Novel zearalenone degrading enzymes and uses thereof

Abstract

The invention relates to a novel zearalenone degrading enzyme and application thereof.

Description

Technical Field

[0001] This disclosure relates to a novel zearalenone-degrading enzyme and its uses. Background Technology

[0002] Among the mycotoxins that contaminate grains, zearalenone (ZEN) is a mycotoxin produced by fungi of the genus *Fusarium*. Zearalenone is a nonsteroidal estrogenic mycotoxin, also known as F-2 mycotoxin or FES (fermented estrogen substance). Its chemical structure and properties are similar to estrogen (a female hormone), which can cause endocrine disorders in humans and livestock. For example, a recent study reported that receptors disrupted by ZEN inhibited estrogen in mouse mammary tissue (Ecotoxicology and Environmental Safety, Vol. 241, August 2022, 113826). Furthermore, ZEN has been reported to stimulate the growth of human breast cancer cells with estrogen-responsive receptors (Toxins 2019, 11(8), 481).

[0003] In particular, zearalenone poisoning is difficult to detect in its early stages because it exhibits weak acute toxicity and does not show toxicity for a short period. Long-term ingestion of grains contaminated with zearalenone can lead to infertility in pigs, and poisoning in humans and livestock can cause cancer or mutations. Although zearalenone is frequently detected in grains imported from South Korea, there have been no reports of acute toxicity; furthermore, due to its heat stability, it can remain in processed foods and feed made from contaminated grains, posing a hazard through accumulation in humans and livestock.

[0004] Biological methods for removing zearalenone from grains contaminated with mycotoxins include methods using microorganisms to degrade the toxins. For example, WO 2006 / 053357 discloses fumonisin, fumonisin derivatives, and microorganisms exhibiting zearalenone-degrading activity; Korean Patent No. 1280811 discloses Bacillus subtilis strain GB-501 (a mutant strain exhibiting zearalenone-degrading activity); and Korean Patent No. 1494624 discloses Agromyces sp. M15 strain with zearalenone-degrading activity. However, since methods using microorganisms are merely an additional effect produced by substances secreted by the strains, they are limited by the low level of degradation activity of zearalenone itself.

[0005] Therefore, there is a need for effective compositions or methods for the effective degradation and removal of the toxicity of zearalenone. Summary of the Invention

[0006] [Technical Issues]

[0007] The problem to be solved by this disclosure is to provide a composition comprising one or more of the following: a polypeptide of SEQ ID NO: 1, a polynucleotide encoding said polypeptide, a vector comprising said polynucleotide and a host cell expressing the polypeptide of SEQ ID NO: 1; its use; and a method for degrading zearalenone.

[0008] [Technical Solution]

[0009] The purpose of this disclosure is to provide a composition for degrading zearalenone, comprising one or more of the following: a polypeptide having zearalenone-degrading activity, a polynucleotide encoding the polypeptide, a vector containing the polynucleotide, and a host cell expressing the polypeptide.

[0010] Another object of this disclosure is to provide a composition for removing the toxicity of zearalenone present in food, feed, or both, comprising one or more of the following: a polypeptide having zearalenone-degrading activity, a polynucleotide encoding said polypeptide, a carrier containing said polynucleotide, and a host cell expressing said polypeptide.

[0011] Another object of this disclosure is to provide a feed additive composition comprising one or more of the following: a polypeptide having zearalenone degradation activity, a polynucleotide encoding the polypeptide, a carrier containing the polynucleotide, and a host cell expressing the polypeptide.

[0012] Another object of this disclosure is to provide a method for degrading zearalenone, comprising contacting zearalenone with one or more of the following: a polypeptide having zearalenone-degrading activity and a host cell expressing said polypeptide.

[0013] Another object of this disclosure is to provide a method for degrading zearalenone, comprising treating zearalenone with a composition comprising one or more of the following: a polypeptide having zearalenone-degrading activity and a host cell expressing said polypeptide.

[0014] Another object of this disclosure is to provide a method for removing the toxicity of zearalenone, comprising treating food or feed containing zearalenone with a composition comprising one or more of the following: a polypeptide having zearalenone-degrading activity and a host cell expressing said polypeptide.

