrid polynucleotides depends on the length and complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., above).

[0175] Another aspect of this disclosure provides a vector comprising a polynucleotide encoding a modified polypeptide of the present disclosure having DON degradation activity. The modified polypeptide and polynucleotide are as described in other aspects.

[0176] As used herein, the term "vector" refers to a DNA construct containing a nucleotide sequence of a polynucleotide encoding a desired polypeptide, operatively linked to a suitable expression regulatory region (expression regulatory sequence) to enable expression of the desired polypeptide in a suitable host cell. The expression regulatory region may contain a promoter capable of initiating transcription, any operon sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating transcription and translation termination. Once transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or it can integrate into its genome.

[0177] For example, a polynucleotide encoding a desired protein can be replaced with a mutated polynucleotide in the chromosome using a vector for intracellular chromosomal insertion. Insertion of the polynucleotide into the chromosome can be performed by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may also contain selection markers to confirm insertion into the chromosome. These selection markers are used to select cells transformed by the vector, i.e., to confirm the insertion of the desired nucleic acid molecule, and may use markers that provide selectable phenotypes (such as drug resistance, auxotrophic phenotype, cytotoxic agent resistance, or surface peptide expression). Only cells expressing the selection markers are able to survive or exhibit a different phenotype under conditions treated with a selection agent, thus allowing for the selection of transformed cells.

[0178] There are no particular limitations on the carriers used in this disclosure; any carrier known in the art may be used.

[0179] Examples of commonly used vectors in prokaryotic cells include pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A as phage or granular vectors; and those based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET as plasmid vectors. Specifically, vectors such as pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pDCM2 can be used.

[0180] Examples of vectors used in eukaryotic cells may include integrative yeast plasmids (YIp) and extrachromosomal plasmid vectors as yeast expression vectors. Extrachromosomal plasmid vectors may include augmentative yeast plasmids (YEp), replicative yeast plasmids (YRp), and yeast centromere plasmids (YCp). Alternatively, artificial yeast chromosomes (YAC) may also be used as vectors in this disclosure. Specific examples of available vectors may 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, P TEF-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, pYJ404, pYJ405 and pYJ406, but not limited to these.

[0181] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target protein into a host cell or microorganism, thereby enabling the expression of the protein encoded by that polynucleotide within the host cell. The transformed polynucleotide can be localized by insertion into the host cell's chromosome or located extrachromosomally, regardless of its position, as long as it can be expressed within the host cell. Furthermore, the polynucleotide includes DNA and RNA encoding the desired protein. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, a gene construct containing all the elements necessary for autonomous expression. Expression cassettes typically include a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the polynucleotide. Expression cassettes can be in the form of a self-replicating expression vector. Furthermore, the polynucleotide can be introduced into the host cell in its own form and operably linked to the sequence required for expression within the host cell, but is not limited thereto.

[0182] Furthermore, the term "operably linked" refers to the functional linking of a gene sequence with a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding the polypeptide desired in this disclosure.

[0183] Methods for transforming the vectors disclosed herein include any method of introducing nucleic acids into cells and can be performed by selecting appropriate standard techniques known in the art according to the host cell. For example, transformation methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) technology, DEAE-dextran technology, cationic liposome technology, lithium acetate-DMSO technology, etc.

[0184] Another aspect of this disclosure provides microorganisms comprising one or more of the following: a modified polypeptide of the present disclosure having DON degradation activity; and a polynucleotide encoding said modified polypeptide.

[0185] The microorganism may contain the aforementioned modified polypeptide, the polynucleotide encoding it, a nucleic acid construct containing the polynucleotide, and / or a vector. The nucleic acid construct or vector may be integrated into the chromosome or retained as a self-replicating extrachromosomal vector.

[0186] The microorganisms disclosed herein may include any microorganism without limitation, as long as they are capable of expressing the modified polypeptides of this disclosure that have DON-degrading activity. For example, the microorganisms may include any cells that can be used for the recombinant production of the modified polypeptides of this disclosure that have DON-degrading activity. For example, the microorganisms may be prokaryotic or eukaryotic cells. As another example, examples of the microorganisms may include fungi and bacteria.

