Novel fumonisin-degrading enzyme and uses thereof

Abstract

The present application relates to a novel fumonisin-degrading enzyme and uses thereof.

Description

[Technical Field]

[0001] The present application relates to a novel fumonisin-degrading enzyme and uses thereof. [Background technology]

[0002] Fumonisins are mycotoxins primarily produced by the plant pathogenic fungi Fusarium verticillioides and Fusarium proliferatum. These fungi often contaminate corn and corn-based products, causing various diseases when ingested by humans and animals through food or feed. Fumonisins are generally known to be hepatotoxic and nephrotoxic in animals and to be associated with esophageal cancer and neural tube defects in humans. Therefore, fumonisin B1, the most commonly found type of fumonisin, is classified as Group 2B by the International Agency for Research on Cancer (IARC), which indicates it may cause cancer in humans.

[0003] Therefore, there is currently a need for effective compositions or methods for detoxifying fumonisins present in corn or corn-based products. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2022-243722 A1 [Non-patent literature]

[0005] [Non-Patent Document 1] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]:2444 [Non-patent document 2] Rice et al., 2000, Trends Genet. 16:276-277 [Non-licensed document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0006] The present application relates to a novel fumonisin-degrading enzyme and uses thereof. [Means for solving the problem]

[0007] An object of the present application is to provide polypeptides having fumonisin-degrading activity.

[0008] Another object of the present application is to provide a polynucleotide encoding said polypeptide.

[0009] Another object of the present application is to provide a vector containing said polynucleotide.

[0010] Another object of the present application is to provide a composition for decomposing fumonisins, comprising one or more of: a polypeptide having fumonisin-decomposing activity; a polynucleotide encoding the polypeptide; a vector containing the polynucleotide; and a host cell expressing the polypeptide.

[0011] Another object of the present application is to provide a composition for detoxifying fumonisins present in food, feed, or both the food and feed, comprising one or more of: a polypeptide having fumonisin-degrading activity; a polynucleotide encoding the polypeptide; a vector containing the polynucleotide; and a host cell expressing the polypeptide.

[0012] Another object of the present application is to provide a feed additive composition comprising 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] Another object of the present application is to provide a method for decomposing fumonisins, comprising the step of contacting one or more of a polypeptide having fumonisin-decomposing activity and a host cell expressing said polypeptide with a fumonisin.

[0014] Another object of the present application is to provide a method for producing a polypeptide having fumonisin decomposition activity, the method comprising culturing a host cell containing one or more of a polypeptide having fumonisin decomposition activity, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0015] Another object of the present application is to provide use of the polypeptide of SEQ ID NO: 1 as a fumonisin-degrading enzyme. [Effects of the Invention]

[0016] The polypeptide of the present application having fumonisin-degrading activity can be used to effectively degrade fumonisins, which are fungal toxins. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows the results of confirming the novel fumonisin-degrading enzyme activity of the present application using thin layer chromatography (TLC). [Figure 2] 1 shows the results of confirming the novel fumonisin-degrading enzyme activity of the present application using LC-FLD. [Figure 3] 1 shows the results of confirming the novel fumonisin-degrading enzyme activity of the present application using LC-TOF / MS. DETAILED DESCRIPTION OF THE INVENTION

[0018] This will be explained in more detail as follows: Meanwhile, each description and embodiment disclosed in this application also applies to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions described below are not considered to limit the category of this application.

[0019] Additionally, 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 invention described herein and such equivalents are intended to be encompassed by this application.

[0020] Furthermore, throughout this specification, numerous papers and patent documents are referenced and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0021] One aspect of the present application is a polypeptide having fumonisin-degrading activity as set forth in SEQ ID NO: 1. The polypeptide is also referred to as a fumonisin-degrading enzyme.

[0022] On the other hand, degradation of fumonisins can be used synonymously with detoxification of fumonisins, inactivation of fumonisins, and decontaminating of fumonisins.

[0023] The fumonisins of the present application include fumonisin B1, fumonisin B2, fumonisin B3, fumonisin B4, fumonisin A1, and fumonisin A2; and derivatives thereof. As an example, the fumonisin of the present application may be selected from fumonisin B1, fumonisin B2, fumonisin B3, fumonisin B4, fumonisin A1, and fumonisin A2. As an example, the fumonisin of the present application may be fumonisin B1.

[0024] Fumonisin B1 (FB1) and its hydrolysis product HFB1 have the structure shown in Chemical Formula 1 below.

[0025] [ka]

[0026] As an example, fumonisin-degrading activity can be confirmed by detecting hydrolysis products of fumonisins.

