Use of a trichothecene-transforming alcohol dehydrogenase, method for transforming trichothecenes and trichothecene-transforming additive

Description

[0001] The present invention relates to the use of a trichothecene transforming alcohol dehydrogenase, a method for transforming trichothecenes and a trichothecene transforming additive.

[0002] Trichothecenes represent a common group of mycotoxins, including deoxynivalenol (DON, CAS No. 51481-10-8), T-2 toxin (CAS No. 21259-20-1), HT-2 toxin (CAS No. 26934-87-2), nivalenol (CAS No. 23282-20-4), fuseranone X (CAS No. 23255-69-8), scribentriol, 15-acetoxyscirpenol (CAS No. 2623-22-5), 4,15-diacetoxyscirpenol (CAS No. 2270-40-8), trichodermol (CAS No. 2198-93-8), verrucarin A (CAS No. 3148-09-2), Verrucarin J (CAS No. 4643-58-7), Isotrichodermin (CAS No. 91423-90-4), Hydroxyisotrichodermin (CAS No. 344781-02-8), Calonectrin (CAS No. 38818-51-8), T-2 Tetraol (CAS No. 34114-99-3), Deacetylneosolaniol (CAS No. 74833-39-9), Neosolaniol (CAS No. 36519-25-2), Acetylneosolaniol (CAS No. 65041-92-1), Sporotrichiol (CAS No. 101401-89-2), Trichotriol (CAS No. 109890-37-1), Sambucinol (CAS No. 90044-33-0) and Culmorin (CAS No. 18374-83-9).Trichothecenes, particularly DON, also known as vomitoxin, can be produced by a variety of Fusarium fungi, especially . F. graminearum and F. culmorum, These fungi infect crops such as corn and various grains such as wheat, oats, or barley. Fungal infestation usually occurs before harvest, and fungal growth or mycotoxin formation can occur before or, if stored improperly, after harvest.

[0003] The Food and Agriculture Organization (FAO) estimates that 25% of agricultural products worldwide are contaminated with mycotoxins, resulting in significant economic losses. In a more recent global study by I. Rodrigues and K. Naehrer, Toxins, 2012, 4, 663-675, a total of 23,781 samples were analyzed between January 2009 and December 2011. 81% tested positive for at least one mycotoxin and 59% tested positive for trichothecenes, particularly DON. Trichothecenes, particularly DON, were found in all regions of the world and in all tested grain and feed classes, such as corn, soybean meal, wheat, wheat bran, DDGS (dry distillers' grains), and in ready-to-use feed mixtures, with a frequency of up to 100%.Apart from unprocessed staple foods, trichothecenes have also been detected in processed foods such as flour, breakfast cereals, pasta, bread, pastries, and cereal-based infant and baby foods.

[0004] Trichothecenes have the following structural formula, The different substitution residues R1 to R5 vary depending on the trichothecene type. It is known that, in addition to the epoxy group, an intact α-hydroxy group at the C-3 atom of trichothecenes is also responsible for their toxic effects. Trichothecene types with a hydroxy group at the C-3 atom include deoxynivalenol, T-2 toxin, HT-2 toxin, nivalenol, fuseranone X, 15-acetoxyscirpenol, 4,15-diacetoxyscirpenol, trichodermol, T-2 tetraol, deacetylneosolaniol, acetylneosolaniol, sporotrichiol, trichotriol, sambucinol, and culmorin.

[0005] Deoynivalenol (DON) has a characteristic carbonyl group at the C-8 atom and has the following structural formula and the IUPAC designation (3α,7α)-3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one. Several toxic DON subtypes with a hydroxy group at C-3 also occur naturally. These include acetylated DON (e.g., 15-acyl-DON), glycosylated DON, sulfonated DON (e.g., DONS-1, DONS-2), or DON sulfates (DON-15-sulfate). These DON subtypes are also classified as trichothecene types with a hydroxy group or substituted hydroxy group at C-3.

[0006] Due to the toxic effects of DON, the relevant authorities have defined limit values ​​and guideline values ​​for food and feed. The European Union has regulated the DON content in food (EC No. 1881 / 2006, EC No. 1126 / 2007) and recommended guideline values ​​for feed (2006 / 576 / EC). In the USA, the FDA has published recommended maximum guideline values.

[0007] Diseases caused by the ingestion of mycotoxins in humans or animals are referred to as mycotoxicoses. In the case of trichothecenes or trichothecene types, these are also referred to as trichothecene mycotoxicoses, more specifically as mycotoxicoses caused by trichothecenes containing a hydroxy group at the C-3 atom, or even more specifically as DON mycotoxicoses. The toxic effects of trichothecenes in animals and humans are known to be based on several factors. These include the inhibition of protein biosynthesis, possible interactions with serotonin and dopamine receptors, and the upregulation of pro-inflammatory cytokines (EFSA Journal 2004, 73, 1-41). DON mycotoxicoses also cause changes in biomarkers, such as an increase in IgA concentration in blood, an increase in SOCS3 concentration in the liver, or a reduction in IGFALS levels in plasma (Pestka et al. 2004, Toxicol. Lett.153, 61-73) and the reduction of claudin concentration in the intestine (Pinton et al. 2009, Tox. Appl. Pharmacol. 237, 41-48).

[0008] In pigs, for example, trichothecene mycotoxicoses manifest themselves in reduced feed intake, reduced growth, vomiting and diarrhea, as well as impaired immune function and nutrient absorption in the intestine. In poultry, trichothecene mycotoxicoses cause, among other things, impaired feed intake and weight gain, diarrhea, and reduced eggshell weight. In ruminants, reduced feed intake and reduced milk production have been described. In aquaculture, trichothecene mycotoxicoses in fish (e.g., salmon, catfish, or trout) and shrimp cause, among other things, impaired feed intake and growth rates (Binder et al., Guide to Mycotoxins; ISBN 978-0-9573721-0-8). Toxic effects in dogs and cats have also been described (EFSA Journal 2004, 73, 1-41).In humans, trichothecene mycotoxicoses can cause, among other things, nausea, vomiting, diarrhea, abdominal pain, headache or fever (Sobrova et al, Interdisc. Toxicol. 2010, 3 (3), 94-99).

[0009] The primary strategy for reducing trichothecene or DON contamination of food or feed is to limit fungal infestation, for example, by adhering to "good agricultural practices." This includes, among other things, the use of seeds that are free of pests and fungal infestation or the plowing in of crop residues. Furthermore, the correct use of fungicides can reduce fungal growth in the field. After harvest, the crop should be stored at a residual moisture content of less than 15% and at a low temperature to prevent fungal growth. Likewise, any material contaminated by fungal infestation should be removed before further processing. Despite this list of measures, I. Rodriges and K. Naehrer (2012) reported that even in regions with the highest agricultural standards, such as the USA and Central Europe, 79% and 72% of all corn samples tested were contaminated with DON between 2009 and 2011, respectively.By 2011, 79% and 72% of all corn samples tested were contaminated with DON, respectively. From the document MUKHERJEE PRANAB K ET AL: "Alcohol dehydrogenase restricts the ability of the pathogen Canadida albicans to form a biofilm on catheter surfaces through an ethanol-based mechanism," INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, Vol. 74, No. 7, July 1, 2006 (2006-07-01), it has become known that an alcohol dehydrogenase is capable of enhancing the ability of Canadida albicans to form biofilms.