[0187] In one embodiment, the microorganism disclosed herein may be a microorganism of the genus *Escherichia*, and may be *Escherichia coli*, *E. coli*, *Escherichia albertii*, *Escherichia fergusonii*, *Escherichia hermannii*, *Escherichia vulneris*, or *Escherichia blattae*. In any of the foregoing embodiments, the microorganism may be *Escherichia coli*, but is not limited thereto.

[0188] Another aspect of this disclosure provides compositions for degrading deoxynivalenol (DON), said compositions comprising one or more of the following: a modified polypeptide of the present disclosure having DON-degrading activity; and a microorganism expressing said modified polypeptide.

[0189] Another aspect of this disclosure provides a method for degrading deoxynivalenol (DON), the method comprising the step of reacting DON with any one or more of the following: a modified polypeptide of the present disclosure having DON-degrading activity; and a microorganism expressing the modified polypeptide.

[0190] The modified polypeptides with DON degradation activity disclosed herein and / or microorganisms expressing the modified polypeptides can be used to degrade substances containing DON.

[0191] In one specific embodiment, the disclosed polypeptide can be used for the degradation of DON toxin, the detoxification of DON, the conversion of DON (deoxynivalenol) to iso-DON (isodeoxynivalenol), and / or the induction of degradation through the conversion of iso-DON.

[0192] Furthermore, in another embodiment of this disclosure, the polypeptide of this disclosure can be used for the detoxification of food contaminated with DON, the removal (decontamination) of contaminants, or detoxification (e.g., degradation).

[0193] In one embodiment, in addition to the modified polypeptides of this disclosure, the composition provided in this disclosure for degrading DON may also contain naturally occurring or non-naturally occurring substances.

[0194] In one embodiment, the composition for degrading DON provided in this disclosure may further contain any component suitable for various industrial applications such as animal feed, baking, biomass saccharification, pulp bleaching, etc.

[0195] Examples of substances that can be added include, but are not limited to, stabilizers, surfactants, building blocks, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, binders, lubricants, disintegrants, excipients, solubilizers, suspending agents, colorants, flavorings, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, diluents, lubricants, and preservatives.

[0196] Another aspect of this disclosure provides a feed additive composition comprising one or more of the following: a modified polypeptide of the present disclosure having DON degradation activity; and a microorganism expressing the modified polypeptide.

[0197] Another aspect of this disclosure provides a method for preparing a feed product, the method comprising the step of mixing feed components with a feed additive composition comprising one or more of the following: a modified polypeptide of the present disclosure having DON degradation activity; and a microorganism expressing the modified polypeptide.

[0198] The modified peptides with DON degradation activity disclosed herein and / or microorganisms expressing said modified peptides can be used for any of the following applications:

[0199] a) Additives in animal feed; and / or

[0200] b) Animal feed supplements; and / or

[0201] c) Decomposition of grain-based materials (e.g., which may be whole grains or partial grains).

[0202] The feed composition disclosed herein may refer to any natural or artificial diet, mono-diet, or component of a mono-diet, which is consumed, ingested, and digested by animals, or is suitable for such diet, and the feed composition may be prepared in various forms known in the art.

[0203] In one implementation, the feed product may be a grain-based material (including whole or partial grains or malted grains, such as wheat, barley, corn, oats, rye, rice, sorghum, etc.).

[0204] Another aspect of this disclosure provides a method for preparing modified peptides with DON degradation activity, the method comprising the following steps:

[0205] Cultivating microorganisms comprising one or more of the following: a modified polypeptide of the present disclosure having DON degradation activity; and a polynucleotide encoding the modified polypeptide; and

[0206] The modified polypeptides of this disclosure with DON degradation activity expressed during the culture step are recovered.

[0207] Cultivation can include the step of culturing microorganisms in a culture medium.

[0208] As used herein, the term "culture" refers to the growth of host cells under appropriately controlled environmental conditions. The culture process of this disclosure can be performed in suitable culture media and culture conditions known in the art. Such a culture process can be readily adapted for use by those skilled in the art according to the strain to be selected. Specifically, the culture can be a batch culture, a continuous culture, or a fed-batch culture, but is not limited thereto.

