Means and methods for modifying trichothecenes

Abstract

The present invention relates to methods of modifying trichothecene, mutant variants of polypeptides, additives, methods of manufacture, uses of said polypeptides, polynucleotides, recombinant host cells and polypeptides for treating, ameliorating and / or preventing symptoms caused by fungal poisoning.

Description

[0001] The present invention relates to a method of modifying trichothecenes, mutant variants of polypeptides, additives, methods of manufacture, uses of the polypeptides, polynucleotides, recombinant host cells and polypeptides for treating, ameliorating and / or preventing symptoms caused by mycotoxicosis.

[0002] Fungal toxins are secondary metabolites produced by fungi, which infest food and feed materials, in particular all types of cereals and crops, or other commodities, such as cotton, coffee, peanuts, dates, spices, etc. Upon fungal growth, the infected material is thus contaminated with fungal toxins, such as aflatoxins, ochratoxins, ergot alkaloids, fumonisins, zearalenone, trichothecenes, etc.

[0003] In more than 17,000 samples, almost three quarters were found to contain at least one fungal toxin, and more than half of all samples contained one or more trichothecenes (Streit et al., 2013. J Sci Food Agric. 93(12): 2892-2899). Deoxynivalenol (DON; also known as vomitoxin; CAS-No. 51481-10-8) is one of the most prevalent trichothecenes and was found to be present worldwide. Feed materials contaminated with deoxynivalenol or other trichothecenes result in a refusal to eat and a reduction in weight gain, thus not only affecting the health and well-being of the animals, but also causing considerable economic losses in animal husbandry.

[0004] Due to the hydrophilic structure of DON to a large extent, removal of DON by binding, such as binding to mineral or clay adsorbents, is inefficient and insufficient.

[0005] As an alternative, it has been suggested to convert DON into the isomeric form 3-epi-DON, e.g. Bracarense et al. 2020. Food Chem Toxicol. 140: 111241. In the case of DON, the hydroxyl group at the 3rd carbon atom is in the R configuration, while in the case of 3-epi-DON, the hydroxyl group at the 3rd carbon atom is in the S configuration. The detoxification conversion from DON to 3-epi-DON has previously been described as a two-step mechanism via the intermediate trichothecene 3-keto-DON (Carere et al. 2018. Microb Biotechnol. 11(6): 1106-1111; Carere et al. 2018. Front Microbiol. 9: 1573; He et al. 2020. Food Chem. 321: 126703). These described mechanisms rely on a pyrroloquinoline quinone (PQQ)-dependent dehydrogenase for the oxidation of DON to form 3-keto-DON with a carbonyl group on the 3rd carbon, and one or more reductases for the reduction of 3-keto-DON to 3-epi-DON. Notably, some enzymes capable of using 3-keto-DON as a substrate catalyze a reduction reaction that at least partially leads back to DON instead of the detoxified isomer 3-epi-DON. Furthermore, the oxidation reaction requires free PQQ, and the reduction reaction relies on the nicotinamide co-factor NADPH (reduced nicotinamide adenine dinucleotide phosphate). While these reactions can be quite efficient, the dependency on two or even three enzymes and at least two additional substrates (PQQ and NADPH) makes these mechanisms rather complex, and thus difficult to be applied, e.g. as a detoxification additive for food or feed. Furthermore, NADPH is an acid-labile and relatively unstable molecule, prone to rapid oxidation, and needs to be supplied in the form of a continuous substance. Thus, even if these systems produce 3-epi-DON, they are accompanied by several hurdles related to applicability.

[0006] Yet another system for the enzymatic modification of trichothecenes to achieve detoxification is described in WO 2020 / 254592 Al. Therein, glutathione-S-transferases (GSTs) were shown to be capable of bioconversion of DON, albeit at a slower catalytic rate. On the other hand, Nakamura et al. (1977. Chem. Pharm. Bull. 25(12): 3410-3414) found that some GST enzymes are not able to modify trichothecenes.

[0007] Thus, in summary, there is a need for new solutions, in particular enzymes capable of modifying and detoxifying trichothecenes at a rate suitable for in vivo applications.

[0008] This object is achieved by providing a method of modifying a trichothecene, said method comprising the steps of: a) forming a reaction mixture by contacting said trichothecene with: (i) at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-36, preferably with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 23, 27, 32, 34 and 36; and (ii) glutathione, preferably reduced glutathione (GSH); and b) incubating said reaction mixture.

[0009] In such a method, the trichothecene can be modified such that glutathione is specifically attached to the C13 atom of the trichothecene, thereby converting e.g. DON to DON13-GSH, 3-ADON to 3-ADON-13-GSH, 15-ADON to 15-ADON-13-GSH, NIV to NIV-13-GSH, DAS to DAS-13-GSH, T-2 to T-2-13-GSH, and / or HT-2 to HT-2-13-GSH. Such C13-glutathionylated trichothecenes are significantly less toxic than their unmodified forms. In contrast, the previously reported enzymes also produce, at least to some extent, trichothecene derivatives in which glutathione is attached to the C10 atom. However, unlike the modification at the C13 atom, the modification at the C10 atom is reversible (Stanic et al. 2016. J. Agric. Food Chem. 64(23): 4777-4785). Thus, C10-glutathionylated trichothecene derivatives cannot be considered effectively detoxified.

[0010] The term "polypeptide" shall be interpreted as commonly used in the art and includes, for example, polypeptides, proteins, peptides, enzymes.

[0011] The term "sequence identity" is used to describe the degree of correlation between two or more nucleic acid sequences (e.g., DNA or RNA polynucleotides) contained in a polynucleotide or two or more amino acid sequences contained in a polypeptide. Sequence identity can be determined by methods commonly known to those skilled in the art. In this document, the preferred method for determining sequence identity between two amino acid sequences contained in two polypeptides is to use the Clustal Omega alignment tool with EMBL-EBI (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ; Sievers et al. 2011. Mol. Syst. Biol. 7: 539) with default settings. Alternatively, the Needleman-Wunsch algorithm for global sequence alignment can be used, such as the algorithm provided by the National Center for Biotechnology Information with default settings (Gap Costs: Existence: 11 Extension 1) ("Needleman-Wunsch Global Align Protein Sequences"). A polypeptide containing an amino acid sequence having at least 70% sequence identity with the target polypeptide can be a polypeptide containing 70% or greater sequence identity with the target polypeptide, such as 70.0%, 70.5%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity. For example, a polypeptide containing an amino acid sequence having 100% sequence identity with SEQ ID NO: 1 and / or a polypeptide composed of an amino acid sequence having 100% sequence identity with SEQ ID NO: 1 is included in the group “polypeptides containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1”.Therefore, polypeptides comprising amino acid sequences having at least 70% sequence identity with amino acid sequences selected from SEQ ID NO: 1-36 and / or polypeptides composed of amino acid sequences having at least 70% sequence identity with amino acid sequences selected from SEQ ID NO: 1-36 include those polypeptides composed of amino acid sequences having at least 70% sequence identity with amino acid sequences selected from SEQ ID NO: 1-36, and those polypeptides wherein an amino acid sequence having at least 70% sequence identity with amino acid sequences selected from SEQ ID NO: 1-36 is linked to one or more additional amino acid sequences. Typically, the term "fusion polypeptide" or "fusion protein" describes two or more polypeptides linked to each other (typically by peptide bonds). For example, in such fusion proteins, two domains that are protein domains are linked to each other by peptide bonds. Furthermore, in some fusion proteins, one or more peptide tags are linked or attached to the N- and / or C-terminus of a polypeptide (e.g., a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequences selected from SEQ ID NO: 1-36 and / or a polypeptide composed of an amino acid sequence having at least 70% sequence identity with the amino acid sequences selected from SEQ ID NO: 1-36), for example, to facilitate easier purification or increase solubility, without substantially altering the enzymatic properties of the tagged polypeptide. Examples of commonly used tags are poly(His) tags (e.g., hexahistidine tags), maltose-binding protein (MBP) tags, Strep tags, Strep II tags, etc. Therefore, the present invention covers enzymatically active fusion polypeptides comprising a polypeptide containing an amino acid sequence having at least 70% sequence identity with the amino acid sequences selected from SEQ ID NO: 1-36. The fusion polypeptide may, for example, contain a hexahistine tag fused to the N-terminus of a polypeptide containing an amino acid sequence having 91% sequence identity with the amino acid sequence of SEQ ID NO: 1; or, for example, contain a maltose-binding protein tag fused to the C-terminus of a polypeptide containing an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 23 via a linker peptide (e.g., GG, GPG, EA or EA repeat sequence, GSG, etc.).In other words, the present invention also covers, as part of the larger polypeptide, larger polypeptides comprising amino acid sequences having at least 70% sequence identity with amino acid sequences selected from SEQ ID NO: 1-36, preferably selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36, and / or larger polypeptides composed of amino acid sequences having at least 70% sequence identity with amino acid sequences selected from SEQ ID NO: 1-36, preferably selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36, provided that the enzymatic activity (i.e., as glutathione transferase) of the amino acid sequences having at least 70% sequence identity with the amino acid sequences selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36 is substantially not eliminated. For clarity only, the enzymatically active polypeptides according to the present invention are capable of and therefore suitable for modifying at least one trichothecene. In particular, the enzymatically active polypeptides according to the present invention are capable of and therefore suitable for modifying at least one trichothecene at the C13 atom. In other words, this invention does not include polypeptides that cannot modify trichothecene.

[0012] In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity) and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity) and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity) and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity) and / or composed thereof.In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity) and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity) and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 36 (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity) and / or composed thereof.

[0013] In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 2 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 3 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 4 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 5 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 6 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 7 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 8 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 9 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 10 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 11 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 12 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 13 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 14 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 15 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 16 and / or composed thereof.In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 17 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 18 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 19 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 20 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 21 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 22 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 23 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 24 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 25 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 26 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 27 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 28 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 29 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 30 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 31 and / or composed thereof.In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 32 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 33 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 34 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 35 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 36 and / or composed thereof.