[0010] US 2006 / 105061 A1 describes a galenic composition for preventing metal-induced liver damage, neoplastic liver disease and tumor induction, comprising Panax pseudoginseng , Eucommiae ulmoides and Polygonati rhizoma.

[0011] WO 2009 / 133461 A1 describes an antifouling composition for inhibiting the formation of a biofilm, comprising an enzyme.

[0012] WO 2014 / 180939 A1 describes a method and means for maintaining and preserving the enzymatic activity of a dehydrogenase.

[0013] Other options for reducing mycotoxin contamination in food or feed include adsorption or transformation. Adsorption requires that the mycotoxin binds to the adsorbent strongly and specifically over a wide pH range and remains stable throughout the entire digestive process in the gastrointestinal tract. Although some non-biological adsorbents, such as activated carbon, silicates, or synthetic polymers such as cholestyramine, can be used efficiently for aflatoxins, their use is not effective for other mycotoxins, particularly trichothecenes. Biological adsorbents, such as yeast or yeast extracts, have also been described in the literature but have similar limitations to non-biological adsorbents. A major disadvantage of adsorbents is their possible non-specific binding of other molecules that may be essential for nutrition.

[0014] The transformation, especially the detoxification of trichothecenes by physical and chemical treatments, is also limited because DON is very stable and remains stable even during temperature treatments of up to 350 °C.

[0015] A possible microbial transformation of DON was described in EP-B 1 042 449, according to which the microorganism BBSH 797 (DSM 11798) is used to detoxify DON. Detoxification is based on the opening of the epoxide ring at C-12 and C-13 of DON. US 2012 / 0263827 A describes the biotransformation of DON to 3-epi-DON by a microorganism with the international Canadian deposit number 040408-1. However, in many feed or food technology processes, the addition of microorganisms or adsorbents is not possible or not permitted by law, so transformation or detoxification of trichothecenes, such as DON or DON subtypes, is not possible.

[0016] Trichothecenes, such as DON and DON subtypes, are rapidly absorbed by the human or animal body in the gastrointestinal tract, which is why rapid and targeted detoxification is important.

[0017] The alcohol dehydrogenase of SEQ ID No. 1 was first described in JP-A 2003 / 159079 for the production of 2-ketogulonic acid. WO 2009 / 133464 describes a process for the oxidation of saccharides using the enzyme of SEQ ID No. 1 in food or feed for the oxidation of starch, particularly in the bakery industry to slow the aging process of bread. The alcohol dehydrogenase is used for the oxidation of hydroxyl groups of carbohydrates.

[0018] The alcohol dehydrogenases with SEQ ID Nos. 2 and 3 were identified during genome sequencing of microorganisms Devosia sp. identified and are deposited online on the server of the National Center for Biotechnology Information (NCBI) under the identification numbers GI:737041022 and GI:630002266. A more detailed characterization of the alcohol dehydrogenases with SEQ ID Nos. 2 and 3 was not carried out in the course of this work.

[0019] Due to the multitude of toxic effects of trichothecenes and their frequency of occurrence, there is a need for substances or groups of substances, such as enzymes, that can be used for the specific, safe and reliable transformation, in particular detoxification of trichothecenes.

[0020] The present invention aims at the use of a specific alcohol dehydrogenase and variants thereof which enable the transformation of at least one trichothecene having a hydroxy group at the C-3 atom into less toxic products.

[0021] To achieve the object, it has surprisingly been found that the use of an alcohol dehydrogenase of SEQ ID No. 1 containing metal ions and a quinone cofactor or a functional variant having a sequence identity of at least 80%, preferably at least 86%, particularly preferably at least 89%, and at least one redox cofactor for the transformation of at least one trichothecene having a hydroxy group at the C-3 atom makes it possible to specifically and reliably transform trichothecenes having a hydroxy group at the C-3 atom, such as DON, T-2 toxin or nivalenol.

[0022] A transformation is understood to mean when the structure of toxins is changed, whereby the toxins are preferentially converted, i.e. transformed, into non-toxic or less toxic metabolites. In the present case, the structural change occurs in particular at the C-3 atom of the trichothecenes containing a hydroxyl group at the C-3 atom, through the catalytic conversion of the C-3 hydroxy group into a keto group. Surprisingly, the use of alcohol dehydrogenase according to the invention succeeds in transforming trichothecenes containing a hydroxyl group at the C-3 atom, in particular DON, in a wide variety of chemical and biological environments, such as in buffers, feed pulp, saliva, gastric juice containing feed, or intestinal contents containing feed.This is unusual, as parameters important for enzymatic activity, such as pH, protease concentration, ionic strength, and substance matrices, vary enormously from environment to environment. This ensures enzyme activity from the moment the food or feed is mixed with water, through its oral ingestion, and even in the oral / gastrointestinal tract. Surprisingly, for certain environments, the external addition of redox cofactors is not necessary; this applies particularly to feed mixtures, saliva, and gastric juice. The alcohol dehydrogenase of SEQ ID No. 1 is a quinone cofactor-dependent alcohol dehydrogenase. To produce an active holoenzyme or alcohol dehydrogenase, a quinone cofactor, preferably pyrroloquinoline quinone (PCC), must be bound to the enzyme in the presence of a metal ion, preferably Ca2+.The activated alcohol dehydrogenase therefore contains both the quinone cofactor and the metal ion, with the molar ratio of enzyme to quinone cofactor being 1:1. Furthermore, a redox cofactor is required for the catalytic activity of the alcohol dehydrogenase. This can be used either in the form of a synthetically produced redox cofactor in addition to the activated alcohol dehydrogenase, or a redox cofactor already present in food or feed, as well as in animal or human secretions. These natural redox cofactors can be formed, for example, from food or feed during food or feed preparation, processing, or digestion in the oral-gastrointestinal tract of humans or animals, and can be extracted therefrom if necessary. Human or animal secretions containing such a natural redox cofactor are, for example,Digestive secretions such as saliva, gastric juice, intestinal juice, pancreatic juice, bile or rumen juice.

[0023] The terms "polypeptide variant" or "variant" refer to functional polypeptides that possess at least one amino acid substitution compared to SEQ ID No. 1, while retaining the enzymatic function. Enzymatic function is understood to mean the transformation, in particular the oxidation of the hydroxy group at the C-3 atom of trichothecenes to a keto group. Furthermore, a "polypeptide variant" may additionally possess amino acid insertions or deletions, in particular a C- or N-terminally extended or shortened sequence relative to the polypeptide sequence of SEQ ID No. 1. An enzymatic function is "essentially retained" if the enzymatic reaction mechanism remains unchanged, i.e.the trichothecene is oxidized at the same site and the enzymatic activity of the variant is at least 10%, preferably at least 50%, more preferably at least 90%, in particular >100% based on the original, parental polypeptide of SEQ ID No. 1.