[0209] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients required for culturing host cells as its main components, providing nutrients and growth factors, as well as water necessary for survival and growth. Specifically, the culture medium and other culture conditions used to culture the host cells of this disclosure can include any culture medium commonly used for culturing host cells without any particular limitation. However, the host cells of this disclosure can be cultured under aerobic conditions in a general culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while simultaneously adjusting temperature, pH, etc.

[0210] In this disclosure, carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, molasses, rice bran, cassava, cane molasses, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used. Furthermore, various other carbon sources may be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0211] Nitrogen sources can 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, and glutamine; and organic nitrogen sources, such as peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its decomposition products. These nitrogen sources can be used alone or in combination of two or more of them, but are not limited thereto.

[0212] Phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric 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 culture medium in batches or continuously, but are not limited thereto.

[0213] Furthermore, during the appropriate culture of host cells, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid. Additionally, foam formation can be prevented during culture by using antifoaming agents such as polyethylene glycol fatty acids. Moreover, oxygen or oxygen-containing gases can be injected into the culture medium to maintain aerobic conditions; alternatively, no gas injection may be necessary, or nitrogen, hydrogen, or carbon dioxide gas may be injected to maintain anaerobic or micro-aerobic conditions, but these are not limited to these methods.

[0214] The temperature of the culture medium can be from 20°C to 55°C, specifically from 25°C to 40°C, but is not limited thereto. The culture can continue until the desired amount of useful material is obtained, and can specifically be carried out from 24 hours to 196 hours, but is not limited thereto.

[0215] In one embodiment, the modified polypeptide with DON-degrading activity expressed during the culture step can be recovered using methods known in the art to which this disclosure pertains. For example, the modified polypeptide can be recovered from the culture medium using conventional procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0216] Recovery methods may involve collecting peptides using the microbial culture methods disclosed herein, for example, using suitable methods known in the art according to batch culture, continuous culture, or fed-batch culture methods. For example, methods such as centrifugation, filtration, treatment with a protein crystallizing precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, and combinations thereof can be used, and suitable methods known in the art can be used to recover the modified peptides of this disclosure from the culture medium or host cells.

[0217] In another embodiment, the modified peptide expressed by the host cell during the culture step may not be recovered. In this embodiment, the host cell expressing the modified peptide can itself be used as a source of the modified peptide.

[0218] Another aspect of this disclosure provides the use of the modified peptides of this disclosure for the degradation of DON; and the use of DON by one or more microorganisms expressing the modified peptides.

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

[0220] Methods of implementing the present invention

[0221] The present disclosure will be described in more detail below with reference to embodiments and experimental examples. However, these embodiments and experimental examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these embodiments and experimental examples.

[0222] Example 1: Preparation of GhSPG-216M2 variant

[0223] Example 1-1. Preparation of GhSPG-216M2

[0224] A variant (GhSPG-216; SEQ ID NO: 21) was synthesized by Cosmo Genetech, in which 35 mutations were introduced into a putative protein sequence (SEQ ID NO: 23) derived from Gossypium harknessii. The polynucleotide (SEQ ID NO: 2) encoding a variant with two additional mutations (hereinafter referred to as GhSPG-216M2, SEQ ID NO: 1) was then cloned into the pET vector (Novagen) to prepare an expression vector.

[0225] Examples 1-2. Preparation of GhSPG-216M2 point mutants and combinatorial variants

[0226] To improve the activity, thermal stability, and acid resistance of GhSPG-216M2, mutation sites were selected and primers were designed to prepare nine point mutations and combinatorial variants (hereinafter referred to as 216M3, 216M4, 216M5, 216M6, 216M7, 216M8, 216M9, 216M10, and 216M11, SEQ ID NO: 3, 5, 7, 9, 11, 13, 31, 33, and 35). The mutation sites based on the amino acid sequence of SEQ ID NO: 1, the mutated amino acids, and the primer sequences used to prepare the variants are listed in Table 1 below.