[0014] In some embodiments, the polypeptide according to the invention is a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO: 23 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO: 27 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO: 32 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO: 34 and / or composed thereof. In some embodiments, the polypeptide according to the invention is a polypeptide comprising the amino acid sequence of SEQ ID NO: 36 and / or composed thereof.

[0015] In some embodiments, the method according to the invention includes the step of incubating the reaction mixture at a temperature of at least 5°C. In some embodiments, the method according to the invention includes the step of incubating the reaction mixture at a temperature of up to 40°C. In some embodiments, the method according to the invention includes the step of incubating the reaction mixture at a temperature of at least 5°C and up to 40°C. In some embodiments, the method according to the invention includes the step of incubating the reaction mixture at a temperature of 5°C to 40°C, preferably 20°C to 40°C. In a preferred embodiment, the reaction mixture is incubated at a temperature of 30-40°C, more preferably 35-40°C.

[0016] Preferably, the incubation step of the reaction mixture in the method according to the invention is carried out for at least 0.1 seconds, preferably at least 1, 5, 10, 20, or 60 seconds, preferably at least 1, 2, 3, 4, or 5 minutes, and preferably at least 10 minutes (e.g., at least 15 minutes, 30 minutes, 60 minutes, etc.). The longer the incubation step of the reaction mixture is carried out, the more trichothecene will be modified. Nevertheless, those skilled in the art will recognize that the enzymatic type of reaction as described herein can also be carried out outside of the preferred conditions. For clarity only, it is considered that incubation should begin immediately upon contact of the polypeptide according to the invention with trichothecene and glutathione. Those skilled in the art are fully capable of selecting incubation conditions suitable for changing the concentration of trichothecene in the composition to the desired level. Nevertheless, particularly preferred trichothecene modification and thus detoxification can be achieved when the incubation step of the reaction mixture is carried out at a temperature of 5-40°C and / or for at least 5 minutes.

[0017] In some embodiments, trichothecene is selected from deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV), T-2 toxin (T-2; sometimes also written as T2), HT-2 toxin (HT-2; sometimes also written as HT2), and diacetylfusinol (DAS). In particular, the high prevalence of deoxynivalenol in food and / or feed materials poses a considerable threat to humans and non-human animals. Therefore, in a preferred embodiment, trichothecene is deoxynivalenol (DON; sometimes also called vomitoxin; 3α,7α,15-trihydroxy-12,13-epoxy-thomyl-9-en-8-one; or (2R,2'S,3R,5R,5aR,6S,9aR)-3,8-dihydroxy-5a-(hydroxymethyl)-5,6-dimethylspiro[[2,5]methane[1]benzoxazon-10,2'-epoxyethylene]-7(6H)-one; CAS No. 51481-10-8).

[0018] In some embodiments of the method according to the invention, GSH is provided or present at a concentration of at least 0.1 mM, preferably at least 1 mM, more preferably at least 5 mM. In some embodiments of the method according to the invention, GSH is provided or present at a concentration of at least 0.1 mmol / kg food or feed, preferably at least 1 mmol / kg food or feed, more preferably at least 5 mmol / kg food or feed. In some embodiments, at a concentration of at least 0.1 μM, preferably at least 1 μM, or at least 0.1 μmol / kg food or feed, preferably at least 1 μmol / kg food or feed, at least at least at least 1 μmol / kg food or feed, at least at least at least at least 1 μmol / kg food or feed, at least at least at least 1 μmol / kg food or feed, at least at least at least 1 μmol / kg food or feed, at least at least 1 polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34, and 36, and / or composed thereof.

[0019] In some embodiments of the method for modifying trichothecene according to the present invention, the trichothecene is contained in a nutritional composition. Such a nutritional composition is a composition containing one or more components with nutritional value. Typically, such components provide energy to the consumer of the nutritional composition. The nutritional composition may be wholly or at least partially based on herbs or plants, such as commonly used animal feed compositions. The forage or feed may, for example, include, corn, hay, straw bedding, soybeans, or products derived therefrom, or consist of, corn, hay, straw bedding, soybeans, or products derived therefrom. Furthermore, the forage or feed may contain or consist of extruded feed products (e.g., pellets).

[0020] On the other hand, the present invention relates to a mutant variant of a polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34, and 36; wherein the mutant variant comprises at least one amino acid mutation, substitution, deletion, and / or insertion. Such mutant variants are polypeptide variants resulting from the recombination of polypeptides comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34, and 36. In particular, such mutant variants comprise, and / or consist of, an amino acid sequence as a synthetic amino acid sequence. In other words, such a synthetic amino acid sequence does not exist in nature. Instead, the synthetic amino acid sequence is the result of human intervention, wherein at least one amino acid of a naturally occurring amino acid sequence is altered. Such an alteration may be the substitution of an amino acid at a certain position with another amino acid that would not exist at that position without human intervention. This change can also involve the deletion of an amino acid at a certain position. Alternatively, one or more amino acids can be inserted into one or more positions in a naturally occurring amino acid sequence.

[0021] For example, the amino acid sequences of SEQ ID NO: 10-22 and 25-26 are synthetic amino acid sequences. Therefore, a polypeptide comprising and / or composed of any one of the amino acid sequences of SEQ ID NO: 10-22 and 25-26 is an example of a mutant variant of a polypeptide comprising and / or composed of an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably selected from SEQ ID NO: 1, 23, 27, 32, 34, and 36. Such mutant variants are typically produced to provide polypeptides with, for example, faster kinetic activity, or (e.g., greater stability at a specific temperature or pH). Therefore, mutant variants are particularly useful in industrial applications.

[0022] Additives used in food, feed, forage, or feed are generally used to improve or enhance the properties of the food, feed, forage, or feed. For example, such additives can be added to improve sensory properties, such as improving the taste, odor, appearance, or color of the food, feed, forage, or feed. Additionally, additives can be added to improve palatability, nutritional availability, or to add probiotic microorganisms to the food, feed, forage, or feed, or to add or enhance the prebiotic activity of the food, feed, forage, or feed. Furthermore, such additives can be added to counteract potentially undesirable effects of the food, feed, forage, or feed, such as removing or reducing one or more undesirable components contained in the food, feed, forage, or feed.

[0023] On the other hand, the present invention relates to an additive for modifying trichothecene (e.g., an additive for feed and / or food; or a feed additive; or a food additive) comprising: (i) at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36, and / or composed thereof; and / or a mutant variant of the polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36, and / or composed thereof; wherein the mutant variant comprises at least one amino acid mutation, substitution, deletion and / or insertion; and (ii) at least one additional component, preferably glutathione (especially reduced glutathione). By providing such an additive according to the invention, a suitable means is provided that allows for convenient detoxification of trichothecene.

[0024] In some embodiments, the at least one additional component of the additive is glutathione. In some preferred embodiments, the additive according to the invention comprises glutathione, particularly reduced glutathione (GSH), at a concentration suitable for achieving an inclusion ratio of at least 0.1 μmol GSH / kg (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 50, 100 μmol GSH / kg). In other words, the concentration of GSH contained in the additive is such that, after mixing the additive with the target composition, the final concentration of GSH in the target composition is 0.1 mmol GSH / kg of the target composition. For example, in one embodiment where the additive is a feed additive, the target composition will be feed. If the feed additive is to be concentrated, for example, by 1000 times, GSH will be contained in the feed additive at a concentration of 100 mmol / kg of feed additive. When the feed additive is mixed with the target composition feed at a ratio of 1:1000, the GSH inclusion ratio will reach 0.1 mmol / kg of feed. Therefore, in this case, GSH will be contained at a concentration of 100 mmol / kg additive.

[0025] In some embodiments, the additive according to the invention further comprises one or more components selected from: dietary supplements; nutritional supplements; pharmaceuticals; vitamins; minerals; amino acids; essential fatty acids; fiber; trace elements; antioxidants; plant extracts; herbal extracts; essential oils; and a carrier, preferably selected from bentonite, silica, and carbohydrates.

[0026] In some embodiments, the additive according to the invention comprises glutathione, particularly reduced glutathione (GSH); preferably, the GSH is contained in the additive at a concentration suitable for achieving an inclusion ratio of at least 0.1 μmol GSH / kg of the target composition (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 50, 100 μmol GSH / kg); and the additive further comprises one or more components selected from: dietary supplements; nutritional supplements; pharmaceuticals; vitamins; minerals; amino acids; essential fatty acids; fiber; trace elements; antioxidants; plant extracts; herbal extracts; essential oils; and a carrier, preferably selected from bentonite, silica, and carbohydrates.

[0027] In some embodiments, the additive according to the invention comprises glutathione, particularly reduced glutathione (GSH); preferably, the GSH is contained in the additive at a concentration suitable for achieving an inclusion ratio of at least 0.1 μmol GSH / kg of the target composition (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 50, 100 μmol GSH / kg); and the additive further comprises an absorbent, preferably clay, more preferably bentonite.

[0028] In some embodiments, the additive according to the invention comprises glutathione, particularly reduced glutathione (GSH); preferably, the GSH is contained in the additive at a concentration suitable for achieving an inclusion ratio of at least 0.1 μmol GSH / kg of the target composition (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 50, 100 μmol GSH / kg); and the additive further comprises a carrier for the enzyme. The carrier for the enzyme can be of inorganic or organic origin. Potential inorganic materials for enzyme immobilization are silica (sol-gel silica, pyrolytic silica, colloidal silica nanoparticles, and silica gel) and oxides (e.g., titanium dioxide, alumina, and zirconium oxide). Furthermore, clay materials (e.g., bentonite, halloysite, kaolinite, montmorillonite, sepiolite, and calcium apatite) can be such carriers. Additionally, carbon-based materials (e.g., activated carbon and charcoal) can be carriers. Organic enzyme carriers can be biopolymers (e.g., carbohydrates, proteins, maltodextrin, trehalose, inulin, collagen, cellulose, keratin, carrageenan, chitosan, and alginate) or synthetic polymers (e.g., polyaniline, polyamide, polystyrene, polyurethane, polypropylene, polyvinyl alcohol, and ion exchange resins). Liquid carriers can be, for example, buffering substances and / or polyols, such as polyepoxides, polyvinyl alcohol, polyethylene-copoly-maleic anhydride, polystyrene-copoly-maleic anhydride, dextran, cellulose, hydrolysates of chitosan, starch, glycogen, sorbitol, agarose and its derivatives, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, hydrolysates of alginate, biopolymers, sorbitol, glycerol, cellobiose, and monopropylene glycol. Additionally or alternatively, the carrier can be an edible component, preferably a non-toxic component, and / or a component that provides texture. In one specific embodiment, the carrier is selected from bentonite, silica, and carbohydrates; preferably, the carrier is bentonite.

