Novel polypeptide with zearalenone-degrading activity

Abstract

The present application relates to a variant polypeptide having zearalenone-degrading activity and uses thereof.

Description

Novel polypeptides having zearalenone decomposition activity

[0001] The present application relates to a mutant polypeptide having zearalenone decomposition activity and its use.

[0002]

[0003] Mycotoxins are substances secreted mainly by Aspergillus, Fusarium, and Penicillium fungi, and when consumed by humans and livestock, they cause various diseases, including cancer, miscarriage, and stillbirth.

[0004] Among the well-known mycotoxins, zearalenone (ZEN) is a mycotoxin produced by the fungus Fusarium genus. Zearalenone is a non-steroidal estrogenic mycotoxin, also known as F-2 mycotoxin or FES (fermentation estrogenic substance). It has a chemical structure and properties similar to estrogen, a type of female hormone, and can cause endocrine disruption in humans. For example, a recent study reported that receptors damaged by ZEN suppressed estrogen hormones in mouse mammary tissue (Ecotoxicology and Environmental Safety Volume 241, August 2022, 113826), and ZEN stimulated the growth of human breast cancer cells containing estrogen-responsive receptors (Toxins 2019, 11(8), 481). Long-term consumption of grains contaminated with zearalenone can cause infertility in pigs, and in humans, exposure to zearalenone can lead to cancer and mutations. Therefore, there is a pressing need for compositions or methods that effectively decompose and detoxify zearalenone.

[0005] Meanwhile, one of the most important considerations in the development of enzymes for feed is the acid stability of the enzyme. This is due to the unique environment of the stomach, a digestive organ. When food enters the stomach, it turns it into a mush and secretes hydrochloric acid to control harmful bacteria. The stomach maintains an acidic pH of approximately 2.0 to 3.0. If acid stability is insufficient, the enzyme will be inactivated during its passage through the stomach, rendering it ineffective in the intestines. Therefore, acid stability is crucial for enzyme activity in the intestines.

[0006] Therefore, there is a need to develop an effective decomposition enzyme that can effectively decompose zearalenone and remain active in the intestine.

[0007]

[0008] The problem to be solved by the present application is to provide a mutant polypeptide having zearalenone (ZEN) decomposition activity; a polynucleotide encoding the mutant polypeptide; a host cell comprising at least one of the mutant polypeptide and the polynucleotide encoding the polypeptide; a composition for decomposing zearalenone comprising at least one of the mutant polypeptide and the host cell expressing the mutant polypeptide; a composition for adding feed; a method for decomposing zearalenone, comprising a step of contacting at least one of the mutant polypeptide and the host cell expressing the mutant polypeptide with zearalenone; and a method for producing the mutant polypeptide having the zearalenone decomposition activity.

[0009]

[0010] The purpose of the present application is to provide a mutant polypeptide having zearalenone decomposition activity.

[0011] Another object of the present application is to provide a polynucleotide encoding a variant polypeptide of the present application.

[0012] Another object of the present application is to provide a host cell comprising at least one of the variant polypeptides of the present application and a polynucleotide encoding the polypeptide.

[0013] Another object of the present application is to provide a composition for decomposing zearalenone, comprising at least one of the mutant polypeptide of the present application and a host cell expressing the mutant polypeptide.

[0014] Another object of the present application is to provide a composition for feed additive comprising at least one of the mutant polypeptide of the present application and a host cell expressing the mutant polypeptide.

[0015] Another object of the present application is to provide a method for decomposing zearalenone, comprising the step of contacting at least one of the variant polypeptide of the present application and a host cell expressing the variant polypeptide with zearalenone.

[0016] Another object of the present application is to provide a method for producing a mutant polypeptide having zearalenone decomposition activity, comprising the steps of: culturing a host cell comprising at least one of the mutant polypeptide of the present application and a polynucleotide encoding the polypeptide; and recovering a mutant polypeptide having zearalenone decomposition activity expressed in the culturing step.

[0017]

[0018] The mutant polypeptide having zearalenone decomposition activity of the present application has improved acid resistance and can effectively decompose zearalenone, a fungal toxin, even in an environment where pH conditions change.

[0019]

[0020] Figure 1 is a diagram showing the results of confirming the zearalenone decomposition activity of the mutant polypeptide of the present application using HPLC-UV.

[0021] Figure 2 is a diagram showing the results of evaluating the enzyme acid stability of the mutant polypeptide of the present application.

[0022]

[0023] 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.

[0024] 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.

[0025]

[0026] As used in the specification and appended claims of this application, the singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless the context clearly dictates otherwise, the singular terms include the plural and the plural terms include the singular. In the specification and appended claims of this application, unless the context clearly dictates otherwise, the use of "or" is intended to include "and / or."

[0027]

[0028] In this application, the term "about" may be used before a specific numerical value. As used herein, the term "about" encompasses not only the exact number described after the term, but also a range that is or is nearly that number. Whether a number is or is nearly the specific number described can be determined based on the context in which it is presented. For example, the term "about" may refer to a range of -10% to +10% of a numerical value. In another example, the term "about" may refer to a range of -5% to +5% of a given numerical value. However, this is not a limitation.

[0029]

[0030] In this application, the terms "first, second, third...", "i), ii), iii)...", or "(a), (b), (c), (d)..." are used to distinguish similar configurations, and these terms do not imply that the steps are performed consecutively or in order. For example, when the terms are used in relation to steps of a method, use, or analysis, these steps may be performed simultaneously, without a time interval, or may be performed at intervals of seconds, minutes, hours, days, or months.

[0031]

[0032] In this application, the term "consisting essentially of" means that a non-specific component may be present if the characteristics of the subject matter claimed in this application are not substantially affected by the presence of the non-specific component.

[0033] In this application, the term "consisting of" means that the proportion of a specific component(s) totals 100%. The components or features listed below the term "consisting of" may be essential or mandatory. In some embodiments, other than the components or features listed below "consisting of," other optional or nonessential components may be excluded.

[0034] In this application, the term "comprising" means the presence of a feature, step, or component described below, and does not exclude the presence or addition of one or more features, steps, or components. The components or features described below "comprising" in this application may be essential or mandatory, but in some embodiments, other optional or non-essential components or features may be further included.

[0035] In this application, the term “comprising” may, in some embodiments, be modified to refer to “consisting essentially of” or “consisting of.”

[0036] In the present application, with respect to an amino acid sequence, even if it is described as a polypeptide “comprising” an amino acid sequence set forth in a specific sequence number, a polypeptide “consisting of” an amino acid sequence set forth in a specific sequence number, or a polypeptide or protein “having” an amino acid sequence set forth in a specific sequence number, it is obvious that a protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted, or added may also be used in the present application, as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number. For example, it may be a case in which the amino acid sequence has an addition of a sequence that does not alter the function of the protein at the N-terminus and / or C-terminus, a mutation that may occur naturally, a silent mutation thereof, or a conservative substitution, but is not limited thereto.

[0037]

[0038] In this application, the term "protein" or "polypeptide" refers to a polymer or oligomer of consecutive amino acid residues. In this application, "polypeptide," "protein," and "peptide" may be used interchangeably with "amino acid sequence."

[0039] In some cases, an amino acid sequence that exhibits activity may be referred to as an "enzyme." In this application, amino acid sequences are described in an N-terminal → C-terminal orientation, unless otherwise indicated.

[0040]

[0041] In relation to a cell, nucleic acid, polypeptide, or vector, the term "recombinant" in this application means that the cell, nucleic acid, polypeptide, or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or by alteration of a native nucleic acid or polypeptide, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell may express a gene not found in the native (non-recombinant) form of the cell, or may express a native gene that is expressed or not expressed at all, or otherwise abnormally expressed.

