Means and methods for modifying trichothecene

Abstract

The present invention relates to a method for modifying a trichothecene, a mutant variant of a polypeptide, an additive, methods for manufacturing, a use of the polypeptide, a polynucleotide, a recombinant host cell, and a polypeptide for use in treatment, amelioration and / or prophylaxis of symptoms caused by mycotoxicosis.

Description

[0001] MEANS AND METHODS FOR MODIFYING TRICHOTHECENE

[0002]

[0001] The present invention relates to a method for modifying a trichothecene, a mutant variant of a polypeptide, an additive, methods for manufacturing, a use of the polypeptide, a polynucleotide, a recombinant host cell, and a polypeptide for use in treatment, amelioration and / or prophylaxis of symptoms caused by mycotoxicosis.

[0003]

[0002] Mycotoxins are secondary metabolites produced by fungi, which infest food and feed material, in particular all types of grains and crops, or other commodities such as cotton, coffee, peanuts, dates, spices etc. Upon fungal growth, the infested material thus gets contaminated with mycotoxins such as aflatoxins, ochratoxin, ergot alkaloids, fumonisins, zearalenone, trichothecenes etc.

[0004]

[0003] Among more than 17,000 samples, almost three quarters were found to contain at least one mycotoxin, 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 referred to as vomitoxin; CAS-No. 51481-10-8) is one of the most prevalent trichothecenes and found to occur globally. Feed material contaminated with deoxynivalenol or other trichothecenes leads to feed refusal and reduced body weight gain and thus not only affects animal health and wellbeing but also causes considerable economic losses in animal farming.

[0005]

[0004] Largely due to its hydrophilic structure, removal of DON by binding e.g. to mineral or clay adsorbents is inefficient and insufficient.

[0006]

[0005] As an alternative, transformation of DON to the isomeric form 3-epi-DON, has been suggested, e.g. Bracarense et al. 2020. Food Chem Toxicol. 140: 111241. In the case of DON, the hydroxy group at carbon atom 3 is in R-configuration, whereas in the case of 3-epi-DON, the hydroxy group at carbon atom 3 is in S-configuration. The detoxification transformation from DON to 3-epi-DON has been described previously 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 DON oxidation to form 3-keto-DON with a carbonyl group on carbon 3, and one or more reductases to reduce 3-keto-DON to 3-epi-DON. Notably, some enzymes capable of using 3-keto-DON as a substrate catalyze a reduction reaction which at least partially leads back to DON rather than to the detoxified isomer 3-epi-DON. In addition, the oxidation reaction requires free PQQ, and the reduction reaction depends on the nicotine amide cofactor NADPH (reduced nicotinamide adenine dinucleotide phosphate). Even though these reactions can be quite efficient, the dependence on two or even three enzymes and at least two further substrates, PQQ and NADPH, renders these mechanisms rather complex and therefore challenging 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 a continuous matter. Thus, even though these systems produce 3-epi-DON, they are accompanied by several obstacles with regard to applicability.

[0007]

[0006] Yet another system for enzymatic modification of trichothecene to achieve detoxification is described in WO 2020 / 254592 A1. Therein, glutathione-S-transferase (GST) is shown to be capable of biotransformation of DON, albeit at slow catalytic rate. On the other hand, Nakamura et al. (1977. Chem. Pharm. Bull. 25(12): 3410-3414) found some GST enzymes not to be capable of modifying trichothecenes.

[0008]

[0007] Taken together, there is thus a need for new solutions, in particular for enzymes which are capable of modifying and detoxifying trichothecenes at a rate that is applicable for in vivo applications.

[0009]

[0008] This aim was achieved by providing a method for modifying a trichothecene, the method comprising the steps of: a) forming a reaction mixture by contacting the 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 SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and (ii) with glutathione, preferably reduced glutathione (GSH); and b) incubating the reaction mixture.

[0010]

[0009] In such a method, a trichothecene can be modified such that glutathione is linked specifically to the C13 atom of the trichothecene, thus transforming 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 markedly less toxic than their non-modified versions. In contrast, previously reported enzymes produce at least to some extent also trichothecene derivatives wherein glutathione is linked to the C10 atom. In contrast to modification at the C13 atom however, modification at the C10 atom is reversible (Stanic et al. 2016. J. Agric. Food Chem. 64(23): 4777- 4785). Therefore, C10-glutathionylated trichothecene derivatives cannot be considered to be effectively detoxified.

[0011]

[0010] The term "polypeptide" is to be construed as commonly used in the art and encompasses e.g. polypeptides, proteins, peptides, enzymes.

[0012]

[0011] The term "sequence identity" is used to describe the degree of relatedness of two or more nucleic acid sequences (e.g. DNA or RNA polynucleotides) comprised in polynucleotides, or of two or more amino acid sequences comprised in polypeptides. Sequence identity can be determined by common methods known to a skilled person. Herein, the preferred method for determination of sequence identity between two amino acid sequences comprised in two polypeptides is the use of the Clustal Omega alignment tool of 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 may be used, e.g. as provided by the National Center for Biotechnology Information (“Needleman- Wunsch Global Align Protein Sequences”) using default settings (Gap Costs: Existence: 11 Extension 1). Polypeptides comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence of a target polypeptide may be polypeptides comprising an amino acid sequence having 70% or more % sequence identity, e.g. 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 to the amino acid sequence of the target polypeptide. For instance, a polypeptide comprising and / or consisting of an amino acid sequence having 100% sequence identity to SEQ ID NO: 1 , is encompassed by the group of "polypeptides comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1". Thus, polypeptides comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, include those polypeptides which consist of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, as well as those polypeptides in which an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36 is connected to one or more further amino acid sequence(s). Typically, the term "fusion polypeptide" or "fusion protein" describes two or more polypeptides linked to one another, typically by peptide bonds. E.g., in such fusion proteins, two domains being protein domains are linked to one another by peptide bond(s). Also, in some fusion proteins, one or more peptide tag(s) are linked or connected to the N- and / or C-terminus of a polypeptide (e.g. a polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36), e.g. in order to facilitate easier purification or to increase solubility, without essentially altering the enzymatic properties of the tagged polypeptide. Examples for commonly used tags are poly(His)-tags (e.g. hexahistidine tag), maltose-binding-protein (MBP)-tag, Strep-tag, Strep ll-tag etc. Thus, enzymatically active fusion polypeptides comprising a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, are encompassed by the present invention. Such fusion polypeptides may e.g. comprise a hexahistidine tag fused to the N-terminus of a polypeptide comprising an amino acid sequence having 91% sequence identity to the amino acid sequence of SEQ ID NO: 1 ; or e.g. comprise a maltose-binding-protein tag fused to the C-terminus of a polypeptide comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 23 via a linker peptide (e.g. GG; GPG; EA or EA repeats; GSG; etc.). In other words, larger polypeptides comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36, as a part of said larger polypeptide are also encompassed by the present invention, as long as the enzymatic activity (i.e. as a glutathione transferase) of said amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1- 36, preferably selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36, is not essentially obliterated. Merely for the sake of clarification, enzymatically active polypeptide(s) according to the invention are capable of and thus suitable for modifying at least one trichothecene. In particular, enzymatically active polypeptide(s) according to the invention are capable of and thus suitable for modifying at least one trichothecene at the C13 atom. In other words, polypeptide(s) not capable of modifying a trichothecene are not encompassed by the present invention.

[0013]

[0012] In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of 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) to an amino acid sequence selected from SEQ ID NOs: 1-36. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of 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) to an amino acid sequence selected from SEQ ID NOs: 1-22. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of 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) to an amino acid sequence selected from SEQ ID NOs: 23-26. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of 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) to an amino acid sequence selected from SEQ ID NOs: 27-31. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of 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) to an amino acid sequence selected from SEQ ID NOs: 32-33. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of 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) to an amino acid sequence selected from SEQ ID NOs: 34-35. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of 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) to SEQ ID NO: 36.

[0014]

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

[0015]

[0014] In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of any one of the amino acid sequences of SEQ ID NOs: 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. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of the amino acid sequence of SEQ ID NO: 32. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of the amino acid sequence of SEQ ID NO: 34. In some embodiments, the polypeptide according to the invention is a polypeptide comprising and / or consisting of the amino acid sequence of SEQ ID NO: 36.

[0016]

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

[0017]

[0016] Preferably, the step of incubating the reaction mixture in a method according to the invention is performed for at least 0.1 seconds, preferably for at least 1 , 5, 10, 20, 60 seconds, preferably for at least 1 , 2, 3, 4, 5 minutes, preferably for at least 10 min (e.g. at least 15 min, 30 min, 60 min etc.). The longer the step of incubating the reaction mixture is performed, the more trichothecene will be modified. Notwithstanding, a skilled person is aware that an enzymatic type of reaction as described herein may also be effected outside said preferred conditions. Merely for the sake of clarity, incubation is considered to start immediately upon contact of a polypeptide according to the invention with the trichothecene and the glutathione. A skilled person is well capable of choosing incubation conditions appropriate to modify a trichothecene concentration in a composition to a desired degree. Notwithstanding, particularly preferred trichothecene modification and thus detoxification will be achievable when performing the step of incubating the reaction mixture at a temperature of 5-40 °C, and / or for at least 5 minutes.