[0227] [Table 1]

[0228]

[0229] Specifically, using the polynucleotide encoding GhSPG-216M2 (SEQ ID NO: 1) prepared in Example 1-1 and cloned into the pET vector as a template, primers (SEQ ID NOs: 15, 16, 17, 18, 19, 20, 25, 26, 27, 28, 29, 30 and combinations thereof in Table 1), and PCR premix (iNtRON, CAT No. 25185), nine point mutations and combinatorial variants of GhSPG-216M2 were prepared by PCR. PCR was performed using an Eppendorf Mastercycler Nexus GX2 under the following reaction conditions:

[0230] Initial denaturation — 94℃, 2 min

[0231] Denaturation—94℃, 20 sec

[0232] Annealing — 50℃, 10 sec

[0233] Extension—72℃, 8 min (15 cycles from denaturation to extension)

[0234] Final extension—72℃, 5 min

[0235] The truncated variants prepared using the QuickChange site-directed mutagenesis kit (Agilent, Cat# 200518) were then transformed into Escherichia coli Dh5α strain, and the presence of sequence mutations was identified by sequencing.

[0236] Example 2: Evaluation of the properties of the GhSPG-216M2 variant

[0237] Example 2-1. Comparative evaluation of the activity of GhSPG-216M2 and its variants

[0238] The expression vectors of GbSPG-216M2 and its nine variants prepared in Examples 1-1 were each transformed into *Escherichia coli* BL21 (DE3), and inoculated into sterile LB medium (BD Difco) and pre-cultured at 37°C and 200 rpm for 16 hours. Subsequently, 1 / 100 of the medium volume was inoculated into flasks containing sterile LB medium and incubated at 37°C and 200 rpm until the absorbance (OD) reached a certain level. 600The concentration of the enzyme was initially set at 0.4 to 0.5, then isopropyl β-D-1-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the cells were cultured for another 16 hours. Cells were then collected by centrifugation. The collected cells were added and resuspended in 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole), and the crude enzyme solution was obtained by sonication and centrifugation. The crude enzyme solution was applied and adsorbed onto Ni-NTA resin (Qiagen, CAT 30230), and the enzyme was purified by sequentially applying wash buffer (containing only 20 mM imidazole in the lysis buffer composition) and elution buffer (containing only 250 mM imidazole in the lysis buffer composition).

[0239] Protein concentration was determined by mixing 5 μl of diluted enzyme solution with 250 μl of Bradford solution (Quick Start™ Bradford 1x dye reagent, #5000205) and measuring absorbance at 595 nm.

[0240] To analyze the activity of purified GhSPG-216M2 and nine GhSPG-216M2 variants against DON, DON (CAS 51481-10-8, deoxynivalenol) dissolved in distilled water and purified enzyme reaction solution (25 mM TrisHCl, 150 mM NaCl, pH 7.4) were prepared and treated with 500 μM NiCl2 as a cofactor. The reaction was carried out at 50 °C for 24 hours and then stopped by incubating at 100 °C for 5 minutes. The residual amount of DON in the reaction product was measured using a high-performance liquid chromatography-ultraviolet (HPLC-UV) detector, and the relative activity of the variants was measured using the ratio of total DON degradation. The results are as follows: Figure 1 and 7 As shown.

[0241] As a result, Figure 1 and 7 The results showed that, when enzyme activity was expressed as a relative ratio, the activities of the nine GhSPG-216M2 variants (216M3, 216M4, 216M5, 216M6, 216M7, 216M8, 216M9, 216M10, and 216M11) were increased by 7% to 227% compared to the activity of the template GhSPG-216M2.

[0242] Example 2-2. Comparative evaluation of the thermal stability of GhSPG-216M2 and its variants

[0243] The GhSPG-216M2 and its six variants 216M3, 216M4, 216M5, 216M6, 216M7 and 216M8 prepared in Example 1 were each transformed into Escherichia coli BL21 (DE3). The enzymes were expressed, purified and reacted using the same methods as described in Example 2-1. The reaction products were then analyzed using HPLC-UV.

[0244] To evaluate the thermostability (residual activity) of GhSPG-216M2 and its six variants, 50 μL of each purified enzyme was incubated at room temperature, 50°C, and 60°C for 10 minutes. The enzyme solutions before and after heat treatment were used to obtain the residual DON content, degradation rate, and residual activity after DON degradation. Figure 2 As shown.