[0015] Another object of this disclosure is to provide a method for preparing a polypeptide of SEQ ID NO: 1 having zearalenone-degrading activity, comprising the step of culturing host cells, said host cells comprising one or more of the following: a polypeptide having zearalenone-degrading activity; a polynucleotide encoding said polypeptide; and a vector comprising said polynucleotide.

[0016] Another object of this disclosure is to provide the use of the polypeptide of SEQ ID NO: 1 as a zearalenone-degrading enzyme.

[0017] Another object of this disclosure is to provide the use of a composition for degrading zearalenone, said composition comprising one or more of the following: a polypeptide having zearalenone-degrading activity, a polynucleotide encoding said polypeptide, a vector containing said polynucleotide, and a host cell expressing said polypeptide.

[0018] [Beneficial Effects]

[0019] Using the polypeptides disclosed herein that have zearalenone-degrading activity, the fungal toxin zearalenone can be effectively degraded. Attached Figure Description

[0020] Figure 1 The activity of the novel zearalenone-degrading enzyme of this disclosure was confirmed by HPLC-UV.

[0021] Figure 2 The activities of the novel zearalenone-degrading enzyme and α / β hydrolase disclosed herein were compared by HPLC-UV. Detailed Implementation

[0022] This disclosure will be described in detail below. Furthermore, each description and embodiment disclosed in this disclosure can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific descriptions below.

[0023] Furthermore, those skilled in the art will recognize, or be able to determine, many equivalents of the specific aspects described in this disclosure using only conventional experiments. Moreover, these equivalents are intended to be included in this disclosure.

[0024] Furthermore, this specification references and cites numerous academic papers and patent documents throughout. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the prior art and the description of this disclosure.

[0025] One aspect of this disclosure is a polypeptide of SEQ ID NO: 1 having zearalenone-degrading activity. This polypeptide may also be referred to as a "zearalenone-degrading enzyme".

[0026] Another aspect of this disclosure is the use of the polypeptide of SEQ ID NO: 1 as a zearalenone-degrading enzyme.

[0027] Another aspect of this disclosure is a composition for degrading zearalenone, comprising one or more of the following: a polypeptide having zearalenone-degrading activity, a polynucleotide encoding the polypeptide, a vector containing the polynucleotide, and a host cell expressing the polypeptide.

[0028] The phrase "degrades zearalenone" can be used interchangeably with "removes the toxicity of zearalenone", "inactivates zearalenone", and "purifies zearalenone".

[0029] The term "zearalenone (ZEN)" in this disclosure refers to a nonsteroidal estrogenic fungal toxin produced by Fusarium fungi, particularly strains of *Fusarium graminearum* (wheat scab), *Fusarium culmorum*, *Fusarium cerealis*, *Fusarium equiseti*, *Fusarium crookwellense*, and *Fusarium semitectum*, and is considered one of the most widely distributed fungal toxins found in cereals worldwide. Zearalenone, also known as F-2 toxin, Fusarium toxin, and FES (fermented estrogen), has approximately 20 isomers, but trans-α-zearalenone is primarily found in naturally occurring cereals. Zearalenone has been reported to cause harm through hormone-like rather than toxic effects. Most cases of poisoning involve excessive hormone secretion, but it can also lead to cancer and mutations. For example, in the livestock industry, there have been reports of zearalenone inducing infertility in pregnant sows; ovarian abnormalities, premature birth, abortion, etc.; and it is known that even a concentration of 50 ppb in feed can cause changes in pig ovaries.

[0030] In one instance, the degradation activity of zearalenone can be confirmed by detecting the residual amount of zearalenone.

[0031] Furthermore, although the polypeptide with zearalenone-degrading activity provided by this invention has been defined as the polypeptide of SEQ ID NO: 1, this does not preclude the addition of non-functional sequences, naturally occurring mutations, silent mutations, or conserved substitutions upstream or downstream of the amino acid sequence of SEQ ID NO: 1. Moreover, it will be apparent to those skilled in the art that any protein exhibiting the same or corresponding activity as the protein composed of SEQ ID NO: 1 corresponds to the polypeptide with zearalenone-degrading activity provided by this disclosure.