[0027] Meanwhile, although the novel polypeptide having fumonisin-degrading activity provided in the present application is defined as the polypeptide of SEQ ID NO: 1, this does not exclude meaningless addition of sequences before or after the amino acid sequence of SEQ ID NO: 1, naturally occurring mutations, silent mutations, or conservative substitutions thereof, and it will be obvious to those skilled in the art that any polypeptide having the same or corresponding activity as a protein consisting of the amino acid sequence of SEQ ID NO: 1 falls within the scope of the polypeptide having fumonisin-degrading activity provided in the present application.

[0028] In other words, even if the present application describes a "protein or polypeptide having an amino acid sequence described in a specific SEQ ID NO," "a protein or polypeptide having an amino acid sequence described in a specific SEQ ID NO," or "a protein or polypeptide comprising an amino acid sequence described in a specific SEQ ID NO," it is obvious that a protein having an amino acid sequence in which part of the sequence has been deleted, modified, substituted, or added is also used in the present application, as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the SEQ ID NO.

[0029] Also included within the scope of the polypeptides provided herein are variant polypeptides that differ from the recited sequence by conservative substitutions and / or modifications of one or more amino acids compared to the amino acid sequence of SEQ ID NO: 1, but that maintain the functions or properties of the protein. Such variants include, for example, variants in which portions are deleted from the N- and / or C-termini of the mature protein.

[0030] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. A polypeptide of the present application can 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 made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0031] In one embodiment, the polypeptides provided herein include SEQ ID NO: 1 or a polypeptide consisting of, comprising, or consisting essentially 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. Furthermore, if a polypeptide has the homology or identity and fumonisin-degrading activity, it is included in the polypeptides provided herein that have fumonisin-degrading activity.

[0032] In one embodiment, the polypeptide provided herein may be derived from a microorganism of the genus Bradyrhizobium, and the fumonisin-degrading enzyme of the present application may be, but is not limited to, a polypeptide having carboxyesterase activity derived from the microorganism.

[0033] As used herein, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0034] Homology or identity of conserved polynucleotide or polypeptide sequences can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing under moderately or highly stringent conditions over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or length. Hybridization obviously also includes polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0035] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, e.g., as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), 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), 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, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0036] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or as described in, e.g., Needleman et al. (1970), J Mol Biol. 48:443. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a unitary matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0037] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. Suitable defined 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).

[0038] Another aspect of the present application is a polynucleotide encoding a polypeptide having fumonisin-degrading activity of the present application.

[0039] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain of a certain length or greater.

[0040] Polynucleotides encoding polypeptides having fumonisin-degrading activity of the present application include, without limitation, polynucleotides that encode the polypeptide of SEQ ID NO: 1 and polypeptides having activity corresponding thereto. For example, polynucleotides encoding polypeptides having fumonisin-degrading activity of the present application may be polynucleotide sequences that encode the amino acid sequence of SEQ ID NO: 1 or polypeptides having at least 60% or more homology or identity thereto.

[0041] A polynucleotide encoding a polypeptide of the present application having fumonisin-degrading activity may have various modifications in the coding region, taking into consideration codon degeneracy or codons preferred in an organism in which the polypeptide is to be expressed, as long as the amino acid sequence of the polypeptide is not changed.

[0042] In one embodiment, a polynucleotide encoding a polypeptide having fumonisin-degrading activity of the present application may consist of, or essentially consist of, a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the sequence of SEQ ID NO: 2, but is not limited thereto. Furthermore, the polynucleotide of the present application may include, without limitation, a probe prepared from a known gene sequence, for example, a sequence that can hybridize under stringent conditions to a complementary sequence to all or a portion of the polynucleotide sequence of the present application.

[0043] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such 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; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, pp. 9.50-9.51, 11.7-11.8). For example, conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 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, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; and conditions under which washing is performed once, or specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, such as 60°C, 1×SSC, and 0.1% SDS, specifically 60°C, 0.1×SSC, and 0.1% SDS, more specifically 68°C, 0.1×SSC, and 0.1% SDS.

[0044] Hybridization requires that two nucleic acids have complementary sequences, even though 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 one another. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0045] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0046] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0047] Another aspect of the present application is a vector comprising a polynucleotide encoding a polypeptide having fumonisin-degrading activity of the present application, wherein the polypeptide and polynucleotide are as described in the other aspects above.

[0048] The term "vector" as used in this application refers to a DNA construct comprising a polynucleotide sequence encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) so as to express the polypeptide in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, a vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0049] For example, a polynucleotide encoding a target protein can be expressed in a chromosome of a cell through a vector for chromosomal integration. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of insertion of the target nucleic acid molecule. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein, is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0050] The vector used in the present application is not particularly limited, and any vector known in the art can be used.