[0042]

[0043] As used herein, the term "isolated" refers to a substance that exists in an environment where it does not occur naturally, or in a form that does not occur naturally. This includes the substance (sequence, enzyme, or nucleic acid) being at least substantially free from at least one other component with which it is naturally associated and found in nature, such as a sequence, enzyme, or nucleic acid.

[0044] For example, the isolated sequences, enzymes or nucleic acids provided in the present application may be provided in a form substantially free of one or more contaminants.

[0045] Examples of isolated substances may include, but are not limited to, i) any non-naturally occurring substance, ii) any substance from which one, more, or all naturally occurring components associated with it in nature have been removed (e.g., an enzyme, variant, nucleic acid, protein, peptide, or cofactor), iii) any substance found in nature that has been artificially modified, or iv) a substance that has been modified to alter the amount of that substance relative to other naturally associated components (e.g., increasing the number of copies of a gene encoding the substance; modifying a promoter naturally associated with a gene encoding the substance to a more active promoter, etc.).

[0046]

[0047] 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.

[0048]

[0049] 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.

[0050]

[0051] 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.

[0052]

[0053] 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.

[0054] 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.

[0055] 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.

[0056]

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061]

[0062] 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.

[0063] The variant differs from the sequence of the parent enzyme by one or more modifications, e.g., amino acid substitutions, deletions and / or insertions.

[0064] 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.

[0065] 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.

[0066] Other variants may include variants in which portions are removed from the N- and / or C-terminus of the mature protein.

[0067] 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.

[0068] 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.

[0069]

[0070] 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.

[0071]

[0072] 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.

[0073]

[0074] Alanine Ala, A Arginine Arg, R

[0075] Asparagine Asn, N Aspartic acid Asp, D

[0076] Cysteine ​​Cys, C Glutamic acid Glu, E

[0077] Glutamine Gln, Q Glycine Gly, G

[0078] Histidine His, H Isoleucine Ile, I

[0079] Leucine Leu, L Lysine Lys, K

[0080] Methionine Met, M Phenylalanine Phe, F

[0081] Proline Pro, P Serine Ser, S

[0082] Threonine Thr, T Tryptophan Trp, W

[0083] Tyrosine Tyr, Y Valine Val, V

[0084]

[0085] Meanwhile, any amino acid can be written as Xaa, X.

[0086] 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.

[0087]

[0088] 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.

[0089] 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.

[0090]

[0091] As used herein, the term "gene" refers to a polynucleotide encoding a polypeptide and a polynucleotide comprising regions preceding and following the coding region. In some embodiments, a gene may have a sequence (intron) inserted between each coding region (exon).

[0092]

[0093] As used herein, the terms "homology" or "identity" refer to the degree of relationship between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

[0094] 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 that are considered codon degenerate.

[0095] 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 ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using, but is not limited to, BLAST of the National Center for Biotechnology Information Database, or ClustalW.

[0096] 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.

[0097] 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 the defined appropriate 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; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, but are not limited thereto).

[0098]

[0099] In this application, the term "mature polypeptide" refers to a polypeptide in a form that lacks a signal sequence or a propeptide sequence. A mature protein / polypeptide / peptide may be a functional form of a protein / polypeptide / peptide. A mature polypeptide may be a final form that has undergone post-translational or post-translational modifications. Examples of post-translational modifications include, but are not limited to, N- or C-terminal modifications, glycosylation, phosphorylation, and leader sequence removal.

[0100]

[0101] The term "nucleic acid construct" in this application means a single or double-stranded nucleic acid molecule that contains one or more regulatory sequences and is artificially synthesized, engineered to contain a specific sequence in a manner that does not exist in nature, or isolated from nature.

[0102]

[0103] As used herein, the term “expression” includes, but is not limited to, any step involved in the production of a polypeptide, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0104] As used herein, the term "expression vector" means a linear or circular nucleic acid molecule comprising a coding sequence and regulatory sequences operably linked thereto for expression thereof.

[0105]

[0106] As used herein, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned appropriately so that the regulatory sequence directs the expression of a coding sequence. Therefore, "operably linked" includes a regulatory region of a functional domain with known or desired activity, such as a promoter, terminator, signal sequence, or enhancer region, attached or linked to a target (gene or polypeptide) so that the expression, secretion, or function of the target can be controlled in accordance with the known or desired activity.

[0107]

[0108] As used herein, the term "cDNA" refers to a DNA sequence that can be produced by reverse transcription from a mature, spliced ​​mRNA molecule, which can be obtained from a eukaryotic or prokaryotic cell. The cDNA sequence does not include intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA before being processed through a series of steps, including splicing, to form the mature, spliced ​​mRNA.

[0109]

[0110] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence necessary for the expression of a coding sequence. Each regulatory sequence may be native to the coding sequence (having the same origin) or foreign (derived from another gene). Examples of the regulatory sequence include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates transcription and translation termination. The minimum unit of the regulatory sequence may include a promoter, a transcription and translation termination sequence.

[0111]

[0112] To describe the variants provided in this application, the following nomenclature is used.

[0113] In this application, reference to a specific position in an amino acid sequence may include reference to an amino acid present or substituted at that position. Reference to an amino acid at a specific position may be described in various ways. For example, "position 022" may be described as "position 22," "amino acid 22," or "22nd amino acid." Furthermore, for example, if the amino acid at position 22 is proline (P), it may be described as "P22" or "Pro22."

[0114] Amino acid substitutions can be expressed by listing the amino acid before substitution, the position, and the amino acid being substituted. These amino acids can be expressed using conventional one-letter and three-letter codes. For example, if proline, the amino acid at position 22 in a specific sequence, is replaced with threonine, it can be described as "P22T" or "Pro22Thr."

[0115] Any amino acid at a particular position can be designated as "X". For example, X22 refers to any amino acid at position 22. Also, when a substituted amino acid is designated as X, it means that it is replaced with an amino acid different from the amino acid present before the substitution. For example, "P22X" indicates that P is replaced with any amino acid other than P at position 22.

[0116] Different alternations can be expressed by listing multiple amino acids simultaneously using the " / " or "," symbols. For example, a substitution of the amino acid (K) at position 43 with A or C can be written interchangeably as K43A / C or K43A,C. As another example, A / T111C means that the amino acid A or T at position 111 before substitution is replaced with C.

[0117] Multiple mutations can be described using a "+" sign. For example, a description such as "A45C+ T111C" means that the amino acid at position 45, alanine, is substituted with cysteine, and the amino acid at position 111, threonine, is substituted with cysteine, respectively.

[0118] Deletions of amino acids can be expressed by listing the amino acid before the deletion, the position, and then *. For example, if alanine, the amino acid corresponding to position 8 of a specific sequence, is deleted, it can be expressed as A45* or (Ala45*).

[0119] Insertion of an amino acid may be described as, for example, Gly8GlyLys or G8GK when a lysine is inserted between the glycine at position 8 and the amino acid at position 9 in a specific sequence. Insertion of more than one amino acid, for example, insertion of a lysine and a valine between the glycine at position 8 and the amino acid at position 9 in a specific sequence, may be described as Gly8GlyLysVal or G8GKV. In this case, the position of the inserted amino acid may be indicated using the amino acid number and the alphabet preceding the inserted amino acid, and for example, in the above case, the inserted lysine and valine may be numbered as 8aK and 8bV.

[0120]

[0121] 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.

[0122] 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.

[0123] 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 zearalenone degrading activity, and unless otherwise indicated in the present application, residues of a specific amino acid sequence are numbered based on SEQ ID NO. 1.

[0124] 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”).

[0125] 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).

