Novel fumonisins degrading enzymes and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a new fumonisin-degrading enzyme and use thereof. BACKGROUND

[0002] Fumonisin is a mycotoxin mainly produced by plant pathogenic fungi Fusarium verticillioides and Fusarium proliferatum. These fungi often contaminate corn or corn-based products, and cause various diseases when ingested by humans or animals through food or feed. In general, fumonisin is known to have hepatotoxicity and nephrotoxicity in animals, and is known to be associated with esophageal cancer and neural tube defects in humans. Therefore, fumonisin B1, which is the most common among fumonisins, is classified as Group 2B by the International Agency for Research on Cancer (IARC), which is possibly carcinogenic to humans.

[0003] Therefore, there is a need for an effective composition or method to detoxify fumonisins present in corn or corn-based products.

[0004] [Related Art Documents]

[0005] (Patent Document 1) WO2022-243722 A1 SUMMARY

[0006] [Technical Problem]

[0007] The present disclosure relates to a new fumonisin-degrading enzyme and use thereof.

[0008] [Technical Solution]

[0009] An object of the present disclosure is to provide a polypeptide having fumonisin-degrading activity.

[0010] Another object of the present disclosure is to provide a polynucleotide encoding the polypeptide.

[0011] Still another object of the present disclosure is to provide a vector comprising the polynucleotide.

[0012] Still another object of the present disclosure is to provide a composition for degrading fumonisins, the composition comprising any one or more of: a polypeptide having fumonisin-degrading activity; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide.

[0013] Still another object of the present disclosure is to provide a composition for detoxifying fumonisins present in food, feed, or both, the composition comprising any one or more of: a polypeptide having fumonisin-degrading activity; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide.

[0014] It is still another object of the present disclosure to provide a feed additive composition comprising any one or more of: a polypeptide having fumonisin-degrading activity; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide.

[0015] It is still another object of the present disclosure to provide a method of degrading fumonisin, the method comprising the step of contacting fumonisin with any one or more of: a polypeptide having fumonisin-degrading activity; and a host cell expressing the polypeptide.

[0016] It is still another object of the present disclosure to provide a method of preparing a polypeptide having fumonisin-degrading activity, the method comprising the step of culturing a host cell comprising any one or more of: a polypeptide having fumonisin-degrading activity; a polynucleotide encoding the polypeptide; and a vector comprising the polynucleotide.

[0017] It is still another object of the present disclosure to provide use of the polypeptide of SEQ ID NO: 1 as a fumonisin-degrading enzyme.

[0018] [Advantages]

[0019] Fungal toxin fumonisin can be effectively degraded by using the polypeptide having fumonisin-degrading activity of the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Results showing detection of activity of the new fumonisin-degrading enzyme of the present disclosure using thin layer chromatography (TLC) are shown;

[0021] Figure 2 Results showing detection of activity of the new fumonisin-degrading enzyme of the present disclosure using LC-FLD are shown; and

[0022] Figure 3 Results showing detection of activity of the new fumonisin-degrading enzyme of the present disclosure using LC-TOF / MS are shown. DETAILED DESCRIPTION

[0023] The present disclosure will be described in detail below. Also, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions described below.

[0024] In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be within the scope of the present disclosure.

[0025] In addition, throughout the specification, many papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter to which the present disclosure pertains.

[0026] One aspect of the present disclosure provides a polypeptide of SEQ ID NO: 1 having fumonisin-degrading activity. The polypeptide can also be referred to as a fumonisin-degrading enzyme.

[0027] Meanwhile, the degradation of fumonisin can be used in the same sense as fumonisin detoxification, fumonisin inactivation, and removal of fumonisin contamination (decontamination).

[0028] The fumonisin of the present disclosure includes fumonisin B1, fumonisin B2, fumonisin B3, fumonisin B4, fumonisin A1, and fumonisin A2; and derivatives thereof. For example, the fumonisin of the present disclosure can be selected from fumonisin B1, fumonisin B2, fumonisin B3, fumonisin B4, fumonisin A1, and fumonisin A2. For example, the fumonisin of the present disclosure can be fumonisin B1.

[0029] Fumonisin B1 (FB1) and its hydrolysis product HFB1 can have the structure shown in Chemical Formula 1 below.