[0032] In other words, even though this disclosure describes "a protein or polypeptide with an amino acid sequence shown in a specific SEQ ID NO", "a protein or polypeptide having an amino acid sequence shown in a specific SEQ ID NO", or "a protein or polypeptide containing an amino acid sequence shown in a specific SEQ ID NO", it is clear that proteins with missing, modified, substituted, or added amino acid sequences in a portion of the sequence can also be used in this invention, as long as they have the same or corresponding activity as the polypeptide composed of the amino acid sequence of the corresponding SEQ ID NO.

[0033] In addition, variant polypeptides that differ from the listed sequence in one or more amino acids by conserved substitutions and / or modifications compared to the amino acid sequence of SEQ ID NO: 1, but retain the function or properties of the protein, are also included within the scope of polypeptides provided in this disclosure. Such modifications may include, for example, modifications that remove a portion from the N- and / or C-terminus of a mature protein.

[0034] The term "conservative substitution" in this disclosure refers to the substitution of one amino acid by another amino acid having similar structure and / or chemical properties. The polypeptides of this disclosure 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 typically occur based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties.

[0035] In one embodiment, the polypeptides provided in this disclosure may include polypeptides that: consist of an amino acid sequence having at least 80% or higher homology or identity with SEQ ID NO: 1, such as at least 85%, 90%, 95%, 97%, or 99% or higher; contain an amino acid sequence having at least 80% or higher homology or identity with SEQ ID NO: 1, such as at least 85%, 90%, 95%, 97%, or 99% or higher; or consist substantially of an amino acid sequence having at least 80% or higher homology or identity with SEQ ID NO: 1, such as at least 85%, 90%, 95%, 97%, or 99% or higher. Furthermore, polypeptides having homology or identity and zearalenone-degrading activity are included in the polypeptides with zearalenone-degrading activity provided in this disclosure.

[0036] In one embodiment, the polypeptides provided in this disclosure may be derived from Sphingomonas sp. microorganisms. In one embodiment, the zearalenone-degrading enzyme of this disclosure may be a polypeptide derived from microorganisms with α / β hydrolase activity, but is not limited thereto.

[0037] As used herein, the terms “homology” or “identity” refer to the degree of correlation between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. The terms “homology” and “identity” are often used interchangeably.

[0038] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms and can be used together with a default gap penalty established by the procedure used. Essentially, under moderately or highly stringent conditions, homologous or identical sequences are generally capable of hybridizing with the entire sequence or a portion corresponding to at least approximately 50%, 60%, 70%, 80%, or 90% of the full length. Clearly, hybridization also includes polynucleotides containing universal codons or codons that account for codon degeneracy in polynucleotides.

[0039] Whether any two polynucleotide or polypeptide (including protein) sequences are homologous, similar, or identical can be determined, for example, by known computer algorithms, such as the “FASTA” program with default parameters as described in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, they can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), such as in the Needleman program (version 5.0.0 or later) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (including the GCG package (Devereux, J., et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, [S.] [F.,] [et al., J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] AcademicPress, San Francisco). The methods described in Diego, 1994 and [CARILLO et al.] (1988) SIAM J Applied Math 48: 1073) can be used, for example, to determine homology, similarity, or identity using BLAST or ClustalW from the National Center for Biotechnology Information.

[0040] Homology, similarity, or identity between polynucleotides or polypeptides (including proteins) can be determined by comparing sequence information, for example, using a GAP computer program (such as Needleman et al., (1970), J Mol Biol. 48:443), as described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program can be defined as a value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter sequence of the two sequences. The default parameters of the GAP procedure may include: (1) a binary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix as disclosed in Schwartz and Dayhoff, Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353–358 (1979) by Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) replacement matrix); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy.

[0041] Furthermore, whether any two polynucleotide or polypeptide (including protein) sequences are homologous, similar, or identical can be confirmed by comparing the sequences under defined strict hybridization conditions by Southern hybridization experiments, and the defined appropriate hybridization conditions are within the scope of this 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 SpringHarbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0042] As used herein, the term "polynucleotide" is a polymer in which nucleotide monomers are covalently linked together to form a long chain of DNA or RNA chains of a certain length or longer.