[0126] Additionally, multiple sequence alignment can be used to identify corresponding amino acid residues in other zearalenone-degrading enzymes. 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.

[0127] 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.

[0128] 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).

[0129] The above methods are examples and are not limiting.

[0130]

[0131] Hereinafter, specific examples of the present application will be described in more detail as follows.

[0132]

[0133] In this application, "zearalenone (ZEN)" refers to a non-steroidal estrogenic mycotoxin produced by strains of fungi of the genus Fusarium, particularly F. graminearum (Gibberella zeae), F. culmorum, F. cerealis, F. equiseti, F. crookwellense, and F. semitectum, and is known as one of the mycotoxins widely distributed in grains worldwide. Zearalenone is also called F-2 toxin, Fusarium toxin, and FES (fermentation extrogenic substance), and has about 20 types of isomers, but trans-a-zearalenone is mainly produced in grains in nature. Zearalenone has been reported to have harmful effects due to its sex hormone-like effects rather than toxicity. Most toxicities manifest as hyperhormonal effects, and it can also cause cancer and mutations. For example, in livestock farming, zearalenone has been reported to cause infertility in pregnant sows, as well as ovarian abnormalities, premature births, and miscarriages. Even a 50 ppb concentration in feed is known to induce ovarian changes in pigs.

[0134]

[0135] In the present application, zearalenone decomposing enzyme means an enzyme having an activity of decomposing the lactone ring ester bond of zearalenone (ZEN), and catalyzing a reaction of converting ZEN into hydrolyzed ZEN (HZEN).

[0136] In the present application, the degradation of zearalenone may be used in the same sense as detoxification of zearalenone, inactivation of zearalenone, and decontaminating of contamination caused by zearalenone.

[0137] In the present application, zearalenone degradation activity can be measured and evaluated using methods known in the art, including the embodiments described in the present application. For example, zearalenone degradation activity can be determined by detecting the residual amount of zearalenone.

[0138]

[0139] In this application, "parent zearalenone degrading enzyme" means a zearalenone degrading enzyme that is modified to produce a variant or mutant polypeptide of the present application. Specifically, the parent zearalenone 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 thereto as long as it is a polypeptide that has zearalenone degrading activity and can be a parent of a variant.

[0140] The parent zearalenone decomposing enzyme provided in the present application may be, but is not limited to, a polypeptide of SEQ ID NO: 1. In addition, as long as it has zearalenone decomposing activity, it may be a polypeptide having a sequence identity of at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the polypeptide of SEQ ID NO: 1, and as long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, it may be included in the scope of the parent zearalenone decomposing enzyme without limitation.

[0141] The parent zearalenone decomposing enzyme of the variant provided in the present application may be derived from the genus Sphingomonas.

[0142] Meanwhile, the above-mentioned microorganism is an example of a microorganism from which the mother zearalenone decomposing enzyme provided in the present application can be derived, and includes a microorganism derived from a taxonomically homologous microorganism, regardless of the name of the microorganism.

[0143] The above-mentioned microorganisms can be obtained from known microorganism depositories such as ATCC, DSMZ, CBS, NRRL, KCTC, and KCCM.

[0144] In the present application, a sequence "derived from" a particular microorganism is not limited to a sequence that is naturally produced or can be produced in the microorganism, but also includes a sequence encoded by a gene that is produced and isolated from the microorganism containing the gene.

[0145] For example, a zearalenone-degrading enzyme from the genus Sphingomonas includes not only an enzyme having zearalenone-degrading activity that is naturally produced in Sphingomonas, but also one produced in a Sphingomonas source, and one produced in another host cell through genetic modification known in the art (e.g., transformation with a sequence encoding the enzyme).

[0146]

[0147] In the present application, the “variant polypeptide having zearalenone decomposing activity” may be a variant of a parent zearalenone decomposing enzyme.

[0148] In this application, the term "variant of parent zearalenone degrading enzyme" or "variant of zearalenone degrading enzyme" refers to a protein having one or more amino acids different from the amino acid sequence of the parent zearalenone degrading enzyme and having zearalenone degrading activity.

[0149] The above “mutant polypeptide having zearalenone decomposing activity”, “mutant of parent zearalenone decomposing enzyme” and “mutant zearalenone decomposing enzyme” can be used interchangeably.

[0150] The variant provided in the present application may have zearalenone decomposing activity and include modification of one or more amino acids in the parent zearalenone decomposing enzyme sequence.

[0151] In addition, the variant is i) a polypeptide having a sequence identity of at least 70% and less than 100% with SEQ ID NO: 1; and / or ii) a polypeptide encoded by a polynucleotide having a sequence identity of at least 70% and less than 100% with a sequence encoding a mature polypeptide of SEQ ID NO: 1; and / or iii) a polypeptide encoded by a polynucleotide that hybridizes with (a) a mature polypeptide coding sequence of SEQ ID NO: 1, (b) a cDNA thereof, or (c) a full-length complement of (a) or (b) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions; and / or iv) a functional fragment of the polypeptide i), ii) or ii) having zearalenone degrading activity,

[0152] It may include a substitution of one or more amino acids at positions 22, 43, 45 and 111 with another amino acid.

[0153] Specifically, the variants provided in the present application may have zearalenone decomposing activity, and may have one or more altered functions or properties compared to the parent zearalenone decomposing enzyme, including modification of one or more amino acids in the parent zearalenone decomposing enzyme sequence.

[0154] In one specific example, the variants provided in the present application have zearalenone degrading activity, and may have one or more altered functions or properties compared to the parent zearalenone degrading enzyme, including modification of one or more amino acids in the parent zearalenone degrading enzyme sequence, and may have one or more conservative substitutions.

[0155]

[0156] The variant provided in the present application is a variant of the parent zearalenone degrading enzyme, and may be a polypeptide having zearalenone degrading enzyme activity.

[0157] In one specific example, the variant provided in the present application may comprise a modification at one or more positions corresponding to positions 22, 43, 45 and 111 of SEQ ID NO: 1.

[0158] 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.

[0159] In one specific example, the variant provided in the present application may comprise a modification of an amino acid corresponding to one or more of P22, K43, A45 and T111 of SEQ ID NO: 1.

[0160]

[0161] In one specific example, the amino acid corresponding to position 22 of the modified sequence number 1 provided in the present application may be proline (P); the amino acid corresponding to position 43 may be lysine (K); the amino acid corresponding to position 45 may be alanine (A); and / or the amino acid corresponding to position 111 may be threonine (T).

[0162]

[0163] In one specific example, the variant provided in the present application may include a substitution of P, an amino acid corresponding to position 22 of SEQ ID NO: 1, with G, V, L, I, M, F, W, S, T, C, Y, N, Q, D, E, K, R or H, for example, with S, T, C, Y, N or Q, for another example, with S, T, N or Q, and for another example, with T.

[0164] In one specific example, the variant provided in the present application may include a substitution of K, an amino acid corresponding to position 43 of SEQ ID NO: 1, with G, A, L, I, M, F, W, P, S, T, C, Y, N, Q, D, E, R, or H, and may include a substitution of G, A, V, L, I, M, F, W, or P as one example, and a substitution of G, A, V, L, I, or M as another example, and a substitution of A as another example.

[0165] In one specific example, the variant provided in the present application may include a substitution of A, an amino acid corresponding to position 45 of SEQ ID NO: 1, with G, V, I, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R or H, and may include a substitution of S, T, C, Y, N or Q as one example, and a substitution of S, T, C, N or Q as another example, and a substitution of C as another example.