[0018]

[0017] In some embodiments, the 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 diacetoxyscirpenol (DAS). In particular the high prevalence of deoxynivalenol in food and / or feed material constitutes a considerable threat for human and non-human animals. Therefore, in a preferred embodiment the trichothecene is deoxynivalenol (DON; sometimes also referred to as vomitoxin; 3a,7a,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one; or (2R,2'S,3R,5R,5aR,6S, 9aR)-3,8-dihydroxy-5a-(hydroxymethyl)-5,6-dimethylspiro[[2,5]methano[1]benzoxepine-10,2'-oxi ran]-7(6H)-one; CAS no. 51481-10-8).

[0019]

[0018] In some embodiments of a 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 a method according to the invention, GSH is provided or present at a concentration of at least 0.1 mmol per kg of food or feed, preferably at least 1 mmol per kg of food or feed, more preferably at least 5 mmol per kg of food or feed. In some embodiments, at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36, is provided at a concentration of at least 0.1 pM, preferably at least 1 pM; or at a concentration of at least 0.1 pmol per kg of food or feed, preferably at least 1 pmol per kg of food or feed.

[0020]

[0019] In some embodiments of the method for modifying a trichothecene according to the invention, the trichothecene is comprised in a nutritional composition. Such a nutritional composition is a composition comprising one or more component(s) having nutritional value. Often such components provide energy to the consumer of the nutritional composition. A nutritional composition may be entirely or at least partly herbal or plant-based, such as commonly used animal feed compositions. Fodder or feed may e.g. comprise or consist of corn, hay, straw litter, soy, or products obtained therefrom. Also, fodder or feed may comprise or consist of extruded feed products, e.g. pellets

[0021]

[0020] In a further aspect, the invention relates to a mutant variant of a polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; wherein the mutant variant comprises at least one amino acid mutation, substitution, deletion, and / or insertion. Such a mutant variant is a recombinantly produced polypeptide variant of a polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36. In particular, such a mutant variant comprises and / or consists of an amino acid sequence that is a synthetic amino acid sequence. In other words, such a synthetic amino acid sequence does not occur in nature. Instead, a 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 altering may be a substitution of an amino acid at a certain position by another amino acid that would not occur at this position without the human intervention. Such altering may also be the deletion of an amino acid at a certain position. Alternatively, one or several amino acids may be inserted at one or several positions in an otherwise naturally occurring amino acid sequence.

[0022]

[0021] For instance, the amino acid sequences of SEQ ID NOs: 10-22, 25-26 are synthetic amino acid sequences. Thus, polypeptides comprising and / or consisting of any one of the amino acid sequences of SEQ ID NOs: 10-22, 25-26 are examples for mutant variants of a polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36. Production of such mutant variants is typically performed to provide polypeptides having e.g. faster kinetic activity, higher stability e.g. towards a certain temperature or pH. As a consequence, mutant variants are particularly useful in industrial applications.

[0023]

[0022] Additives for food, foodstuff, fodder or feed are often used to improve or add properties to the food, foodstuff, fodder or feed. For instance, such additives may be added to improve organoleptic properties, e.g. to improve taste, smell, appearance, color of the food, foodstuff, fodder or feed. Also, additives may be added to improve palatability, nutrient availability, or to add probiotic microorganisms to the food, foodstuff, fodder or feed, or to add or enhance prebiotic activity of the food, foodstuff, fodder or feed. Also, such additives may be added to counteract potentially undesirable effects of the food, foodstuff, fodder or feed, such as removal or reduction of one or more undesirable components comprised in the food, foodstuff, fodder or feed.

[0024]

[0023] In another aspect, the invention relates to an additive (e.g. an additive for feed and / or food; or a feed additive; or a food additive) for modifying a trichothecene, comprising (i) at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and / or a mutant variant of a polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; 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 (in particular, reduced glutathione). By providing such an additive according to the invention, a suitable means is provided that allows convenient detoxification of trichothecene(s).

[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, in particular reduced glutathione (GSH), at a concentration suitable for achieving an inclusion rate of at least 0.1 pmol GSH per kg (e.g. at least 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 5, 10, 50, 100 pmol per kg). In other words, GSH is comprised in the additive at such a concentration that the final concentration of GSH in a target composition is 0.1 mmol of GSH per kg of target composition, after mixing the additive with the target composition. For instance, in an embodiment where the additive is a feed additive, the target composition would be feed. If said feed additive would be e.g. 1000-fold concentrated, GSH would be comprised in said feed additive at a concentration of 100 mmol per kg of the feed additive. Upon mixing of said feed additive with the target composition feed at a ratio of 1 :1000, the inclusion rate of GSH of 0.1 mmol per kg of feed would be achieved. Thus, in such a scenario, GSH would be comprised at the concentration of 100 mmol per kg of the additive.

[0025]

[0025] In some embodiments, the additive according to the invention further comprises one or more component(s) selected from dietary supplement(s); nutraceutical(s); pharmaceutical(s); vitamin(s); mineral(s); amino acid(s); essential fatty acid(s); fiber(s); trace element(s); antioxidant(s); plant extract(s); herbal extract(s); essential oil(s); carrier(s), preferably selected from bentonite, silica, and carbohydrates.

[0026]

[0026] In some embodiments, the additive according to the invention comprises glutathione, in particular reduced glutathione (GSH); preferably wherein GSH is comprised in the additive at a concentration suitable for achieving an inclusion rate of at least 0.1 pmol of GSH per kg of target composition (e.g. at least 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 5, 10, 50, 100 pmol of GSH per kg); and the additive further comprises one or more component(s) selected from dietary supplement(s); nutraceutical(s); pharmaceutical(s); vitamin(s); mineral(s); amino acid(s); essential fatty acid(s); fiber(s); trace element(s); antioxidant(s); plant extract(s); herbal extract(s); essential oil(s); carrier(s), preferably selected from bentonite, silica, and carbohydrates.

[0027]

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

[0028]

[0028] In some embodiments, the additive according to the invention comprises glutathione, in particular reduced glutathione (GSH); preferably wherein GSH is comprised in the additive at a concentration suitable for achieving an inclusion rate of at least 0.1 pmol of GSH per kg of target composition (e.g. at least 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 5, 10, 50, 100 pmol of GSH per kg); and the additive further comprises a carrier for enzymes. Carriers for enzymes can be both of inorganic and organic origin. Potential inorganic materials used for the immobilization of enzymes are silica (sol-gel silica, fumed silica, colloidal silica nanoparticles and silica gels) and oxides such as titanium oxide, aluminum oxide and zirconium oxide. Furthermore, clay materials such as bentonite, halloysite, kaolinite, montmorillonite, sepiolite and calcium apatite may be such carriers. Additionally, carbon-based materials such as activated carbons and charcoal can be carriers. Organic enzyme carriers may be biopolymers (e.g. carbohydrates, proteins, maltodextrin, trehalose, inulin, collagen, cellulose, keratins, carrageenan, chitin, chitosan and alginate) or synthetic polymers (e.g. polyaniline, polyamides, polystyrene, polyurethane, polypropylene, polyvinyl alcohol and ion exchange resins). Liquid carriers could be e.g. buffer substances and / or polyalcohols such as polyalkylene oxides, polyvinyl alcohols, polyethylene-co- maleic acid anhydrides, polystyrene-co-malic acid anhydrides, dextrans, celluloses, hydrolyzates of chitosan, starches, glycogen, sorbitol, agarose and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolyzates, bio-polymers, sorbitol, glycerol, cellobiose, and mono propylene glycol. The carrier may additionally or alternatively be an eatable component, preferably a non-toxic component and / or a component providing for a texture. In a particular embodiment, the carrier is selected from bentonite, silica, and carbohydrates; preferably the carrier is bentonite.

[0029]

[0029] In another aspect, the invention relates to a method for manufacturing an additive for feed and / or food, additive for feed composition and / or food composition, or additive for pharmaceutical composition, 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 to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and b) contacting the at least one polypeptide with one or more components of the additive for feed and / or food, additive for feed composition and / or food composition, or additive for pharmaceutical composition.