[0030] [Chemical Formula 1]

[0031]

[0032] For example, fumonisin-degrading activity can be determined by detecting a hydrolysis product of fumonisin.

[0033] Meanwhile, although the new polypeptide having fumonisin-degrading activity provided in the present disclosure is defined as a polypeptide of SEQ ID NO: 1, it does not exclude the addition of a meaningless sequence, a naturally occurring mutation, or a silent mutation or conservative substitution thereof upstream or downstream of the amino acid sequence of SEQ ID NO: 1, and it is obvious to one skilled in the art that, as long as the protein has the same or corresponding activity to the activity of the protein consisting of the amino acid sequence of SEQ ID NO: 1, it can belong to the polypeptide having fumonisin-degrading activity provided in the present disclosure.

[0034] In other words, although described as a "protein or polypeptide of an amino acid sequence represented by a specific sequence number", "protein or polypeptide having an amino acid sequence represented by a specific sequence number", or "protein or polypeptide comprising an amino acid sequence represented by a specific sequence number" in the present disclosure, it is obvious that any protein having an amino acid sequence with deletion, modification, substitution, or addition in a partial sequence can also be used in the present application, as long as the protein can have the same or corresponding activity as the polypeptide consisting of the amino acid sequence of the corresponding sequence number.

[0035] In addition, a variant polypeptide that differs from the listed sequence by conservative substitutions and / or modifications at one or more amino acids, but that retains the function or property of the protein, compared to the amino acid sequence of SEQ ID NO: 1, also falls within the scope of the polypeptide provided in the present disclosure. Such modifications can include, for example, modifications that remove a portion from the N- and / or C-terminus of the mature protein.

[0036] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structure and / or chemical properties. The polypeptides of the present disclosure can have, for example, one or more conservative substitutions while still retaining one or more biological activities possessed by the polypeptide of SEQ ID NO: 1. Such amino acid substitutions are typically made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues.

[0037] In one embodiment, the polypeptide provided in the present application can comprise a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, or a polypeptide consisting of an amino acid sequence having at least 60% or more, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the amino acid sequence of SEQ ID NO: 1, or comprising or consisting essentially of the amino acid sequence. In addition, as long as the polypeptide has the above homology or identity and has fumonisin-degrading activity, it is included in the polypeptide having fumonisin-degrading activity provided in the present disclosure.

[0038] In one embodiment, the polypeptide provided in the present disclosure can be derived from a microorganism of the genus Bradyrhizobium. In one embodiment, the fumonisin-degrading enzyme of the present disclosure can be a polypeptide having carboxylesterase activity derived from a microorganism, but is not limited thereto.

[0039] As used herein, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid sequences or nucleotide sequences and can be expressed in percentage. The terms homology and identity are often used interchangeably with each other.

[0040] Conserved sequence homology or identity of polynucleotides or polypeptides can be determined by standard alignment algorithms and can be used with the default gap penalties established by the program being used. Essentially, homologous or identical sequences will typically hybridize over the full length or at least about 50%, 60%, 70%, 80%, or 90% of the full length under conditions of moderate or high stringency. Obviously, hybridization also encompasses hybridization of a polynucleotide to a polynucleotide comprising codons commonly used or codons that take into account codon degeneracy.

[0041] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using, for example, known computer algorithms, such as the "FASTA" program using the default parameters 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) can be used, such as the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), using the default parameters (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ET AL, J Mol 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 the BLAST or ClustalW of the National Center for Biotechnology Information.

[0042] Homology, similarity or identity of polynucleotides or polypeptides can be ascertained, e.g., by using sequence information comparing the sequences using, for example, the GAP computer program (as described in Needleman et al. (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482). Briefly, the GAP program defines homology, similarity or identity as the number of similar aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a weight matrix containing values of 1 for identity and 0 for non-identity, and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or the EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4)); (2) a penalty of 3.0 for each gap, and an extra 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.

[0043] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity to each other can be ascertained by comparing the sequences to each other under defined stringent conditions in Southern hybridization experiments, and defined suitable hybridization conditions are within the skill in 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, 2nded., 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).