[0166] In one specific example, the variant provided in the present application may include a substitution of T, an amino acid corresponding to position 111 of SEQ ID NO: 1, with G, A, V, L, I, F, W, P, S, C, Y, N, Q, D, E, K, R or H, and may include a substitution of S, C, Y, N or Q as one example, and a substitution of S, C, N or Q as another example, and a substitution of C as another example.

[0167]

[0168] As a specific example, the variant provided in the present application may comprise a substitution of the amino acid corresponding to position 22 of SEQ ID NO: 1 with a polar or hydrophilic amino acid.

[0169] As one specific example, the variant provided in the present application may comprise a substitution of the amino acid corresponding to position 43 of SEQ ID NO: 1 with a non-polar amino acid.

[0170] As one specific example, the variant provided in the present application may comprise a substitution of the amino acid corresponding to position 45 of SEQ ID NO: 1 with a polar or hydrophilic amino acid.

[0171] As one specific example, the variant provided in the present application may comprise a substitution of the amino acid corresponding to position 111 of SEQ ID NO: 1 with a polar or hydrophilic amino acid.

[0172] In one specific example, the variant provided in the present application may comprise one or more substitutions of P22T, K43A, A45C, and T111C of SEQ ID NO: 1. In one specific example, the variant provided in the present application may comprise one, two, three, or four amino acid substitutions of a combination of the amino acid substitutions listed above.

[0173] In one specific example, the variants provided in the present application include all possible combinations of the modifications described above.

[0174] For example, the variant may comprise a modification of an amino acid at a position selected from i) to xv) in a combination of the above-described modifications: i) position 22; ii) position 43; iii) position 45; iv) position 111; v) positions 22+43; vi) positions 22+45; vii) positions 22+111; viii) positions 43+45; ix) positions 43+111; x) positions 45+111; xi) positions 22+43+45; xii) positions 22+43+111; xiii) positions 22+45+111; xiv) positions 43+45+111; and xv) positions 22+43+45+111.

[0175] As another example, the variant may comprise, but is not limited to, one or more of the following substitutions: i) substitution of amino acid 22 with threonine; ii) substitution of amino acid 43 with alanine; iii) substitution of amino acid 45 with cysteine; and iv) substitution of amino acid 111 with cysteine.

[0176] As another example, the variant may include, but is not limited to, one or more modifications selected from the following:

[0177] P22T

[0178] K43A

[0179] A45C

[0180] T111C

[0181] P22T+K43A

[0182] P22T+A45C

[0183] P22T+T111C

[0184] K43A+A45C

[0185] K43A+T111C

[0186] A45C+T111C

[0187] P22T+K43A+A45C

[0188] P22T+K43A+T111C

[0189] P22T+A45C+T111C

[0190] K43A+A45C+T111C

[0191] P22T+K43A+A45C+T111C

[0192]

[0193] In one specific example, the variant provided in the present application may have a sequence identity of at least about 60%, for example, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% and less than 100% to a parent zearalenone degrading enzyme; its mature polypeptide or a functional fragment thereof.

[0194] In one specific example, the variant provided in the present application may have a sequence identity of at least about 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100% with SEQ ID NO: 1.

[0195] In one specific example, the variant provided in the present application may be a polypeptide encoded by a polynucleotide having a sequence identity of at least about 60%, for example, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% and less than 100%, with a sequence encoding a mature polypeptide of SEQ ID NO: 1.

[0196] In one specific example, the variant provided in the present application may have a sequence identity of at least about 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100% with a functional fragment of SEQ ID NO: 1.

[0197]

[0198] Specifically, a variant comprising the A45C and T111C substitutions of SEQ ID NO: 1 provided in the present application may be represented by SEQ ID NO: 5, a variant comprising the K43A substitution of SEQ ID NO: 1 may be represented by SEQ ID NO: 6, and a variant comprising the P22T substitution of SEQ ID NO: 1 may be represented by SEQ ID NO: 7.

[0199] Additionally, variant polypeptides that differ from the recited sequence by conservative substitution and / or modification of one or more amino acids compared to any one of the amino acid sequences of SEQ ID NOs: 5 to 7, but which retain the functions or properties of the protein, are also included within the scope of the polypeptides provided by the present application. Such modifications may include, for example, modifications in which a portion is removed from the N- and / or C-terminus of the mature protein.

[0200] The polypeptide of the present invention may have, for example, one or more conservative substitutions while still retaining one or more biological activities of the polypeptide of any one of SEQ ID NOs: 5 to 7. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

[0201] In one embodiment, the polypeptide provided in the present application may comprise a polypeptide consisting of an amino acid sequence having at least 80%, for example at least 85%, 90%, 95%, 97% or 99% homology or identity with any one of SEQ ID NOs: 5 to 7, or a polypeptide comprising said amino acid sequence or consisting essentially of said amino acid sequence. In addition, if it has said homology or identity and has zearalenone degrading activity, it is included in the polypeptide having zearalenone degrading activity provided in the present application.

[0202]

[0203] The variants provided in the present application may have one or more altered properties or attributes of the polypeptide that can be selected or detected compared to other zearalenone degrading enzymes, such as wild-type zearalenone degrading enzymes, parent zearalenone degrading enzymes, other zearalenone degrading enzyme variants, etc.

[0204] The above properties or attributes include, but are not limited to, oxidative stability, substrate specificity, catalytic activity, thermal stability, acid stability, alkaline stability, pH activity profile, resistance to proteolysis, Km, kcat, kcat / Km ratio, protein folding, induction of an immune response, ability to bind a ligand, ability to bind a receptor, ability to be secreted, ability to be displayed on the surface of cells, ability to form oligomers, ability to signal, ability to promote cell proliferation, ability to inhibit cell proliferation, ability to induce apoptosis, ability to be modified by phosphorylation or glycosylation, and / or ability to treat a disease.

[0205]

[0206] Specifically, the variants provided in the present application may have pH stability or have activity with increased pH stability compared to the parent sequence.

[0207] 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 defined conditions. This comparison allows for the expression of enzyme stability, or the level at which enzyme activity is lost. Factors that affect enzyme activity include, for example, pH, heat, and the presence of other substances (e.g., oxidizing agents, chelating agents).

[0208] As used herein, the term "pH stability" refers to the ability of a protein to function within a specific pH range. In one specific example, the variants provided in the present application may be active at a pH ranging from about 2.0 to about 12.0, but are not limited thereto.

[0209] A protein can be defined as having "pH stability" if it maintains its function over a specific pH range, and can be defined as having "acid resistance (acid stability)", "alkali resistance", etc., depending on the pH range.

[0210] Increased stability includes retention of high enzymatic activity compared to other enzymes, e.g., wild-type enzyme, parent enzyme and / or other variants; increased pH range over which the protein remains functional.

[0211] As a specific example, a mutant polypeptide in which the amino acids corresponding to positions 45 and 111 of SEQ ID NO: 1 are substituted with cysteine; a mutant polypeptide in which the amino acid corresponding to position 43 of SEQ ID NO: 1 is substituted with alanine; or a mutant polypeptide in which the amino acid corresponding to position 22 of SEQ ID NO: 1 is substituted with threonine; may have acid resistance or increased acid resistance (acid stability) compared to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0212] Specifically, the acid resistance may be, but is not limited to, a decomposition rate at acidic pH of 50% or more, specifically 60% or more, and more specifically 60% to 90% compared to the decomposition rate at optimal pH. The optimal pH may be the optimal pH of the parent sequence or variant, and may be specifically pH 8. The acidic pH may be pH 2 to 4, and specifically pH 3.

[0213] As a specific example, a variant polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 5 to 7 may have acid resistance or increased acid resistance (acid stability) compared to a polypeptide comprising an amino acid sequence of SEQ ID NO: 1.