[0043] The polynucleotide encoding the polypeptide with zearalenone-degrading activity disclosed herein may, without limitation, include the polynucleotide encoding the polypeptide of SEQ ID NO: 1 and the polynucleotide encoding the polypeptide having the corresponding activity. For example, the polynucleotide encoding the polypeptide with zearalenone-degrading activity disclosed herein may be a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or a polynucleotide sequence encoding a polypeptide having at least 80% or higher homology or identity with it.

[0044] In the polynucleotide encoding the disclosed polypeptide with zearalenone degradation activity, various modifications can be made to the coding region, taking into account codon degeneracy or preferred codons in the organism intended to express the polypeptide, as long as the amino acid sequence of the polypeptide remains unchanged.

[0045] In one embodiment, the polynucleotide encoding the polypeptide of the present disclosure having zearalenone-degrading activity may consist of, or substantially consist of, a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity with SEQ ID NO: 2, but is not limited thereto. Furthermore, the polynucleotide of the present disclosure may contain a probe, which may be prepared from a known gene sequence (e.g., any sequence capable of hybridizing under stringent conditions with all or part of the complementary sequence of the polynucleotide sequence of the present disclosure, without limitation).

[0046] The term "strict conditions" refers to conditions that enable specific hybridization between polynucleotides. These conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples of stringent conditions may be conditions in which polynucleotides with high homology or identity of 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher hybridize with each other, but polynucleotides with low homology or identity do not hybridize with each other; or washing conditions typical of Southern hybridization, i.e., washing once, or specifically two to three times, at a salt concentration and temperature corresponding to 60°C 1×SSC, 0.1% SDS, specifically 60°C 0.1×SSC, 0.1% SDS, or more specifically 68°C 0.1×SSC, 0.1% SDS.

[0047] Hybridization requires two nucleic acids to have complementary sequences, although mismatches between bases can be possible depending on the strictness of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure may also include substantially similar nucleic acid sequences as well as separate nucleic acid fragments complementary to the whole sequence.

[0048] Specifically, polynucleotides homologous to or identical with the polynucleotides disclosed herein can be detected by applying hybridization conditions comprising a hybridization step at a Tm of 55°C using the conditions described above. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art.

[0049] The appropriate stringency of hybrid polynucleotides depends on the length and complementarity of the polynucleotides, and the variables are well known in the relevant technical fields (e.g., J. Sambrook et al., see above).

[0050] In this disclosure, the term "vector" refers to a DNA construct containing the base sequence of a polynucleotide encoding a desired polypeptide, said polynucleotide being operatively linked to a suitable expression regulatory region (or expression control sequence) such that the desired polypeptide can be expressed in a suitable host. The expression regulatory region may include a promoter capable of initiating transcription, any operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling 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 integrate into the genome itself.

[0051] For example, a polynucleotide encoding a target protein can be expressed in a chromosome using a vector for insertion into the chromosome within a cell. The polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may also contain a selection marker for confirming insertion into the chromosome. The selection marker is used to select cells transformed by the vector, i.e., to confirm the insertion of the desired nucleic acid molecule, and may use markers that confer selectable phenotypes such as drug resistance, auxotrophic phenotype, cytotoxic agent resistance, or surface protein expression. In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit different phenotypic traits, thereby enabling the selection of transformed cells.

[0052] There are no particular limitations on the carriers used in this disclosure, and any carrier known in the art may be used.

[0053] Examples of vectors commonly used in prokaryotic cells include pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, which can be used as phage vectors or granular vectors; and the pDZ, pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET systems, which can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0054] As examples of vectors used in eukaryotic cells, yeast expression vectors can be integrative yeast plasmids (YIp) or extrachromosomal plasmid vectors. Extrachromosomal plasmid vectors can include episome yeast plasmids (YEp), replicative yeast plasmids (YRp), and yeast centromere plasmids (YCp). Furthermore, artificial yeast chromosomes (YAC) can also be used as vectors in this disclosure. Specific examples of vectors that can be used 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 (pYD1 Yeast Display Vector), pYES2, pYES2 / CT, pYES2 / NT A, pYES2 / NT B, and pYES2 / NT. C. pYES2 / CT, pYES2.1, pYES-DEST52, pTEF1 / Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, P TEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron type A The following are examples of pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405, and pYJ406, but not limited to these.