[0051] Examples of vectors commonly used in prokaryotic cells include phage or cosmid vectors such as pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, and plasmid vectors such as pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pDCM2 vectors can be used.

[0052] Examples of vectors used in eukaryotic cells include yeast expression vectors, such as integrative yeast plasmids (YIp) and extrachromosomal plasmid vectors. The extrachromosomal plasmid vectors include episomal yeast plasmids (YEp), replicative yeast plasmids (YRp), and yeast centromere plasmids (YCp). Yeast artificial chromosomes (YACs) can also be used as vectors in this application. As specific examples, available vectors include pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPαA, pGAPαB, pGAPαC, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6αA, pPIC6αB, pPIC6αC, pPIC9K, pYC2 / CT, pYD1 Yeast Display Vector, pYES2, pYES2 / CT, pYES2 / NT A, pYES2 / NT B, pYES2 / NT C, pYES2 / CT, pYES2.1, pYES-DEST52, pTEF1 / Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, P TEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A Forms include, but are not limited to, pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405 and pYJ406.

[0053] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target protein into a host cell or microorganism, thereby enabling the expression of the protein encoded by the polynucleotide in the host cell. A transformed polynucleotide may include any polynucleotide, whether it is located intrachromosomally or extrachromosomally, as long as it is expressible in the host cell. The polynucleotide may also include DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form that can be introduced and expressed in the host cell. 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 its own expression. The expression cassette typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0054] In addition, the term "operably linked" means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target polypeptide of the present application.

[0055] Methods for transforming the vectors of the present application include any method for introducing nucleic acids into cells, and can be carried out by selecting a suitable standard technique as known in the art depending on the host cell, such as, but 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.

[0056] Another aspect of the present application is a host cell expressing a polypeptide having fumonisin-degrading activity of the present application.

[0057] The host cell may comprise one or more of: a polypeptide having fumonisin-degrading activity of the present application; a polynucleotide encoding the polypeptide; and a vector comprising the polynucleotide.

[0058] In one embodiment, the vector may be integrated into a chromosome or maintained as an autonomously replicating extrachromosomal vector, as described above.

[0059] The host cell of the present application can be any cell, e.g., a prokaryotic or eukaryotic cell, useful 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 bacterium.

[0060] As an example, the host cell may be a microorganism, for example, but not limited to, E. coli.

[0061] Another aspect of the present application is a composition for decomposing fumonisins, comprising one or more of the polypeptide of the present application having fumonisin-decomposing activity; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide having fumonisin-decomposing activity.

[0062] The polypeptides having fumonisin-degrading activity of the present application; polynucleotides encoding the polypeptides; vectors containing the polynucleotides; and / or host cells expressing the polypeptides having fumonisin-degrading activity can be used to degrade fumonisins.

[0063] In one embodiment, the fumonisin may be present in a food product.

[0064] In one embodiment, the fumonisin may be present in feed.

[0065] The compositions of the present application can be used to degrade and detoxify fumonisins present in food and / or feed.

[0066] Accordingly, another aspect of the present application is a composition for detoxifying fumonisins present in food, feed, or both the food and feed, comprising one or more of the polypeptide having fumonisin-degrading activity of the present application; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide having fumonisin-degrading activity.

[0067] Another aspect of the present application is a feed additive composition comprising one or more of the polypeptide of the present application having fumonisin-degrading activity; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and a host cell expressing the polypeptide having fumonisin-degrading activity.

[0068] In one embodiment, the composition of the present application may further include a naturally occurring or non-naturally occurring substance.

[0069] Examples of substances that may be added include, but are not limited to, stabilizers, surfactants, builders, chelating agents, dispersing agents, enzymes, enzyme stabilizers, catalysts, activators, carriers, compounding agents, lubricants, disintegrants, excipients, solubilizers, suspending agents, dyes, flavorings, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, diluents, lubricants, preservatives, and the like.

[0070] Another aspect of the present application is a method for decomposing a fumonisin, comprising the step of contacting a polypeptide of the present application having fumonisin-decomposing activity and / or a host cell expressing the polypeptide having fumonisin-decomposing activity with a fumonisin.

[0071] Another aspect of the present application is a method for producing a polypeptide having fumonisin decomposition activity, the method comprising culturing a host cell containing one or more of the polypeptide having fumonisin decomposition activity of the present application, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide.

[0072] In the present application, the term "culturing" refers to growing the host cells under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using an appropriate medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, or fed-batch culture.