[0214] In one embodiment of the present application, as a result of evaluating the acid resistance (acid stability) of the mutant polypeptide, it was confirmed that it had acid resistance and had superior zearalenone decomposition activity under acidic conditions compared to the polypeptide of sequence number 1, and thus, it was confirmed that the acid resistance (acid stability) was improved.

[0215]

[0216] Another aspect of the present application provides a polynucleotide encoding a variant polypeptide having zearalenone decomposition activity of the present application.

[0217] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the variant.

[0218] A polynucleotide encoding a variant of the present invention may comprise the coding sequence of the aforementioned variant. The polynucleotide may undergo various modifications to the coding region, without altering the amino acid sequence of the polypeptide, due to codon degeneracy or in consideration of codon preference in the organism intended to express the polypeptide.

[0219] 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 hybridizes under stringent conditions with a complementary sequence to all or part of the base sequence, so long as it encodes a variant of the present application.

[0220] The above "stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0221] For example, conditions under which polynucleotides having a high degree of homology or identity hybridize with each other, specifically at least 40%, specifically at least 90%, more specifically at least 95%, at least 96%, at least 97%, at least 98%, and even more specifically at least 99%, and polynucleotides having a lower degree of homology or identity than that hybridize, or conditions under which washing is performed once, specifically twice or three times, at a salt concentration and temperature corresponding to 60°C, 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, and more specifically 68°C, 0.1ХSSC, 0.1% SDS, which are washing conditions of typical southern hybridization, can be listed.

[0222] 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, adenosine 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.

[0223] Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the 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 those skilled in the art depending on the purpose.

[0224] The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0225] For example, “high stringency” may occur at about 5 to 10°C below the Tm of the probe; “medium stringency” may occur at about 10 to 20°C below the Tm of the probe; and “low stringency” may occur at about 20 to 25°C below the Tm, but is not limited thereto.

[0226] For example, “low stringency conditions” can be prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 25% formamide for 12-24 hours, for a probe of at least 100 nucleotides in length, according to standard Southern blotting procedures. The carrier material can be finally washed two to three times for 15 minutes each with 2 X SSC, 0.1 to 0.2% SDS at 50°C.

[0227] For example, “medium stringency conditions” can be prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide for 12-24 hours, for a probe of at least 100 nucleotides in length, according to standard Southern blotting procedures. The carrier material can be finally washed two to three times for 15 minutes each with 2 X SSC, 0.1 to 0.2% SDS at 55°C. For example, “medium-high stringency conditions” can be prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide for 12-24 hours, for a probe of at least 100 nucleotides in length, according to standard Southern blotting procedures. The carrier material can be finally washed two to three times for 15 minutes each with 1 to 2 X SSC, 0.1 to 0.2% SDS at 60°C.

[0228] For example, “high stringency conditions” can be prehybridization and hybridization at 42°C in 5 X SSPE, 0.3% SDS, 200 micrograms / ml of sheared and denatured salmon sperm DNA, and 35% formamide for 12-24 hours, for a probe of at least 100 nucleotides in length, according to standard Southern blotting procedures. The carrier material can be finally washed two to three times for 15 minutes each with 2 X SSC, 0.1 to 0.2% SDS at 65°C.

[0229]

[0230] The polynucleotide encoding the variant polypeptide having zearalenone decomposition activity of the present application may include, without limitation, any polynucleotide encoding any one of SEQ ID NOs: 5 to 7 and a polypeptide having a corresponding activity. For example, the polynucleotide encoding the polypeptide having zearalenone decomposition activity of the present application may be any one of SEQ ID NOs: 5 to 7, or a polynucleotide sequence encoding a polypeptide having at least 80% homology or identity therewith.

[0231] In one embodiment, a polynucleotide encoding a variant polypeptide having zearalenone degrading activity of the present application may consist of, or consist essentially of, a base sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with any one of SEQ ID NOs: 8 to 10, but is not limited thereto.

[0232]

[0233] The nucleic acid construct provided in the present application comprises a polynucleotide encoding a variant provided in the present application, operably linked to one or more regulatory sequences that direct expression of the coding sequence in a suitable host cell under conditions suitable for the regulatory sequences.

[0234] Polynucleotides can be manipulated in a variety of ways to enable expression of variants. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide before inserting it into the vector. Such manipulations can be performed using methods known in the art.

[0235]

[0236] 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 outside the chromosome, 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.

[0237] 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.

[0238]

[0239] The "vector" provided in this application refers to a DNA construct containing a base sequence of a polynucleotide encoding a variant of the present application, operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the variant in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.

[0240] The vector that can be used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A 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 can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used.

[0241] For example, a polynucleotide encoding a variant provided in the present application can be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. 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 polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that transformed cells can be selected.

[0242]

[0243] The host cell of the present application may include, without limitation, any cell capable of expressing the variant of the present application.

[0244] The host cell of the present application may comprise the above-described variant, a polynucleotide encoding the variant, a nucleic acid construct comprising the same, and / or a vector.

[0245] The nucleic acid construct or vector may be integrated into a chromosome as described above, or may be maintained as an extrachromosomal vector that replicates autonomously.

[0246] The host cell of the present invention includes any progeny of the parent cell that are not identical to the parent cell due to mutations that occur during replication.

[0247] The host cell can be any cell useful for recombinant production of variants, e.g., a prokaryotic or eukaryotic cell.

[0248] The prokaryotic host cell can be any gram-positive or gram-negative bacterium.

[0249] Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.

[0250] Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli (E. coli), Flavobacterium, Fusobacterium, Helicobacter, Iliobacter, Neisseria, Pseudomonas, Salmonella, Vibrio (e.g., Vibrio natriegens), and Ureaplasma.

[0251] In one specific example, the bacterial host cell can be a Bacillus genus host cell, specifically including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis cells.

[0252] In one specific example, the bacterial host cell can be a Streptococcus genus host cell, specifically including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis and Streptococcus equi subspecies Zooepidemicus cells.

[0253] In one specific example, the bacterial host cell can be a host cell of the genus Streptomyces, specifically including but not limited to Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicol, Streptomyces griseus and Streptomyces lividans cells.

[0254] In one specific example, the bacterial host cell may be a host cell of the genus Corynebacterium, such as Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes. ammoniagenes), Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0255] The host cell may be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

[0256] The host cell may be a fungal cell. In the present application, "fungus" includes the Ascomycota, Basidiomycota, Fasciomycota, and Zygomycota, as well as the Oomycota and all imperfect fungi.

[0257] The fungal host cell may be a yeast cell. The term "yeast" in the present application includes yeasts belonging to the order ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts, and Fungi imperfecti (Blastomycetes). However, this classification may vary and may be defined according to the Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0258] The yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Komagataella, Saccharomyces, Schizosaccharomyces or Yarrowia cell, for example, Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces These may be cells of Saccharomyces norbensis, Saccharomyces oviformis, Komagataella phaffii or Yarrowia lipolytica.

[0259] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the phylum Eumycota and the subphylum Oomycota (as defined in the above reference (Hawksworth et al., 1995)). Filamentous fungi are typically characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbonation is strictly aerobic. In contrast, vegetative growth in yeasts, such as Saccharomyces cerevisiae, is by germination of a unicellular thallus, and carbonation may be fermentative.

[0260] The filamentous fungal host cells are Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycellioptora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, It may be a cell of Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or Trichoderma.

[0261] For example, the filamentous fungal host cells include Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Mysellioptora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum,It may be a cell of Trichoderma reesei or Trichoderma viride, but is not limited thereto.

[0262] In one embodiment of the present invention, Escherichia coli (E. coli) was used as the host cell, but the present invention is not limited thereto.