[0055] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a desired protein into a host cell or microorganism so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can include polynucleotides inserted into and located within the host cell's chromosome and extrachromosomal polynucleotides, provided that the protein encoded by the polynucleotide can be expressed in the host cell. Furthermore, the polynucleotide contains DNA and / or RNA encoding the desired protein. The polynucleotide can be introduced into the host cell in any form, as long as it can be expressed in the host cell. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements required for self-expression. The expression cassette often includes a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the polynucleotide. The expression cassette can be in the form of a self-replicating expression vector. Furthermore, the polynucleotide can be introduced into the host cell in its own form and operably linked to the sequence required for expression in the host cell, but is not limited thereto.

[0056] Furthermore, as used herein, the term "operably linked" refers to the functional linking of a polynucleotide encoding the polypeptide desired by the present disclosure to a promoter sequence that initiates and mediates polynucleotide transcription.

[0057] Methods for converting the vectors of this disclosure include any method of introducing nucleic acids into cells, and can be performed by selecting appropriate standard techniques known in the art, depending on the host cell. For example, methods such as electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) methods, DEAE-dextran methods, cationic liposome methods, lithium acetate-DMSO methods, etc., can be used, but are not limited thereto.

[0058] The host cell expressing the polypeptide of the present disclosure having zearalenone degradation activity can be a host cell comprising one or more of the following: the polypeptide of the present disclosure having zearalenone degradation activity; a polynucleotide encoding the polypeptide; and a vector comprising the polynucleotide.

[0059] In one embodiment, as described above, the vector can be integrated into the chromosome or retained as a self-replicating extrachromosomal vector.

[0060] The host cell disclosed herein can be any cell used for the recombinant production of polypeptides with zearalenone-degrading activity, such as prokaryotic or eukaryotic cells. In one example, the host cell can be a fungal cell. In one example, the prokaryotic host cell can be any Gram-positive or Gram-negative bacterium.

[0061] In one instance, the host cell may be a microorganism. For example, the host cell may be Escherichia coli (E. coli), but is not limited thereto.

[0062] The present disclosure includes a polypeptide with zearalenone-degrading activity; a polynucleotide encoding the polypeptide; a vector containing the polynucleotide; and / or a host cell expressing the polypeptide with zearalenone-degrading activity that can be used to degrade zearalenone.

[0063] In one implementation, zearalenone can be present in food.

[0064] In one implementation, zearalenone can be present in feed.

[0065] The compositions disclosed herein can be used to degrade and remove the toxicity of zearalenone present in food and / or feed.

[0066] Another aspect of this disclosure is a composition for removing the toxicity of zearalenone present in food, feed, or both, comprising one or more of the following: a polypeptide of the present disclosure having zearalenone-degrading activity; a polynucleotide encoding the polypeptide; a carrier containing the polynucleotide; and a host cell expressing the polypeptide having zearalenone-degrading activity.

[0067] Another aspect of this disclosure is a feed additive composition comprising one or more of the following: a polypeptide of the present disclosure having zearalenone-degrading activity; a polynucleotide encoding the polypeptide; a carrier comprising the polynucleotide; and a host cell expressing the polypeptide having zearalenone-degrading activity.

[0068] In one embodiment, the compositions disclosed herein may further comprise naturally occurring or non-naturally occurring substances.

[0069] Examples of substances that can be added include, but are not limited to, stabilizers, surfactants, detergent builders, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, binders, lubricants, disintegrants, excipients, solubilizers, suspending agents, colorants, flavorings, buffers, preservatives, analgesics, isotonic agents, diluents, lubricants, etc.

[0070] Another aspect of this disclosure is a method for degrading zearalenone, comprising contacting a polypeptide of the present disclosure having zearalenone-degrading activity and / or a host cell expressing a polypeptide having zearalenone-degrading activity with zearalenone.

[0071] The polypeptide with zearalenone degradation activity and the host cell expressing it are described above.

[0072] Another aspect of this disclosure is a method for degrading zearalenone, comprising treating zearalenone with a composition comprising one or more of the following: a polypeptide of the present disclosure having zearalenone-degrading activity and a host cell expressing a polypeptide having zearalenone-degrading activity.