[0030]

[0030] In a further aspect, the invention relates to a method for manufacturing of feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; 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 to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and b) contacting the at least one polypeptide; and / or an additive according to the invention, with one or more components of feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

[0031] GSH is a natural component that can be readily available e.g. in food, feed, human or nonhuman animals, for the enzymatic modification reaction described herein. It was found by the inventors, that by increasing GSH availability, said enzymatic modification reaction can be accelerated. Therefore, in some preferred embodiments, a method for manufacturing according to the invention, also comprises the step of c) providing GSH at a concentration of at least 0.1 mmol per kg of the additive for feed and / or food, additive for feed and / or food composition, additive for pharmaceutical composition; feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

[0031]

[0032] In further aspects, the invention relates to a use of at least one polypeptide in one or more of the following: a) modifying a 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 of feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; wherein the at least one polypeptide comprises and / or consists of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

[0032]

[0033] In another aspect, the invention relates to a polynucleotide encoding a polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and / or a mutant variant according to the invention. In some embodiments, the invention relates to an isolated polynucleotide encoding a polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and / or a mutant variant according to the invention. Merely for clarification, an isolated polynucleotide is not considered to occur in nature, where polynucleotides only exist in the context of a biological cell, i.e. in a non-isolated context. In contrast, an isolated polynucleotide occurs only in the context of molecular biological steps being performed by a human experimenter. For instance, isolated polynucleotides are being produced and handled in cloning activities which are necessary e.g. for achieving later recombinant protein production.

[0033]

[0034] In a further aspect, the invention relates to a recombinant host cell comprising at least one polypeptide; and / or at least one polynucleotide encoding the at least one polypeptide and / or a mutant variant thereof as referred to herein, wherein the at least one polypeptide comprises and / or consists an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

[0034]

[0035] Such a host cell according to the invention allows production, in particular recombinant, heterologous production of the polypeptide(s) referred to herein.

[0035]

[0036] For the sake of clarity, the term "recombinant" means that the host cell comprising the at least one polypeptide and / or the at least one polynucleotide encoding the at least polypeptide would not comprise said at least one polypeptide and / or polynucleotide naturally, but only as a consequence of a human experimenter introducing said at least one polypeptide and / or polynucleotide into said host cell by technical means, such as e.g. means of molecular biology, biotechnology, and / or synthetic biology.

[0036]

[0037] In some embodiments, the recombinant host cell according to the invention is selected from Escherichia coir, Bacillus sp. such as Bacillus subtilis or Bacillus amyloliquefaciens; Saccharomyces sp. such as Saccharomyces cerevisiae; Pichia sp. such as Pichia pastoris or Pichia stipitis; Kluyveromyces sp. such as Kluyveromyces I act is; Schizosaccharomyces sp. such as Schizosaccharomyces pombe; Hansenula sp. such as Hansenula polymorpha; Streptomyces sp.; Yarrowia sp.; Trichoderma sp.; Lactobacillus sp.; Aspergillus sp.; plant cells; and / or spores of Bacillus sp., Trichoderma sp. or Aspergillus sp. In some preferred embodiments, the recombinant host cell according to the invention is a bacterial cell, preferably Escherichia coli; Bacillus sp. such as Bacillus subtilis or Bacillus amyloliquefaciens.

[0037]

[0038] Strains commonly associated with "Pichia pastoris" are sometimes referred to as Komagataella pastoris, Komagataella phaffii or Komagataella pseudopastoris, depending on the respective taxonomic classification at the time of reference. In any case, the term "Pichia pastoris" as used herein is therefore to be interpreted to encompass all of these strains, even in cases where they are not referred to as Pichia pastoris but as Komagataella pastoris, Komagataella phaffii or Komagataella pseudopastoris.

[0038]

[0039] In another aspect, the invention relates to a polypeptide for use in treatment, amelioration and / or prophylaxis of symptoms caused by mycotoxicosis, in particular by trichothecene mycotoxicosis, wherein the polypeptide comprises and / or consists of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1- 36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36. The use of a polypeptide according to the invention for such a purpose is of particular relevance e.g. in the field of animal husbandry. In order to prevent symptoms caused by trichothecenes, e.g. by vomitoxin, in animals (e.g. in weaning piglets, grower pigs, or finisher pigs), prophylactic application of a polypeptide according to the invention will lead to detoxification of the trichothecene(s) prior to consumption. In cases where the trichothecene(s) have already been consumed, application of a polypeptide according to the invention will treat and ameliorate the symptoms, thus minimizing animal sickness and promoting recovery.

[0039]

[0040] The invention is further characterized by the following items:

[0040]

[0041] Item 1 : Method for modifying a trichothecene, the method comprising the steps of a) forming a reaction mixture by contacting the trichothecene with

[0041] (i) at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and

[0042] (ii) glutathione; and b) incubating the reaction mixture.

[0043]

[0042] Item 2: Method for modifying a trichothecene, the method comprising the steps of a) forming a reaction mixture by contacting the trichothecene with

[0044] (i) at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-22, preferably SEQ ID NO: 1; and

[0045] (ii) glutathione; and b) incubating the reaction mixture.

[0046]

[0043] Item 3: Method for modifying a trichothecene, the method comprising the steps of a) forming a reaction mixture by contacting the trichothecene with

[0047] (i) at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 23-26, preferably SEQ ID NO: 23; and

[0048] (ii) glutathione; and b) incubating the reaction mixture.

[0049]

[0044] Item 4: Method for modifying a trichothecene, the method comprising the steps of a) forming a reaction mixture by contacting the trichothecene with

[0050] (i) at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 27-31, preferably SEQ ID NO: 27; and

[0051] (ii) glutathione; and b) incubating the reaction mixture.

[0052]

[0045] Item 5: Method for modifying a trichothecene, the method comprising the steps of a) forming a reaction mixture by contacting the trichothecene with

[0053] (i) at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 32-33, preferably SEQ ID NO: 32; and (ii) glutathione; and b) incubating the reaction mixture.

[0054]

[0046] Item 6: Method for modifying a trichothecene, the method comprising the steps of a) forming a reaction mixture by contacting the trichothecene with

[0055] (i) at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 34-35, preferably SEQ ID NO: 34; and

[0056] (ii) glutathione; and b) incubating the reaction mixture.

[0057]

[0047] Item 7: Method for modifying a trichothecene, the method comprising the steps of a) forming a reaction mixture by contacting the trichothecene with

[0058] (i) at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 36; and

[0059] (ii) glutathione; and b) incubating the reaction mixture.

[0060]

[0048] Item 8: The method according to any one of items 1-7, wherein step b) is performed at a temperature of 5 - 40 °C, preferably at a temperature of 20 - 40 °C.

[0061]

[0049] Item 9: The method according to any one of items 1-8, wherein step b) is performed for at least 5 minutes, preferably for at least 10 minutes.

[0062]

[0050] Item 10: The method according to any one of items 1-9, wherein the trichothecene is selected from deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, and diacetoxyscirpenol; preferably deoxynivalenol.

[0063]

[0051] Item 11 : Polypeptide comprising and / or consisting of an amino acid sequence, wherein the amino acid sequence has at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and wherein the amino acid sequence has less than 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1- 36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

[0064]

[0052] Item 12: The polypeptide according to item 11 , wherein the polypeptide comprises at least one amino acid mutation, substitution, deletion, and / or insertion compared to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

[0065]

[0053] Item 13: Mutant variant, in particular a recombinant mutant variant, of a polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; wherein the mutant variant comprises at least one amino acid mutation, substitution, deletion, and / or insertion.

[0066]

[0054] Item 14: Additive, preferably food additive and / or feed additive, for modifying a trichothecene, comprising

[0067] (i) at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and / or a polypeptide according to any one of items 11 and 12; and / or a mutant variant according to item 13; and

[0068] (ii) at least one additional component, preferably glutathione, in particular reduced glutathione (GSH).

[0069]

[0055] Item 15: The additive according to item 14, wherein the at least one additional component is glutathione, in particular reduced glutathione (GSH).

[0070]

[0056] Item 16: The additive according to item 15, wherein the glutathione is comprised in the additive at a concentration suitable for achieving an inclusion rate of at least 0.1 pmol of GSH per kg of target composition (e.g. at least 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 5, 10, 50, 100 pmol of GSH per kg).

[0071]

[0057] Item 17: The additive according to item 16, wherein the target material is food and / or feed.

[0058] Item 18: The additive according to any one of items 14-17, wherein the additive further comprises one or more component(s) selected from dietary supplement(s); nutraceutical(s); pharmaceutical(s); vitamin(s); mineral(s); amino acid(s); essential fatty acid(s); fiber(s); trace element(s); antioxidant(s); plant extract(s); herbal extract(s); essential oil(s); carrier(s), preferably selected from bentonite, silica, and carbohydrates.