[0029] In another aspect, the present invention relates to a method for manufacturing an additive for feed and / or food, for feed compositions and / or food compositions, or for pharmaceutical compositions, the method comprising the steps of: a) providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and b) contacting said at least one polypeptide with one or more components of the additive for feed and / or food, the additive for feed compositions and / or food compositions, or the additive for pharmaceutical compositions.

[0030] In another aspect, the present invention relates to a method for manufacturing feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch, the method comprising the steps of: a) providing at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36, and / or being composed thereof; and b) contacting the at least one polypeptide and / or an additive according to the invention with one or more components of the feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch.

[0031] GSH is a natural component used in the enzymatic modification reactions described herein, and it is readily available (in, for example, food, feed, and in humans or non-human animals). The inventors have discovered that the enzymatic modification reactions can be accelerated by increasing the availability of GSH. Therefore, in some preferred embodiments, the method for manufacturing according to the invention further includes the step of: c) providing GSH at a concentration of at least 0.1 mmol / kg for feed and / or food additives, for feed and / or food compositions, for pharmaceutical compositions, feed, food, feed compositions, food compositions, pharmaceutical compositions, biogas, bioethanol, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch, and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch.

[0032] In a further aspect, the present invention relates to the use of at least one polypeptide in one or more of the following: a) modifying trichothecene; b) manufacturing an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive for pharmaceutical composition; and / or c) manufacturing feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch, and / or silage, wherein the sugar is preferably sugar derived from sugarcane or sugar beets, and the starch is particularly corn starch; wherein the at least one polypeptide comprises, and / or is composed of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34, and 36.

[0033] In another aspect, the present invention relates to polynucleotides encoding polypeptides comprising, and / or consisting of, amino acid sequences having at least 70% sequence identity with amino acid sequences selected from SEQ ID NO: 1-36, preferably with amino acid sequences selected from SEQ ID NO: 1, 23, 27, 32, 34, and 36; and / or mutant variants according to the invention. In some embodiments, the present invention relates to isolated polynucleotides encoding polypeptides comprising, and / or consisting of, amino acid sequences having at least 70% sequence identity with amino acid sequences selected from SEQ ID NO: 1-36, preferably with amino acid sequences selected from SEQ ID NO: 1, 23, 27, 32, 34, and 36; and / or mutant variants according to the invention. For clarification only, isolated polynucleotides are not considered to exist in nature, wherein the polynucleotides exist only in the environment of biological cells, i.e., in a non-isolated environment. Rather, isolated polynucleotides only occur in the context of molecular biological steps performed by human experimenters. For example, isolated polynucleotides are produced and processed in cloning activities, which are necessary, for example, to enable the subsequent production of recombinant proteins.

[0034] In another aspect, the present invention relates to recombinant host cells comprising: at least one polypeptide; and / or at least one polynucleotide encoding at least one polypeptide and / or a mutant variant thereof as described herein, wherein the at least one polypeptide comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36, and / or constitutes thereof.

[0035] The host cell according to the invention allows for the production of the polypeptides mentioned herein, particularly recombinant heterologous production of the polypeptides mentioned herein.

[0036] For clarity, the term "recombination" means that a host cell containing at least one polypeptide and / or at least one polynucleotide encoding at least one polypeptide does not naturally contain said at least one polypeptide and / or polynucleotide, but is only the result of a human experimenter introducing said at least one polypeptide and / or polynucleotide into the host cell by technical means (e.g., molecular biology, biotechnology and / or synthetic biology).

[0037] In some embodiments, the recombinant host cells according to the present invention are selected from: Escherichia coli ; Bacillus sp. ,For example Bacillus subtilis or Bacillus amyloliquefaciens Saccharomycessp., for example Saccharomyces cerevisiae ; Pichia sp. ,For example Shepherd's pie or Peach logs ; Kluyveromyces sp. ,For example Kluyveromyces lactis ; Schizosaccharomyces sp. .,For example Schizosaccharomyces pombe ; Hansenula sp. ,For example Hansenula polymorpha ; Streptomyces sp. .; Yarrowia sp. .; Trichoderma sp. Lactobacillus sp. Aspergillus sp. Plant cells; and / or Bacillus sp. , Trichoderma sp. or Aspergillus sp. The spores. In some preferred embodiments, the recombinant host cell according to the invention is a bacterial cell, preferably... Escherichia coli ; Bacillus sp. ,For example Bacillus subtilis or Bacillus amyloliquefaciens .

[0038] Usually with " Shepherd's pie The relevant strains are sometimes referred to as Shepherd's Komagataella , Komagataella phaffii or Komagataella pseudopastoris This depends on the respective taxonomic classifications at the time of reference. Therefore, in any case, as used herein, the term " Shepherd's pie "This should be interpreted as including all of these strains, even those not referred to as such." Shepherd's pie, But called Shepherd's Komagataella , Komagataella phaffi or Komagataella pseudopastoris The same applies at times.

[0039] In another aspect, the present invention relates to polypeptides for use in treating, improving, and / or preventing symptoms caused by fungal poisoning, particularly by trichothecene fungal poisoning, wherein the polypeptide comprises, and / or is composed of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34, and 36. The use of the polypeptides according to the invention for this purpose is particularly relevant, for example, in the livestock industry. Prophylactic application of the polypeptides according to the invention will result in the detoxification of trichothecene (e.g., vomitoxin) in animals (e.g., weaned piglets, growing pigs, or finishing pigs) to prevent symptoms caused by trichothecene (e.g., vomitoxin). In cases where trichothecene has already been consumed, application of the polypeptides according to the invention will treat and improve symptoms, thereby minimizing animal disease and promoting recovery.

[0040] A further feature of the present invention is the following item: Item 1: A method for modifying trichothecene, the method comprising the following steps: a) A reaction mixture is formed by contacting the trichothecene with the following substances: (i) at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and (ii) glutathione; and b) Incubate the reaction mixture.

[0041] Item 2: A method for modifying trichothecene, the method comprising the following steps: a) A reaction mixture is formed by contacting the trichothecene with the following substances: (i) at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-22, preferably SEQ ID NO: 1; and (ii) glutathione; and b) Incubate the reaction mixture.

[0042] Item 3: A method for modifying trichothecene, the method comprising the following steps: a) A reaction mixture is formed by contacting the trichothecene with the following substances: (i) at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 23-26, preferably SEQ ID NO: 23; and (ii) glutathione; and b) Incubate the reaction mixture.

[0043] Item 4: A method for modifying trichothecene, the method comprising the following steps: a) A reaction mixture is formed by contacting the trichothecene with the following substances: (i) at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 27-31, preferably SEQ ID NO: 27; and (ii) glutathione; and b) Incubate the reaction mixture.

[0044] Item 5: A method for modifying trichothecene, the method comprising the following steps: a) A reaction mixture is formed by contacting the trichothecene with the following substances: (i) at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 32-33, preferably SEQ ID NO: 32; and (ii) glutathione; and b) Incubate the reaction mixture.

[0045] Item 6: A method for modifying trichothecene, the method comprising the following steps: a) A reaction mixture is formed by contacting the trichothecene with the following substances: (i) at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 34-35, preferably SEQ ID NO: 34; and (ii) glutathione; and b) Incubate the reaction mixture.

[0046] Item 7: A method for modifying trichothecene, the method comprising the following steps: a) A reaction mixture is formed by contacting the trichothecene with the following substances: (i) at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 36; and (ii) glutathione; and b) Incubate the reaction mixture.

[0047] Item 8: The method according to any one of Items 1 to 7, wherein step b) is carried out at a temperature of 5-40°C, preferably at a temperature of 20-40°C.

[0048] Item 9: The method according to any one of Items 1 to 8, wherein step b) is performed for at least 5 minutes, preferably at least 10 minutes.

[0049] Item 10: The method according to any one of Items 1 to 9, wherein the trichothecene is selected from deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin and diacetylfusarenol; preferably deoxynivalenol.

[0050] Item 11: A polypeptide comprising an amino acid sequence and / or thereof, wherein the amino acid sequence has at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and wherein the amino acid sequence has less than 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36.

[0051] Item 12: The peptide according to Item 11, wherein the polypeptide comprises at least one amino acid mutation, substitution, deletion and / or insertion compared with an amino acid sequence selected from SEQ ID NO: 1-36, preferably compared with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36.

[0052] Item 13: A mutant variant of a polypeptide, particularly a recombinant mutant variant, said polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; said mutant variant comprising at least one amino acid mutation, substitution, deletion and / or insertion.

[0053] Item 14: An additive for modifying trichothecene, preferably a food additive and / or feed additive, comprising: (i) at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and / or a polypeptide according to any one of entries 11 to 12; and / or a mutant variant according to entry 13; and (ii) at least one additional component, preferably glutathione, especially reduced glutathione (GSH).

[0054] Item 15: The additive according to Item 14, wherein the at least one additional component is glutathione, particularly reduced glutathione (GSH).