[0024] The term "sequence identity" refers to a percentage sequence identity. For amino acid sequences and nucleotide sequences, sequence identity can be determined visually, but is preferably calculated using a computer program. The amino acid sequence of SEQ ID No. 1 is defined as the reference sequence. Sequence comparison is also performed within sequence segments, where a segment is understood to be a continuous sequence of the reference sequence. The length of the sequence segments for peptide sequences is normally 3 to 200, preferably 15 to 65, and most preferably 30 to 50 amino acids. There are numerous commercially or freely available bioinformatics programs that can be used for homology determination and are continually being developed. Examples include the GCG Wisconsin Bestfit package (Devereux et al. 1984), BLAST (Altschul et al. 1990), and BLAST 2 (Tatusova and Madden 1999).Due to the different configuration options of these algorithms, it is possible that they produce different results for the same input sequences. Therefore, the search algorithm and its associated settings must be defined. In this case, sequence identity was determined using the NCBI BLAST (Basic Local Alignment Search Tool) program, specifically BLASTP for polypeptides, which is available on the website of the National Center for Biotechnology Information (NCBI; http: / / www.ncbi.nlm.nih.gov / ). This allows two or more sequences to be compared using the algorithm of Altschul et al., 1997 (Nucleic Acids Res., 25:3389-3402). The program version dated August 12, 2014, was used.The basic settings were used as program settings, especially for the amino acid sequence comparison: "max target sequence" = 100; "expected threshold" = 10; "word size" = 3; "matrix" = BLOSOM62; "gap costs" = "Existence: 11; Extention: 1"; "computational adjustment" = "Conditional compositional score matrix adjustment".

[0025] By the inventive use of the alcohol dehydrogenase containing metal ions and a quinone cofactor or a functional variant thereof, it is possible to transform at least 20%, preferably at least 50%, in particular at least 90% of at least one trichothecene having a hydroxy group at the C-3 atom, in particular DON, it being sufficient for this purpose to bring the alcohol dehydrogenase containing metal ions and a quinone cofactor or a functional variant thereof into contact with at least one trichothecene having a hydroxy group at the C-3 atom for at least one minute, preferably at least 5 minutes, in particular at least 60 minutes.

[0026] According to a further development of the invention, the amino acid sequence of the functional variant selected from the group of SEQ ID Nos. 2 to 4 is used. With these functional variants, which have a sequence identity of at least 86% to the alcohol dehydrogenase of SEQ ID No. 1, it is possible to transform trichothecenes containing a hydroxyl group at the C-3 atom, in particular DON, with consistently good results.

[0027] According to a further development of the invention, the quinone cofactor is selected from the group PCC, TTC, TPC, LTC, and CTC, preferably PCC. By using one of the quinone cofactors pyrroloquinoline quinone (PCC, CAS No. 72909-34-3), tryptophan tryptophylquinone (TTQ, CAS No. 134645-25-3), topoquinone (TPC, CAS No. 64192-68-3), lysine tyrosyl quinone (LTQ, CAS No. 178989-72-5), or cysteine ​​tryptophylquinone (CTC, CAS No. 400616-72-0) in the alcohol dehydrogenases, it is possible to transform trichothecenes containing a hydroxy group at the C-3 atom, such as DON, into non-toxic or toxicologically safe derivatives.

[0028] A particularly rapid and complete binding of the quinone cofactor to the alcohol dehydrogenase is achieved by binding it by means of at least one metal ion selected from the group Li +< , Na +< , K +< , Mg 2+< , Ca 2+< , Zn 2+< , Zn 3+< , Mn 2+< , Mn 3+< , Fe 2+< , Fe 3+< , Cu 2+< , Cu 3+< , Co 2+< and Co 3< , preferably Ca 2+< and Mg 2+<.

[0029] By additionally using at least one redox cofactor selected from the group consisting of phenazine methosulfate (PMS), PMS derivatives, potassium hexacyanidoferrate (III), sodium hexacyanidoferrate (III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), preferably phenazine methosulfate (PMS, CAS No.: 299-11-6), coenzyme Q1 and coenzyme Q10, as corresponds to a further development of the invention, a complete and rapid transformation of the trichothecenes is achieved exclusively in the presence of moisture, so that it can be ensured, for example, that trichothecenes contained in feed components are transformed into non-toxic derivatives during the production of feed and in any case before their use in animals.PMS derivatives are, for example, 1-hydroxyphenazine, 2-(pentaprenyloxy)dihydrophenazine, 5,10-dihydro-9-dimethylallylphenazine-1-carboxylic acid, 5,10-dihydrophenazine-1-carboxylic acid, 5-methylphenazinium methylsulfate, 6-acetophenazine-1-carboxylic acid, benthophoenin, clofazimine, dihydromethanophenazine, esmeraldic acid, esmeraldine B, izumiphenazine A - C, Janus Green B cation, methanophenazine pelagiomicin A, phenazine, phenazine-1,6-dicarboxylic acid, phenazine-1-carboxamide, phenazine-1-carboxylic acid, phenosafranin, pyocyanin, saphenamycin, saphenic acid or saphenic acid methyl ester.By carrying out the transformation of trichothecenes having a hydroxyl group at the C-3 atom in food or feed, in particular feed for pigs, poultry, cattle, horses, fish, aquaculture and pets as well as in plant raw materials used for the production or processing of food or feed, it is possible to avoid damage to the health of animals and humans through the use according to the invention.

[0030] The present invention further aims to provide a process by which trichothecenes, in particular trichothecenes having a hydroxy group at the C-3 atom, can be safely and reliably transformed into less toxic products, regardless of whether the agricultural products in which they are present are in a processed state or not.

[0031] To achieve this object, the process according to the invention for the enzymatic transformation of trichothecenes is essentially characterized in that at least one trichothecene having a hydroxy group at the C-3 atom is brought into contact with an alcohol dehydrogenase of SEQ ID No. 1 containing metal ions and a quinone cofactor, or with a functional variant having a sequence identity of at least 80%, preferably of at least 86%, particularly preferably of at least 89%, with at least one redox cofactor and water and optionally additionally with at least one auxiliary substance. By bringing a trichothecene having a hydroxy group at the C-3 atom into contact with metal ions and an alcohol dehydrogenase of SEQ ID No. 1 containing a quinone cofactor,1 and furthermore at least one redox cofactor and water, it is possible to oxidize the hydroxy group present at the C-3 atom of the trichothecenes into a ketone, whereby the trichothecene itself is detoxified and transformed into a non-toxic or low-toxic compound.

[0032] By further using a functional variant thereof, which is selected from the group of SEQ ID Nos. 2 to 4, instead of the amino acid sequence of SEQ ID No. 1, identical advantages can be achieved as by using the alcohol dehydrogenase of SEQ ID No. 1 and, in particular, a rapid and reliable transformation of the trichothecenes contained in the food and / or feed, regardless of their processing state, ie whether they are already processed agricultural products or not, can be achieved.

