Novel zearalenone-degrading enzyme and uses thereof

Abstract

The present application relates to a novel zearalenone-degrading enzyme and a use thereof.

Description

Novel zearalenone-degrading enzyme and its use

[0001] The present application relates to a novel zearalenone decomposing enzyme and its use.

[0002] Among the mycotoxins that contaminate grains, zearalenone (ZEN) is a mycotoxin produced by the fungus Fusarium. 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 and animals. For example, a recent study reported that receptors damaged by ZEN suppressed estrogen hormones in rat mammary tissue (Ecotoxicology and Environmental Safety Volume 241, August 2022, 113826), and it was reported that ZEN stimulated the growth of human breast cancer cells containing estrogen-responsive receptors (Toxins 2019, 11(8), 481).

[0003] Zearalenone poisoning is difficult to detect in the early stages, as its acute toxicity is weak and does not appear in a short period of time. Long-term consumption of grains contaminated with zearalenone can cause infertility in pigs, and can also cause cancer or mutations in humans and livestock. Zearalenone is frequently detected in grains imported into Korea, but acute toxicity has not been reported. Zearalenone is heat-stable, so it can remain in processed foods and feed made from contaminated grains, accumulating in humans and livestock, causing problems.

[0004] Biological methods for removing zearalenone from grains contaminated with mycotoxins include methods that utilize microorganisms to decompose the toxins. For example, WO 2006 / 053357 discloses fumonisin, fumonisin derivatives, and microorganisms exhibiting zearalenone-decomposing activity. Korean Patent No. 1280811 discloses Bacillus subtilis strain GB-501, a mutant strain exhibiting zearalenone-decomposing activity. Korean Patent No. 1494624 discloses Agromyces genus M15 strain exhibiting zearalenone-decomposing activity. However, methods utilizing microorganisms have the limitation that their zearalenone-decomposing activity itself is low, as their only additional effect is due to substances secreted from the strains.

[0005] Therefore, there is a need for an effective composition or method for effectively decomposing and detoxifying zearalenone.

[0006] The problem to be solved by the present application is to provide a composition comprising at least one of a polypeptide of SEQ ID NO: 1, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, and a host cell expressing the polypeptide of SEQ ID NO: 1, a use thereof, and a method for decomposing zearalenone.

[0007] The purpose of the present application is to provide a composition for decomposing zearalenone, comprising at least one of a polypeptide having zearalenone decomposition activity; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide.

[0008] Another object of the present application is to provide a composition for detoxifying zearalenone present in a food, feed, or both a food and a feed, comprising at least one of a polypeptide having zearalenone decomposition activity; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide.

[0009] Another object of the present application is to provide a composition for feed additive comprising at least one of a polypeptide having zearalenone decomposition activity; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide.

[0010] Another object of the present application is to provide a method for decomposing zearalenone, comprising the step of contacting a polypeptide having zearalenone decomposing activity and at least one host cell expressing the polypeptide with zearalenone.

[0011] Another object of the present application is to provide a method for decomposing zearalenone, comprising treating zearalenone with a composition comprising at least one of a polypeptide having zearalenone decomposing activity and a host cell expressing the polypeptide.

[0012] Another object of the present application is to provide a method for detoxifying zearalenone, comprising treating a food or feed containing zearalenone with a composition comprising at least one of a polypeptide having zearalenone decomposition activity and a host cell expressing the polypeptide.

[0013] Another object of the present application is to provide a method for producing a polypeptide having zearalenone decomposition activity, having SEQ ID NO: 1, comprising a step of culturing a host cell comprising at least one of a polypeptide having zearalenone decomposition activity, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide.

[0014] Another object of the present application is to provide a use of the polypeptide of SEQ ID NO: 1 as a zearalenone degrading enzyme.

[0015] Another object of the present application is to provide a use of a composition for degrading zearalenone, the composition comprising at least one of a polypeptide having zearalenone degrading activity, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, and a host cell expressing the polypeptide.

[0016]

[0017] Zearalenone, a fungal toxin, can be effectively decomposed using the polypeptide having zearalenone decomposition activity of the present application.

[0018]

[0019] Figure 1 shows the results of confirming the activity of the novel zearalenone decomposing enzyme of the present application using HPLC-UV.

[0020] Figure 2 shows the results of comparing the activities of the novel zearalenone decomposing enzyme and α / β hydrolase enzyme of the present application using HPLC-UV.

[0021]

[0022] 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.

[0023] Furthermore, those skilled in the art will recognize or be able to ascertain, using only routine experimentation, numerous equivalents to the specific embodiments of the present application described herein. Furthermore, such equivalents are intended to be encompassed by this application.