[0263] A host cell expressing a variant polypeptide of the present application may be a host cell comprising at least one of the variant polypeptide of the present application; a polynucleotide encoding the polypeptide; and a vector comprising the polynucleotide.

[0264]

[0265] Another aspect of the present application provides a composition comprising at least one of: a variant polypeptide of the present application; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide.

[0266] The composition of the present application can be used to convert a substrate of a zearalenone-decomposing enzyme into a final product, and can be, but is not limited to, a composition for decomposing zearalenone, a composition for detoxifying zearalenone present in food and / or feed, or a composition for adding to feed.

[0267] The variant polypeptide of the present application; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and / or a host cell expressing the polypeptide can be used to degrade zearalenone.

[0268] The terms used herein are as described above, and degradation of zearalenone can be used interchangeably with detoxification of zearalenone, inactivation of zearalenone, and decontaminating of contamination caused by zearalenone.

[0269] In one embodiment, the zearalenone may be present in a food.

[0270] In one embodiment, the zearalenone may be present in the feed.

[0271] In one embodiment, the composition of the present application can be used to decompose and detoxify zearalenone present in food and / or feed.

[0272]

[0273] The composition of the present application may further include other components in addition to the variants provided in the present application. Those skilled in the art can appropriately select the components to be added to the composition of the present application.

[0274] In one specific example, the composition of the present application may further comprise any component suitable for application in converting a substrate of a zearalenone degrading enzyme into a final product.

[0275] 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.

[0276] In one specific example, the composition provided in the present application may further include a naturally occurring substance or a non-naturally occurring substance in addition to the variant provided in the present application.

[0277] In one specific example, the composition provided in the present application may further comprise an additional enzyme used for decomposing zearalenone in addition to the variant provided in the present application.

[0278] In one embodiment, the vector may be integrated into the chromosome as described above, or may be maintained as an extrachromosomal vector that replicates autonomously.

[0279] In the composition of the present application, the mutant polypeptide may be recovered from a microorganism expressing the polypeptide, or the host cell expressing the polypeptide itself may be used as a source of the polypeptide without being recovered.

[0280]

[0281] Another aspect of the present application provides a method for degrading zearalenone, comprising the step of contacting at least one of: a variant polypeptide of the present application; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide with zearalenone.

[0282] The terms used herein are as described above.

[0283] In the method of the present application, the mutant polypeptide may be recovered from a microorganism expressing the polypeptide, or the host cell itself expressing the polypeptide may be used as a source of the polypeptide without being recovered.

[0284]

[0285] A method for producing a variant of the present invention may include a step of culturing a host cell; and a step of recovering a variant expressed in the culturing step.

[0286] 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.

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] In one specific example, the mutant expressed in the above-described culturing step can be recovered using methods known in the art. For example, the mutant can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0294] The above recovery method may be to collect the mutant using a suitable method known in the art according to the culture method of the host cell of the present application, such as 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 may be recovered from the medium or host cell using a suitable method known in the art.

[0295] In another specific embodiment, the variant expressed by the host cell during the culture step may not be recovered. In this specific embodiment, the host cell expressing the variant itself may be used as a source of the variant.

[0296]

[0297] Another aspect of the present application provides use of the mutant polypeptide of the present application as a zearalenone degrading enzyme.

[0298] The terms used herein are as described above.

[0299] Another aspect of the present application provides a use of a composition comprising at least one of a variant polypeptide of the present application and a host cell expressing the variant polypeptide for degrading zearalenone.

[0300] The terms used herein are as described above.

[0301]

[0302] The specific details for implementing the invention are as follows. Furthermore, each description and embodiment disclosed in this application can 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.

[0303] 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.

[0304]

[0305] Example 1. Preparation of expression vectors for zearalenone-degrading enzyme ZOR_7 and its variants

[0306]

[0307] Example 1-1. Production of a vector expressing zearalenone-degrading enzyme ZOR_7.

[0308]

[0309] A polynucleotide (SEQ ID NO: 2) encoding alpha / beta hydrolase (hereinafter referred to as ZOR_7, SEQ ID NO: 1) derived from Sphingomonas sp. was synthesized by Cosmo Genetech and cloned into a pET vector (Novagen). This was used as a template, and the hydrolase sequence, its polynucleotide sequence, and primer sequences used are as shown in Tables 1 and 2 below.

[0310]

[0311]

[0312] Sequence ContentSequence NumberNamePrimer Sequence (5'→3')ZOR73ZOR7-FgatataccatggggATGACGAGCGAAACCGGCG4ZOR7-RcgagtgcggccgcCGCATCCGGCAGGGTCG

[0313]

[0314] Specifically, ZOR_7 was produced by PCR using the constructed gene construct, primers (SEQ ID NOs. 3 and 4 in Table 2), and PCR premix (iNtRON, cat. no. 25185). PCR was performed using an Eppendorf Mastercycler Nexus GX2, and the reaction conditions were as follows.

[0315] Initial denaturation - 94℃, 2 min

[0316] Denaturation - 94℃ 20sec

[0317] Annealing - 55℃ 10sec

[0318] Extension - 72℃ 2 min (25 cycles from denaturation to extension)

[0319] Final Extension - 72℃ 5min

[0320]

[0321] After the PCR product and vector were treated with restriction enzymes (NcoI, NotI), ligation was performed using T4 DNA ligase (NEB, Cata# M0202S), and then transformed into E. coli Dh5α strain to confirm sequence mutations through sequencing.

[0322]

[0323] Example 1-2. Production of a mutant expression vector of zearalenone-degrading enzyme ZOR7.

[0324]

[0325] To improve the acid stability of the zearalenone-decomposing enzyme ZOR_7, mutation sites were selected, and primers were designed to produce three point mutations and three multiple mutations (hereinafter referred to as ZOR7_M1, ZOR7_M2, and ZOR7_M3, SEQ ID NOs: 5, 6, and 7). The sequence mutation positions refer to the mutation positions based on the sequence of SEQ ID NO: 1, and the amino acid sequences of the mutants, their polynucleotide sequences, and primer sequences for producing the mutants are described in Tables 3 and 4 below.

[0326]