[0073] The polypeptide with zearalenone degradation activity and the host cell expressing it are described above.

[0074] Another aspect of this disclosure is a method for removing the toxicity of zearalenone, comprising treating food or feed containing zearalenone with a composition comprising one or more of the following: a polypeptide of this disclosure having zearalenone-degrading activity and a host cell expressing a polypeptide having zearalenone-degrading activity.

[0075] The polypeptide with zearalenone degradation activity and the host cell expressing it are described above.

[0076] In the compositions and methods disclosed herein, the polypeptide having zearalenone degradation activity may be recovered from the microorganism expressing the polypeptide, or may not be recovered, and the host cell expressing the polypeptide may itself be used as a source of the polypeptide.

[0077] Another aspect of this disclosure is a method for preparing a polypeptide of SEQ ID NO: 1 having zearalenone-degrading activity, comprising the step of culturing host cells, said host cells comprising one or more of the following: the polypeptide of this disclosure having zearalenone-degrading activity; a polynucleotide encoding said polypeptide; and a vector comprising said polynucleotide.

[0078] The polypeptide with zearalenone degradation activity and the host cell expressing it are described above.

[0079] Another aspect of this disclosure is to provide the use of the polypeptide of SEQ ID NO: 1 as a zearalenone-degrading enzyme.

[0080] The polypeptide of SEQ ID NO: 1 is as described above.

[0081] Another aspect of this disclosure is to provide the use of a composition for degrading zearalenone, said composition comprising one or more of the following: a polypeptide of the present disclosure having zearalenone-degrading activity, a polynucleotide encoding said polypeptide, a vector containing said polynucleotide, and a host cell expressing said polypeptide.

[0082] The polypeptide with zearalenone degradation activity and the host cell expressing it are described above.

[0083] [Modes for Implementing the Invention]

[0084] The present disclosure will be described in more detail below by way of examples and experimental examples. However, the following examples and experimental examples are for illustrative purposes only, and therefore the scope of the disclosure is not limited thereto.

[0085] Example 1: Preparation of a novel expression vector for the ZEN-degrading enzyme ZOR_7

[0086] A polynucleotide (SEQ ID NO: 2) encoding an α / β hydrolase derived from the genus Sphingomonas (hereinafter referred to as ZOR_7, SEQ ID NO:1) was synthesized by Cosmo Genetech and cloned into the pET vector (Novagen) using the primers shown in Table 1.

[0087] [Table 1]

[0088]

[0089] Specifically, ZOR_7 was synthesized by PCR using the gene construct, primers (SEQ ID NO: 3 and 4 in Table 1), and PCR premix (iNtRON, catalog number 25185). PCR was performed using an Eppendorf Mastercycler Nexus GX2, and the reaction conditions are as follows:

[0090] Initial denaturation -94℃, 2 minutes

[0091] - Transformation - 94℃, 20 seconds

[0092] Annealing at -55℃ for 10 seconds

[0093] -Extension - 72℃, 2 minutes (25 cycles of denaturation to extension)

[0094] -Final extension-72℃, 5 minutes

[0095] The PCR product and the obtained vector were treated with restriction enzymes (NcoI and NotI), then ligated with T4 DNA ligase (NEB, Cata# M0202S), and then transformed into Escherichia coli DH5α strain. Sequencing was used to verify that there were no sequence variations.

[0096] Example 2: Expression and purification of a novel ZEN-degrading enzyme, ZOR_7, for activity evaluation

[0097] The ZOR_7 prepared in Example 1 was transformed into Escherichia coli BL21 (DE3) and inoculated into sterile LB medium (BD Difco), then pre-cultured at 37°C and 200 rpm for 16 hours. Then, 1 / 100 of the medium volume was inoculated into a flask containing sterile LB medium and incubated at 37°C and 200 rpm until the absorbance (OD) reached a certain level. 600 The concentration was increased to 0.4-0.5. Isopropyl β-D-1-thiogalactoside (IPTG) was then added to a final concentration of 1 mM, and the cells were cultured for another 16 hours, after which they were recovered by centrifugation. The recovered cells were resuspended in 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole) and the crude enzyme extract was obtained by sonication and centrifugation. The crude enzyme extract was adsorbed onto Ni-NTA resin (Qiagen, catalog number 30230), and the enzyme was purified by sequentially passing it through wash buffer (the lysis buffer composition described above, containing 20 mM imidazole) and elution buffer (the lysis buffer composition described above, containing 250 mM imidazole).