[0033] A particularly rapid and complete transformation of a trichothecene having a hydroxy group at the C-3 atom is achieved with the process according to the invention at a temperature between 5 °C and 55 °C, preferably between 10 °C and 50 °C, particularly preferably between 28 °C and 45 °C. Because the process according to the invention can be carried out in such a broad temperature range, the use of the alcohol dehydrogenases of SEQ ID No. 1 or their functional variants, which have at least a sequence identity of at least 80% to SEQ ID No. 1, is possible in a wide variety of applications, such as in aquaculture or in technological processes with elevated temperatures.Such technological processes in which the transformation of trichothecenes at elevated temperatures is important include, for example, animal feed processing, the production of pasta and other corn products such as polenta, popcorn, corn flakes, cornbread, or tortillas, as well as starch liquefaction processes, saccharification processes, or fermentation processes, such as mashing or fermentation, particularly in bioethanol production. It is important to ensure that the food or feed produced by these processes do not contain harmful amounts of trichothecenes containing a hydroxyl group at the C-3 atom.

[0034] According to a further development of the process according to the invention, the process is carried out in such a way that the at least one trichothecene having a hydroxy group at the C-3 atom is brought into contact with the alcohol dehydrogenase containing metal ions and a quinone cofactor, or at least one functional variant thereof, the redox cofactor, water, and optionally the excipient for at least one minute, preferably for at least 5 minutes, and particularly preferably for at least 60 minutes. Since contact times between 1 minute and more than 60 minutes are sufficient to achieve sufficient transformation of the trichothecenes into non-toxic or low-toxic derivatives, the process according to the invention can be used, for example, in a processing process for agricultural food or feed raw materials.On the other hand, it can also be carried out directly by the farmer, for example, immediately before feeding, by adding water to a feed and allowing it to stand for between 1 minute and about 1 hour at a temperature between 5 °C and 55 °C before feeding, which initiates a transformation of the trichothecenes into non-toxic products.

[0035] A particularly rapid and complete transformation is achieved if, as is the case with a further development of the process according to the invention, the quinone cofactor is selected from the group consisting of PCC, TTC, PTC, LTC, and CTC, preferably PCC. Such a quinone cofactor enables the alcohol dehydrogenases to rapidly and reliably attack the hydroxy group at the C-3 atom of the trichothecenes and convert them into the non-toxic derivative containing a keto group.

[0036] A further completion of the reaction and in particular an acceleration of the reaction is achieved if, in the process according to the invention, the cofactor is bound to the alcohol dehydrogenase by means of at least one metal ion selected from the group Li +< , Na +< , K +< , Mg 2+< , Ca 2+< , Zn 2+< , Zn 3+< , Mn 2+< , Mn 3+< , Fe 2+< , Fe 3+< , Cu 2+< , Cu 3+< , Co 2+< and Co 3+< , preferably Ca 2+< and Mg 2+<. Such a process not only causes a strong binding of the quinone cofactor to the alcohol dehydrogenase but also allows a rapid and reliable transformation of trichothecenes.

[0037] To further improve the transformation of the trichothecenes, in particular to complete the transformation reaction, the process according to the invention is further developed by using a redox cofactor selected from the group consisting of PMS, PMS derivatives, potassium hexacyanidoferrate (III), sodium hexacyanidoferrate (III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue, and TMPD, preferably PMS, coenzyme Q1, and coenzyme Q10. By adding such a redox cofactor, it is possible to carry out the transformation of the trichothecenes containing a hydroxyl group at the C-3 atom, for example, in an aqueous medium, such as in a feed slurry or a feed administered to the animals in aquaculture, without the need to add redox cofactors, which could be obtained, for example, from saliva, gastric juice, or intestinal juice.must be present, or the animal must have already ingested the feed slurry or the feed, which can prevent absorption of trichothecenes by the animals ingesting the feed.

[0038] Finally, the invention aims to provide a trichothecene transforming additive with which it is possible to transform trichothecenes in a feed or foodstuff safely and reliably into non-toxic derivatives.

[0039] To achieve this object, the additive according to the invention is essentially characterized in that it contains an alcohol dehydrogenase of SEQ ID No. 1 containing metal ions and a quinone cofactor or a functional variant having a sequence identity of at least 80%, preferably of at least 86%, particularly preferably of at least 89%, and optionally additionally at least one further component selected from the group consisting of a synthetic redox cofactor and at least one excipient.Such additives can be added to conventional feed in low concentrations, such as about 10 g to 1 kg per tonne of feed, and in such a low concentration they make it possible to transform trichothecenes containing a hydroxyl group at the C-3 atom into non-toxic derivatives, so that the overall health and performance of animals fed with such feed is improved, for example, and thus not only can failure rates be reduced, but also, for example, feed conversion is improved.

[0040] Consistently good results can be achieved with an additive according to the invention which contains, instead of the alcohol dehydrogenase of SEQ ID No. 1, a functional variant thereof selected from the group of SEQ ID Nos. 2 to 4.

[0041] For a substantially complete transformation of the hydroxy group present on the C-3 atom of trichothecenes by the additive according to the invention, said additive is further developed in such a way that it contains a quinone cofactor selected from the group PCC, TTC, TPC, LTC and CTC, as well as at least one metal ion selected from the group Li +< , Na +< , K +< , Mg 2+< , Ca 2+< , Zn 2+< , Zn 3+< , Mn 2+< , Mn 3+< , Fe 2+< , Fe 3+< , Cu 2+< , Cu 3+< , Co 2+< and Co 3+< . Through such further development, on the one hand, it is possible to bind the quinone cofactor safely and reliably to the alcohol dehydrogenase and, on the other hand, with an alcohol dehydrogenase containing such supplements, a complete transformation of trichothecenes, such as deoxynivalenol, which have a hydroxy group at the C-3 atom of the molecule, can be achieved.

[0042] In order to be able to carry out such a reaction even without the presence of redox cofactors naturally occurring in saliva, gastric or intestinal juice or the like, the additive according to the invention is further developed in such a way that a synthetic redox cofactor is additionally selected as a further redox cofactor, from the group PMS, PMS derivatives, potassium hexacyanidoferrate (III), sodium hexacyanidoferrate (III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue and TMPD, preferably PMS, coenzyme Q1 and coenzyme Q10.

[0043] According to a further development of the invention, the additive is designed such that the excipient is selected from the group of inert carriers, vitamins, minerals, phytogenic substances, enzymes and other components for the detoxification of mycotoxins, such as mycotoxin-degrading enzymes, in particular aflatoxin oxidases, ergotamine hydrolases, ergotamine amidases, zearalenone esterases, zearalenone lactonases, zearalenone hydrolases, ochratoxin amidases, fumonisin aminotransferases, fumonisin carboxyltransferases, aminopolyol amine oxidases, deoxynivalenol epoxide hydrolases, deoxynivalenol dehydrogenases, deoxynivalenol oxidases, trichothecene dehydrogenases, trichothecene oxidases; and mycotoxin-transforming microorganisms, such as DSM 11798; and mycotoxin-binding substances, for example microbial cell walls or inorganic materials, such as bentonites, especially smectites.By using such an additive, it can be ensured, for example, in feed or foodstuffs that any amounts of trichothecenes containing a hydroxyl group at the C-3 atom and any other mycotoxins such as fusarium toxins, ergotamines, ochratoxins contained therein are detoxified to such an extent that the mycotoxins do not have a harmful effect on the organism of the subject consuming this feed or foodstuff.