[0024] Additionally, numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety, thereby providing a clearer understanding of the technical field to which this application pertains and the content of this application.

[0025]

[0026] One embodiment of the present application is a polypeptide having SEQ ID NO: 1 having zearalenone degrading activity. The polypeptide may also be referred to as a "zearalenone degrading enzyme."

[0027] Another aspect of the present application is the use of the polypeptide of SEQ ID NO: 1 as a zearalenone decomposing enzyme.

[0028] Another aspect of the present application is a composition for decomposing zearalenone, comprising at least one of: a polypeptide having zearalenone decomposition activity; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide.

[0029]

[0030] Degradation of zearalenone can be used synonymously with detoxification of zearalenone, inactivation of zearalenone and decontaminating of zearalenone.

[0031] In this application, the term "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.

[0032]

[0033] For example, zearalenone degradation activity can be confirmed by detecting the residual amount of zearalenone.

[0034] Meanwhile, although the polypeptide having zearalenone decomposition activity provided in the present application is defined as the polypeptide of SEQ ID NO: 1, it does not exclude meaningless sequence additions before and after the amino acid sequence of SEQ ID NO: 1, mutations that may occur naturally, silent mutations thereof, or conservative substitutions, and it is obvious to those skilled in the art that if it has the same or corresponding activity as a protein consisting of the amino acid sequence of SEQ ID NO: 1, it corresponds to the polypeptide having zearalenone decomposition activity provided in the present application.

[0035] That is, even if the present application describes “a protein or polypeptide having an amino acid sequence described by a specific sequence number,” “a protein or polypeptide having an amino acid sequence described by a specific sequence number,” or “a protein or polypeptide comprising an amino acid sequence described by a specific sequence number,” it is clear that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, or added can also be used in the present application if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number.

[0036] Additionally, variant polypeptides that differ from the recited sequence by conservative substitution and / or modification of one or more amino acids compared to the amino acid sequence of SEQ ID NO: 1, but 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.

[0037] The term "conservative substitution" in this application refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. The polypeptide of the present application may have, for example, one or more conservative substitutions while still retaining one or more biological activities of the polypeptide of SEQ ID NO: 1. Such amino acid substitutions may generally be based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0038] 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 SEQ ID NO: 1 or an amino acid sequence of SEQ ID NO: 1, or a polypeptide comprising or consisting essentially of said amino acid sequence. In addition, if it has the homology or identity and has zearalenone degrading activity, it is included in the polypeptide having zearalenone degrading activity provided in the present application.

[0039] In one embodiment, the polypeptide provided in the present application may be derived from a microorganism of the genus Sphingomonas. In one embodiment, the zearalenone degrading enzyme of the present application may be, but is not limited to, a polypeptide having α / β hydrolase activity derived from the microorganism.

[0040]

[0041] In this application, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or base sequences are related, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0042] 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.

[0043] Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO et al.](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information database.

[0044] 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). The default parameters for the GAP program are (1) a unitary matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a 1-bit integer matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0045] Additionally, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and appropriate hybridization conditions are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0046]

[0047] In this application, the term "polynucleotide" means a DNA or RNA strand of a certain length or longer, which is a polymer of nucleotides in which nucleotide units (monomers) are linked in a long chain shape by covalent bonds.

[0048] The polynucleotide encoding a polypeptide having zearalenone decomposition activity of the present application may include, without limitation, a polynucleotide encoding a polypeptide of SEQ ID NO: 1 and a polypeptide having a corresponding activity. For example, the polynucleotide encoding a polypeptide having zearalenone decomposition activity of the present application may be a polynucleotide sequence encoding an amino acid sequence of SEQ ID NO: 1, or a polypeptide having at least 80% homology or identity therewith.

[0049] The polynucleotide encoding the polypeptide having zearalenone decomposition activity of the present application can have various modifications made to the coding region within a range that does not change the amino acid sequence of the polypeptide due to the degeneracy of the codon or in consideration of the codon preferred in the organism to which the polypeptide is to be expressed.

[0050] In one embodiment, the polynucleotide encoding the polypeptide having zearalenone degrading activity of the present application may consist of or consist essentially of a base sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the sequence of SEQ ID NO: 2, but is not limited thereto. In addition, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known gene sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application.

[0051] The above "stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions in which polynucleotides having high homology or identity hybridize with each other, polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 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, are performed.

[0052] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

[0053] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0054] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

[0055] The term "vector" as used herein refers to a DNA construct containing a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or may be integrated into the genome itself.