[0098] Protein concentration was determined by mixing 5 μL of diluted enzyme solution with 250 μL of Bradford solution (Quick Start™ Bradford 1x staining reagent, #5000205) and measuring the absorbance at 595 nm.

[0099] Example 3: Activity evaluation using HPLC-UV

[0100] To analyze the activity of ZOR_7 prepared in Example 2 against 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) dissolved in acetonitrile. The reaction was carried out at 37°C for 3 hours and then stopped by treatment with methanol.

[0101] The residual amount of ZEN in the reaction product was determined using high-performance liquid chromatography-ultraviolet (HPLC-UV). Detailed analytical conditions are as follows:

[0102] (1) Chromatography: Agilent Technologies 1260 Infinity II HPLC system

[0103] (2) Column: Inertsil ODS-3 C18 5 μm 4.6 x 250 mm

[0104] (3) Column temperature: 30℃

[0105] (4) Flow rate: 1 mL / min

[0106] (5) Sample injection: 20.0 μL

[0107] (6) Mobile phase: A: Water, B: Acetonitrile

[0108] (7) Elution conditions: isocratic elution (40% water / 60% acetonitrile), detection wavelength 272 nm

[0109] Complete degradation of ZEN was observed in ZOR_7. Detailed results are as follows: Figure 1 As shown.

[0110] Therefore, it is confirmed that the ZOR_7 disclosed herein has the ability to degrade zearalenone.

[0111] Example 4: Comparison of the activities of the new ZEN-degrading enzyme ZOR_7 and α / β hydrolases

[0112] Example 4-1: Preparation of expression vector for control α / β hydrolases derived from Sphingomonas spp.

[0113] A polynucleotide (SEQ ID NO: 6) encoding a control α / β hydrolase derived from Sphingomonas (hereinafter referred to as NC_ZOR_7, SEQ ID NO:5) was synthesized by Cosmo Genetech and cloned into the pET vector (Novagen) using the primers shown in Table 2.

[0114] [Table 2]

[0115]

[0116] Specifically, ZOR_7 was synthesized by PCR using the gene construct, primers (SEQ ID NO: 7 and 8 in Table 2), and PCR premix (iNtRON, catalog number 25185). PCR was performed using an Eppendorf Mastercycler Nexus GX2, and the reaction conditions are as follows:

[0117] Initial denaturation -94℃, 2 minutes

[0118] - Transformation - 94℃, 20 seconds

[0119] Annealing at -55℃ for 10 seconds

[0120] -Extension - 72℃, 2 minutes (25 cycles of denaturation to extension)

[0121] -Final extension-72℃, 5 minutes

[0122] The PCR product and the obtained vector were treated with restriction enzymes (NcoI and NotI), then ligated with T4 DNA ligase (NEB, Cata# M0202S), and then transformed into Escherichia coli DH5α strain. Sequencing was used to verify that there were no sequence variations.

[0123] Example 4-2: Expression and purification of control α / β hydrolases derived from Sphingomonas spp.

[0124] The NC_ZOR_7 prepared in Example 4-1 was transformed into Escherichia coli BL21 (DE3) and inoculated into sterile LB medium (BD Difco), then pre-cultured at 37°C and 200 rpm for 16 hours. Then, 1 / 100 of the medium volume was inoculated into a flask containing sterile LB medium and incubated at 37°C and 200 rpm until the absorbance (OD) reached a certain level. 600 The concentration was increased to 0.4-0.5. Isopropyl β-D-1-thiogalactoside (IPTG) was then added to a final concentration of 1 mM, and the cells were cultured for another 16 hours, after which they were recovered by centrifugation. The recovered cells were resuspended in 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole) and the crude enzyme extract was obtained by sonication and centrifugation. The crude enzyme extract was adsorbed onto Ni-NTA resin (Qiagen, catalog number 30230), and the enzyme was purified by sequentially passing it through wash buffer (the lysis buffer composition described above, containing 20 mM imidazole) and elution buffer (the lysis buffer composition described above, containing 250 mM imidazole).