[0044] Further areas of application of the invention are additives which, in addition to at least one alcohol dehydrogenase according to the invention, additionally contain at least one enzyme which is involved, for example, in the degradation of proteins, such as proteases, or which is involved in the metabolism of starch or fiber or fat or glycogen, such as amylase, cellulase or glucanase, as well as, for example, hydrolases, lipolytic enzymes, mannosidases, oxidases, oxidoreductases, phytases or xylanases.

[0045] It goes without saying that the additive can of course be in encapsulated or coated form, whereby standard methods such as those described in WO 92 / 12645 can be used for encapsulation or coating. Encapsulation or coating makes it possible to transport the additive to its place of use without alteration, in particular without degradation or damage, so that the polypeptide only begins to act after the protective shell has dissolved, for example in the digestive tract of animals. This enables an even more targeted, rapid and complete degradation of trichothecenes containing a hydroxy group at the C-3 atom, even in acidic, protease-rich and anaerobic environment. Furthermore, encapsulation or coating also makes it possible to increase the temperature stability of the alcohol dehydrogenases in the additive, thereby improving its use, for example, in the pelleting process for animal feed.

[0046] The additive according to the invention can be used in a variety of ways, such as for the production of a preparation for the prophylaxis and / or treatment of trichothecene mycotoxicoses, preferably mycotoxicoses caused by trichothecenes that have a hydroxy group at the C-3 atom, such as in particular deoxynivalenol mycotoxicoses. Such mycotoxicoses have serious consequences for humans and animals. By using the additive in this way, it is possible in the case of prophylaxis to maintain the health status of humans or animals essentially at the level corresponding to that without or with a reduced oral intake of the toxins, despite oral intake of trichothecenes, in particular trichothecenes that have a hydroxy group at the C-3 atom, in particular deoxynivalenol.In the case of the treatment of mycotoxicoses, it is possible to alleviate the symptoms of such a disease and, in particular, to normalize the SOCS3 concentration in the liver or the IGFALS levels in the plasma as well as the claudin concentration in the intestine.

[0047] Furthermore, such use makes it possible to improve the performance of farm animals, in particular feed utilization and weight gain, and to reduce the mortality rate.

[0048] The invention is explained in more detail below using exemplary embodiments and a drawing. In the drawing: Fig. 1 the temporal transformation of deoxynivalenol for the activated alcohol dehydrogenase of SEQ ID No. 1 and a control (CTR) as comparison, and Fig. 2 the representation of the temporal transformation of DON with the activated alcohol dehydrogenases of SEQ ID Nos. 1 to 4 and a control (CTR) for comparison. Example 1: Cloning of the genes and purification of alcohol dehydrogenase

[0049] The codon-optimized nucleotide sequences of the alcohol dehydrogenases with SEQ ID Nos. 1 to 4 for the respective host cell were obtained from DNA2.0 and contained restriction sites at the 5' and 3' ends of the sequence, as well as a C- or N-terminal 6xHis tag at the amino acid level. These nucleotide sequences were transformed into expression vectors using standard methods for expression in Escherichia coli or Komagataella pastoris integrated, and in E. coli or K. pastoris transformed, as well as in E. coli or K. pastoris expressed (JM Cregg, Pichia Protocols, second Edition, ISBN-10: 1588294293, 2007; J. Sambrook et al. 2012, Molecular Cloning, A Laboratory Manual 4th Edition, Cold Spring Harbor).

[0050] The alcohol dehydrogenases with SEQ ID Nos. 1-4 were isolated from soluble cell lysates in the case of expression in E. coliand from intracellular expression in K. pastoris or from the culture supernatant in the case of extracellular expression in K. pastorisSelectively enriched using standard methods chromatographically on nickel-Sepharose columns. The selectively enriched eluates were incubated in the presence of metal ions and quinone cofactors and activated; "activate" means that the alcohol dehydrogenases contain both the metal ion and the quinone cofactor. These activated alcohol dehydrogenases were used to determine the enzymatic properties of the alcohol dehydrogenases with SEQ ID Nos. 1 to 4 in Examples 3 to 7 below. The total protein concentration was determined photometrically using the Bradford reagent (Sigma # B6916), with the absorption being measured in a microtiter plate photometer (plate reader, Biotek, Synergy HT) at a wavelength of 595 nm.The protein concentration was determined using a calibration curve obtained by measuring Bovine Serum Albumin (BSA, Sigma #A4919) solutions with concentrations up to 1500 µg / ml using the Bradford assay. Example 2: Determination of sequence identity

[0051] The determination of the percentage sequence identity across the entire length of the amino acid sequence of the alcohol dehydrogenases with SEQ ID Nos. 1-4 relative to each other was performed using the BLAST (Basic Local Alignment Search Tool) program, specifically BLASTP, which can be accessed on the website of the National Center for Biotechnology Information (NCBI; http: / / www.ncbi.nlm.nih.gov / ). BLASTP allows two or more sequences to be compared using the algorithm of Altschul et al., 1997 (Nucleic Acids Res. (1997) 25:3389-3402). The basic program settings were used, specifically: "max target sequence" = 100; "expected threshold" = 10; "word size" = 3; "matrix" = BLOSOM62; "gap costs" = "Existence: 11; Extention: 1"; "computational adjustment" = "Conditional compositional score matrix adjustment". The percentage identities of the amino acid sequences to each other are shown in Table 1. Table 1: SEQ ID No. 1 SEQ ID No. 2 SEQ ID No. 3 SEQ ID No. 4 SEQ ID No. 1 100% 87% 89% 86% SEQ ID No. 2 87% 100% 99% 90% SEQ ID No. 3 89% 99% 100% 91% SEQ ID No. 4 86% 90% 91% 100% Example 3: Transformation of trichothecene containing a hydroxy group at the C-3 atom

[0052] To determine their ability to transform trichothecenes containing a hydroxy group at the C-3 atom, in particular DON, nivalenol and T-2 toxin, the alcohol dehydrogenases of SEQ ID Nos. 1-4 were transformed with a C-terminal 6xHis tag into E. coli, prepared as described in Example 1.

[0053] A transformation occurs when the amount of a trichothecene containing a hydroxy group at the C-3 atom is reduced by bringing it into contact with an activated alcohol dehydrogenase containing metal ions and a quinone cofactor.

[0054] 100 ml each of a E. coliCultures with an optical density (OD600 nm) of 2.0–2.5 were harvested by centrifugation at 4°C and resuspended in 20 ml of potassium phosphate buffer. The cell suspensions were lysed by three treatments with a French press at 20,000 psi. The cell lysates were separated into soluble and insoluble fractions by centrifugation. The supernatant was sterile filtered, and alcohol dehydrogenase was selectively enriched for chromatography on nickel-Sepharose columns using standard methods. Buffer exchange was then performed by dialysis using specific tubes with a cutoff of ten kilodaltons. The resulting total protein concentration was measured using the Bradford assay.