[0044] Another aspect of the present disclosure provides a polynucleotide encoding a polypeptide having fumonisin-degrading activity of the present disclosure.

[0045] As used herein, the term "polynucleotide" is a long chain polymer of nucleotides formed by covalent linkage of nucleotide monomers, and refers to a DNA or RNA chain having a predetermined length or longer.

[0046] The polynucleotide encoding the polypeptide having fumonisin-degrading activity of the present disclosure can include any polynucleotide without limitation, as long as it is a polynucleotide encoding the polypeptide of SEQ ID NO: 1 or a polypeptide having activity corresponding thereto. For example, the polynucleotide encoding the polypeptide having fumonisin-degrading activity of the present disclosure can be a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or a polypeptide having homology or identity of at least 60% or more thereto.

[0047] The polynucleotide encoding the polypeptide having fumonisin-degrading activity of the present disclosure can be subjected to various modifications in the coding region within the range that does not change the amino acid sequence of the polypeptide due to codon degeneracy or in consideration of codons preferred in an organism in which the polypeptide is to be expressed.

[0048] In one embodiment, the polynucleotide encoding the polypeptide having fumonisin-degrading activity of the present disclosure can consist of or consist essentially of a nucleotide sequence having homology or identity of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more to the sequence of SEQ ID NO: 2, but is not limited thereto. In addition, the polynucleotide of the present disclosure can include a probe, which can be prepared from a known gene sequence, for example, any sequence capable of hybridizing to the complement of all or a portion of the polynucleotide sequence of the present disclosure under stringent conditions without limitation.

[0049] The term "stringent conditions" refers to conditions under which specific hybridization between polynucleotides can occur. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nded., 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, 9.50-9.51, 11.7-11.8). For example, the stringent conditions can include conditions under which polynucleotides having high homology or identity to each other hybridize to each other, polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize to each other, and polynucleotides having lower homology or identity than the above do not hybridize to each other, or washing conditions of a general Southern hybridization, i.e., washing once, specifically twice or three times, at a salt concentration and temperature corresponding to 60°C, 1xSSC, 0.1% SDS, specifically 60°C, 0.1xSSC, 0.1% SDS, and more specifically 68°C, 0.1xSSC, 0.1% SDS.

[0050] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present disclosure can include isolated nucleic acid fragments that are complementary to the entire sequence as well as nucleic acid sequences that are substantially similar thereto.

[0051] Specifically, the polynucleotides having homology or identity to the polynucleotides of the present disclosure can be detected using hybridization conditions including a Tm value of 55°C, a hybridization step under the above conditions. In addition, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by a person skilled in the art according to the purpose thereof.

[0052] The appropriate stringency of polynucleotide hybridization depends on the length and complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., supra).

[0053] Yet another aspect of the present disclosure provides a vector comprising a polynucleotide encoding a polypeptide having fumonisin-degrading activity of the present disclosure. The polypeptide and polynucleotide are as described in other aspects.

[0054] As used herein, the term "vector" refers to a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a desired polypeptide operably linked to suitable expression control regions (expression control sequences) so as to express the desired polypeptide in a suitable host cell. The expression control regions can include a promoter capable of initiating transcription, any operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences for controlling termination of transcription and translation. Once transformed into a suitable host cell, the vector can replicate independently of the host genome or function as an episome, or can integrate into the host genome.

[0055] For example, the polynucleotide encoding the desired protein can be expressed in a chromosome by a vector for intracellular chromosomal insertion. The polynucleotide can be inserted into the chromosome by any method known in the art, for example, by homologous recombination, but is not limited thereto. The vector can further comprise a selection marker to confirm insertion into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the desired nucleic acid molecule is inserted, and a marker that provides a selectable phenotype, such as drug resistance, auxotrophy, resistance to a cytotoxic agent, or expression of a surface protein, can be used. Only cells expressing the selection marker can survive in an environment treated with a selection agent or exhibit a different phenotype, and thus transformed cells can be selected.

[0056] The vector used in the present disclosure is not particularly limited, and any vector known in the art can be used.

[0057] Examples of vectors commonly used in prokaryotic cells can include pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc., as phage vectors or cosmid vectors, and those based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET, etc., as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pDCM2 vector, etc., can be used.