[0055] Item 16: The additive according to Item 15, wherein glutathione is contained in the additive at a concentration suitable for achieving an inclusion ratio of at least 0.1 μmol GSH / kg of the target composition (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 50, 100 μmol GSH / kg).

[0056] Item 17: Additives as described in Item 16, wherein the target material is food and / or feed.

[0057] Item 18: An additive according to any one of Items 14 to 17, wherein the additive further comprises one or more components selected from: dietary supplements; nutritional supplements; pharmaceuticals; vitamins; minerals; amino acids; essential fatty acids; fiber; trace elements; antioxidants; plant extracts; herbal extracts; essential oils; and a carrier, preferably selected from bentonite, silica, and carbohydrates.

[0058] Item 19: An additive according to any one of Items 14 to 18, wherein the at least one additional component is glutathione, particularly reduced glutathione (GSH); and wherein the additive further comprises one or more components selected from: dietary supplements; nutritional supplements; pharmaceuticals; vitamins; minerals; amino acids; essential fatty acids; fiber; trace elements; antioxidants; plant extracts; herbal extracts; essential oils; and a carrier, preferably selected from bentonite, silica, and carbohydrates.

[0059] Item 20: An additive according to any one of Items 14 to 18, wherein the at least one additional component is glutathione, particularly reduced glutathione (GSH); and wherein the additive further comprises an absorbent.

[0060] Item 21: The additive according to Item 20, wherein the absorbent is clay, preferably bentonite.

[0061] Item 22: Methods for manufacturing one or more of the following a) Additives used in feed and / or food; b) Additives used in feed compositions and / or food compositions; c) Additives used in pharmaceutical compositions; d) Feed and / or food; e) Feed compositions and / or food compositions; f) Pharmaceutical composition; g) Biogas; h) Bioethanol; i) Wet Distillers' Grains (WDG); j) Dried distiller's grains (DDGS) containing solubles; k) Sugar, preferably sugar derived from sugarcane or sugar beets; l) Corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch, especially corn starch; and / or m) Silage; The method includes the following steps: i) Providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence selected from SEQ ID NO: 1-36, SEQ ID NO: 1-22, SEQ ID NO: 23-26, SEQ ID NO: 27-31, SEQ ID NO: 32-33, SEQ ID NO: 34-35 and SEQ ID NO: 36, preferably having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and ii) Contact the at least one polypeptide (e.g., a polypeptide according to any of entries 11-12 and / or a mutant variant according to entry 13) and / or an additive according to any of entries 14-21 with one or more of the following components: additives for feed and / or food; additives for feed compositions and / or food compositions; additives for pharmaceutical compositions; feed and / or food; feed compositions and / or compositions; pharmaceutical compositions; biogas; bioethanol; wet distillers grains (WDG); dried distillers grains (DDGS) with solubles; sugar, preferably sugar derived from sugarcane or sugar beets; corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch, especially corn starch; and / or silage.

[0062] Item 23: A method for manufacturing one or more of the following: an additive for use in feed and / or food, an additive for use in feed compositions and / or food compositions, or an additive for use in pharmaceutical compositions, said method comprising the following steps: i) Providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-22, preferably SEQ ID NO: 1; and ii) Contact the at least one polypeptide (e.g., a polypeptide according to any of entries 11-12 and / or a mutant variant according to entry 13) with one or more of the following components: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive for pharmaceutical composition.

[0063] Item 24: A method for manufacturing one or more of the following: an additive for use in feed and / or food, an additive for use in feed compositions and / or food compositions, or an additive for use in pharmaceutical compositions, said method comprising the following steps: i) Providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 23-26, preferably SEQ ID NO: 23; and ii) Contact the at least one polypeptide (e.g., a polypeptide according to any of entries 11-12 and / or a mutant variant according to entry 13) with one or more of the following components: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive for pharmaceutical composition.

[0064] Item 25: A method for manufacturing one or more of the following: an additive for use in feed and / or food, an additive for use in feed compositions and / or food compositions, or an additive for use in pharmaceutical compositions, said method comprising the following steps: i) Providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 27-31, preferably SEQ ID NO: 27; and ii) Contact the at least one polypeptide (e.g., a polypeptide according to any of entries 11-12 and / or a mutant variant according to entry 13) with one or more of the following components: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive for pharmaceutical composition.

[0065] Item 26: A method for manufacturing one or more of the following: an additive for use in feed and / or food, an additive for use in feed compositions and / or food compositions, or an additive for use in pharmaceutical compositions, said method comprising the following steps: i) Providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 32-33, preferably selected from SEQ ID NO: 32; and ii) Contact the at least one polypeptide (e.g., a polypeptide according to any of entries 11-12 and / or a mutant variant according to entry 13) with one or more of the following components: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive for pharmaceutical composition.

[0066] Item 27: A method for manufacturing one or more of the following: an additive for use in feed and / or food, an additive for use in feed compositions and / or food compositions, or an additive for use in pharmaceutical compositions, said method comprising the following steps: i) Providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 34-35, preferably SEQ ID NO: 34; and ii) Contact the at least one polypeptide (e.g., a polypeptide according to any of entries 11-12 and / or a mutant variant according to entry 13) with one or more of the following components: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive for pharmaceutical composition.

[0067] Item 28: A method for manufacturing one or more of the following: an additive for use in feed and / or food, an additive for use in feed compositions and / or food compositions, or an additive for use in pharmaceutical compositions, said method comprising the following steps: i) Providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 36; and ii) Contact the at least one polypeptide (e.g., a polypeptide according to any of entries 11-12 and / or a mutant variant according to entry 13) with one or more of the following components: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive for pharmaceutical composition.

[0068] Item 29: A method for producing one or more of the following: feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch, the method comprising the following steps: (i) providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-22, preferably SEQ ID NO: 1; and (ii) Contacting the at least one polypeptide and / or the additive according to any of the entries 14-21 with one or more of the following components: feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably sugar derived from sugarcane or sugar beets, and the starch is particularly corn starch.

[0069] Item 30: A method for producing one or more of the following: feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch, the method comprising the following steps: (i) providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 23-26, preferably SEQ ID NO: 23; and (ii) Contacting the at least one polypeptide and / or the additive according to any one of entries 14-21 with one or more or all of the components of feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol distillers grains, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably sugar derived from sugarcane or sugar beets, and the starch is particularly corn starch.

[0070] Item 31: A method for producing one or more of the following: feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch, the method comprising the following steps: (i) providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 27-31, preferably SEQ ID NO: 27; and (ii) Contacting the at least one polypeptide and / or the additive according to any one of entries 14-21 with one or more or all of the components of feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol distillers grains, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably sugar derived from sugarcane or sugar beets, and the starch is particularly corn starch.

[0071] Item 32: A method for producing one or more of the following: feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch, the method comprising the following steps: (i) providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 32-33, preferably selected from SEQ ID NO: 32; and (ii) Contacting the at least one polypeptide and / or the additive according to any one of entries 14-21 with one or more or all of the components of feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol distillers grains, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably sugar derived from sugarcane or sugar beets, and the starch is particularly corn starch.

[0072] Item 33: A method for producing one or more of the following: feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch, the method comprising the following steps: (i) providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 34-35, preferably SEQ ID NO: 34; and (ii) Contacting the at least one polypeptide and / or the additive according to any one of entries 14-21 with one or more or all of the components of feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol distillers grains, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably sugar derived from sugarcane or sugar beets, and the starch is particularly corn starch.

[0073] Item 34: A method for producing one or more of the following: feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch, the method comprising the following steps: (i) providing at least one polypeptide comprising, and / or consisting of, an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 36; and (ii) Contacting the at least one polypeptide and / or the additive according to any one of entries 14-21 with one or more or all of the components of feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol distillers grains, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably sugar derived from sugarcane or sugar beets, and the starch is particularly corn starch.

[0074] Item 35: Use of at least one polypeptide in the following: a) Modification of trichothecene; b) Manufacturing additives for use in feed and / or food, additives for use in feed compositions and / or food compositions, or additives for use in pharmaceutical compositions; and / or c) Manufacturing feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, WDG, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch; The at least one polypeptide comprises an amino acid sequence that is at least 70% sequence identical to an amino acid sequence selected from SEQ ID NO: 1-36, SEQ ID NO: 1-22, SEQ ID NO: 23-26, SEQ ID NO: 27-31, SEQ ID NO: 32-33, SEQ ID NO: 34-35 and SEQ ID NO: 36, and / or consists of an amino acid sequence that is at least 70% sequence identical to an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36.

[0075] Item 36: Polynucleotides, particularly isolated polynucleotides, encoding: polypeptides comprising amino acid sequences having at least 70% sequence identity with amino acid sequences selected from SEQ ID NO: 1-36, preferably with amino acid sequences selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and / or polypeptides as described in any of Items 11 to 12; and / or mutant variants as described in Item 13.

[0076] Item 37: A recombinant host cell comprising at least one polypeptide and / or at least one polynucleotide encoding said at least one polypeptide, wherein said at least one polypeptide comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, SEQ ID NO: 1-22, SEQ ID NO: 23-26, SEQ ID NO: 27-31, SEQ ID NO: 32-33, SEQ ID NO: 34-35 and SEQ ID NO: 36, preferably with an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36.

[0077] Item 38: The recombinant host cell described in Item 37, wherein the recombinant host cell is selected from: Escherichia to be cultivated ; Bacillus sp., for example Bacillus subtilis or Bacillus amyloliquefaciens ; Saccharomyces sp., For example Saccharomyces cerevisiae ; Pichia sp. ,For example Shepherd's pie or Pichia stump ; Kluyveromyces sp. .,For example Kluyveromyces lactis ; Schizosaccharomyces sp. .,For example Schizosaccharomyces pombe ; Hansenula sp. .,For example Hansenula polymorpha ; Streptomyces sp. ; Yarrowia sp. ; Trichoderma sp. ; Lactobacillus sp. ; Aspergillus sp. Plant cells; and / or Bacillus sp. , Trichoderma sp. or Aspergillus sp. spores.