[0125] Protein concentration was determined by mixing 5 μL of diluted enzyme solution with 250 μL of Bradford solution (Quick Start™ Bradford 1x staining reagent, #5000205) and measuring the absorbance at 595 nm.

[0126] Example 4-3: Activity evaluation using HPLC-UV

[0127] To compare the activity of ZOR_7 prepared in Example 2 and the control NC_ZOR_7 prepared in Example 4-2 against ZEN, the purified enzyme reaction solutions from Example 2 or Example 4-2 were treated with zearalenone (CAS 17924-92-4) dissolved in acetonitrile (50 mM Tris HCl, 150 mM NaCl, pH 7.4). The reaction was carried out at 37°C for 3 hours and then stopped by treatment with methanol.

[0128] The residual amount of ZEN in the reaction product was determined using high-performance liquid chromatography-ultraviolet (HPLC-UV). Detailed analytical conditions are as follows:

[0129] (1) Chromatography: Agilent Technologies 1260 Infinity II HPLC system

[0130] (2) Column: Inertsil ODS-3 C18 5 μm 4.6 x 250 mm

[0131] (3) Column temperature: 30°C

[0132] (4) Flow rate: 1 mL / min

[0133] (5) Sample injection: 20.0 μL

[0134] (6) Mobile phase: A: Water, B: Acetonitrile

[0135] (7) Elution conditions: isocratic elution (40% water / 60% acetonitrile), detection wavelength 272 nm

[0136] It was observed that when ZOR_7 completely degraded ZEN, the control NC_ZOR_7 did not degrade ZEN. Detailed results are as follows... Figure 2 As shown.

[0137] Therefore, it has been confirmed that not all α / β hydrolases derived from the genus *Sphingomonas* possess ZEN degradation capabilities. In fact, among the α / β hydrolases derived from *Sphingomonas*, only one specific enzyme, namely ZOR_7 disclosed herein, possesses a unique zearalenone degradation capability.

[0138] Based on the foregoing description, those skilled in the art to which this disclosure pertains will understand that this disclosure may be implemented in other specific forms without modifying the technical concept or essential features of this disclosure. In this regard, the above embodiments should be understood as illustrative in every respect, not restrictive. The scope of this disclosure should be interpreted as the meaning and scope of the appended claims, not the meaning and scope of the detailed description, and all changes or variations derived from equivalent concepts fall within the scope of this disclosure.

Claims

1. A composition for degrading zearalenone, comprising one or more of the following: The polypeptide of SEQ ID NO: 1; The polynucleotide encoding the polypeptide; A carrier containing the polynucleotide; and Host cells expressing the polypeptide of SEQ ID NO:

1.

2. A composition for removing the toxicity of zearalenone present in food, feed, or both, comprising one or more of the following: The polypeptide of SEQ ID NO: 1; The polynucleotide encoding the polypeptide; A carrier containing the polynucleotide; and Host cells expressing the polypeptide of SEQ ID NO:

1.

3. A feed additive composition comprising one or more of the following: The polypeptide of SEQ ID NO: 1; The polynucleotide encoding the polypeptide; A carrier containing the polynucleotide; and Host cells expressing the polypeptide of SEQ ID NO:

1.

4. A method for degrading zearalenone, comprising contacting zearalenone with one or more of the following: The polypeptide of SEQ ID NO: 1; The polynucleotide encoding the polypeptide; A carrier containing the polynucleotide; and Host cells expressing the polypeptide of SEQ ID NO:

1.

5. A method for preparing a polypeptide of SEQ ID NO: 1 having zearalenone degradation activity, comprising the step of culturing host cells, said host cells comprising one or more of the following: the polypeptide of SEQ ID NO: 1; a polynucleotide encoding said polypeptide; and a vector comprising said polynucleotide.

6. Use of a composition for degrading zearalenone, said composition comprising one or more of the following: a polypeptide of SEQ ID NO: 1; a polynucleotide encoding said polypeptide; a vector comprising said polynucleotide; and a host cell expressing the polypeptide of SEQ ID NO: 1.

Images