[0245] As a result, after being placed at 50°C for 10 minutes, the residual activity of GhSPG-216M2, 216M3, 216M4, 216M5, 216M6, 216M7, and 216M8 compared to the residual activity before heat treatment were 89.4%, 102.8%, 100.8%, 90.7%, 103.1%, 96.5%, and 101.6%, respectively, indicating that the residual activity of all variants was improved. After being placed at 60°C for 10 minutes, the residual activity of GhSPG-216M2, 216M3, 216M4, 216M6, 216M7, and 216M8 compared to the residual activity before heat treatment were 60.3%, 79.9%, 92.6%, 98.3%, 63.3%, and 96.3%, respectively, indicating that the residual activity of all variants was improved.

[0246] Examples 2-3. Comparative evaluation of the acid resistance of GhSPG-216 M2 and its variants

[0247] The GhSPG-216M2 and six variants 216M3, 216M4, 216M5, 216M6, 216M7 and 216M8 prepared in Example 1 were each transformed into Escherichia coli BL21 (DE3). The enzymes were expressed, purified and reacted using the same methods as described in Example 2-1. The reaction products were then analyzed using HPLC-UV.

[0248] To evaluate the acid tolerance (residual activity) of GhSPG-216M2 and six variants, 47.5 μL of each purified enzyme was mixed with 2.5 μL of glycine-HCl (1 M, pH 3.0), sodium acetate (1 M, pH 5.0), and Tris-HCl (1 M, pH 7.0) and Tris-HCl (1 M, pH 9.0), and incubated at 37°C for 60 min. The residual activity of each acid-treated enzyme solution at pH 3 relative to the DON degradation at the optimum pH was obtained after DON degradation. Figure 3 As shown.

[0249] As a result, after being placed at pH 3 and 37°C for 60 minutes, the residual activities of GhSPG-216M2, 216M3, 216M4, 216M5, 216M6, 216M7, and 216M8 relative to the residual activities at the optimal pH (maximum DON degradation) were 32.73%, 55.17%, 70.91%, 100.0%, 80.69%, 48.41%, and 47.58%, respectively. This confirmed that 216M5 (a point variant) exhibited the highest residual activity, and the residual activities of the other five variants were all improved.

[0250] Examples 2-4. Comparative evaluation of the activity, acid resistance, and heat resistance of variants with site saturation mutations at position 101.

[0251] To compare and evaluate the activity, acid resistance, and heat resistance of variants with saturation mutations at induced sites in the GhSPG-216M2 sequence, primers were designed, and 19 point mutation and combinatorial variants were prepared (hereinafter referred to as 216M3, 216M4, 216M5, 216M6, 216M7, 216M8, 216M9, 216M10, and 216M11, SEQ ID NO: 3, 5, 7, 9, 11, 13, 31, 33, and 35). The mutation sites, mutated amino acids, and primer sequences used to prepare the variants based on the amino acid sequence of SEQ ID NO: 1 are listed in Table 2 below.

[0252] [Table 2]

[0253]

[0254]

[0255] Specifically, using the polynucleotide encoding GhSPG-216M2 (SEQ ID NO: 1) prepared in Example 1-1 and cloned into the pET vector as a template, primers (SEQ ID NO: 37 to 74 in Table 2), and PCR premix (iNtRON, CAT No. 25185), 19 point mutations at position 101 were prepared by PCR. PCR was performed using an Eppendorf Mastercycler Nexus GX2 under the following conditions:

[0256] Initial denaturation — 94℃, 2 min

[0257] Denaturation—94℃, 20 sec

[0258] Annealing — 50℃, 10 sec

[0259] Extension—72℃, 8 min (15 cycles from denaturation to extension)

[0260] Final extension—72℃, 5 min

[0261] The truncated variants prepared using the QuickChange site-directed mutagenesis kit (Agilent, Cat# 200518) were then transformed into Escherichia coli Dh5α strain, and the presence of sequence mutations was identified by sequencing.

[0262] The activity, heat resistance, and acid resistance of the 19 variants were evaluated using the same methods as in Examples 2-1, 2-2, and 2-3, and the results are as follows: Figures 4 to 6 As shown.

[0263] Specifically, in Figure 4 In the study, it was confirmed that when the amino acid (K; Lys) at position 101 of GhSPG-216 M2 was replaced with another amino acid, the activity of the variant increased by approximately 0.9% to 12% when the enzyme activity was expressed as a relative ratio.