[0078] Item 39: A polypeptide for use in the treatment, improvement and / or prevention of symptoms caused by fungal poisoning, particularly by trichothecene fungi, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, SEQ ID NO: 1-22, SEQ ID NO: 23-26, SEQ ID NO: 27-31, SEQ ID NO: 32-33, SEQ ID NO: 34-35 and SEQ ID NO: 36, preferably having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36.

[0079] The invention is further described below by way of non-limiting embodiments, which should not be construed as limiting embodiments of the invention, which is defined only by the claims.

[0080] Example The present invention disclosed herein is not limited to the specific embodiments, drawings, methodologies, examples, and solutions described herein, but is defined solely by the claims.

[0081] Example 1 After screening hundreds of glutathione S-transferases (also known as glutathione-S-transferases, GSTs), 36 polypeptides were identified that can modify deoxynivalenol (DON) by conjugation with reduced glutathione (GSH) to form the metabolite DON-13-GSH, in which GSH is covalently bonded to the C13 atom of DON. These polypeptides contain each of the amino acid sequences in SEQ ID NO: 1-36, i.e., the first polypeptide contains the amino acid sequence of SEQ ID NO: 1, the second polypeptide contains the amino acid sequence of SEQ ID NO: 2, and so on. For sequence analysis purposes, the amino acid sequences of SEQ ID NO: 1-36 were aligned with the previously described additional GST sequences (SEQ ID NO: 37-45) using the Clustal Omega alignment tool of EMBL-EBI with default settings (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ; Sievers et al. 2011. Mol. Syst. Biol. 7: 539) using the default EMBL-EBI tool. The amino acid sequences of SEQ ID NO: 1-45 and their identification with each other are shown in Tables 1 and 2, respectively.

[0082] Table 1. Amino acid sequences of SEQ ID NO: 1-45.

[0083] Table 2: Sequence identity matrix of SEQ ID NO: 1-45.

[0084] Example 2 To produce and purify the protein, the nucleotide sequence encoding a polypeptide comprising any one of SEQ ID NO: 1-45 is cloned into a plasmid for use in a suitable host organism (e.g., Escherichia coli Recombinant production was carried out in pET. These genes were sequenced as synthetic genes and cloned into pET-based systems. E. coliThe gene was cloned into an expression vector. For ease of subsequent purification, the gene was cloned into pET-28a(+) (NdeI / XhoI clone) containing an N-terminal 6xHis tag and a thrombin cleavage site. To verify the independence of enzymatic activity from tags fused to other peptides, GST domains without any tags (as shown in SEQ IDN NO: 1-45); GST domains with a C-terminal 6xHis tag; GST domains with an N-terminal 6xHis tag plus a thrombin cleavage site in addition to the C-terminal 6xHis tag; and GST domains with an N-terminal 6xHis tag plus a maltose-binding protein (MBP) solubility tag plus a thrombin cleavage site, all with the N-terminus fused to the GST domain. Ultimately, the untagged enzyme exhibited substantially the same enzymatic activity compared to enzymes fused with any of the aforementioned exemplary tags.

[0085] Transformed using recombinant expression vectors E. Coli BL21(DE3) strain. Recombinant gene expression was induced using OvernightExpress Instant TB medium (Novagen) containing 100 mg / L kanamycin for inducing IPTG-inducible promoters. However, any medium suitable for growth can also be used. E. coli And the culture medium for inducing IPTG-inducible promoters.

[0086] Cells are harvested by centrifugation and then lysed by sonication. Alternatively, bacterial cells can be opened (especially...). E. coli (cells) and any other suitable method to produce crude cell lysates. The lysates were removed by centrifugation, and the supernatant containing soluble recombinant proteins was used to determine the catalytic properties of the resulting peptides.

[0087] The active His-labeled enzyme was purified using a suitable affinity chromatography column (Protino Ni-IDA 2000 column, Macherey-Nagel) for purifying His-labeled proteins. Elution was performed from the affinity chromatography column with an imidazole-containing buffer. Subsequently, buffer exchange was performed using a centrifuge filter unit (Amicon Ultra-4) with a 10 kDa cutoff, transferring the imidazole-containing elution buffer to 100 mM sodium phosphate buffer at pH 6.5. Protein quantification of the thus purified enzyme was performed using the Qubit Protein Assay Kit (nvitrogen #Q33212), a Qubit 4 fluorometer, and a LabChip GXII HT Touch.

[0088] Example 3 To test the ability of the polypeptide containing any one of the GST domains having the amino acid sequences of SEQ ID NO: 1-45, generated as described above, to conjugate DON with reduced GSH, the polypeptide was tested in an activity assay as described below.

[0089] Typically, the specific activity of a peptide is tested under the following conditions: 100 mM sodium phosphate buffer, pH 6.5, containing 0.1 mg / mL bovine serum albumin (BSA), 20 ppm (=68 µM) DON, 1.7 mM GSH, and diluted peptide, at a temperature of 25 or 37 °C.

[0090] The reference method was used to assess pH and temperature-dependent stability and activity at variable (co)substrate concentrations of 2–2000 ppm DON and 67.5 µM–30 mM GSH, with appropriate adjustments made. As a buffer, McIlvaine buffer (McIlvaine. 1921. Journal of Biological Chemistry 49(1): 183–186) was used to assess pH-dependent stability or activity, and reaction temperatures of 25–60 °C were used to assess temperature-dependent activity. All assays were performed in Eppendorf Protein LoBind tubes or plates with diluted protein.

[0091] Prepare fresh GSH stock solutions or store aliquots at -20°C in 100 mM sodium phosphate buffer (pH 6.5) to minimize oxidation. Check the pH of the glutathione solution in the buffer and readjust after GSH dissolution. Typically, prepare a master mixture containing DON and GSH shortly before starting the reaction to minimize background. The reaction is typically carried out for 2 hours. In the case of enzymes with lower activity, extend the assay to 24 hours. Collect samples at regular intervals (0, 10, 20, 30, 40, 50, 60, 120 minutes after reaction start) to monitor reaction progress. Terminate the reaction by adding twice the volume of methanol to the aliquots. Then centrifuge the samples at full speed for 10 minutes at 4°C (21,000 xg for tubes or 3,200 xg for plates). Dilute the supernatant with distilled water to fall within the calibration range for LC-MS / MS analysis. These samples are then analyzed using the LC-MS / MS method described below.

[0092] Specific activity was calculated based on product formation within a linear range at the start of the reaction. When testing the labeled enzyme, the specific activity per mg of GST domain was calculated. Activity data are summarized in Table 3 below. Notably, the specific activities of the enzymes described in the literature (SEQ ID NO: 37-45) were found to be significantly lower than those of the enzymes described in SEQ ID NO: 1-36. Furthermore, the GST enzymes described in the literature are not specific for the production of DON-13-GSH, but also form the Michael adduct DON-10-GSH.

[0093] Table 3: Specific activity of the peptides of SEQ ID NO: 1-45 is expressed in units (U) / mg; normalized to the mass of the GST domain; 1 U is defined as the amount of enzyme activity required to modify 1 µmol of substrate in 1 minute. Activity data were recorded using 20 ppm DON, 1.7 mM MGSH, 0.1 mg / mL BSA, 100 mM sodium phosphate, pH 6.5, and 37°C.

[0094] Example 4 For LC-MS / MS analysis, DON and its enzyme-modified derivatives were separated on an Agilent 1290 series HPLC system with a 150 mm x 2.1 mm Phenomenex Kinetex biphenyl column with a particle size of 2.6 μm and a column temperature of 40 °C. The mobile phase consisted of a mixture of solvent A (ultrapure water and 1% acetic acid v / v) and solvent B (methanol and 1% acetic acid v / v) at a flow rate of 0.5 mL / min. The following gradients were used: 0–0.3 min: 0% B; 0.3–2.3 min: linear gradient to 70% B; 2.31–2.6 min: 100% B; 2.6–2.8 min: linear gradient to 0% B; 2.8–4 min: 0% B. Ions were generated using electrospray ionization (ESI) in negative ionization mode. Quantification was performed using a SCIEX QTrap 5500 or QTRAP 6500+ mass spectrometer from Sciex or a similar instrument. Dilute the sample with water to fall within the linear range of this method, which is analyte concentration in the injected sample from 1 ppb to 500 ppb DON or 1 ppb to 1800 ppb DON-13-GSH. Injection volume is 5 µL. LOQ is 1 ppb; the lower limit is set to... <LOQ。

[0095] Table 4: m / z transitions of DON and DON-13-GSH Example 5 Further testing was conducted on the ability of peptides containing amino acids of SEQ ID NO: 1-36 to modify other trichothecenes besides DON, such as 3-acetyldeoxynivalenol (3-ADON, CAS 50722-38-8), 15-acetyldeoxynivalenol (15-ADON, CAS 88337-96-6), nivalenol (NIV, CAS 23282-20-4), T-2 toxin (CAS 21259-20-1), HT-2 toxin (CAS 26934-87-2), and diacetylfusoxanthioneol (DAS, CAS: 2270-40-8). Therefore, the reaction was carried out at 25°C in 100 mM sodium phosphate buffer (pH 6.5) containing 0.1 mg / mL BSA, 1.7 mM GSH, 5 ppm trichothecene (3-ADON, 15-ADON, NIV, T-2; HT-2 or DAS), and enzyme concentrations ranging from 20 to 300 nM. Enzyme concentrations were selected based on previously recorded DON activity; concentrations were chosen to achieve near-complete DON conversion within 30–120 minutes. Sample sizes were minimized using trichothecene mixtures containing 5 ppm of each trichothecene. Mixture 1 contained DON + 3-ADON + T-2 + HT-2; Mixture 2 contained DON + 15-ADON + NIV + DAS. Each mixture also contained DON to allow for relative activity comparisons. Samples were taken at 0, 10, 20, 30, 60, 2, 4.5, and 24 hours after the start of the reaction. Subsequently, 20 μL of the aliquot sample was mixed with 40 μL of methanol to stop the reaction. The sample was centrifuged at 3,200 xg and 4°C for 10 minutes, then diluted 1:10 with distilled water, and measured using QTOF in both positive and negative modes, as described in Example 6 below.