[0058] Examples of vectors used in eukaryotic cells can include integrative yeast plasmids (YIp) and extrachromosomal plasmid vectors as yeast expression vectors. The extrachromosomal plasmid vectors can include episomal yeast plasmids (YEp), replicative yeast plasmids (YRp), and yeast centromeric plasmids (YCp). In addition, artificial yeast chromosomes (YACs) can also be used as vectors of the present disclosure. Specific examples of available vectors can include pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPaA, pGAPaB, pGAPaC, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6aA, pPIC6aB, pPIC6aC, 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, pYJ404, pYJ405, and pYJ406, but are not limited thereto.

[0059] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target protein into a host cell or microorganism so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can be inserted into the chromosome of the host cell or located at an extrachromosomal, irrelevant site, as long as it can be expressed in the host cell. Furthermore, the polynucleotide contains DNA and RNA encoding the desired protein. The polynucleotide can be introduced in any form, as long as it can be introduced and 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 contains 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.

[0060] Furthermore, the term "operably linked" refers to the functional linking of a gene sequence with a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a desired polypeptide disclosed herein.

[0061] Methods for transforming the vectors disclosed herein include any method of introducing nucleic acids into cells, and can be performed by selecting appropriate standard techniques known in the art according to the host cell. For example, transformation methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) technology, DEAE-dextran technology, cationic liposome technology, lithium acetate-DMSO technology, etc.

[0062] Another aspect of this disclosure provides host cells expressing the polypeptides of this disclosure that have fumonisin degradation activity.

[0063] The host cell may be a host cell comprising one or more of the following: a polypeptide of the present disclosure having fumonisin degradation activity; a polynucleotide encoding the polypeptide; and a vector comprising the polynucleotide.

[0064] In one embodiment, the vector can be integrated into the chromosome as described above, or it can be retained as a self-replicating extrachromosomal vector.

[0065] The host cell of the present disclosure can be any cell, e.g., a prokaryotic or eukaryotic cell, used for the recombinant production of a polypeptide having fumonisin-degrading activity. For example, the host cell can be a fungal cell. For example, the prokaryotic host cell can be any gram-positive or gram-negative bacteria.

[0066] For example, the host cell can be a microorganism. For example, the host cell can be E. coli, but is not limited thereto.

[0067] Yet another aspect of the present disclosure provides a composition for degrading fumonisin, the composition comprising any one or more of: a polypeptide having fumonisin-degrading activity of the present disclosure; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing a polypeptide having fumonisin-degrading activity.

[0068] The polypeptide having fumonisin-degrading activity of the present disclosure; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and / or a host cell expressing a polypeptide having fumonisin-degrading activity can be used for degrading fumonisin.

[0069] In one embodiment, the fumonisin can be present in food.

[0070] In one embodiment, the fumonisin can be present in feed.

[0071] The composition of the present disclosure can be used for degrading and detoxifying fumonisin present in food and / or feed.

[0072] Accordingly, yet another aspect of the present disclosure provides a composition for detoxifying fumonisin present in food, feed, or both, the composition comprising any one or more of: a polypeptide having fumonisin-degrading activity of the present disclosure; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing a polypeptide having fumonisin-degrading activity.

[0073] Yet another aspect of the present disclosure provides a feed additive composition comprising any one or more of: a polypeptide having fumonisin-degrading activity of the present disclosure; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing a polypeptide having fumonisin-degrading activity.

[0074] In one embodiment, the composition of the present disclosure can further comprise a naturally-occurring substance or a non-naturally-occurring substance.

[0075] Examples of the substance that can be added include stabilizers, surfactants, builders, chelating agents, dispersants, enzymes, enzyme stabilizers, catalysts, activators, carriers, binders, lubricants, disintegrants, excipients, solubilizers, suspending agents, colorants, flavoring agents, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, diluents, lubricants, preservatives, etc., but are not limited thereto.

[0076] Another aspect of the present disclosure provides a method of degrading fumonisin, the method comprising the step of contacting fumonisin with a polypeptide having fumonisin-degrading activity of the present disclosure; and / or a host cell expressing a polypeptide having fumonisin-degrading activity.