[0056] For example, a polynucleotide encoding a target protein can be expressed within a chromosome through a vector for intracellular chromosome 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 protein, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic characteristics, so that transformed cells can be selected.

[0057] The vector used in this application is not particularly limited, and any vector known in the art can be used.

[0058] Examples of vectors commonly used in prokaryotes include pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, which can be used as phage vectors or cosmid vectors, and pDZ series, pDC series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series, which can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0059] As an example of a vector used in a eukaryotic cell, the yeast expression vector can be both an integrative yeast plasmid (YIp) and an extrachromosomal plasmid vector. The extrachromosomal plasmid vector can include an episomal yeast plasmid (YEp), a replicative yeast plasmid (YRp), and a yeast centromer plasmid (YCp). In addition, artificial yeast chromosomes (YACs) can also be used as the vector of the present application. As specific examples, available vectors include pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPα A, pGAPα B, pGAPα C, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2 / CT, pYD1 Yeast Display Vector, pYES2, pYES2 / CT, pYES2 / NT A, pYES2 / NT B, pYES2 / NT C, pYES2 / CT, pYES2.1, pYES-DEST52, pTEF1 / Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form, pRS403, pRS404, pRS405, pRS406, pYJ403, Including but not limited to pYJ404, pYJ405 and pYJ406.

[0060] 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.

[0061] Additionally, the term "operably linked" as used herein means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target polypeptide of the present application.

[0062] 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.

[0063]

[0064] The host cell expressing the polypeptide having zearalenone decomposition activity of the present application may be a host cell comprising at least one of: the polypeptide having zearalenone decomposition activity of the present application; a polynucleotide encoding the polypeptide; and a vector comprising the polynucleotide.

[0065] 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.

[0066] The host cell of the present application may be any cell, such as a prokaryotic cell or a eukaryotic cell, useful for the recombinant production of a polypeptide having zearalenone degrading activity. For example, the host cell may be a fungal cell. For example, the prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.

[0067] For example, the host cell may be a microorganism. For example, the host cell may be, but is not limited to, Escherichia coli.

[0068]

[0069] The polypeptide having zearalenone decomposition activity of the present application; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and / or a host cell expressing the polypeptide having zearalenone decomposition activity can be used to decompose zearalenone.

[0070] In one embodiment, the zearalenone may be present in a food.

[0071] In one embodiment, the zearalenone may be present in the feed.

[0072] The composition of the present application can be used to decompose and detoxify zearalenone present in food and / or feed.

[0073]

[0074] Another aspect of the present application is a composition for detoxifying zearalenone present in a food, feed, or both a food and a feed, comprising at least one of a polypeptide having zearalenone decomposition activity of the present application; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide having zearalenone decomposition activity.

[0075] Another aspect of the present application is a composition for feed additive comprising at least one of: a polypeptide having zearalenone decomposition activity of the present application; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide having zearalenone decomposition activity.

[0076]

[0077] In one embodiment, the composition of the present application may further comprise a naturally occurring substance or a non-naturally occurring substance.

[0078] 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.

[0079]

[0080] Another aspect of the present application is a method for degrading zearalenone, comprising the step of contacting a polypeptide having zearalenone degrading activity of the present application and / or a host cell expressing the polypeptide having zearalenone degrading activity with zearalenone.

[0081] The polypeptide having the above zearalenone decomposition activity and the host cell expressing the same are as described above.

[0082]

[0083] Another aspect of the present application is a method for decomposing zearalenone, comprising treating zearalenone with a composition comprising at least one of a polypeptide having zearalenone decomposition activity of the present application and a host cell expressing the polypeptide having zearalenone decomposition activity.

[0084] The polypeptide having the above zearalenone decomposition activity and the host cell expressing the same are as described above.

[0085] Another aspect of the present application is a method for detoxifying zearalenone, comprising treating a food or feed containing zearalenone with a composition comprising at least one of a polypeptide having zearalenone decomposition activity of the present application and a host cell expressing the polypeptide having zearalenone decomposition activity.

[0086] The polypeptide having the above zearalenone decomposition activity and the host cell expressing the same are as described above.

[0087]

[0088] In the composition and method of the present application, the polypeptide having zearalenone decomposition activity can be recovered from a microorganism expressing the polypeptide, or the host cell expressing the polypeptide itself can be used as a source of the polypeptide without being recovered.

[0089]

[0090] Another aspect of the present application is a method for producing a polypeptide having zearalenone decomposition activity, having SEQ ID NO: 1, comprising the step of culturing a host cell comprising at least one of a polypeptide having zearalenone decomposition activity of the present application, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide.