[0264] In addition, Figure 5 The study confirmed that when the amino acid at position 101 of GhSPG-216 M2 was replaced with another amino acid, the thermal stability at 50°C increased by about 4% to 22%, and the thermal stability at 60°C increased by about 10% to 75%.

[0265] exist Figure 6 The study confirmed that when the amino acid at position 101 of GhSPG-216 M2 was replaced with another amino acid, the acid resistance increased by 3% to 51%.

[0266] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in different specific forms without altering its technical spirit or essential features. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all respects. The scope of this disclosure is defined by the appended claims and not by the description that follows them; therefore, all changes and modifications falling within the boundaries and scope of the claims, or equivalent substitutions of those boundaries and scopes, are intended to be covered by the claims.

[0267] [Sequence List]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

Claims

1. A modified polypeptide having deoxynivalenol (DON) degrading activity, wherein an amino acid at a position corresponding to any one or more positions selected from the group consisting of positions 28, 29, 87, 91, 101, and 152 of SEQ ID NO: 1 is replaced with another amino acid.

2. The modified polypeptide of claim 1, wherein the amino acid corresponding to position 28 of SEQ ID NO: 1 is replaced with isoleucine; the amino acid corresponding to position 29 of SEQ ID NO: 1 is replaced with leucine; the amino acid corresponding to position 87 of SEQ ID NO: 1 is replaced with isoleucine; the amino acid corresponding to position 91 of SEQ ID NO: 1 is replaced with serine; the amino acid corresponding to position 101 of SEQ ID NO: 1 is replaced with phenylalanine; or the amino acid corresponding to position 152 of SEQ ID NO: 1 is replaced with glycine.

3. The modified polypeptide of claim 1, wherein the amino acid corresponding to position 28 of SEQ ID NO: 1 is replaced with isoleucine; and the amino acid corresponding to position 101 of SEQ ID NO: 1 is replaced with phenylalanine.

4. The modified polypeptide of claim 1, wherein the amino acid corresponding to position 28 of SEQ ID NO: 1 is replaced with isoleucine; and the amino acid corresponding to position 152 of SEQ ID NO: 1 is replaced with glycine.

5. The modified polypeptide of claim 1, wherein the amino acid corresponding to position 28 of SEQ ID NO: 1 is replaced with isoleucine; the amino acid corresponding to position 101 of SEQ ID NO: 1 is replaced with phenylalanine; and the amino acid corresponding to position 152 of SEQ ID NO: 1 is replaced with glycine.

6. The modified polypeptide of claim 1, comprising any one selected from the group consisting 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: 31, SEQ ID NO: 33, and SEQ ID NO:

35.

7. The modified polypeptide of claim 1, wherein one or more properties selected from the group consisting of DON degrading activity, heat tolerance, thermal stability, and acid tolerance are improved compared to a polypeptide consisting of the amino acid sequence of SEQ ID NO:

1.

8. A polynucleotide encoding the modified polypeptide of any one of claims 1 to 7.

9. A microorganism comprising any one or more of: the modified polypeptide of any one of claims 1 to 7; and a polynucleotide encoding the modified polypeptide.

10. A composition for degrading deoxynivalenol (DON), the composition comprising any one or more of: the modified polypeptide of any one of claims 1 to 7; and a microorganism expressing the modified polypeptide.

11. A feed additive composition comprising any one or more of: the modified polypeptide according to any one of claims 1 to 7; and a microorganism expressing the modified polypeptide.

12. A method of degrading deoxynivalenol (DON), the method comprising the step of reacting DON with any one or more of: the modified polypeptide according to any one of claims 1 to 7; and a microorganism expressing the modified polypeptide.

13. A method of preparing a feed product, the method comprising the step of mixing a feed component with the feed additive composition according to claim 11.

14. A method of preparing a modified polypeptide having DON degrading activity, the method comprising the steps of: culturing the microorganism according to claim 9; and recovering the modified polypeptide having DON degrading activity according to any one of claims 1 to 7 expressed in the culturing step.

15. Use of the modified polypeptide according to any one of claims 1 to 7 for degrading DON; and use of one or more microorganisms expressing the modified polypeptide for degrading DON. ​

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