[0055] Binding of the quinone cofactors and metal ions to the alcohol dehydrogenases was achieved by incubation in an aqueous solution. The quinone cofactors, such as pyrroloquinoline quinone (PCC, CAS No. 72909-34-3), tryptophan tryptophylquinone (TTC, CAS No. 134645-25-3), topoquinone (TPC, CAS No. 64192-68-3), lysine tyrosylquinone (LTC, CAS No. 178989-72-5), and cysteine ​​tryptophylquinone (CTC, CAS No. 400616-72-0), were added as an aqueous solution in an approximately 20-fold molar excess to the total protein concentration. The metal ions selected from Li +< , Na +< , K +< , Mg 2+< , Ca 2+< , Zn 2+< , Zn 3+< , Mn 2+< , Mn 3+< , Fe 2+< , Fe 3+< , Cu 2+< , Cu 3+< , Co 2+< and Co 3+< are used as an aqueous solution or a salt thereof. Unless otherwise stated, the alcohol dehydrogenases were activated with PCC (Sigma Aldrich #D7783) and Ca 2+< ; used as a 5 mM CaCl 2 solution. The enzymes purified and activated in this way were used for in vitroTransformation approaches using a trichothecene containing a hydroxy group at C-3 are used. Unless otherwise stated, the terms "enzyme" or "alcohol dehydrogenase" always refer to the corresponding alcohol dehydrogenase containing activated metal ions and a quinone cofactor.

[0056] The transformation mixtures were carried out in aqueous solution with the following components: 100 mM Tris-HCl pH 7.5 or 10% Teorell Stenhagen pH 7.5; synthetic redox cofactor selected from the group consisting of 1 mM phenazine methosulfate PMS (Sigma Aldrich #P9625), 1 mM methylene blue (Sigma #M9140), 1 mM coenzyme Q10 (Sigma #C9538), 1 mM coenzyme Q1 (Sigma #C9538), and 20 mM potassium hexacyanidoferrate(III) PFC(III) (Fluka #60300); 10 ppm to a maximum of 100 ppm of a trichothecene having a hydroxy group at the C-3 atom by adding the desired amount of a toxin substrate stock solution; and 10 nM to 100 nM, with a maximum of 300 nM, of an activated metal ion and quinone cofactor-containing alcohol dehydrogenase of SEQ ID No. 1, 2, 3, or 4. Unless otherwise stated, the Tris-HCl buffer, the redox cofactor PMS, DON, and the alcohol dehydrogenase of SEQ ID No. 1 were used as standard. Each transformation mixture was carried out in a 1.5 ml brown Eppendorf tube.The reaction mixtures were incubated at 30 °C on a thermoblock for up to 120 min, with a minimum of 40 min. At time points 0, 10, 20, 30, and 40 min, a 0.1 ml sample was taken and mixed with 0.1 ml of methanol. The samples were stored at -20 °C or, alternatively, immediately analyzed by LC-MS / MS or HPLC.

[0057] A sterile-filtered, aqueous 2000 ppm DON solution served as the DON substrate stock solution. To prepare this solution, DON in crystalline form (Biopure Standard from Romer Labs, Art. No. 001050, purity at least 98%) was weighed and dissolved.

[0058] To quantify the trichothecenes containing a hydroxy group at the C-3 atom and their transformation metabolites, HPLC analyses were performed. The substances were chromatographically separated using a Phenomenex C18 Gemini-NX column with dimensions of 150 mm x 4.6 mm and a particle size of 5 µm. A methanol-water mixture with an ammonium acetate concentration of 5 mM served as the mobile phase. The UV signal at 220 nm was recorded and evaluated. For quantification by LC-MS / MS analyses, the substances were chromatographically separated using a Zorbax eclipse C8 column with dimensions of 150 mm x 4.6 mm and a particle size of 5 µm. A methanol-water mixture with an ammonium acetate concentration of 5 mM served as the mobile phase. The UV signal at 220 nm was recorded. Electrospray ionization (ESI) served as the ionization source.The trichothecenes containing a hydroxy group at the C-3 atom were quantified using QTrap / LC / MS / MS (triple quadrupole, Applied Biosystems) in enhanced mode.

[0059] The negative slopes of the transformation curves (= decrease in toxin concentration over time) in the linear range were used as a measure of alcohol dehydrogenase activity. To determine the residual activities, the measured activities at different parameters were related to the baseline activity measured under standard conditions, specifically 30 °C and pH 7.5, and are generally expressed as percentages. Fig. 1 is the temporal transformation of DON for the activated alcohol dehydrogenase of SEQ ID No. 1 and in Fig. 2The temporal transformation of DON is shown for the activated alcohol dehydrogenases of SEQ ID Nos. 2-4 (Fig. 1B). It is clearly evident from the figures that DON transformation occurs, as the concentration of DON decreased as a function of real time.

[0060] Here, Fig. 1 The transformation of DON with the alcohol dehydrogenase of SEQ ID No. 1 in 100 mM Tris HCl pH 7.5 in the presence of 50 ppm DON and 1 mM PMS is shown. The measurement results were obtained by LC-MS / MS analyses (A), and the transformation of DON with the alcohol dehydrogenases of SEQ ID Nos. 1-4 is shown in Fig. 2 The measurement results were obtained by HPLC analysis (B). CTR served as a negative control in the experiments, containing all components of the transformation mixture except for the alcohol dehydrogenases of SEQ ID Nos. 1-4.

[0061] To compare the efficiency of the quinone cofactors, 10 nM of alcohol dehydrogenase (SEQ ID No. 1) activated with the quinone cofactors PCC, TTC, TPC, LTC, and CTC, 10 ppm DON, and 1 mM synthetic redox cofactor PMS were mixed in 100 mM Tris-HCl pH 7.5 in transformation mixtures and incubated at 30 °C. DON concentrations were determined after 30 minutes by LC-MS / MS; the results are presented in Table 2.

[0062] To compare the efficiency of the synthetic redox cofactors, 10 nM activated enzyme (alcohol dehydrogenase of SEQ ID No. 1), 10 ppm DON, and 1 mM or 20 mM of the synthetic redox cofactors to be tested were mixed in 100 mM Tris-HCl pH 7.5 in transformation mixtures and incubated at 30 °C. DON concentrations were determined after 30 minutes by LC-MS / MS, with the results presented in Table 2. Table 2: Quinone cofactor DON [ppm] Redox cofactor DON [ppm] PCC 1,94 1 mM PMS 1,95 TTQ 2,32 20 mM PFC(III) 2,11 TPQ 2,41 1 mM coenzyme Q1 8,58 LTQ 2,04 1 mM methylene blue 6,88

[0063] To test the influence of metal ions in the activated enzyme on transformation, the activation of alcohol dehydrogenase of SEQ ID No. 1 and PCC was carried out, but with different metal ions, namely Mg 2+< , Ca 2+< , Zn 2+< , Mn 2+< , Fe 2+< and Cu 2+<. The transformation mixtures each contained 10 nM activated alcohol dehydrogenase, 10 ppm DON and 1 mM PMS in 100 mM Tris-HCl pH 7.5 and were incubated at 30 °C. The DON concentrations were determined after 30 minutes by LC-MS / MS, and the results are shown in Table 3. Table 3: Metal ion DON [ppm] Metal ion DON [ppm] Mg2+< 1,90 Mn2+< 2,57 Ca 2+< 1,98 Fe 2+< 2,17 Zn2+< 2,46 Cu 2+< 2,61

[0064] Analogous to the DON transformation assays described above, transformation assays were carried out with other trichothecenes containing a hydroxy group at the C-3 atom. In these assays, 50 ppm T-2 toxin or 50 ppm nivalenol were used instead of 50 ppm DON. All four alcohol dehydrogenases of SEQ ID Nos. 1 to 4 containing metal ions and quinone cofactors were also capable of transforming T-2 toxin and nivalenol, with more than half of the original toxin being transformed within 30 minutes. Example 4: Measuring the activity areas

[0065] To determine the ability of the alcohol dehydrogenases of SEQ ID Nos. 1-4 to transform DON under different conditions, the alcohol dehydrogenase of SEQ ID No. 1 was used as an example.