[0073] In the present application, the term "culture medium" refers to a mixture of nutrients required for culturing the host cells, including water as the main component, and provides nutrients and growth factors essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the host cells of the present application can be any medium used for culturing conventional host cells without any particular limitations. The host cells of the present application can be cultured in a conventional culture medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins under aerobic conditions while controlling the temperature, pH, etc.

[0074] In one embodiment, the method may further include recovering the polypeptide having fumonisin-degrading activity expressed in the culturing step.

[0075] In the recovering step, the polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0076] The recovery method may involve collecting the polypeptide using a suitable method known in the art based on the host cell culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods can be used, and the polypeptide can be recovered from the medium or host cells using a suitable method known in the art.

[0077] In other embodiments, the polypeptide expressed by the host cells during the culturing step may not be recovered, and the host cells expressing the polypeptide may themselves be used as the source of the polypeptide.

[0078] Another aspect of the present application is the use of the polypeptide of SEQ ID NO: 1 as a fumonisin-degrading enzyme.

[0079] For example, the present invention provides the use of SEQ ID NO: 1 or a polypeptide consisting of, comprising, or consisting essentially of an amino acid sequence having at least 60% or more homology or identity to the amino acid sequence of SEQ ID NO: 1, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, as a fumonisin-degrading enzyme. [Example]

[0080] The present application will be described in more detail below through examples and experimental examples. However, these examples and experimental examples are intended to exemplify the present application and are not intended to limit the scope of the present application.

[0081] Example 1. Construction of a novel fumonisin-degrading enzyme EFB1-9 expression vector A polynucleotide (SEQ ID NO: 2) encoding a carboxylesterase family protein derived 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.

[0082] [Table 1]

[0083] Specifically, EFB1-9 was constructed by PCR using the 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 under the following reaction conditions:

[0084] Initial denaturation - 94℃, 2 min Denaturation - 94℃, 20 seconds Annealing - 56℃, 10 seconds Extension - 72°C, 2 min (35 cycles from denaturation to extension) Final Extension - 72℃, 5min

[0085] The resulting PCR product and vector were treated with restriction enzymes (NdeI, NotI) and then ligated using T4 DNA ligase (NEB, Cata# M0202S). The resulting product was then transformed into E. coli Dh5α strain and sequence mutations were confirmed by sequencing.

[0086] Example 2. Expression and purification of novel FUM-degrading enzyme EFB1-9 for activity evaluation E. coli BL21(DE3) was transformed with EFB1-9 prepared in Example 1 and inoculated into sterilized LB medium (BD Difco) and pre-cultured at 37°C and 200 rpm for 16 hours. One-hundredth of the medium volume was then inoculated into a flask containing sterilized LB medium and cultured at 37°C and 200 rpm until the absorbance (OD600) reached 0.4-0.5. IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 1 mM, and the culture was further continued for 16 hours. The cells were then harvested by centrifugation. The harvested cells were re-suspended in 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole) and sonicated and centrifuged to obtain the coenzyme solution. The coenzyme solution was applied to Ni-NTA resin (Qiagen, Cat. No. 30230) for adsorption, and then the enzyme was purified by sequentially applying a wash buffer (lysis buffer containing 20 mM imidazole only) and an elution buffer (lysis buffer containing 250 mM imidazole only).

[0087] The protein concentration was determined by mixing 5 μl of the diluted enzyme solution with 250 μl of Bradford solution (Quick Start™ Bradford 1× Dye Reagent, #5000205) and then measuring the absorbance at 595 nm.

[0088] Example 3. Activity evaluation using TLC To analyze the activity of the purified EFB1-9 against 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 1:1 solution of acetonitrile and distilled water, and the reaction was allowed to proceed at room temperature for 24 hours, and then the reaction was stopped by leaving it at 95°C for 10 minutes.

[0089] The reaction product was applied to one edge of a silica gel-coated aluminum TLC plate (Merck, TLC Silica gel 60 F254 20x20cm, Cat# 105554) and placed vertically in a sealed chamber containing a developing solution of acetonitrile and distilled water (7:3) for 6 minutes and 30 seconds. After drying, the plate was immersed in a 0.2% ninhydrin developer in ethanol and dried in a dryer at 140°C for 10 minutes.

[0090] As a result, it was confirmed that the FB1 spot (Rf = 0.59) became fainter than the control group on the plate, and the HFB1 spot (Rf = 0.81) was formed. Detailed results are shown in Figure 1 and Table 2.

[0091] [Table 2]

[0092] Example 4. Activity evaluation using LC-FLD and LC-TOF / MS To confirm the concentration of Fumonisin B1 remaining after the reaction and the composition of the degradation products, LC-FLD and LC-TOF / MS analyses were performed.