[0072]

[0059] Item 19: The additive according to any one of items 14-18, wherein the at least one additional component is glutathione, in particular reduced glutathione (GSH); and wherein the additive further comprises one or more component(s) selected from dietary supplement(s); nutraceutical(s); pharmaceutical(s); vitamin(s); mineral(s); amino acid(s); essential fatty acid(s); fiber(s); trace element(s); antioxidant(s); plant extract(s); herbal extract(s); essential oil(s); carrier(s), preferably selected from bentonite, silica, and carbohydrates.

[0073]

[0060] Item 20: The additive according to any one of items 14-18, wherein the at least one additional component is glutathione, in particular reduced glutathione (GSH); and wherein the additive further comprises an absorbent.

[0074]

[0061] Item 21 : The additive according to item 20, wherein the absorbent is a clay, preferably bentonite.

[0075]

[0062] Item 22: Method for manufacturing one or more of a) additive for feed and / or food; b) additive for feed composition and / or food composition; c) additive for pharmaceutical composition; d) feed and / or food; e) feed composition and / or food composition; f) pharmaceutical compositiong g) biogas; h) bioethanol; i) wet distillers grains (WDG); j) dried distillers grains with solubles (DDGS); k) sugar, preferably from sugar cane or sugar beets; l) corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or m) silage; the method comprising the steps of i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, SEQ ID NOs: 1-22, SEQ ID NOs: 23-26, SEQ ID NOs: 27- 31 , SEQ ID NOs: 32-33, SEQ ID NOs: 34-35, and SEQ ID NO: 36; preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and ii) contacting the at least one polypeptide (e.g. a polypeptide according to any one of items 11-12 and / or a mutant variant according to item 13), and / or an additive according to any one of items 14-21 , with one or more or all components of: additive for feed and / or food; additive for feed composition and / or food composition; additive for pharmaceutical composition; feed and / or food; feed composition and / or food composition; pharmaceutical composition; biogas; bioethanol; wet distillers grains (WDG); dried distillers grains with solubles (DDGS); sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

[0076]

[0063] Item 23: Method for manufacturing one or more of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition, the method comprising the steps of i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-22; preferably SEQ ID NO: 1 ; and ii) contacting the at least one polypeptide (e.g. a polypeptide according to any one of items 11-12 and / or a mutant variant according to item 13), with one or more or all components of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition.

[0064] Item 24: Method for manufacturing one or more of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition, the method comprising the steps of i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 23-26; preferably SEQ ID NO: 23; and ii) contacting the at least one polypeptide (e.g. a polypeptide according to any one of items 11-12 and / or a mutant variant according to item 13), with one or more or all components of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition.

[0077]

[0065] Item 25: Method for manufacturing one or more of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition, the method comprising the steps of i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 27-31 ; preferably SEQ ID NO: 27; and ii) contacting the at least one polypeptide (e.g. a polypeptide according to any one of items 11-12 and / or a mutant variant according to item 13), with one or more or all components of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition.

[0078]

[0066] Item 26: Method for manufacturing one or more of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition, the method comprising the steps of i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 32-33; preferably selected from SEQ ID NO: 32; and ii) contacting the at least one polypeptide (e.g. a polypeptide according to any one of items 11-12 and / or a mutant variant according to item 13), with one or more or all components of: an additive for feed and / or food, an additive for feed and / or food composition, or an additive pharmaceutical composition.

[0079]

[0067] Item 27: Method for manufacturing one or more of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition, the method comprising the steps of i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 34-35; preferably SEQ ID NO: 34; and ii) contacting the at least one polypeptide (e.g. a polypeptide according to any one of items 11-12 and / or a mutant variant according to item 13), with one or more or all components of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition.

[0080]

[0068] Item 28: Method for manufacturing one or more of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition, the method comprising the steps of i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence of SEQ ID NO: 36; and ii) contacting the at least one polypeptide (e.g. a polypeptide according to any one of items 11-12 and / or a mutant variant according to item 13), with one or more or all components of: an additive for feed and / or food, an additive for feed composition and / or food composition, or an additive pharmaceutical composition.

[0081]

[0069] Item 29: Method for manufacturing one or more of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; the method comprising the steps of

[0082] (i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-22; preferably SEQ ID NO: 1 ; and

[0083] (ii) contacting the at least one polypeptide; and / or an additive according to any one of items 14-21 , with one or more or all components of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

[0084]

[0070] Item 30: Method for manufacturing one or more of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; the method comprising the steps of

[0085] (i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 23-26; preferably SEQ ID NO: 23; and

[0086] (ii) contacting the at least one polypeptide; and / or an additive according to any one of items 14-21 , with one or more or all components of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

[0071] Item 31 : Method for manufacturing one or more of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; the method comprising the steps of

[0087] (i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 27-31 ; preferably SEQ ID NO: 27; and

[0088] (ii) contacting the at least one polypeptide; and / or an additive according to any one of items 14-21 , with one or more or all components of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

[0089]

[0072] Item 32: Method for manufacturing one or more of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; the method comprising the steps of

[0090] (i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 32-33; preferably selected from SEQ ID NO: 32; and

[0091] (ii) contacting the at least one polypeptide; and / or an additive according to any one of items 14-21 , with one or more or all components of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

[0092]

[0073] Item 33: Method for manufacturing one or more of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; the method comprising the steps of

[0093] (i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 34-35; preferably SEQ ID NO: 34; and

[0094] (ii) contacting the at least one polypeptide; and / or an additive according to any one of items 14-21 , with one or more or all components of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

[0095]

[0074] Item 34: Method for manufacturing one or more of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; the method comprising the steps of

[0096] (i) providing at least one polypeptide comprising and / or consisting of an amino acid sequence having at least 70% sequence identity to an amino acid sequence of SEQ ID NO: 36; and

[0097] (ii) contacting the at least one polypeptide; and / or an additive according to any one of items 14-21 , with one or more or all components of: feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

[0098]

[0075] Item 35: Use of at least one polypeptide in a) modifying a trichothecene; b) manufacturing an additive for feed and / or food, an additive for feed composition and / or for food composition, or an additive for pharmaceutical composition; and / or c) manufacturing of feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; WDG, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; wherein the at least one polypeptide comprises and / or consists of an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, SEQ ID NOs: 1-22, SEQ ID NOs: 23-26, SEQ ID NOs: 27-31 , SEQ ID NOs: 32-33, SEQ ID NOs: 34-35, and SEQ ID NO: 36; preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

[0099]

[0076] Item 36: Polynucleotide, in particular isolated polynucleotide, encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and / or a polypeptide according to any one of items 11-12; and / or a mutant variant according to item 13.

[0100]

[0077] Item 37: Recombinant host cell comprising at least one polypeptide and / or at least one polynucleotide encoding the at least one polypeptide, wherein the at least one polypeptide comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, SEQ ID NOs: 1-22, SEQ ID NOs: 23-26, SEQ ID NOs: 27-31 , SEQ ID NOs: 32-33, SEQ ID NOs: 34-35, and SEQ ID NO: 36; preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

[0101]

[0078] Item 38: The recombinant host cell of item 37, wherein the recombinant host cell is selected from Escherichia coir, Bacillus sp. such as Bacillus subtilis or Bacillus amyloliquefaciens; Saccharomyces sp. such as Saccharomyces cerevisiae; Pichia sp. such as Pichia pastoris or Pichia stipitis; Kluyveromyces sp. such as Kluyveromyces I act is; Schizosaccharomyces sp. such as Schizosaccharomyces pom be; Hansenula sp. such as Hansenula polymorpha; Streptomyces sp.; Yarrowia sp.; Trichoderma sp.; Lactobacillus sp.; Aspergillus sp.; plant cells; and / or spores of Bacillus sp., Trichoderma sp. or Aspergillus sp.

[0102]

[0079] Item 39: Polypeptide for use in treatment, amelioration and / or prophylaxis of symptoms caused by mycotoxicosis, in particular by trichothecene mycotoxicosis, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, SEQ ID NOs: 1-22, SEQ ID NOs: 23-26, SEQ ID NOs: 27-31 , SEQ ID NOs: 32-33, SEQ ID NOs: 34-35, and SEQ ID NO: 36; preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

[0103]

[0080] In the following, the present invention is further described by non-limiting examples that are not to be construed as limiting embodiments of the present invention, which is solely defined by the claims.

[0104] Examples

[0105]

[0081] The present invention as disclosed herein is not limited to specific embodiments, figures, methodology, examples, protocols etc. described herein but solely defined by the claims.