[0077] Still another aspect of the present disclosure provides a method of preparing a polypeptide having fumonisin-degrading activity, the method comprising the step of culturing a host cell comprising any one or more of a polypeptide having fumonisin-degrading activity of the present disclosure, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide.

[0078] As used herein, the term "culturing" refers to the growth of a host cell under suitably controlled environmental conditions. The culturing process of the present disclosure can be performed under suitable culture media and culture conditions known in the art. Such a culturing process can be easily adjusted for use by a person skilled in the art depending on the strain to be selected. Specifically, the culturing can be batch culture, continuous culture, or fed-batch culture, but is not limited thereto.

[0079] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients necessary for culturing the host cell as a main ingredient, which provides nutrients and growth factors necessary for survival and growth, and water. Specifically, the culture medium and other culture conditions for culturing the host cell of the present disclosure can include any culture medium commonly used for culturing host cells, without any particular limitation. However, the host cell of the present disclosure can be cultured in a general culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin under aerobic conditions, while adjusting the temperature, pH, etc.

[0080] In one embodiment, the preparation method can further include the step of recovering the polypeptide having fumonisin-degrading activity expressed in the culturing step.

[0081] In the recovery step, the polypeptide can be recovered using a method known in the art to which the present disclosure pertains. For example, the polypeptide can be recovered from the nutrient culture medium by a conventional method, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0082] The recovery method can be collecting the polypeptide using the method of culturing the host cell of the present disclosure, for example, using a suitable method known in the art according to a batch culture, a continuous culture, or a fed-batch culture method. For example, methods such as centrifugation, filtration, treatment with a protein crystallization precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, etc., HPLC, and combinations of these methods can be used, and a suitable method known in the art can be used to recover the polypeptide from the culture medium or the host cell.

[0083] In another embodiment, the polypeptide expressed by the host cell in the culturing step can not be recovered. The host cell expressing the polypeptide itself can be used as a source of the polypeptide.

[0084] Still another aspect of the present disclosure provides use of the polypeptide of SEQ ID NO: 1 as a fumonisin-degrading enzyme.

[0085] For example, use of the polypeptide of SEQ ID NO: 1 or a polypeptide consisting of an amino acid sequence having at least 60% or more, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the amino acid sequence of SEQ ID NO: 1, a polypeptide comprising the amino acid sequence, or a polypeptide consisting essentially of the amino acid sequence as a fumonisin-degrading enzyme is provided.

[0086] [Mode for carrying out the invention]

[0087] Hereinafter, the present disclosure will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustration of the present disclosure only, and the scope of the present disclosure is not intended to be limited by these Examples and Experimental Examples.

[0088] Example 1. Construction of a vector expressing a new fumonisin-degrading enzyme EFB1-9

[0089] A polynucleotide (SEQ ID NO: 2) encoding a carboxyesterase family protein from Bradyrhizobium sp. (hereinafter referred to as EFB1-9, SEQ ID NO: 1) was synthesized by Cosmo Genetech and cloned into a pET vector (Novagen) using the primers listed in Table 1.

[0090] [Table 1]

[0091]

[0092] In detail, EFB1-9 was prepared by PCR using a gene construct, primers (SEQ ID NO: 3 and 4 in Table 1) and a PCR master mix (iNtRON, Cat. No. 25185). PCR was performed using an Eppendorf Mastercycler Nexus GX2, and the reaction conditions were as follows.

[0093] Initial denaturation - 94°C, 2 min

[0094] Denaturation - 94°C, 20 sec

[0095] Annealing - 56°C, 10 sec

[0096] Extension - 72°C, 2 min (35 cycles from denaturation to extension)

[0097] Final extension - 72°C, 5 min

[0098] After treating the PCR product and the vector with restriction enzymes (NdeI, NotI), ligation was performed using T4 DNA ligase (NEB, Cat. No. M0202S), and then transformed into the E. coli Dh5α strain, and sequence mutations were identified by sequencing.