[0093] Example 4-1. LC-FLD analysis conditions The analysis conditions for fumonisin B1 concentration are as follows. Liquid chromatography-fluorescence detection analysis (LC-FLD) was performed using a Waters Acquity UPLC and a fluorescence detector. After separating fumonisin B1 on a column, it was analyzed by post-column derivatization with OPA (o-phthalaldehyde) reagent before entering the fluorescence detector. The detailed analysis conditions are as follows:

[0094] (1) Chromatography: Waters Acquity UPLC System (2) Column: Waters Acquity UPLC BEH C18 1.7um 2.1x150mm (3) Column temperature: 40°C (4)Flow rate: 0.25mL / min (5) Sample injection: 10.0 μL (6) Mobile phase: A:13.7 Mm Octanesulfonic acid + 25 Mm Potassium dihydrogen phosphate in DW(Ph 2.1,by H3PO4), B:13.7 Mm Octanesulfonic acid + 25 Mm Potassium dihydrogen phosphate in 50% Acetonitrile(Ph 2.1,by H3PO4) (7) Elution conditions: [Table 3] (8) Detection wavelength: Excitation wavelength 338 nm, Emission wavelength 425 nm (9) Post-column derivatization method Reagent:5.2mM o-phthalaldehyde (OPA) in borate buffer Reagent flow rate: 0.25mL / min Reactor temperature: 40℃

[0095] Example 4-2. LC-TOF / MS analysis conditions Liquid chromatography-mass spectrometry (LC-TOF / MS) was performed using a Waters Acquity UPLC and Xe-vo G2-XS Q-Tof mass spectrometer under the following analytical conditions:

[0096] (1) Chromatography: Waters Acquity UPLC System (2) Column: Waters Acquity UPLC BEH C18 1.7um 2.1x150mm (3) Column temperature: 40°C (4)Flow rate: 0.20mL / min (5) Sample injection: 1.0 μL (6) Mobile phase: A:5mM Ammonium Formate w. 0.1% Formic acid in DW B:5mM Ammonium Formate w. 0.1% Formic acid in Methanol (7) Elution conditions: [Table 4] (8) Mass spectrometer: Waters Xe-vo G2-XS Q-Tof Ionization mode: ESI Positive Capillary voltage: 2.5 kV Cone voltage: 30 V Source Temperature: 120℃ Desolvation Temperature:400℃ Mass scan range: 50 to 1000 m / z

[0097] Example 4-3. Experimental results As in Example 4-1, it was confirmed that the concentration of Fumonisin B1 decreased from 6.40 ppm to 4.60 ppm through the reaction of purified EFB1-9, and a peak presumed to be a degradation product was generated (FIG. 2).

[0098] When the same sample was analyzed by LC-TOF / MS as in Example 4-2, it was confirmed that the proportion of Fumonisin B1 (m / z 722.39) in the negative control reaction product decreased to 70%, while that of HFB1 (m / z 406.35) increased to 30% (Figure 3).

[0099] The detailed analysis results are summarized in Table 5 below.

[0100] [Table 5]

[0101] This confirmed that EFB1-9 of the present application has the ability to decompose fumonisin.

[0102] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A polypeptide of SEQ ID NO: 1 having fumonisin-degrading activity.

2. A polynucleotide encoding the polypeptide of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A host cell comprising at least one of a polypeptide of SEQ ID NO: 1 having fumonisin-degrading activity, a polynucleotide encoding said polypeptide, and a vector comprising said polynucleotide.

5. The polypeptide of SEQ ID NO: 1; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and A composition for decomposing fumonisins, comprising one or more host cells expressing the polypeptide of SEQ ID NO:

1.

6. The polypeptide of SEQ ID NO: 1; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and Any one or more host cells expressing the polypeptide of SEQ ID NO: 1, A composition for detoxifying fumonisins present in food, feed or both said food and feed.

7. The polypeptide of SEQ ID NO: 1; a polynucleotide encoding the polypeptide; a vector comprising the polynucleotide; and A feed additive composition comprising one or more host cells expressing the polypeptide of SEQ ID NO:

1.

8. The polypeptide of SEQ ID NO: 1; and A method for decomposing fumonisin, comprising contacting any one or more host cells expressing the polypeptide of SEQ ID NO: 1 with fumonisin.

9. Culturing a host cell containing one or more of the polypeptide of SEQ ID NO: 1, a polynucleotide encoding said polypeptide, and a vector containing said polynucleotide, A method for producing the polypeptide according to claim 1, which has fumonisin-degrading activity.

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

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