[0106] Example 1

[0107]

[0082] Upon screening hundreds of glutathione transferase enzymes (also referred to as glutathione-S-transferase, GST), 36 polypeptides were identified to be capable of modifying deoxynivalenol (DON) by conjugation with reduced glutathione (GSH) to form the metabolite DON-13-GSH, wherein GSH is covalently bonded to the C13 atom of DON. These polypeptides comprise each one of the amino acid sequence of SEQ ID NOs: 1-36, i.e. a first polypeptide comprises the amino acid sequence of SEQ ID NO: 1 , a second polypeptide comprises the amino acid sequence of SEQ ID NO: 2, etc. For the purpose of sequence analysis, the amino acid sequences of SEQ ID NOs: 1-36 as well as additional GST sequences that were previously described (SEQ ID NOs: 37-45) were aligned to one another using the Clustal Omega alignment tool of EMBL-EBI (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ; Sievers et al. 2011. Mol. Syst. Biol. 7: 539) with default settings. The amino acid sequences of SEQ ID NOs: 1-45 and the identities of these amino acid sequences to one another are shown in Tables 1 and 2, respectively.

[0108] Table 1. Amino acid sequences of SEQ ID NOs: 1-45.

[0109] Table 2: Sequence identity matrix of SEQ ID NOs 1-45.

[0110] Example 2

[0111]

[0083] For protein production and purification, nucleotide sequences encoding the polypeptides comprising any of SEQ ID NOs: 1-45 were cloned into plasmids for recombinant production in a suitable host organism, such as Escherichia coli. The genes were ordered as synthetic genes and cloned into pET-based E. coli expression vectors. To allow convenient later purification, genes were cloned into pET-28a(+) with N-terminal 6xHis tag and thrombin cleavage site (Ndel / Xhol cloning). To verify independence of the enzymatic activity from tags fused to other polypeptides, GST domains (as shown in SEQ IDN NOs: 1-45) were also produced without any tag(s); with a C-terminal 6xHis tag; with N-terminal 6xHis tag plus thrombin cleavage site in addition to a C- terminal 6xHis tag; as well as N-terminal 6xHis tag plus a maltose binding protein (MBP) solubility tag plus a thrombin cleavage site, all N-terminally fused to the GST domain. Ultimately, enzymatic activities of untagged enzymes as compared to enzymes fused with any of the above listed exemplary tags were essentially the same.

[0112]

[0084] An E. coli BL21(DE3) strain was transformed with the recombinant expression vectors. Recombinant gene expression was induced using OvernightExpress Instant TB medium (Novagen) for induction of IPTG-inducible promoters, comprising 100 mg / L kanamycin. However, any media suitable for growing E. coli and inducing IPTG-inducible promoters can be used.

[0113]

[0085] The cells were harvested by centrifugation and then lysed by ultrasound treatment. Alternatively, any other suitable method to open bacterial cells, in particular E. coli cells, and to produce crude cell lysates may be applied. The lysates were cleared by centrifugation and the supernatant comprising the soluble recombinant protein was used to determine the catalytic properties of the produced polypeptides.

[0114]

[0086] Purification of active, His-tagged enzymes was performed using a suitable affinity chromatography column for purifying His-tagged proteins (Protino Ni-IDA 2000 columns, Macherey-Nagel). Elution from the affinity chromatography column was achieved with buffer comprising imidazol. Thereafter, buffer exchange from the imidazol-containing elution buffer to 100 mM sodium phosphate buffer pH 6.5 was done with centrifugal filter units having a 10 kDa cutoff (Amicon Ultra-4). Protein quantification of thus purified enzymes was done with Qubit Protein assay kit (Invitrogen #Q33212) with Qubit 4 fluorimeter and LabChip GXII HT Touch.

[0115] Example 3

[0116]

[0087] To test for the ability of the polypeptides comprising either one of the GST domains having an amino acid sequence of SEQ ID NOs: 1-45, which were produced as described above, to conjugate DON with reduced GSH, the polypeptides were tested in an activity assay as described below.

[0117]

[0088] The specific activities of the polypeptides were typically tested under the following conditions: 100 mM sodium phosphate buffer, pH 6.5, comprising 0.1 mg / mL bovine serum albumin (BSA), 20 ppm (=68 pM) DON, 1.7 mM GSH, and diluted polypeptide, at 25 or 37 °C.

[0118]

[0089] The reference method was used to perform assays with variable (co-)substrate concentrations of 2-2000 ppm DON and 67.5 pM-30 mM GSH, to assess pH- and temperature dependent stability and activity and adapted accordingly. As buffer, Mcllvaine buffer was used (Mcllvaine. 1921. Journal of Biological Chemistry 49(1): 183-186) to assess pH dependent stability or activity between pH 3 and 8, and reaction temperatures between 25 and 60 °C to assess the temperature dependent activity. All assays with diluted proteins were performed in Eppendorf Protein LoBind tubes or plates.

[0090] GSH stock solutions in 100 mM sodium phosphate buffer, pH 6.5, were either prepared fresh or stored in aliquots at -20 °C to minimize oxidation. The pH of glutathione solutions in buffer was checked and re-adjusted after GSH dissolution. Typically, a master mix containing DON and GSH was prepared shortly before starting the reaction to minimize background. Reactions typically proceeded for 2 hours. In case of less active enzymes, the assay was prolonged to 24 hours. Samples were taken in regular intervals (0, 10, 20, 30, 40, 50, 60, 120 min after reaction start) to monitor reaction progress. The reaction was stopped by addition of the double volume of methanol to the aliquots. Samples were then centrifuged for 10 min at full speed (21 ,000 xg for tubes, or 3,200 xg for plates, at 4 °C). The supernatant was diluted with distilled water to fall within the calibration range of the LC-MS / MS analysis. These samples were subsequently analyzed using the LC-MS / MS method described below.

[0119]

[0091] Specific activities were calculated based on product formation within the linear range at the beginning of the reaction. When tagged enzymes were tested, specific activities were calculated per mg of the GST domain. Activity data is summarized in Table 3 below. Notably, the specific activity of the enzymes of SEQ ID NOs 37-45 as described in literature was found considerably lower than of the enzymes of SEQ ID NOs 1-36. Also, GST enzymes described in literature were not specific for the production of DON-13-GSH, but also formed the Michael adduct DON-10-GSH.

[0120] Table 3: Specific activities of polypeptides of SEQ ID NOs: 1-45 shown in units (II) per mg; normalized to the mass of the GST domain; 1 II is defined as the amount of enzyme acitivty required to modify 1 pmol of substrate within 1 min. Activity data was recorded with 20 ppm DON, 1.7 mM GSH, 0.1 mg / mL BSA, 100 mM sodium phosphate, pH 6.5, 37 °C.

[0121] Example 4

[0122]

[0092] For LC-MS / MS analyses, DON and its enzymatically modified derivatives were separated on an HPLC Agilent 1290 series device with a 150 mm x 2.1 mm Phenomenex Kinetex Biphenyl column with a particle size of 2.6 pm at 40 °C column temperature. The mobile phase consisted of a mixture of solvent A (ultrapure water with 1% acetic acid v / v) and solvent B (methanol with 1 % acetic acid v / v), the flow rate was 0.5 mL / min. The following gradient was 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 electro spray ionization (ESI) in negative ionization mode. The quantification was done using a SCI EX QTrap 5500 or QTRAP 6500+ mass spectrometer from Sciex or similar devices. Samples were diluted with water to fall into the linear range of this method which ranges from analyte concentrations of 1 ppb to 500 ppb DON or 1 ppb to 1800 ppb DON-13-GSH in the injected sample. The injection volume was 5 pL. The LOQ was 1 ppb; lower values were set to <LOQ.

[0123] Table 4: m / z transitions for DON and DON-13-GSH

[0124] Example 5

[0125]

[0093] Polypeptides comprising an amino acid of SEQ ID NOs: 1-36 were further tested for capability of modifying other trichothecenes than 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 diacetoxyscirpenol (DAS, CAS: 2270-40-8). To this end, reactions were performed at 25 °C in 100 mM sodium phosphate buffer, pH 6.5, comprising 0.1 mg / mL BSA, with 1.7 mM GSH, 5 ppm trichothecene (3-ADON, 15-ADON, NIV, T-2; HT-2, or DAS) and enzyme concentrations of 20-300 nM. Enzyme concentrations were chosen based on the already recorded activity with DON; concentrations were chosen to get a nearly complete conversion of DON within 30-120 min. Trichothecene mixtures containing 5 ppm of each trichothecene were used to minimize the number of samples. 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 relative comparisons of the activities. Sampling was done at 0, 10, 20, 30, 60 min, 2, 4.5 and 24 h after start of the reaction. Thereafter, 20 pL aliquots was mixed with 40 pL methanol to stop the reactions. Samples were centrifuged for 10 min at 3,200 xg, 4 °C and then diluted 1 :10 with distilled water prior to the measurements with QTOF in positive and negative mode as described in Example 6 below.