[0091] The polypeptide having the above zearalenone decomposition activity and the host cell expressing the same are as described above.

[0092]

[0093] Another aspect of the present application provides a use of the polypeptide of SEQ ID NO: 1 as a zearalenone degrading enzyme.

[0094] The polypeptide of the above sequence number 1 is as described above.

[0095]

[0096] Another aspect of the present application provides a use of a composition for degrading zearalenone, the composition comprising at least one of a polypeptide having zearalenone degrading activity of the present application, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, and a host cell expressing the polypeptide.

[0097] The polypeptide having the above zearalenone decomposition activity and the host cell expressing the same are as described above.

[0098]

[0099] Hereinafter, this application will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify this application and the scope of this application is not limited to these examples and experimental examples.

[0100]

[0101] Example 1. Preparation of a novel ZEN degrading enzyme ZOR_7 expression vector

[0102]

[0103] A polynucleotide (SEQ ID NO: 2) encoding α / β 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) using the primers described in Table 1.

[0104]

[0105] Sequence number name primer sequence (5'→3') ZOR_73ZOR-7-FgatataccatggggATGACGAGCGAAACCGGCG4ZOR-7-RcgagtgcggccgcCGCATCCGGCAGGGTCG

[0106]

[0107] Specifically, ZOR_7 was produced by PCR using a gene construct, primers (SEQ ID NOs. 3 and 4 in Table 1), and PCR premix (iNtRON, cat. no. 25185). PCR was performed using an Eppendorf Mastercycler Nexus GX2, and the reaction conditions were as follows: Initial denaturation - 94°C, 2 min

[0108] Denaturation - 94℃, 20sec

[0109] Annealing - 55℃, 10sec

[0110] Extension - 72℃, 2 min (25 cycles from denaturation to extension)

[0111] Final Extension - 72℃, 5min

[0112]

[0113] 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.

[0114]

[0115] Example 2. Expression and purification for evaluating the activity of the novel ZEN degrading enzyme ZOR_7.

[0116]

[0117] ZOR_7 produced in the above Example 1 was transformed into E. coli BL21 (DE3), inoculated into sterilized LB medium (BD Difco), and pre-cultured at 37°C and 200 rpm for 16 hours. Afterwards, 1 / 100 of the medium volume was inoculated into a flask containing sterilized LB medium, and then the absorbance (OD 600 ) was cultured until the confluency was between 0.4 and 0.5, then IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 1 mM, and cultured for an additional 16 hours. Then, the cells were harvested by centrifugation. 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole) was added to the harvested cells, redispersed, and 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).

[0118] 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.

[0119]

[0120] Example 3. Activity evaluation using HPLC-UV

[0121]

[0122] In order to analyze the activity of ZOR_7 produced in Example 2 on ZEN, the purified enzyme reaction solution (50 mM Tris HCl, 150 mM NaCl, pH 7.4) from Example 2 was treated with zearalenone (CAS 17924-92-4, Zearalenone) dissolved in acetonitrile, and then the reaction was stopped by treating with methanol after 3 hours of reaction at 37°C.

[0123] The residual amount of ZEN in the above reaction product was measured using HPLC-UV (High-performance liquid chromatography-Ultraviolet). Detailed analysis conditions are as follows.

[0124]

[0125] (1) Chromatography: Agilent Technologies 1260 Infinity II HPLC System

[0126] (2) Column: Inertsil ODS-3 C18 5um 4.6x250mm

[0127] (3) Column temperature: 30 ℃

[0128] (4) Flow rate: 1 mL / min

[0129] (5) Sample injection: 20.0 μL

[0130] (6) Mobile phase: A: Water, B: Acetonitrile

[0131] (7) Elution conditions: Isocratic elution (40% water / 60% Acetonitrile), detection wavelength 272 nm

[0132]

[0133] As a result, it was confirmed that ZOR_7 completely disassembled ZEN. The detailed results are shown in Figure 1.

[0134] Through this, it was confirmed that ZOR_7 of the present application has the ability to decompose zearalenone.

[0135]

[0136] Example 4. Comparison of the activities of the novel ZEN degrading enzyme ZOR_7 and α / β hydrolase enzymes.

[0137]

[0138] Example 4-1. Preparation of α / β hydrolase expression vector derived from control strain Sphingonas sp.