[0099] Example 2. Expression and purification for evaluation of activity of new FUM-degrading enzyme EFB1-9

[0100] EFB1-9 prepared in Example 1 was transformed into E. coli BL21(DE3) and inoculated into sterile LB medium (BD Difco), and then pre-cultured at 37°C, 200 rpm for 16 hours. Thereafter, 1 / 100 of the medium volume was inoculated into a flask containing sterile LB medium, and cultured at 37°C, 200 rpm until the absorbance (OD 600 ) reached 0.4 to 0.5. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and further cultured for another 16 hours. Then, the cells were recovered by centrifugation. To the recovered cells, 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole) was added, and then resuspended, and then a crude enzyme solution was obtained by ultrasonication and centrifugation. The crude enzyme solution was applied and adsorbed to Ni-NTA resin (Qiagen, Cat. No. 30230), and then the enzyme was purified by sequentially applying a washing buffer (20 mM imidazole concentration in the lysis buffer composition) and an elution buffer (250 mM imidazole concentration in the lysis buffer composition).

[0101] Bradford solution (Quick Start Bradford Protein Assay Kit, Bio-Rad, Cat. No. 500-0205) was mixed, and then the absorbance was measured at 595 nm. The protein concentration was calculated using the following equation: Protein concentration (mg / ml) = (A595 - A650) x dilution factor x 1.25 x 0.1 TMBradford 1x dye reagent, #5000205) and measuring the absorbance at 595 nm to determine the protein concentration.

[0102] Example 3. Evaluation of activity using TLC

[0103] To analyze the activity of the purified EFB1-9 on FUM, the purified enzyme reaction solution (50 mM Tris-HCl, pH 7.4) was treated with fumonisin (CAS 116355-83-0, fumonisin B1 from Fusarium moniliforme) dissolved in a solution of acetonitrile and distilled water in a ratio of 1 : 1 and reacted at room temperature for 24 hours, and then stopped by placing at 95°C for 10 minutes.

[0104] The reaction product was spotted on one end of a TLC plate (Merck, TLC Silica gel 60 F254 20 X 20 cm, catalog number 105554) coated with silica gel on an aluminum surface and placed vertically in a sealed chamber containing a developing solution of acetonitrile and distilled water in a ratio of 7:3 for 6 minutes and 30 seconds. The plate was dried and then immersed in a 0.2% ninhydrin developer dissolved in ethanol and dried in a desiccator at 140°C for 10 minutes.

[0105] It was found that, compared to the control group, the FB1 spot (Rf = 0.59) became brighter and a HFB1 spot (Rf = 0.81) appeared on the plate. The results are shown in Figure 1 and Figure 2 .

[0106] [Table 2]

[0107] No. Sample FB1 HFB1 1 Negative control O X 2 EFB1-9 X O

[0108] Example 4. Evaluation of activity using LC-FLD, LC-TOF / MS

[0109] To detect the concentration of fumonisin B1 remaining after the above reaction and the composition of the degradation products, LC-FLD and LC-TOF / MS analysis was performed.

[0110] Example 4-1. LC-FLD analysis conditions

[0111] The conditions for analyzing the concentration of fumonisin B1 were as follows. Liquid chromatography-fluorescence detection (LC-FLD) was performed using Acquity UPLC of Waters and a fluorescence detector. Fumonisin B1 was separated by column and then analyzed by post-column derivatization with o-phthalaldehyde (OPA) reagent before entering the fluorescence detector. The detailed analysis conditions were as follows.

[0112] (1) Chromatography: Waters Acquity UPLC system

[0113] (2) Column: Waters Acquity UPLC BEH C18 1.7 μm 2.1 x 150 mm

[0114] (3) Column temperature: 40 °C

[0115] (4) Flow rate: 0.25 mL / min

[0116] (5) Sample injection: 10.0 μL

[0117] (6) Mobile phase:

[0118] A: 13.7 mM Octane sulfonic acid + 25 mM Potassium dihydrogen phosphate in DW (pH 2.1 by H3PO4),

[0119] B: 13.7 mM Octane sulfonic acid + 25 mM Potassium dihydrogen phosphate in 50% Acetonitrile (pH 2.1 by H3PO4)

[0120] (7) Elution conditions:

[0121] [Table 3]

[0122] Time (min) ％A ％B 0.0 25 75 8.0 25 75 8.1 0 100 10.5 0 100 10.6 25 75 16.0 25 75

[0123] (8) Detection wavelength

[0124] Excitation wavelength 338 nm, Emission wavelength 425 nm

[0125] (9) Post-column derivatization method

[0126] Reagent: 5.2 mM o-Phthalaldehyde (OPA) in Borate buffer

[0127] Reagent flow rate: 0.25 mL / min

[0128] Reactor temperature: 40 °C

[0129] Example 4-2. LC-TOF / FLD analysis conditions

[0130] Liquid chromatography-time of flight mass spectrometry (LC-TOF / MS) was performed using Waters' Acquity UPLC Xe-VO G2-XS Q-TOF mass spectrometer with the following analysis conditions.