[0126]

[0094] Reaction rates with either of the non-DON trichothecenes were compared with the reaction rates with DON. Certain polypeptides were selected as exemplary polypeptides. All these polypeptides quickly conjugated 15-ADON and DAS with GSH, faster than DON. Polypeptides of either 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 comparably fast with 3-ADON as with DON. Polypeptides of either SEQ ID NO: 29 or SEQ ID NO: 7 reacted more slowly with 3-ADON than with DON. Conversion of NIV was approximately as fast as with DON. Polypeptides of either 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. Polypeptides of either SEQ ID NO: 36, SEQ ID NO: 2 or SEQ ID NO: 4 showed similar reaction rates with both, T-2 and DON. Polypeptide of SEQ ID NO: 29 reacted faster with HT-2 toxin than with DON. Polypeptides of either SEQ ID NO: 1 , SEQ ID NO: 7 or SEQ ID NO: 23 showed comparable reaction rates with DON and HT-2 toxin. Polypeptides of either 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. Results are summarized in Table 5. Taken together, it was concluded that enzymes containing an amino acid sequence of either one of SEQ ID NOs: 1-36 have a broad substrate range and can be used for detoxification of many different trichothecenes.

[0127] Table 5: Enzymatic modification of trichothecenes. Data shows the time in h until conversion of at lesat 95% of the initial substate concentration (peak areas).

[0128] Example 6

[0095] For analysis of non-DON trichothecenes and their modified derivatives, samples from Example 5 were analyzed with a high-resolution mass spectrometry (HRMS) system consisting of an Agilent 1290 Infinity II liquid chromatography system together with a SCIEX X500R QTOF mass spectrometer. An Agilent Zorbax RRHD Eclipse plus C18 50x2.1 mm 1.8pm column was used at a flow rate of 0.5 mL / min at 40 °C column temperature. Solvent A was water with 0.1 % formic acid, solvent B was methanol with 0.1 % formic acid and following gradient was used: 0-1 min = 0% B; 1-6 min = linear gradient from 0 to 100% B; 6-8 min = 100% B; 8-8.1 min = linear gradient from 100 to 0% B. Mass spectrometric detection was done in positive and negative mode with a mass scan from 200-1200 Da. 3-ADON, 15-ADON, NIV, DAS, T-2, and HT-2 were transformed by the polypeptides 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 the glutathione conjugates were available (except for DON-13-GSH). Thus, the GSH conjugates were identified from the QTOF spectra by their expected masses.

[0129] Example 7

[0130]

[0096] To determine the toxicity of DON-glutathione-related conjugates, standards of DON-13- GSH and DON-13-Cys were prepared. DON-13-Cys has been described to be a major breakdown product of DON-13-GSH in vivo (Stanic et al. 2016. J. Agric. Food Chem. 64(23): 4777-4785). Forthat purpose, DON and the respective thiol (reduced L-glutathione or L-cysteine) were allowed to react in carbonate buffer, pH 10.7, at 37 °C 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.

[0131]

[0097] The toxicity of the thiol conjugates was assessed by 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). The luciferase T7 control DNA (Promega #L4821) was used as reporter gene to quantify ribosome inhibition. The amount of produced luciferase was then assessed with a Luciferase Assay System (Promega E1500). The assay was performed in chemiluminescence 96-well plates in a microplate reader with dispense module. Measurements were done in duplicates: 2 pL in-vitro translation reaction (stored at -20 °C) was pipetted into 96-well plates, 100 pL luciferase assay reagent was added by the dispenser, mixed for 10 s, followed by a 2 s delay and 10 s measurement.

[0132]

[0098] Luminescence reads of reactions without toxin were used as reference and defined as 100% translation efficiency. Binding of inhibitors (such as DON) to the ribosomes lead to lower levels of produced luciferase and thus to lower chemiluminescence. Results are shown in Table 6 below. DON showed inhibition of translation at all concentrations, which was the expected behavior since DON is known for its inhibiting action on eukaryotic ribosomes. On the other hand, DON-13-GSH and DON-13-Cys did not show significant inhibition of luciferase production. It was thus concluded that both, DON-13-GSH and DON-13-Cys have much lower binding affinity to ribosomes than DON. Therefore, modification of DON to DON-13-GSH by the polypeptides according to the invention is an efficient means to detoxify DON.

[0133] Table 6: Ribosomal inhibition by DON, DON-13-GSH, or DON-13-Cys.

[0134] Example 8

[0135]

[0099] To verify activity of the polypeptides comprising any one of SEQ ID NOs: 1-36 in vivo, an E. coli strain producing the polypeptide of SEQ ID NO: 7 was cultivated in a DASGIP parallel bioreactor system, at 37 °C, pH 7.0, applying level control with antifoam reagent (10% PPG 2000), and with dissolved oxygen monitoring. The bioreactors were prepared according to common procedures for the cultivation of E. coli BL21 (DE3) strains, followed by inoculation of the batch medium (6.4 g / L potassium dihydrogen phosphate, 2.1 g / L phosphoric acid, 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 AICI3*6H2O, 7,3 g / L CoCI2*6H2O, 2 g / L ZnSO4*7H2O, 1 g / L CuCI2*2H2O, 2 g / L Na2MoO4*2H2O, 0,5 g / L H3BO3), 3.06 g / L ammonium sulfate, 39.6 g / L glucose) with an overnight culture of the strain at QD600=2.0. For 450 g feed medium per reactor, the following components were mixed: 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 hours batch phase, the reactors were run at 37 °C, with a feed rate of 12.3 mL of feed medium per h and per 500 mL batch volume for 15 hours. Recombinant gene expression was induced after 27 hours by addition of 15 pmol IPTG per g 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 end of fermentation (after 17 hours). Thereafter, the cultivation broth was subjected to centrifugation to harvest the cell mass. Cells were lysed according to the method described in example 2. Recombinant protein was purified with affinity chromatography from the resulting cell pellet analogous to the purification described in Example 2. Elution fractions containing the enzyme were pooled, the buffer was changed to 100 mM sodium phosphate buffer, pH 6.5, on a crossflow filtration system. 2.8 g of recombinant enzyme in 111 mL 100 mM sodium phosphate buffer, pH 6.5, was diluted with distilled water to 1 L, further containing 2 mM GSH and 100 g / L maltodextrin. The entire mixture was freeze-dried, and the lyophilized product was further used for feeding trials.

[0136] Example 9

[0137]

[0100] To assess the functionality of the enzymes in piglets, standard piglet feed (886 g / kg dry matter, 204 g / kg crude protein, 40 g / kg crude fiber, 663 g / kg nitrogen-free extractives, 515 g / kg starch, 35 g / kg crude fat, 59 g / kg crude ash, 15,26 MJ / kg metabolizable energy) was used as base diet. To the base diet, different components were added according to the respective test group (Table 7). The base diet already contained low DON levels of approximately 400 ppb. The background control group (A) received the base diet, the DON control group (B) received the base diet supplemented with 3 ppm DON, test group 1 (C) received the base diet supplemented with DON and enzyme formulated with GSH as described in Example 8; and test group 2 (D) received the base 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 the base diet supplemented with 8 ppm DON-13-GSH. DON-13-GSH was used at higher concentration than DON to have equimolar concentrations of DON and DON-13-GSH.

[0138] Table 7: Groups and experimental diets.

[0139]

[0101] Each trial group encompassed 4 piglets, in total 16 animals (8 females, 8 males; Austrian genotype 0-HYB-F1 [(Landrace x Large White) x Pietrain]). During the entire trial, the animals were housed in pairs in 8 metabolic pens (two pens per group) to allow for pen-based urine collection. The animals selected for the trial weighed around 8 kg and were 27-29 days old at weaning.

[0140]

[0102] The trial included a ten-day adaptation period starting with day 0 upon arrival of the animals at the facility. After the adaptation period the animals received experimental diets under a restricted feeding regime (Table 8) for three consecutive days from day 11-13, followed by two days (days 14-15), where they again received piglet base diet. During the trial period, animals were restrictively fed in the morning and afternoon to be able to record the feed intake. Urine samples were collected per pen in the morning and in the evening prior to feeding on days 11-15. The amount of urine in mL was recorded for each pen and representative samples were collected. Blood for later dried blood spot analyses was collected 2 hours after the morning meal on days 11-14 from single animals by vein puncturing.

[0141] Table 8: Trial set up and duration.

[0142]

[0103] All samples were analyzed by LC-MS for presence of DON in urine samples to determine whether a reduction of DON compared to the background control group was achieved in vivo. Results are shown in Table 9.

[0143] Table 9: DON concentrations pg / mmol in urine samples in creatinine.

[0144]

[0104] DON was detected in blood and urine samples of all groups (A-E), due to DON being ubiquitously present in feed stuff at approximately 400 ppb. In the blood samples of group A animals, 5-8 ng / mL of DON were detected, whereas the level was higher (10-16 ng / mL) in the blood samples of Group B. DON levels in the blood samples of group C animals were similar to group B, whereas DON in blood of group D animals was 1-3 ng / mL on average. Animals from group E had DON levels in their blood similar to animals from group A (5-8 ng / mL), see Table 10.