[0139]

[0140] A polynucleotide (SEQ ID NO: 6) encoding a control α / β hydrolase derived from Sphingomonas sp. (hereinafter referred to as NC_ZOR_7, SEQ ID NO: 5) was synthesized by Cosmo genetech and cloned into a pET vector (Novagen) using the primers described in Table 2.

[0141]

[0142] Sequence number Name Primer sequence (5'→3') NC_ZOR_77NC_ZOR_7_FgatataccatggggATGAAGTTGCGCCTGACGATATTG8NC_ZOR_7_RctcgagtgcggccgcTTTGCGACCATGTCCTAAATAACG

[0143]

[0144] Specifically, ZOR_7 was produced by PCR using a gene construct, primers (SEQ ID NOs. 7 and 8 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: Initial denaturation - 94°C, 2 min

[0145] Denaturation - 94℃, 20sec

[0146] Annealing - 55℃, 10sec

[0147] Extension - 72℃, 2 min (25 cycles from denaturation to extension)

[0148] Final Extension - 72℃, 5min

[0149]

[0150] 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.

[0151]

[0152] Example 4-2. Control: Purification of α / β hydrolase expression from Sphingonas sp.

[0153]

[0154] NC_ZOR_7 produced in the above Example 4-1 was transformed into E. coli BL21 (DE3), inoculated into sterilized LB medium (BD Difco), and pre-cultured at 37°C and 200 rpm for 16 hours. Afterwards, 1 / 100 of the medium volume was inoculated into a flask containing sterilized LB medium, and then the absorbance (OD 600) was cultured until the confluency was between 0.4 and 0.5, then IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 1 mM, and cultured for an additional 16 hours. Then, the cells were harvested by centrifugation. 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole) was added to the harvested cells, redispersed, and 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).

[0155] Protein concentration was determined by mixing 5 ㎕ of diluted enzyme solution and 250 ㎕ of Bradford solution (Quick Start Bradford 1x Dye Reagent, #5000205) and measuring the absorbance at 595 nm.

[0156]

[0157] Example 4-3. Activity evaluation using HPLC-UV

[0158]

[0159] In order to compare and analyze the activity of ZOR_7 produced in the above Example 2 and the control NC_ZOR_7 produced in the above Example 4-2 against ZEN, each enzyme reaction solution (50 mM Tris HCl, 150 mM NaCl, pH 7.4) purified in the above Example 2 or the above Example 4-2 was treated with zearalenone (CAS 17924-92-4, Zearalenone) dissolved in acetonitrile solution, and then the reaction was stopped by treating with methanol after 3 hours of reaction at 37°C.

[0160] The residual amount of ZEN in the reaction product was measured using HPLC-UV (High performance liquid chromatography-Ultraviolet) in the same manner as in Example 3.

[0161]

[0162] As a result, it was confirmed that ZOR_7 completely decomposes ZEN, whereas the control group, NC_ZOR_7, does not decompose ZEN. Detailed results are shown in Fig. 2.

[0163] Through this, it was confirmed that not all α / β hydrolases derived from the genus Sphingomonas have ZEN decomposition activity, and even among the α / β hydrolases derived from the genus Sphingomonas, it was confirmed that only a specific enzyme, i.e., ZOR_7 of the present application, has a specific ability to decompose zearalenone.

[0164]

[0165] 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. Polypeptide of sequence number 1; A polynucleotide encoding the above polypeptide; A vector comprising the polynucleotide; and A composition for decomposing zearalenone, comprising at least one host cell expressing the polypeptide of the above sequence number 1.

2. Polypeptide of sequence number 1; A polynucleotide encoding the above polypeptide; A vector comprising the polynucleotide; and Comprising at least one host cell expressing the polypeptide of the above sequence number 1, A composition for detoxifying zearalenone present in food, feed or both said food and feed.

3. Polypeptide of sequence number 1; A polynucleotide encoding the above polypeptide; A vector comprising the polynucleotide; and A composition for feed additive comprising at least one host cell expressing the polypeptide of the above sequence number 1.

4. Polypeptide of sequence number 1; A polynucleotide encoding the above polypeptide; A vector comprising the polynucleotide; and A method for decomposing zearalenone, comprising the step of contacting at least one host cell expressing the polypeptide of the above sequence number 1 with zearalenone.

5. A method for producing a polypeptide of SEQ ID NO: 1 having zearalenone decomposition activity, comprising the step of culturing a host cell comprising at least one of a polypeptide of SEQ ID NO: 1, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide.

6. Use of a composition comprising at least one of a polypeptide of SEQ ID NO: 1, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, and a host cell expressing the polypeptide of SEQ ID NO: 1 for decomposing zearalenone.

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