[0131] (1) Chromatography: Waters Acquity UPLC system

[0132] (2) Column: Waters Acquity UPLC BEH C18 1.7 μm 2.1 x 150 mm

[0133] (3) Column temperature: 40 °C

[0134] (4) Flow rate: 0.20 mL / min

[0135] (5) Sample injection: 1.0 μL

[0136] (6) Mobile phase:

[0137] A: 5 mM ammonium formate with 0.1 wt% formic acid in DW

[0138] B: 5 mM ammonium formate with 0.1 wt% formic acid in methanol

[0139] (7) Elution conditions:

[0140] [Table 4]

[0141] Time (min) ％A ％B 0.0 45 55 6.2 45 55 8.2 0 100 9.2 0 100 9.3 45 55 13.0 45 55

[0142] (8) Mass spectrometer: Waters Xe-vo G2-XS Q-Tof

[0143] Ionization mode: ESI positive

[0144] Capillary voltage: 2.5 kV

[0145] Cones hole voltage: 30 V

[0146] Source temperature: 120 °C

[0147] Desolvation gas temperature: 400 °C

[0148] Mass scan range: 50 m / z to 1000 m / z

[0149] Example 4-3. Experimental results

[0150] As in Example 4-1, it was confirmed that the concentration of fumonisin B1 was reduced from 6.40 ppm to 4.60 ppm by the purified EFB1-9 reaction, and peaks considered to be degradation products were produced Figure 2 ).

[0151] As in Example 4-2, it was confirmed that when the same sample was analyzed by LC-TOF / MS, the proportion of fumonisin B1 (m / z 722.39) in the reaction product was reduced to 70% compared to the negative control, while HFB1 (m / z 406.35) was increased to 30% Figure 3 ).

[0152] The detailed analysis results are summarized in Table 3 below.

[0153] [Table 5]

[0154]

[0155]

[0156] Thus, it is confirmed that the EFB1-9 of the present disclosure has fumonisin-degrading ability.

[0157] Based on the above description, those skilled in the art will understand that the present disclosure can be implemented in different specific forms without changing its technical spirit or basic characteristics. In this regard, it should be understood that the above-mentioned embodiments are not restrictive but are illustrative in all aspects. The scope of the present disclosure is defined by the appended claims rather than by the description before them, and thus all changes and modifications falling within the boundaries and scope of the claims, or equivalents of these boundaries and scope, are included in the claims.

Claims

1. A polypeptide of SEQ ID NO:1 with fumonisin degradation activity.

2. A polynucleotide encoding the polypeptide according to claim 1.

3. A vector comprising the polynucleotide according to claim 2.

4. A host cell comprising any one or more of a polypeptide of SEQ ID NO:1 having fumonisin degradation activity, a polynucleotide encoding said polypeptide, and a carrier comprising said polynucleotide.

5. A composition for degrading fumonisin, said composition comprising any 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.

6. A composition for detoxifying fumonisin present in food, feed, or both, said composition comprising any 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.

7. A feed additive composition comprising any 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.

8. A method for degrading fumonisin, the method comprising the step of contacting fumonisin with any one or more of the following: a polypeptide of SEQ ID NO:1; and a host cell expressing the polypeptide of SEQ ID NO:

1.

9. A method for preparing the polypeptide with fumonisin degradation activity according to claim 1, the method comprising the following steps: Cultivate host cells containing any one or more of the polypeptide comprising SEQ ID NO:1, the polynucleotide encoding the polypeptide, and the vector containing the polynucleotide.

10. Use of the polypeptide of SEQ ID NO:1 as a fumonisin-degrading enzyme.

Images