[0145] Table 10: DON concentrations in ng / mL in blood samples taken from individual animals. Data shows the average of the individuals per group and time point ± standard deviation.

[0146]

[0105] DON is known to be mainly excreted via urine. In urine samples of group A animals, DON concentration increased from 40-70 pg / mmol creatinine, whereas in group B animals, an increase to approximately 150 pg / mmol creatinine was observed. The increase in DON concentration correlated with the feeding of the experimental diet. DON concentrations in urine samples in group C animals were 50-150 pg / mmol creatinine. DON levels in urine samples of group D animals were initially at 9-32 pg / mmol creatinine and then increased to 57 pg / mmol creatinine after supplementation with enzyme and GSH was stopped on day 14. DON levels in group E animals were comparable to DON levels observed in group A animals, indicating that DON-13-GSH was not metabolized to DON by the animal. Consequently, opening of the DON epoxide ring upon formation of DON-13-GSH is irreversible. In addition, DON-13-GSH and its derivative DON-13- Cys (as described by Stanic et al. 2016. J. Agric. Food Chem. 64(23): 4777-4785) were also detected in urine samples in group D and E. Thus, polypeptide preparations as described herein, and as produced e.g. as described in Example 8 are functional and applicable in vivo.

[0147] Example 10

[0148]

[0106] In the following, methods for analyses of the urine samples from the in vivo trial described above are shown. Creatinine content of the urine samples was determined for normalization of the trichothecene results to a certain creatinine value, thus accounting for differences in sample volume. Prior to analysis, 10 pL of urine was diluted with 990 pL of ultrapure water tube, the mixture was mixed thoroughly and 10 pL was then transferred to an HPLC vial containing 990 pL of ultrapure water. The vial was mixed again prior to analysis.

[0149]

[0107] A 1290 Infinity II HPLC system from Agilent Technologies coupled to a TripleQuad 5500 mass spectrometer from Sciex equipped with an electrospray source was used 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 reversed phase column Gemini 5 pm C18 110 A, 150 x 4.6 mm from Phenomenex by increasing mobile phase B from initially 0% (0-1.60 min) to 100% (1.65 min). This proportion of eluent B was maintained until 2.00 min and then changed again to the starting conditions of 0% B (2.05 min). The total run time of the method was 3 minutes. The measured m / z transitions are described in Table 11. Each transition was measured for 30 ms. The injection volume was 2 pL and the column temperature was 30 °C. Table 11 : measured m / z transitions for creatinine.

[0150]

[0108] The creatinine content was determined based on a 1 / x weighted linear calibration function generated from a standard curve prepared in neat solvent. For the further process of determination of DON and DON-13-GSH, all urine sample were diluted individually to a creatinine concentration of 2 mM with ultrapure water.

[0151]

[0109] Prior to analysis, 20 pL of creatinine-adjusted urine samples was transferred to a reaction tube containing 180 pL methanol / water (20 / 80, v / v). Samples were vortexed, placed at -20 °C for 30 min, centrifuged at 19,000 xg for 10 min and supernatants were transferred to HPLC vials with inserts and closed with snap caps. LC-MS / MS analysis was performed using a 1290 Infinity II HPLC system from Agilent Technologies coupled to a QTRAP 5500 mass spectrometer from Sciex equipped with an electrospray source. The system was operated in negative ionization mode.

[0152]

[0110] For quantification of DON, mobile phase A consisted of acetonitrile / water (5 / 95, v / v); mobile phase B was 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 reversed phase column Kinetex C18 150x2.1 mm, 2.6 pm from Phenomenex by increasing mobile phase B from initially 2% (0-0.1 min) to 25% (2.2 min) and finally 100% (2.3 min). This proportion of eluent B was maintained until 2.8 min, then decreased to the starting conditions of 2% B (2.85 min). The total run time of the 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 pL and the column temperature was 30 °C. The concentrations were determined based on a 1 / x weighted linear calibration function generated from a standard curve prepared in neat solvent in combination with 13C-labelled internal standard correction.

[0153]

[0111] For quantification of DON-13-GSH mobile phase A consisted of water / acetic acid (99 / 1 , v / v); mobile phase B was methanol / acetic acid (99 / 1 , v / v). Chromatographic separation was achieved on a reversed phase column Kinetex Biphenyl 150x2.1 mm, 2.6 pm from Phenomenex by increasing mobile phase B from initially 0% (0-0.30 min) to 70% (2.30 min) and finally 100% (2.31 min). This proportion of eluent B was maintained until 2.60 min, and then decreased to the starting conditions of 0% B (2.80 min). The total run time of the method was 4 minutes. The measured m / z transitions are described in Table 12. Each transition was measured for 10 ms. The injection volume was 5 pL and the column temperature was 40 °C. The concentrations were determined based on a 1 / x weighted linear calibration function generated from a standard curve prepared in neat solvent.

[0154] Table 12: measured m / z transitions for D0N-13-GSH.

[0155] Example 11

[0156]

[0112] In order to identify in vivo metabolites in urine samples, the following QTOF analytical method was applied. Urine samples were diluted to 7 mM creatinine with 20% methanol and analyzed with a High-resolution mass spectrometry (HRMS) system consisting of an Agilent 1290 Infinity II liquid chromatography system together with a SCIEX X500R QTOF mass spectrometer. Samples were measured in negative mode according to the method described in example 6.

[0157] Example 12

[0158]

[0113] To determine the concentration of DON and DON glucuronide in whole blood as obtained from the feeding trial of Example 9, 50 pL whole blood was placed on a filter card and dried at room temperature. The whole spot was cut and placed in a reaction tube. Afterwards, 10 pL of 100 pbb of isotopically labelled 13C DON internal standard and 800 pL extraction solvent (H2O / ACN 30 / 70 v / v) was added and mixed for 60 min on an end-over-end shaker. Then 2 aliquots (300 pL each) were dried under reduced pressure. One aliquot was reconstituted in 37.5 pL H2O / ACN (95 / 5 v / v) for 10 min and centrifuged for 10 min at 19,000 xg. The supernatant was transferred in a silanized HPLC vial and measured. The second aliquot was reconstituted in 150 pL 1x PBS for 10 min. Thereafter, 150 pL p-glucuronidase-solution was added resulting in a final enzyme concentration of approximately 3000 U / mL. The samples were incubated over night at 37 °C and extracted with 300 pL ethyl acetate for 5 min. After extraction the samples were centrifuged for 5 min at 19,000 xg; the supernatant was collected in a fresh reaction tube. Extraction with ethyl acetate was done in total three times and the supernatants were combined and dried under reduced pressure. Finally, the samples were reconstituted in 37.5 pL H20 / ACN (95 / 5 v / v) for 10 min and centrifuged for 10 min at 19,000 xg. The supernatant was transferred in a silanized HPLC vial and measured using LC-MS / MS:

[0159]

[0114] For quantification of DON and DON-13-GSH, LC-MS / MS analysis was performed using a 1290 Infinity II HPLC system from Agilent Technologies coupled to a QTRAP 6500+ mass spectrometer from Sciex equipped with an electrospray source. The LC-MS / MS was operated in negative ionization mode. Mobile phase A consisted of acetonitrile / water (5 / 95 v / v); mobile phase B was acetonitrile / water (95 / 5 v / v). Acetic acid was added to both eluents to a concentration of 0.1 %. Separation was achieved on a reversed phase column Kinetex C18 150x2.1 mm, 2.6 pm from Phenomenex by increasing mobile phase B from initially 2% (0.1 min) to 25 % (2.2 min), and then to 100% (2.3 min). This proportion of eluent B was maintained until 2.8 min, and then decreased to the starting condition of 2% (2.85 min). The total run time of the 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 pL and the column temperature was 30 °C.

[0160] Table 13: m / z transitions for DON and DON-13-GSH.

[0161]

[0115] Quantification was either based on standards in neat solvent in combination with ISC- labelled internal standard correction for DON, or based on spiked blood samples as processed standards for DON-13-GSH. For processed standards, blank blood was spiked with defined concentrations of DON-13-GSH, dried and extracted as described above.