[0096] The reaction rates with any non-DON trichothecene were compared to the reaction rates with DON. Certain peptides were selected as exemplary peptides. All of these peptides rapidly conjugated 15-ADON and DAS to GSH faster than DON. Peptides of SEQ ID NO: 36, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 1, or SEQ ID NO: 23 reacted rapidly with 3-ADON at a rate comparable to that with DON. Peptide of SEQ ID NO: 29 or SEQ ID NO: 7 reacted more slowly with 3-ADON than with DON. NIV conversion was approximately as fast as with DON. Peptides of SEQ ID NO: 29, SEQ ID NO: 5, SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 23 reacted faster with T-2 toxin than with DON. Peptides of SEQ ID NO: 36, SEQ ID NO: 2, or SEQ ID NO: 4 showed similar reaction rates with both T-2 and DON. The peptide of SEQ ID NO: 29 reacted faster with HT-2 toxin than with DON. Peptides of SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 23 showed reaction rates comparable to DON and HT-2 toxin. Peptides of SEQ ID NO: 36, SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5 reacted slower with HT-2 toxin than with DON. The results are summarized in Table 5. In conclusion, enzymes containing any one of the amino acid sequences of SEQ ID NO: 1-36 have a broad substrate range and can be used for the detoxification of many different trichothecetes.

[0097] Table 5: Enzymatic modification of trichothecene. The data show the time until at least 95% of the initial substate concentration (peak area) is achieved, in hours.

[0098] Example 6 To analyze non-DON trichothecenes and their modified derivatives, samples from Example 5 were analyzed using a high-resolution mass spectrometry (HRMS) system consisting of an Agilent 1290 Infinity II liquid chromatography system and a SCIEX X500R QTOF mass spectrometer. An Agilent Zorbax RRHD Eclipse plus C18 50x2.1 mm 1.8 µm column was used at a flow rate of 0.5 mL / min and a column temperature of 40 °C. Solvent A was water containing 0.1% formic acid, and solvent B was methanol containing 0.1% formic acid, using the following gradients: 0–1 min = 0% B; 1–6 min = 0 to 100% B (linear gradient); 6–8 min = 100% B; 8–8.1 min = 100% to 0% B (linear gradient). Mass spectrometry was performed in both positive and negative modes, with a mass scan range of 200–1200 Da. 3-ADON, 15-ADON, NIV, DAS, T-2, and HT-2 were converted with peptides to form 3-ADON-13-GSH, 15-ADON-13-GSH, NIV-13-GSH, DAS-13-GSH, T-2-13-GSH, and HT-2-13-GSH, respectively. No standards for glutathione conjugates were available (except for DON-13-GSH). Therefore, GSH conjugates were identified from QTOF spectra by their expected quality.

[0099] Example 7 To determine the toxicity of DON-glutathione-related conjugates, standards for DON-13-GSH and DON-13-Cys were prepared. DON-13-Cys has been described as the major degradation product of DON-13-GSH in vivo (Stanic et al. 2016. J. Agric. Food Chem. 64(23): 4777-4785). For this purpose, DON and the corresponding thiols (reduced L-glutathione or L-cysteine) were allowed to react at 37 °C in carbonate buffer at pH 10.7 for several days or weeks and purified by preparative HPLC, essentially as described by Stanic et al. 2016. J. Agric. Food Chem. 64(23): 4777-4785.

[0100] The toxicity of the thiol conjugate was assessed using an in vitro transcription-translation assay based on rabbit reticulocyte lysates, using an in vitro translation kit (TNT® Coupled Reticulocyte Lysate Systems kit; Promega #L4611). A luciferase T7 control DNA (Promega #L4821) was used as a reporter gene to quantify ribosome inhibition. The amount of luciferase produced was then assessed using a luciferase assay system (Promega E1500). Assays were performed in chemiluminescent 96-well plates in a microplate reader with a dispensing module. Measurements were performed in duplicate: 2 μL of the in vitro translation reaction (stored at -20°C) was pipetteed into a 96-well plate, 100 μL of the luciferase assay reagent was added via dispenser, mixed for 10 s, then delayed for 2 s and measured for 10 s.

[0101] The luminescence readings of the reaction without toxins were used as a reference and defined as 100% translation efficiency. The binding of inhibitors (e.g., DON) to the ribosome resulted in a decrease in the level of luciferase produced, thereby reducing chemiluminescence. The results are shown in Table 6 below. DON showed inhibition of translation at all concentrations, which is expected behavior given DON's reputation for its inhibitory effect on eukaryotic ribosomes. On the other hand, DON-13-GSH and DON-13-Cys did not show significant inhibition of luciferase production. Therefore, it can be concluded that DON-13-GSH and DON-13-Cys have much lower affinity for ribosome binding than DON. Therefore, modifying DON to DON-13-GSH using the peptide according to the invention is an effective means of detoxifying DON.

[0102] Table 6: Ribosomal inhibition of DON, DON-13-GSH or DON-13-Cys.

[0103] Example 8 To verify the in vivo activity of the peptide containing any one of SEQ ID NO: 1-36, the peptide of SEQ ID NO: 7 was generated. E. coli The strain was cultured in a DASGIP parallel bioreactor system at 37°C and pH 7.0, with level control achieved using an antifoaming agent (10% PPG 2000) and dissolved oxygen monitored. The bioreactor was designed for cultivation. E. ColiThe BL21(DE3) strain was prepared using a common procedure, followed by inoculation with batch culture medium (6.4 g / L potassium dihydrogen phosphate, 2.1 g / L phosphate, 1.78 g / L yeast extract, 2.78 g / L sodium citrate, 0.55 g / L magnesium chloride hexahydrate, 0.24 g / L calcium chloride dihydrate, 0.61 ml / L trace element solution (TES; 40 g / L FeSO4*7H2O, 10 g / L MnSO4*H2O, 10 g / L AlCl3*6H2O, 7.3 g / L CoCl2*6H2O, 2 g / L ZnSO4*7H2O, 1 g / L CuCl2*2H2O, 2 g / L Na2MoO4*2H2O, 0.5 g / L H3BO3), 3.06 g / L ammonium sulfate, and 39.6 ml / L... The strain was cultured overnight at OD600 = 2.0 (g / L glucose). For each reactor, 450 g of feed medium was prepared by mixing the following components: 231 g glucose, 3.22 g magnesium chloride hexahydrate, 1.414 g calcium chloride dihydrate, 3.5 g TES, and 211 g water. After a 12-hour batch phase, the reactor was run at 37°C for 15 hours at a feed rate of 12.3 mL feed medium / hour and 500 mL batch volume. After 27 hours, recombinant gene expression was induced by adding 15 μmol IPTG per g of cell dry weight. After induction, the temperature was lowered to 28°C, and the feed rate was set to 8.6 mL / h per 500 mL batch volume to produce protein until the end of fermentation (17 hours later). The culture was then centrifuged to harvest cell pellets. Cells were lysed according to the method described in Example 2. The recombinant protein was purified from the resulting cell pellet by affinity chromatography, similar to the purification described in Example 2. The elution fractions containing the enzyme were combined, and the buffer was changed to 100 mM sodium phosphate buffer, pH 6.5, on a cross-flow filtration system. 2.8 g of the recombinant enzyme was diluted to 1 L with distilled water in 111 ml of 100 mM sodium phosphate buffer (pH 6.5), further containing 2 mM GSH and 100 g / L maltodextrin. The entire mixture was lyophilized, and the lyophilized product was further used for feed trials.

[0104] Example 9 To assess the function of the enzyme in piglets, a standard piglet diet (886 g / kg dry matter, 204 g / kg crude protein, 40 g / kg crude fiber, 663 g / kg nitrogen-free extract, 515 g / kg starch, 35 g / kg crude fat, 59 g / kg crude ash, 15.26 MJ / kg metabolizable energy) was used as the basal diet. Different components were added to the basal diet according to each experimental group (Table 7). The basal diet already contained a low DON level of approximately 400 ppb. The background control group (A) received the basal diet, the DON control group (B) received a basal diet supplemented with 3 ppm DON, experimental group 1 (C) received a basal diet supplemented with DON and the enzyme formulated with GSH as described in Example 8; and experimental group 2 (D) received a basal diet supplemented with DON (the enzyme formulated with GSH as described in Example 8) plus additional GSH. The DON-13-GSH control group (E) received a basal diet supplemented with 8 ppm DON-13-GSH. DON-13-GSH is used at a higher concentration than DON, with DON and DON-13-GSH having equimolar concentrations.

[0105] Table 7: Grouping and experimental diets.

[0106] Each experimental group consisted of 4 piglets, for a total of 16 animals (8 females and 8 males; Austrian genotype). -HYB-F1 [(Landrace x Large White) x Pietrain]). Throughout the experiment, animals were housed in pairs in eight metabolic pens (two pens per group) to allow for pen-based urine collection. Animals selected for the experiment weighed approximately 8 kg and were weaned at 27–29 days of age.

[0107] The experiment included a 10-day acclimatization period beginning on day 0 after the animals arrived at the facility. Following the acclimatization period, from days 11–13, the animals were fed an experimental diet under a restricted feeding protocol (Table 8) for three consecutive days, followed by two days (days 14–15) during which they were again fed a basal piglet diet. During the experiment, the animals were restricted-fed in the morning and afternoon to allow for recording feed intake. Urine samples were collected from each pen on the morning and evening before feeding on days 11–15. The urine volume (mL) for each pen was recorded, and representative samples were collected. Two hours after breakfast on days 11–14, blood was collected from individual animals via venipuncture for later dried blood spot analysis.