[0327] <h2 style=";text-align:left;direction:ltr">5ZOR7_M1 AA(A45C+T111C)MTSETGAPHPAHRNIFVSHSFPEQQVDLGEVTLNYVEAGDAGKPCLLLVPEQTGSWWSYEPSIAMLAENFHVFAVDMRGQGRSDWTPRRYSLDNFGNDLVRFIALVIKRPCIVAGNSSGGVLAAWLSAYAMPGQIRGALCEDAPFFASELTPAYGHPIRQAAGPAFALMRDYLGDQWSVANWEGFAAAAKASSSPIARLFFATEEAPQNLKEYDPEWGRAFYEGTVALHCPHDRMLAQVKKPMLLTHHAHHIDPETGDLVGALSDFQAQKVQEIVRSTGVRIDYQSFPDALHMMHLFDPALYTQVLREWAATLPDA6ZOR7_M2 AA(K43A)MTSETGAPHPAHRNIFVSHSFPEQQVDLGEVTLNYVEAGDAGAPALLLVPEQTGSWWSYEPSIAMLAENFHVFAVDMRGQGRSDWTPRRYSLDNFGNDLVRFIALVIKRPTIVAGNSSGGVLAAWLSAYAMPGQIRGALCEDAPFFASELTPAYGHPI RQAAGPAFALMRDYLGDQWSVANWEGFAAAAKASSSPIARLFFATEEAPQNLKEYDPEWGRAFYEGTVALHCPHDRMLAQVKKPMLLTHHAHHIDPETGDLVGALSDFQAQKVQEIVRSTGVRIDYQSFPDALHMMHLFDPALYTQVLREWAATLPDA7ZOR7_M3AA(P22T)MTSETGAPHPAHRNIFVSHSFTEQQVDLGEVTLNYVEAGDAGKPALLLVPEQTGSWWSYEPSIAMLAENFHVFAVDMRGQGRSDWTPRRYSLDNFGNDLVRFIALVIKRPTIVAGNSSGGVLAAWLSAYAMPGQIRGALCEDAPFFASELTPAYGHPIRQAAGPAFALMRDYLGDQWSVANWEGFAAAAKASSSPIARLFFATEEAPQNLKEYDPEWGRAFYEGTVALHCPHDRMLAQVKKPMLLTHHAHHIDPETGDLVGALSDFQAQKVQEIVRSTGVRIDYQSFPDALHMMHLFDPALYTQVLREWAATLPDA8ZOR7_M1NT(A45C+T111C)ATGACGAGCGAAACCGGCGCGCCGCATCCGGCGCATCGCAACATTTTTGTGAGCCATAGCTTTCCGGAACAGCAAGTGGATCTGGGCGAAGTGACCCTGAACTATGTGGAAGCGGGCGATGCGGGCAAACCGtgtCTGCTGCTGGTGCCGGAACAGACCGGCAGCTGGTGGAGCTATGAACCGAGCATTGCGATGCTGGCGGAAAACTTTCATGTGTTTGCGGTGGATATGCGCGGCCAAGGCCGCAGCGATTGGACCCCGCGCCGCTATAGCCTGGATAACTTTGGCAACGATCTGGTGCGCTTTATTGCGCTGGTGATTAAGCGCCCTtgtATCGTGGCTGGTAACAGCAGCGGGGGTGTCTTGGCTGCGTGGCTGAGCGCGTATGCGATGCCGGGTCAGATTCGCGGCGCGCTGTGCGAAGATGCGCCGTTTTTTGCGAGCGAACTGACCCCGGCGTATGGCCATCCGATTCGCCAAGCGGCGGGCCCGGCGTTTGCGCTGATGCGCGATTATCTGGGCGATCAGTGGAGCGTGGCGAACTGGGAAGGCTTTGCGGCGGCGGCGAAAGCGAGCAGCAGCCCGATTGCGCGCCTGTTTTTTGCGACCGAAGAAGCGCCGCAGAACCTGAAAGAATATGATCCGGAATGGGGCCGCGCGTTTTATGAAGGCACCGTGGCGCTGCATTGCCCGCATGATCGCATGCTGGCGCAAGTGAAAAAACCGATGCTGCTGACCCATCATGCGCATCATATTGATCCGGAAACCGGCGATCTGGTGGGCGCGCTGAGCGATTTTCAAGCGCAGAAAGTGCAAGAAATTGTGCGCAGCACCGGCGTGCGCATTGATTATCAGAGCTTTCCGGATGCGCTGCACATGATGCATCTGTTTGATCCGGCGCTGTATACCCAAGTGCTGCGCGAATGGGCGGCGACCCTGCCGGATGCG9ZOR7_M2NT(K43A)ATGACGAGCGAAACCGGCGCGCCGCATCCGGCGCATCGCAACATTTTTGTGAGCCATAGCTTTCCGGAACAGCAAGTGGATCTGGGCGAAGTGACCCTGAACTATGTGGAAGCGGGCGATGCGGGCgcgCCGGCGCTGCTGCTGGTGCCGGAACAGACCGGCAGCTGGTGGAGCTATGAACCGAGCATTGCGATGCTGGCGGAAAACTTTCATGTGTTTGCGGTGGATATGCGCGGCCAAGGCCGCAGCGATTGGACCCCGCGCCGCTATAGCCTGGATAACTTTGGCAACGATCTGGTGCGCTTTATTGCGCTGGTGATTAAGCGCCCTACCATCGTGGCTGGTAACAGCAGCGGGGGTGTCTTGGCTGCGTGGCTGAGCGCGTATGCGATGCCGGGTCAGATTCGCGGCGCGCTGTGCGAAGATGCGCCGTTTTTTGCGAGCGAACTGACCCCGGCGTATGGCCATCCGATTCGCCAAGCGGCGGGCCCGGCGTTTGCGCTGATGCGCGATTATCTGGGCGATCAGTGGAGCGTGGCGAACTGGGAAGGCTTTGCGGCGGCGGCGAAAGCGAGCAGCAGCCCGATTGCGCGCCTGTTTTTTGCGACCGAAGAAGCGCCGCAGAACCTGAAAGAATATGATCCGGAATGGGGCCGCGCGTTTTATGAAGGCACCGTGGCGCTGCATTGCCCGCATGATCGCATGCTGGCGCAAGTGAAAAAACCGATGCTGCTGACCCATCATGCGCATCATATTGATCCGGAAACCGGCGATCTGGTGGGCGCGCTGAGCGATTTTCAAGCGCAGAAAGTGCAAGAAATTGTGCGCAGCACCGGCGTGCGCATTGATTATCAGAGCTTTCCGGATGCGCTGCACATGATGCATCTGTTTGATCCGGCGCTGTATACCCAAGTGCTGCGCGAATGGGCGGCGACCCTGCCGGATGCG10ZOR7_M3NT(P22T)ATGACGAGCGAAACCGGCGCGCCGCATCCGGCGCATCGCAACATTTTTGTGAGCCATAGCTTTaccGAACAGCAAGTGGATCTGGGCGAAGTGACCCTGAACTATGTGGAAGCGGGCGATGCGGGCAAACCGGCGCTGCTGCTGGTGCCGGAACAGACCGGCAGCTGGTGGAGCTATGAACCGAGCATTGCGATGCTGGCGGAAAACTTTCATGTGTTTGCGGTGGATATGCGCGGCCAAGGCCGCAGCGATTGGACCCCGCGCCGCTATAGCCTGGATAACTTTGGCAACGATCTGGTGCGCTTTATTGCGCTGGTGATTAAGCGCCCTACCATCGTGGCTGGTAACAGCAGCGGGGGTGTCTTGGCTGCGTGGCTGAGCGCGTATGCGATGCCGGGTCAGATTCGCGGCGCGCTGTGCGAAGATGCGCCGTTTTTTGCGAGCGAACTGACCCCGGCGTATGGCCATCCGATTCGCCAAGCGGCGGGCCCGGCGTTTGCGCTGATGCGCGATTATCTGGGCGATCAGTGGAGCGTGGCGAACTGGGAAGGCTTTGCGGCGGCGGCGAAAGCGAGCAGCAGCCCGATTGCGCGCCTGTTTTTTGCGACCGAAGAAGCGCCGCAGAACCTGAAAGAATATGATCCGGAATGGGGCCGCGCGTTTTATGAAGGCACCGTGGCGCTGCATTGCCCGCATGATCGCATGCTGGCGCAAGTGAAAAAACCGATGCTGCTGACCCATCATGCGCATCATATTGATCCGGAAACCGGCGATCTGGTGGGCGCGCTGAGCGATTTTCAAGCGCAGAAAGTGCAAGAAATTGTGCGCAGCACCGGCGTGCGCATTGATTATCAGAGCTTTCCGGATGCGCTGCACATGATGCATCTGTTTGATCCGGCGCTGTATACCCAAGTGCTGCGCGAATGGGCGGCGACCCTGCCGGATGCG

[0328]

[0329] Variant Name Mutation Content Sequence Number Primer Sequence (5' → 3')ZOR7_M1A45CForward11GATGCGGGCAAACCGtgtCTGCTGCTGGTGCCGReverse12acaCGGTTTGCCCGCATCGCCT111CForward13GTGATTAAGCGCCCCTtgtATCGTGGCTGGTAACReverse14acaAGGGCGCTTAATCACCAGZOR7_M2 K43AForward15GCGGGCGATGCGGGCgcgCCGGGCCTGCTGReverse16cgcGCCCGCATCGCCCGCTTCCACZOR7_M3P22TForward17GTGAGCCATAGCTTTaccGAACAGCAAGTGGATReverse18ggtAAAGCTATGGCTCACAAAAATGTTGCG

[0330] Specifically, three ZOR_7 variants were produced by PCR using the template, primers (SEQ ID NOs: 11 to 18 in Table 4), and PCR premix (iNtRON, cat no. 25185) produced in Example 1-1. PCR was performed using an Eppendorf Mastercycler Nexus GX2, and the reaction conditions were as follows.