[0066] The alcohol dehydrogenase of SEQ ID No. 1 was prepared as described in Example 3 and activated with Ca 2+ and PCC. To determine the activity of the enzyme over a temperature range of 10 °C to 50 °C and a pH range of 3.0 to 9.0, a 10% Teorell Stenhagen buffer was used instead of the 100 mM Tris-HCl pH 7.5 buffer.

[0067] The transformation experiments to determine the activities at different temperatures were carried out in aqueous solution with the following components: 10% Teorell Stenhagen pH 7.5, 1 mM synthetic redox cofactor PMS, 50 ppm DON, and 10 nM activated alcohol dehydrogenase of SEQ ID No. 1. The transformation mixtures were incubated for up to 60 min in a thermocycler (Eppendorf) with a temperature gradient from 10 °C to 50 °C. At times 0, 10, 20, 30, 40, and 60 min, a 0.05 ml sample was taken and mixed with 0.05 ml methanol to stop the reaction and stored at -20 °C. The samples were prepared for LC-MS / MS as described in Example 3 and analyzed by LC-MS / MS. The course of DON reduction was determined at each temperature and the activity was calculated as described in Example 3.The slope of the linear portion of the transformation curve at 30 °C served as a reference value for calculating the residual activity at the other temperatures. Table 4 lists the temperatures in °C and the corresponding residual activities in percent. Surprisingly, alcohol dehydrogenase with SEQ ID No. 1 was found to be active over a wide temperature range. A residual activity of 48% was measured at 10 °C, and 67% at approximately 50 °C. Table 4: Temperature [°C] Residual activity [%] Temperature [°C] Residual activity [%] 10,0 48 32,8 105 12,7 60 33 108 15 69 35,3 105 17,6 73 38,4 120 20,5 86 40,7 116 23,3 89 43,2 108 26,2 82 45,9 96 28,3 100 48,2 89 30,2 100 49,8 67

[0068] The transformation experiments to determine the activity in a pH range of 4.0 to 9.0 were carried out in aqueous solution with the following components: 10% Teorell Stenhagen pH 4.0 to pH 10.0, 20 mM synthetic redox cofactor PFC(III), 100 ppm DON, and 20 nM activated alcohol dehydrogenase of SEQ ID No. 1. The transformation mixtures were incubated for up to 60 min in a thermoblock at 30 °C. At times 0, 10, 20, 30, 40, and 60 min, a 0.05 ml sample was taken and mixed with 0.05 ml methanol to stop the reaction and stored at -20 °C. The samples were diluted as described in Example 3 and analyzed by LC-MS / MS. The course of DON reduction was determined at each pH value and the activity was calculated as described in Example 3. The slope of the linear portion of the transformation line at pH 7.5 served as a reference value for calculating the residual activity at the other pH values.Table 5 shows the pH values ​​and the corresponding residual activities (DON reduction relative to the reference pH value of 7.5) in percent. Table 5: pH Residual activity pH Residual activity 4,0 10 % 7,0 105 % 5,0 18 % 7,5 100 % 6,0 20 % 8,0 69 % 6,5 52 % 9,0 105 % Example 5: Determination of temperature stability

[0069] The temperature stability of the alcohol dehydrogenase of SEQ ID No. 1 was determined over a range of 30°C to 55°C. For this purpose, the activated alcohol dehydrogenase was incubated in 100 mM Tris-HCl buffer, pH 7.5, for up to 60 min at a specific temperature in a thermocycler (Eppendorf). At time points of 0, 5, 10, 15, 20, 30, 40, and 60 min, an aliquot of the alcohol dehydrogenase was taken, and the activity was determined in a DON transformation mixture as described in Example 3. The transformation mixtures contained the following components: 100 mM Tris-HCl, pH 7.5, 1 mM PMS, 50 ppm DON, and 10 nM activated alcohol dehydrogenase of SEQ ID No. 1. The reactions were incubated as described in Example 3, and samples were taken for activity determination after 0, 10, 20, 30, 40, and 60 min. The course of DON reduction was determined at each temperature for each incubation time.To determine temperature stability, the slope of the linear region of the DON transformation line was calculated. The slope of the linear region of the DON transformation line for each temperature at time t = 0 min was used as a reference value for calculating residual activities. Table 6 shows the temperatures in °C, the incubation time in minutes, and the corresponding residual activities in percent. Alcohol dehydrogenase of SEQ ID No. 1 was most stable after one hour of storage at temperatures of 30 °C and 37 °C. In comparison, alcohol dehydrogenase at 40 °C still showed 73% residual activity after one hour of storage. After storage at 45 °C for 30 min, 50% residual activity was measured. Surprisingly, 84% residual activity was detected after 5 min of storage at 50 °C. Table 6: incubation period 0 min 5 minutes 10 minutes 15 minutes 20 minutes 30 minutes 40 minutes 60 minutes 30 °C 100 % 99 % 98 % 94 % 88 % 99 % 100 % 95 % 37 °C 100 % 92 % 94 % 92 % 90 % 91 % 47 % 79 % 40 °C 100 % 90 % 83 % 77 % 75 % 83 % 82 % 73 % 45 °C 100 % 85 % 78 % 77 % 60 % 57 % 47 % 19 % 50 °C 100 % 84 % 30 % 36 % 13 % 12 % 10 % 0 % Example 6: Determination of pH stability

[0070] The pH stability of the activated alcohol dehydrogenase of SEQ ID No. 1 was determined over a range of pH 4.0 to pH 10.0. For this purpose, a 10-fold concentration of the activated alcohol dehydrogenase (100 nM) was stored in 10% Teorell Stenhagen buffer pH 4.0 to pH 10.0 for up to 120 minutes at a temperature of 30 °C. At times 0, 60 and 120 min, an aliquot of the alcohol dehydrogenase was taken and the activity determined in a transformation mixture and carried out as described in Example 3 with the following components at 30 °C. Samples for activity determination were taken after 0, 10, 20, 30, and 40 minutes. The DON reduction curve was determined at each pH value for each time point. To determine stability, the slope of the linear portion of the DON transformation curve was calculated at the respective pH value at each time point.The slope of the linear region of the DON transformation curve for each pH value at time t=0 min was used as a reference value for calculating the activities for the following incubation times. Table 7 shows the pH values, the pH incubation time in minutes, and the corresponding residual activities in percent. The alcohol dehydrogenase of SEQ ID No. 1 was stable after a 60-minute incubation at pH 5.0 to pH 9.0. Surprisingly, the alcohol dehydrogenase showed particularly good stability in acidic environments (no loss of activity at pH 5.0) and in strongly basic environments (barely any loss of activity after a 120-minute incubation at pH 9.0). Table 7: incubation period 60 minutes 120 minutes pH 4.0 72 % 51 % pH 5.0 111 % 109 % pH 6.0 92 % 88 % pH 7.0 87 % 85 % pH 8.0 83 % 73 % pH 9.0 93 % 60 % pH 10.0 69 % 55 % Example 7: Transformation of DON in complex matrices

[0071] To determine the ability of activated alcohol dehydrogenases to transform trichothecenes in complex matrices even without the external addition of synthetic redox cofactors, the activated alcohol dehydrogenase of SEQ ID No. 1 was produced as described in Example 3, and DON transformation experiments were conducted in complex matrices. Complex matrices include, among others, bovine rumen fluid, intestinal contents from the jejunum of pigs, gastric juice from pigs, human and pig saliva, granulated piglet rearing feed (FAF), and granulated piglet rearing feed mixed with saliva, rumen fluid, or intestinal contents. To provide a comparison to the buffer system, controls with Tris-HCl were included, as described in Example 3. A standard feed based on corn, soy, and barley was used as the FAF.