[0162] Example 13

[0163]

[0116] In a further feeding trial, 48 piglets (24 females, 24 males; Austrian genotype O-HYB-F1 [(Landrace x Large White) x Pietrain]) were allocated to respective groups and pens. During the entire trial, the animals were housed in pairs in metabolic pens. One experimental group consisted of four animals or two pens. Animals selected for the trial were 27-29 days old at weaning. The adaptation period started on day 0 upon arrival of the animals at the facility. During the first days, the piglets received NuristartSweet, and NuristartSweet mixed with standard piglet feed. DON background level of the feed was 180 ppb. During the experimental period, the piglets received the standard piglet feed as base diet, and the experimental diets for three consecutive days per week following a restrictive feeding pattern with a distinct amount of feed per piglet in the morning and in the evening. Feed intake was recorded and left-over feeds were removed. Experimental diet was artificially contaminated with DON culture material to mimic a 4 ppm DON contamination. Between weeks 1 and 2, the animals received standard diet for several days (wash out period). Blood samples were collected from individual animals within 1-2 hours after the morning meal. The amount of excreted urine was recorded, and aliquots were collected per pen prior to feeding in the morning and in the evening, including the “wash out” period. As exemplary enzymes capable of modifying trichothecenes, polypeptides having either SEQ ID NO: 7 or 8 were used for this trial.

[0164]

[0117] In week 1 of the trial, GSH was tested at varying concentrations, at constant enzyme concentration of 6 pmol / kg. In week 2 of the trial, different sources of GSH were tested.

[0165]

[0118] Trial schedule and setup are shown in Table 14 and follow the general scheme described above, with the exception that blood was drawn only on two days in the experimental period and on one day in the wash out phase. Consequently, lower DON levels were expected on day 7 in all test groups, as there was no DON fed prior to blood collection.

[0166] Table 14: Feeding trial setup. Feeding and sampling regime.

[0167]

[0119] All experimental diets contained standard piglet feed. DON culture material was added for groups 2-12, 14-19. Where indicated, 6 pmol / kg enzyme (SEQ ID NO: 7 or 8) was added. The feed was supplemented with 0, 1.25, 2.5, 5, 10, or 20 mmol / kg reduced glutathione (GSH) by adding pure GSH from either of two different suppliers. Group 1 served as background control (N), group 2 served as DON control (DON). These groups were run in both trial weeks. The test groups are shown in table 15. Table 15: Diets for test groups.

[0168]

[0120] Blood spots were collected from individual animals (four blood spots per group) and urine samples were collected per pen from two animals (two urine samples per group). The samples were analyzed via LC-MS as described herein. Creatinine content of urine samples was determined to normalize all samples to 2 mM creatinine. Dried blood spots were extracted using a standard method. All samples were measured with LC-MS / MS and internal standards were used to compensate for matrix effects, as described herein.

[0169]

[0121] Results from week 1 showed effective DON removal from blood upon administration of either of the two tested enzymes according to the invention. Under the tested conditions, a GSH concentration of at least 2.5 mmol / kg was found recommendable for DON removal from blood. Even better effects were observed when supplementing 5 mmol or more of GSH per kg. When supplementing 10 or 20 mmol of GSH per kg, comparable DON removal was observed. In detail, at day 4, DON concentration of the DON control group was 15 ng / mL, compared to 1-5 ng / mL in any of groups 3, 4, 8, 9. At day 7, DON concentration of the DON control group was approximately 9 ng / mL, compared to 1-2 ng / mL in any of groups 3, 4, 8, 9; and approximately 5 ng / mL in any of groups 5-7, 10-12. Analyses of the urine samples confirmed these observations. Results from week 2 showed the enzymes according to the invention performing equally well independent from the source of GSH (i.e. from either supplier 1 or 2).

[0170] Example 14

[0122] In another feeding trial, 48 piglets (24 females, 24 males; Austrian genotype 0-HYB-F1 [(Landrace x Large White) x Pietrain]) were allocated to respective groups and pens. During the entire trial, the animals were housed in pairs in metabolic pens and one experimental group consisted of four animals or two pens. Animals selected for the trial were 27-29 days old at weaning. The adaptation period started on day 0 upon arrival of the animals at the facility. During the first days, the piglets received Nuri startSweet and Nuri startSweet mixed with standard piglet feed. DON background level of the feed was 78 ppb. During the experimental period the piglets received the standard piglet feed as base diet, and the experimental diets for three consecutive days per week following a restrictive feeding pattern with a distinct amount of feed per piglet in the morning and in the evening. Feed intake was recorded and left-over feeds were removed. Experimental diet was artificially contaminated with DON culture material to mimic a 4 ppm DON contamination. Between weeks 1 and 2, the animals received standard diet for several days (wash out period). Blood samples were collected from individual animals within 1-2 hours after the morning meal. The amount of excreted urine was recorded, and aliquots were collected per pen prior to feeding in the morning and in the evening, including the “wash out” period. As exemplary enzymes capable of modifying trichothecenes, polypeptides having either SEQ ID NO: 7, 8 or 35 were used for this trial.

[0171]

[0123] In week 1 of the trial, the polypeptide having SEQ ID NO: 7 was used at 6 - 0.1 pmol / kg, the polypeptide having SEQ ID NO: 8 was used at 6 - 1 pmol / kg. In week 2, the polypeptide having SEQ ID NO: 8 was used at 6, 0.5, and 0.1 pmol / kg, the polypeptide having SEQ ID NO: 35 was used at 0.1-6 pmol / kg. GSH from supplier 2 was used in this trial. The trial schedule and set up are depicted in Table 16 .

[0172] Table 16: Feeding trial setup. Feeding and sampling regime.

[0173]

[0124] All experimental diets contained standard piglet feed. DON culture material was added for all groups except for the background control group. Where indicated, enzyme (SEQ ID NO: 7, 8 or 35) was added. The feed was supplemented with 10 mmol / kg GSH, and either 0.1 , 0.5, 1 , 3, or 6 pmol / kg enzyme. Group 1 served as background control (N), group 2 as DON control (DON).

[0174] These groups were run in both trial weeks. The test groups are shown in table 17.

[0175] Table 17: Diets for test groups.

[0176]

[0125] Again, four blood spot samples and two urine samples were collected per group. The samples were analyzed as described above. Addition of any of the enzymes of the invention effected modification, in particular removal of DON in blood and urine. Under the tested conditions, DON reduction was observed at enzyme concentrations higher than 0.1 pmol / kg. Increasing the enzyme concentration from 0.5 pmol / kg to 1 pmol / kg, to 3 pmol / kg or even to 6 pmol / kg achieved increasing DON removal, as observed in reduced DON concentrations in urine and blood samples depending on the administered enzyme concentration. Ultimately, all tested enzymes confirmed in vivo applicability at varying concentrations.

Claims

Claims1 . Method for modifying a trichothecene, the method comprising the steps of a) forming a reaction mixture by contacting the 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 SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and(ii) glutathione; and b) incubating the reaction mixture.

2. The method of claim 1 , wherein step b) is performed 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 for 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 diacetoxyscirpenol; preferably deoxynivalenol.

5. Mutant variant of a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; wherein the mutant variant comprises at least one amino acid mutation, substitution, deletion, and / or insertion.

6. Additive for modifying a trichothecene, comprising (i) at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 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 comprised in the additive at a concentration suitable for achieving an inclusion rate of at least 0.1 pmol per kg (e.g. at least 0.1 , 0.2, 0.3, 0.4, 0.5, 1 , 5, 10, 50, 100 pmol per kg).

8. The additive of claim 6 or 7, wherein the additive further comprises one or more component(s) selected from dietary supplement(s); nutraceutical(s); pharmaceutical(s); vitamin(s); mineral(s); amino acid(s); essential fatty acid(s); fiber(s); trace element(s); antioxidant(s); plant extract(s); herbal extract(s); essential oil(s); carrier(s), preferably selected from bentonite, silica, and carbohydrates.

9. Method for manufacturing an additive for feed and / or food, for feed composition and / or food composition, or for pharmaceutical composition, the method comprising the steps of a) providing at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and b) contacting the at least one polypeptide with one or more components of the additive for feed, for food, for feed composition, for food composition, and / or for pharmaceutical composition.

10. Method for manufacturing of feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol, DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; the method comprising the steps of a) providing at least one polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and b) contacting the at least one polypeptide; and / or an 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, preferably from sugarcane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage.

11. Use of at least one polypeptide in a) modifying a trichothecene; b) manufacturing an additive for feed and / or food, for feed composition and / or food composition, or for pharmaceutical composition; and / or c) manufacturing of feed; food; feed composition; food composition; pharmaceutical composition; biogas; bioethanol; DDGS; sugar, preferably from sugar cane or sugar beets; corn oil, corn germs, corn germ meal, corn fibers, corn gluten, starch, in particular corn starch; and / or silage; wherein the at least one polypeptide comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

12. Isolated polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36; and / or a mutant variant according to claim 5.

13. 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 to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

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

15. Polypeptide for use in treatment, amelioration and / or prophylaxis of symptoms caused by mycotoxicosis, in particular by trichothecene mycotoxicosis, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1-36, preferably to an amino acid sequence selected from SEQ ID NOs: 1 , 23, 27, 32, 34 and 36.

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