[0331] Initial denaturation - 94℃, 2 min

[0332] Denaturation - 94℃ 20sec

[0333] Annealing - 55℃ 10sec

[0334] Extension - 72℃ 2 min (25 cycles from denaturation to extension)

[0335] Final Extension - 72℃ 5min

[0336]

[0337] 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.

[0338]

[0339] Example 2. Expression and purification of zearalenone-degrading enzyme ZOR7 and its variants.

[0340]

[0341] The vectors of ZOR7 and three mutants produced in Example 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 the absorbance (OD) was measured at 37°C and 200 rpm. 600) After culturing until the confluency was between 0.4 and 0.5, IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 1 mM, and the culture was further performed for 16 hours, and 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, and 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). The purified enzyme solution was placed in an Amicon Ultra-15 Centrifugal Filter Unit (UFC901024, Merck) and concentrated by centrifugation at 4°C, 4000 rpm, and 20 minutes. 50 mM Tris-Hcl pH 7.4 buffer was added to the concentrate, and the centrifugation was repeated 2 to 3 times to perform buffer changes. The final concentrate was used as an enzyme solution.

[0342] Protein concentration was determined by mixing 5 μl of diluted enzyme solution and 250 μl of Bradford solution (Quick Start Bradford 1x Dye Reagent, #5000205) and measuring the absorbance at 595 nm.

[0343]

[0344] Example 3. Evaluation of the activity of zearalenone-degrading enzyme ZOR7 and its variants.

[0345]

[0346] Example 3-1. Evaluation of zearalenone degradation activity of ZOR7 and its variants

[0347]

[0348] In order to analyze the activity of ZOR7 and its three mutants produced in Example 2 on zearalenone (ZEN), zearalenone (CAS 17924-92-4, Zearalenone) dissolved in acetonitrile and an enzyme solution were prepared, reacted at pH 8 and 37°C for 4 minutes, and then the reaction was stopped by treating with methanol. The residual amount of ZEN in the reaction product was measured using HPLC-UV (High performance liquid chromatography-Ultraviolet), and the enzyme degradation activity was calculated using the ratio of the total degraded ZEN. The HPLC-UV analysis conditions are as follows.

[0349] As a result, when the relative activity was compared based on the activity of ZOR7, it was confirmed that all three mutants showed a slight increase in zearalenone decomposition activity (Table 5 and Fig. 1).

[0350] VariantsZEN Remaining(%)ZEN Degraded(%)Relative Activity(%)ZOR728.771.3100.0M126.973.1102.5M227.672.4101.5M328.471.6100.4

[0351]

[0352] Example 3-2. Acid stability evaluation of ZOR7 and its variants

[0353]

[0354] To evaluate the acid stability of ZOR7 and its three mutants produced in Example 2, 47.5 μL of purified enzyme was mixed with 2.5 μL of Glycine-HCl (1 M, pH 3.0) and acid-treated at 37°C for 60 minutes. Thereafter, the reaction conditions, except for pH, were the same as in Example 3-1, and the ZEN decomposition amount at pH 3 was compared to the ZEN decomposition amount at pH 8 after each ZEN decomposition reaction, and is shown in Figure 2.

[0355] Relative activity (%) was calculated by dividing the amount of ZEN decomposition at pH 3 by the amount of ZEN decomposition at pH 8.

[0356] As a result of enzyme residual activity evaluation, it was confirmed that the residual activity of M1, M2, and M3 was improved by more than 20% compared to ZOR7 (Fig. 2).

[0357]

[0358] Through the above results, it was confirmed that three mutants of zearalenone decomposing enzyme ZOR7 (ZOR7_M1 of SEQ ID NO: 5, ZOR7_M2 of SEQ ID NO: 6, and ZOR7_M3 of SEQ ID NO: 7) had improved acid stability compared to the zearalenone decomposing enzyme of SEQ ID NO: 1.

[0359]

[0360] 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.

Claims

1. A mutant polypeptide having zearalenone (ZEN) decomposition activity, i) the mutant polypeptide has a sequence identity of 70% or more and less than 100% with SEQ ID NO: 1; and / or ii) the mutant polypeptide is a polypeptide encoded by a polynucleotide having a sequence identity of 99% or more and less than 100% with the sequence encoding the mature polypeptide of SEQ ID NO: 1; and / or iii) the mutant polypeptide is a polypeptide encoded by a polynucleotide that hybridizes with (a) the mature polypeptide coding sequence of SEQ ID NO: 1, (b) a cDNA thereof, or (c) the full-length complement of (a) or (b) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions; and / or iv) the above mutant polypeptide is a functional fragment of i), ii) or iii) polypeptide having zearalenone (ZEN) degrading activity; and A variant polypeptide comprising any one of the following modifications: Substitution of an amino acid at one or more of positions 22, 43, 45 and 111 with another amino acid; Here, the position number corresponds to the position of the polypeptide of sequence number 1.

2. A mutant polypeptide having zearalenone (ZEN) decomposition activity in the first paragraph, wherein amino acid position 22 before modification is proline (P); amino acid position 43 is lysine (K); amino acid position 45 is alanine (A); and amino acid position 111 is threonine (T).

3. In the first paragraph, the mutant polypeptide comprises a substitution of an amino acid at a position selected from the following i) to iii); i) 45+111; ii) No. 43; and iii) No. 22; Here, the position number corresponds to the position of the polypeptide of sequence number 1.

4. In the first paragraph, the mutant polypeptide comprises at least one substitution among the following i) to iii); i) Substitution of amino acids 45 and 111 with cysteine; ii) substitution of amino acid 43 with alanine; and iii) Substitution of amino acid 22 with threonine, Here, the position number corresponds to the position of the polypeptide of sequence number 1.

5. A mutant polypeptide according to claim 1, wherein the mutant polypeptide has a characteristic of increased pH stability compared to a polypeptide composed of the amino acid sequence of sequence number 1.

6. A polynucleotide encoding a mutant polypeptide of any one of claims 1 to 5.

7. A host cell comprising at least one of the mutant polypeptides of any one of claims 1 to 5 and a polynucleotide encoding the polypeptide.

8. A composition for decomposing zearalenone, comprising at least one of the mutant polypeptides of any one of claims 1 to 5 and a host cell expressing the mutant polypeptide.

9. A composition for feed additive comprising at least one of the mutant polypeptides of any one of claims 1 to 5 and a host cell expressing the mutant polypeptide.

10. / A method for decomposing zearalenone, comprising the step of contacting at least one of the mutant polypeptides of any one of claims 1 to 5 and a host cell expressing the mutant polypeptide with zearalenone.

11. A step of culturing a host cell comprising at least one of the variant polypeptides of any one of claims 1 to 5 and a polynucleotide encoding the polypeptide; and A step of recovering a mutant polypeptide having zearalenone decomposition activity expressed in the above culturing step; comprising; A method for producing a mutant polypeptide having zearalenone decomposition activity.

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