[0108] Table 8: Test setup and duration.

[0109] All samples were analyzed by LC-MS to determine the presence of DON in urine samples, in order to ascertain whether a reduction in DON was achieved in vivo compared to the background control group. The results are shown in Table 9.

[0110] Table 9: DON concentration in creatinine in urine samples (µg / mmol).

[0111] DON was detected in blood and urine samples from all groups (AE), as it is prevalent in feed at a concentration of approximately 400 ppb. In blood samples from group A animals, 5–8 ng / mL of DON was detected, while higher levels (10–16 ng / mL) were found in blood samples from group B animals. DON levels in blood samples from group C animals were similar to those from group B, while the average DON level in blood samples from group D animals was 1–3 ng / mL. DON levels in the blood of animals from group E were similar to those from group A (5–8 ng / mL), see Table 10.

[0112] Table 10: DON concentration (ng / mL) in blood samples taken from individual animals. Data show the mean ± standard deviation for each group and individual at each time point. DON is known to be primarily excreted in urine. In urine samples from group A animals, DON concentrations increased from 40–70 µg / mmol creatinine, while in group B animals, an increase of approximately 150 µg / mmol creatinine was observed. The increase in DON concentration was correlated with the experimental diet. DON concentrations in urine samples from group C animals ranged from 50–150 µg / mmol creatinine. DON levels in urine samples from group D animals initially ranged from 9–32 μg / mmol creatinine, then increased to 57 μg / mmol creatinine after enzyme supplementation and cessation of GSH on day 14. DON levels in group E animals were comparable to those observed in group A animals, indicating that DON-13-GSH was not metabolized to DON by the animals. Therefore, the opening of the DON epoxide ring during the formation of DON-13-GSH is irreversible. Furthermore, DON-13-GSH and its derivative DON-13-Cys (as described in Stanic et al., 2016. J. Agric. Food Chem. 64(23): 4777-4785) were also detected in urine samples from groups D and E. Therefore, the peptide formulations prepared as described herein and (e.g., as described in Example 8) are functional and suitable for in vivo application.

[0113] Example 10 The method used to analyze urine samples from the in vivo tests described above is illustrated below. Creatinine levels in the urine samples were determined to normalize the trichothecete results to a specific creatinine value, thus accounting for variations in sample volume. Prior to analysis, 10 μL of urine was diluted with a 990 μL ultrapure water tube, the mixture was thoroughly mixed, and then 10 μL was transferred to an HPLC vial containing 990 μL of ultrapure water. The vial was mixed again prior to analysis.

[0114] An Agilent Technologies 1290 Infinity II HPLC system was coupled to a Sciex TripleQuad 5500 mass spectrometer equipped with an electrospray ionization source for creatinine quantification. The system was operated in positive ionization mode. Mobile phase A consisted of methanol / water / acetic acid (40 / 59.8 / 0.2, v / v / v); mobile phase B was methanol / acetic acid (99.8 / 0.2, v / v). Separation was achieved on a Phenomenex Gemini 5 µm C18 110A, 150 x 4.6 mm reversed-phase column by increasing mobile phase B from an initial 0% (0–1.60 min) to 100% (1.65 min). This eluent B ratio was maintained for 2.00 min, then the starting conditions were changed back to 0% B (2.05 min). The total run time for this method was 3 min. The measured m / z transitions are described in Table 11. Each transition was measured for 30 ms. The injection volume was 2 μL, and the column temperature was 30℃.

[0115] Table 11: Measurement of creatinine m / z transition.

[0116] Creatinine levels were determined based on a 1 / x weighted linear calibration function generated from a standard curve prepared in pure solvent. To further determine DON and DON-13-GSH, all urine samples were individually diluted with ultrapure water to a creatinine concentration of 2 mM.

[0117] Prior to analysis, 20 µL of creatinine-corrected urine sample was transferred to a reaction tube containing 180 µL of methanol / water (20 / 80, v / v). The sample was vortexed, incubated at -20°C for 30 min, centrifuged at 19,000 xg for 10 min, and the supernatant was transferred to an HPLC vial with inserts and sealed with a snap-on cap. LC-MS / MS analysis was performed using an Agilent Technologies 1290 Infinity II HPLC system coupled to a Sciex QTRAP 5500 mass spectrometer equipped with an electrospray ionization source. The system was operated in negative ionization mode.

[0118] For the quantification of DON, mobile phase A consisted of acetonitrile / water (5 / 95, v / v); mobile phase B consisted of acetonitrile / water (95 / 5, v / v). Acetic acid was added to both eluents to a concentration of 0.1%. Chromatographic separation was achieved on a Phenomenex Kinetex C18150 x 2.1 mm, 2.6 µm reversed-phase column by increasing mobile phase B from an initial 2% (0–0.1 min) to 25% (2.2 min), and finally to 100% (2.3 min). This eluent B concentration was maintained for 2.8 min, then reduced to the initial condition of 2% B (2.85 min). The total run time for this method was 3.5 min. The measured m / z transitions are described in Table 12. Each transition was measured for 10 ms. The injection volume was 1 μL, and the column temperature was 30 °C. The concentration was determined by a 1 / x weighted linear calibration function generated from a standard curve prepared in pure solvent, combined with 13C-labeled internal standard correction.

[0119] To quantify DON-13-GSH, mobile phase A consisted of water / acetic acid (99 / 1, v / v); mobile phase B consisted of methanol / acetic acid (99 / 1, v / v). Chromatographic separation was achieved on a Phenomenex Kinetex Biphenyl 150 x 2.1 mm, 2.6 µm reversed-phase column by increasing mobile phase B from an initial 0% (0–0.30 min) to 70% (2.30 min), and finally to 100% (2.31 min). This eluent B concentration was maintained for 2.60 min and then reduced to the initial condition of 0% B (2.80 min). The total run time for this method was 4 min. The measured m / z transitions are described in Table 12. Each transition was measured for 10 ms. The injection volume was 5 μL, and the column temperature was 40 °C. Concentrations were determined based on a 1 / x weighted linear calibration function generated from a standard curve prepared in pure solvent.

[0120] Table 12: Measured m / z transition of DON-13-GSH.

[0121] Example 11 To identify in vivo metabolites in urine samples, the following QTOF analysis method was applied. Urine samples were diluted to 7 mM creatinine with 20% methanol and analyzed using a high-resolution mass spectrometry (HRMS) system consisting of an Agilent 1290 Infinity II liquid chromatography system and a SCIEX X500R QTOF mass spectrometer. Samples were measured in negative mode according to the method described in Example 6.

[0122] Example 12 To determine the concentrations of DON and DON glucoside in whole blood as obtained from the feeding experiment in Example 9, 50 μL of whole blood was placed on a filter card and dried at room temperature. The entire spot was cut and placed in a reaction tube. Then, 10 μL of 100 PBB isotope-labeled 13C DON internal standard and 800 μL of extraction solvent (H2O / ACN 30 / 70 v / v) were added, and the mixture was stirred on an end-to-end shaker for 60 min. Two aliquots (300 μL each) were then dried under reduced pressure. One aliquot was reconstituted in 37.5 μL of H2O / ACN (95 / 5 v / v) for 10 min and centrifuged at 19,000 x g for 10 min. The supernatant was transferred to a silanized HPLC vial and measured. The second aliquot was reconstituted in 150 μL of 1x PBS for 10 min. Subsequently, 150 μL of β-glucuronidase solution was added, bringing the final enzyme concentration to approximately 3000 U / mL. The sample was incubated overnight at 37°C and extracted with 300 μL of ethyl acetate for 5 minutes. After extraction, the sample was centrifuged at 19,000 xg for 5 minutes; the supernatant was collected in a new reaction tube. Extraction with ethyl acetate was performed a total of three times, and the supernatants were combined and dried under reduced pressure. Finally, the sample was reconstituted in 37.5 µL of H2O / ACN (95 / 5 v / v) for 10 minutes and centrifuged at 19,000 xg for 10 minutes. The supernatant was transferred to a silanized HPLC vial and measured using LC-MS / MS.

[0123] Quantification of DON and DON-13-GSH was performed using an Agilent Technologies 1290 Infinity II HPLC system coupled to a Sciex QTRAP 6500 mass spectrometer equipped with an electrospray ionization source. LC-MS / MS was operated in negative ionization mode. Mobile phase A consisted of acetonitrile / water (5 / 95 v / v); mobile phase B consisted of acetonitrile / water (95 / 5 v / v). Acetic acid was added to both eluents to a concentration of 0.1%. Separation was achieved on a Phenomenex Kinetex C18 150 x 2.1 mm, 2.6 µm reversed-phase column by increasing mobile phase B from an initial 2% (0.1 min) to 25% (2.2 min), and then to 100% (2.3 min). This eluent B concentration was maintained for 2.8 min and then reduced to the initial condition of 2% (2.85 min). The total run time for this method was 3.5 min. The measured m / z transitions are described in Table 13. Each transition was measured for 15 ms. The injection volume was 20 μL, and the column temperature was 30 ℃.

[0124] Table 13: m / z transitions of DON and DON-13-GSH.

[0125] Quantification is based on standards in pure solvents with 13C-labeled DON internal standard correction, or on spiked blood samples treated as DON-13-GSH standards. For treated standards, blank blood is spiked with a specified concentration of DON-13-GSH, dried, and extracted as described above.

[0126] Example 13 In further feeding trials, 48 ​​piglets (24 females and 24 males; Austrian genotype) were included. -HYB-F1 [(Landrace x Large White) x Pietrain]) were assigned to appropriate groups and enclosures. Animals were housed in pairs in metabolic enclosures throughout the experiment. An experimental group consisted of four animals or two enclosures. Animals selected for the experiment were 27–29 days old at weaning. The acclimatization period began on day 0 after the animals arrived at the facility. On day 1, piglets received NuristartSweet mixed with standard piglet feed. The DON background level in the feed was 180 ppb. During the experiment, piglets received standard piglet feed as their basal diet and an experimental diet for three consecutive days each week following a restricted feeding pattern, with different amounts of feed per piglet in the morning and evening. Feed intake was recorded and any uneaten feed was removed. The experimental diet was artificially contaminated with DON culture to simulate 4 ppm DON contamination. Between weeks 1 and 2, animals received a standard diet for several days (washout period). Blood samples were collected from individual animals 1–2 hours after breakfast. Record the amount of urine excreted and collect equal aliquots from each enclosure before morning and evening feedings (including the “flushing” period). As an exemplary enzyme capable of modifying trichothecene, a polypeptide having SEQ ID NO: 7 or 8 was used for this assay.

[0127] In the first week of the experiment, GSH was tested at different concentrations at a constant enzyme concentration of 6 μmol / kg. In the second week of the experiment, GSH from different sources was tested.

[0128] The experimental schedule and setup are shown in Table 14 and follow the general protocol described above, except that blood was drawn only on two days of the experimental period and one day of the washout period. Therefore, lower DON levels were expected on day 7 in all experimental groups because DON was not administered prior to blood collection.

[0129] Table 14: Feeding trial setup. Feeding and sampling procedures.

[0130] All experimental diets contained standard piglet feed. DON culture was added to groups 2–12 and 14–19. At the indicated point, 6 μmol / kg of enzyme (SEQ ID NO: 7 or 8) was added. Reduced glutathione (GSH) was supplemented to the feed at 0, 1.25, 2.5, 5, 10, or 20 mmol / kg by adding purified GSH from two different suppliers. Group 1 served as a background control (N), and Group 2 served as a DON control (DON). These groups were tested for both experimental weeks. The experimental groups are shown in Table 15.

[0131] Table 15: Diet of the experimental group.

[0132] Blood spots were collected from each individual animal (four blood spots per group), and urine samples were collected from two animals in each enclosure (two urine samples per group). Samples were analyzed by LC-MS as described herein. Creatinine levels in urine samples were determined to normalize all samples to 2 mM creatinine. Dried blood spots were extracted using standard methods. All samples were measured by LC-MS / MS as described herein, with an internal standard used to compensate for matrix effects.

[0133] Results from week 1 showed that DON was effectively removed from the blood when either of the two test enzymes according to the invention was administered. Under the test conditions, a GSH concentration of at least 2.5 mmol / kg was found to be recommended for the removal of DON from the blood. Even better results were observed when 5 mmol or more of GSH was supplemented per kg. Comparable DON removal was observed when 10 or 20 mmol of GSH was supplemented per kg. Specifically, on day 4, the DON concentration in the DON control group was 15 ng / mL, compared to 1-5 ng / mL in any of groups 3, 4, 8, and 9. On day 7, the DON concentration in the DON control group was approximately 9 ng / mL, compared to 1-2 ng / mL in any of groups 3, 4, 8, and 9; and approximately 5 ng / mL in any of groups 5-7 and 10-12. Urine sample analysis confirmed these observations. Results from week 2 showed that the enzymes according to the invention performed well regardless of the source of GSH (i.e., from supplier 1 or 2).

[0134] Example 14 In another feeding trial, 48 piglets (24 females, 24 males; Austrian genotype) were included. -HYB-F1 [(Landrace x Large White) x Pietrain]) were assigned to appropriate groups and pens. Throughout the experiment, animals were housed in pairs in metabolic pens, with each experimental group consisting of four animals or two pens. Animals selected for the experiment were 27–29 days old at weaning. The acclimatization period began on day 0 after the animals arrived at the facility. On day 1, piglets received NuristartSweet mixed with standard piglet feed. The DON background level in the feed was 78 ppb. During the experiment, piglets received standard piglet feed as their basal diet and an experimental diet for three consecutive days each week following a restricted feeding pattern, with different amounts of feed per piglet in the morning and evening. Feed intake was recorded and any uneaten feed was removed. The experimental diet was artificially contaminated with DON culture to simulate 4 ppm DON contamination. Between weeks 1 and 2, animals received a standard diet for several days (washout period). Blood samples were collected from individual animals 1–2 hours after breakfast. Record the amount of urine excreted and collect equal aliquots from each enclosure before morning and evening feedings (including the “flushing” period). As an exemplary enzyme capable of modifying trichothecene, a polypeptide having SEQ ID NO: 7, 8, or 35 was used for this assay.

[0135] In week 1 of the experiment, the peptide with SEQ ID NO: 7 was used at 6–0.1 μmol / kg, and the peptide with SEQ ID NO: 8 was used at 6–1 μmol / kg. In week 2, the peptide with SEQ ID NO: 8 was used at 6, 0.5, and 0.1 μmol / kg, and the peptide with SEQ ID NO: 35 was used at 0.1–6 μmol / kg. GSH from supplier 2 was used in this experiment. The experimental schedule and setup are shown in Table 16.

[0136] Table 16: Feeding trial setup. Feeding and sampling procedures.

[0137] All experimental diets contained standard piglet feed. Except for the background control group, all groups were supplemented with DON culture material. Enzymes (SEQ ID NO: 7, 8, or 35) were added at the indicated points. The diets were supplemented with 10 mmol / kg GSH and 0.1, 0.5, 1, 3, or 6 μmol / kg of enzyme. Group 1 served as the background control (N), and Group 2 served as the DON control (DON). These groups were tested for two experimental weeks. The experimental groups are shown in Table 17.

[0138] Table 17: Diet of the experimental group.

[0139] Similarly, four blood spot samples and two urine samples were collected from each group. The samples were analyzed as described herein. Modifications were achieved by adding any enzyme of the present invention, particularly for the removal of DON from blood and urine. Under test conditions, a reduction in DON was observed when the enzyme concentration was above 0.1 µmol / kg. As the decrease in DON concentration observed in urine and blood samples depended on the applied enzyme concentration, increasing the enzyme concentration from 0.5 µmol / kg to 1 µmol / kg, 3 µmol / kg, or even 6 µmol / kg resulted in increased DON removal. Ultimately, all tested enzymes demonstrated in vivo suitability at different concentrations.

Claims

1. A method for modifying trichothecene, the method comprising the following steps: a) A reaction mixture is formed by contacting the trichothecene with the following substances: (i) at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and (ii) glutathione; and b) Incubate the reaction mixture.

2. The method of claim 1, wherein step b) is carried out at a temperature of 5-40°C, preferably at a temperature of 20-40°C.

3. The method of claim 1 or 2, wherein step b) is performed for at least 5 minutes, preferably at least 10 minutes.

4. The method of any one of the preceding claims, wherein the trichothecene is selected from deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin and diacetylfusarenol; preferably deoxynivalenol.

5. A mutant variant of a polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; wherein the mutant variant comprises at least one amino acid mutation, substitution, deletion and / or insertion.

6. An additive for modifying trichothecene, comprising: (i) at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and / or a mutant variant according to claim 5; and (ii) at least one additional component, preferably glutathione.

7. The additive of claim 6, wherein the at least one additional component is glutathione; and wherein the glutathione is contained in the additive at a concentration suitable for achieving an inclusion ratio of at least 0.1 μmol / kg (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 50, 100 μmol / kg).

8. The additive of claim 6 or 7, wherein the additive further comprises one or more components selected from: dietary supplements; nutritional supplements; pharmaceuticals; vitamins; minerals; amino acids; essential fatty acids; fiber; trace elements; antioxidants; plant extracts; herbal extracts; essential oils; and a carrier, preferably selected from bentonite, silica, and carbohydrates.

9. A method for manufacturing an additive for use in feed and / or food, in feed compositions and / or food compositions, or in pharmaceutical compositions, said method comprising the following steps: a) Provides at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and b) Contacting the at least one polypeptide with one or more components of an additive used in feed, food, feed compositions, food compositions, and / or pharmaceutical compositions.

10. A method for manufacturing feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch, and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch, the method comprising the following steps: a) Provides at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and b) Contact the at least one polypeptide and / or the additive according to any one of claims 6-8 with one or more components of feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably sugar derived from sugarcane or sugar beets, and the starch is particularly corn starch.

11. The use of at least one polypeptide in the following: a) Modification of trichothecene; b) Manufacturing additives for use in feed and / or food, feed compositions and / or food compositions, or pharmaceutical compositions; and / or c) Manufacturing feed, food, feed composition, food composition, pharmaceutical composition, biogas, bioethanol, DDGS, sugar, corn oil, corn germ, corn germ powder, corn fiber, corn gluten, starch and / or silage, wherein the sugar is preferably derived from sugarcane or sugar beets, and the starch is particularly corn starch; The at least one polypeptide comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36.

12. An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36; and / or a mutant variant according to claim 5.

13. A recombinant host cell comprising at least one polypeptide and / or at least one polynucleotide according to claim 12, wherein the at least one polypeptide comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36.

14. The recombinant host cell of claim 13, wherein the recombinant host cell is selected from: Escherichia coli ; Bacillus sp., For example Bacillus subtilis or Bacillus amyloliquefaciens ; Saccharomyces sp., For example Saccharomyces cerevisiae ; Pichia sp. ,For example Pichia pastoris or Pichia stipitis ; Kluyveromyces sp .,For example Kluyveromyces lactis ; Schizosaccharomyces sp .,For example Schizosaccharomyces pombe ; Hansenula sp .,For example Hansenula polymorpha ; Streptomyces sp. ; Yarrowia sp. ; Trichoderma sp. ; Lactobacillus sp. ; Aspergillus sp. Plant cells; and / or Bacillus sp. , Trichoderma sp. or Aspergillus sp. spores.

15. A polypeptide for use in the treatment, improvement and / or prevention of symptoms caused by fungal poisoning, particularly by trichothecene fungal poisoning, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with an amino acid sequence selected from SEQ ID NO: 1-36, preferably with an amino acid sequence selected from SEQ ID NO: 1, 23, 27, 32, 34 and 36.

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