[0072] To determine alcohol dehydrogenase activity in rumen fluid (pH 5.9), 1 ml of sterile rumen fluid filtrate was treated with 100, 200, and 300 nM of activated alcohol dehydrogenase of SEQ ID No. 1 and 50 ppm DON. Control assays in aqueous solution were performed as described in Example 3. All transformation assays were incubated at 30 °C on a thermoblock for up to 24 hours. Sampling occurred at time points of 0, 0.5, 1.0, 5.0, and 24.0 hours, with 0.1 ml of sample taken at each time point, and the reaction was stopped with 0.1 ml of methanol. The samples were stored at -20 °C, thawed, and centrifuged for 10 min at 13,000 rpm using an Eppendorf tabletop centrifuge. Sterile filtered using a 0.2 µM Spartan filter. For LC-MS / MS, the samples were diluted as described in Example 3 and analyzed by LC-MS / MS. The DON concentration at time t = 0 h was used as the reference value (100%) for the subsequent values.Table 8 shows the percentage of DON concentration measured at a specific time point relative to time t = 0 h. For activity in Tris-HCl buffer, the presence of an externally added synthetic redox cofactor is necessary, as DON transformation occurs slowly and was only detectable after 24 hours with an alcohol dehydrogenase concentration of 300 nM. Surprisingly, it was shown that DON is transformed in sterile rumen fluid filtrate at a pH of 5.9 without the addition of an external synthetic redox cofactor. This clearly demonstrates that rumen fluid contains substances that serve as natural redox cofactors. At a concentration of 300 nM, only 42% of the initial DON amount is still present in the mixture after 5 hours of incubation. After 24 hours of incubation, DON is only detectable in small amounts at alcohol dehydrogenase concentrations greater than 200 nM. Table 8: Rumen fluid with synthetic redox cofactor Rumen fluid without synthetic redox cofactor Tris-HCl pH 7.5 without synthetic redox cofactor 100 nM 100 nM 200 nM 300 nM 100 nM 200 nM 300 nM 0 h 100 % 100 % 100 % 100 % 100 % 100 % 100 % 0,5 h 0 % 100 % 100 % 87 % 100 % 99 % 95 % 1,0 h 0 % 100 % 100 % 83 % 100 % 99 % 89 % 5,0 h 0 % 94 % 75 % 42 % 99 % 88 % 86 % 24,0 h 0 % 53 % 3 % 0 % 97 % 84 % 67 %

[0073] To determine alcohol dehydrogenase activity in pure porcine gastric juice without feed pulp at a pH of approximately 3, in porcine intestinal contents at a pH of approximately 6, as well as in porcine saliva and human saliva, 300 nM activated alcohol dehydrogenase of SEQ ID No. 1 and approximately 20 ppm DON were mixed with 1 ml each of gastric juice (sterile filtered), 1 ml of pulpy intestinal contents, and 1 ml of saliva. Preparations containing only digestive fluids with 20 ppm DON were run as negative controls, and transformation preparations containing all components, including 20 mM of the synthetic redox cofactor PFC(III), served as positive controls. Sampling was carried out at time points of 0, 3.0, 5.0, and 24.0 hours, with 0.1 ml of sample being taken at each time point, and the reaction was stopped with 0.1 ml of methanol. The samples were stored at -20 °C, thawed and incubated for 10 min at 13 °C.000 rpm using an Eppendorf tabletop centrifuge and sterile filtered (0.2 µM Spartan filter). For LC-MS / MS, the samples were diluted 1:10 in the mobile phase (see Example 3) and analyzed by LC-MS / MS as in Example 3. Table 9 shows the respective DON concentrations measured at the time of sample collection. Surprisingly, a reduction of DON occurred in saliva without externally added synthetic redox cofactor (regardless of species). This clearly shows that the saliva secretions of humans and pigs contain substances that are suitable as natural redox cofactors for the transformation of DON with alcohol dehydrogenase of SEQ ID No. 1. In pure gastric juice without feed pulp, no significant reduction in the DON concentration was measured. In the intestinal contents, a decrease in the DON concentration only occurs with the addition of the synthetic redox cofactor. Table 9: DON [ppm] sample 0 h 3 h 5 h 24 h Saliva (human) negative control 20 19 18 18 0 mM PFC (III) 20 13 12 8 positive control 20 mM PFC (III) 18 0 0 0 Saliva (pig) negative control 20 20 19 18 0 mM PFC(III) 21 10 8 5 positive control 20 mM PFC (III) 20 0 0 0 gastric juice negative control 22 22 21 21 0 mM PFC(III) 22 21 21 20 positive control 20 mM PFC (III) 24 21 19 18 intestinal contents negative control 21 20 20 20 0 mM PFC(III) 24 23 22 22 positive control 20 mM PFC (III) 23 9 8 4

[0074] To determine the activity of alcohol dehydrogenases in piglet rearing feed (FAF), 100 mg of FAF were mixed with 400 µl of 100 mM Tris-HCl buffer, pH 7.5, 400 µl of porcine saliva, 400 µl of sterile porcine gastric juice, or 400 µl of porcine intestinal contents. These FAF suspensions were stored overnight at 4 °C. Afterwards, approximately 20 ppm DON and / or 300 nM activated alcohol dehydrogenase of SEQ ID No. 1 and / or 20 mM of the synthetic redox cofactor PFC(III) were added to all samples. The samples without alcohol dehydrogenase and without the external synthetic redox cofactor served as negative controls. The samples with the addition of alcohol dehydrogenase and the synthetic redox cofactor served as positive controls. Sampling took place at time points 0; 3.0, 5.0, and 24.0 hours. One sample was used in its entirety at each time point. 500 µl of methanol was added to the sample, followed by 30 min of homogenization on a shaker at 300 rpm.The samples were then centrifuged for 15 min (Eppendorf benchtop centrifuge at 13,000 rpm), and the supernatant was filtered through a 0.2 µM Spartan filter using a syringe. The supernatants were stored at -20 °C, thawed, and diluted 1:10 in the mobile phase for LC-MS / MS analysis. The samples were analyzed by LC-MS / MS as described in Example 3.

[0075] Table 10 shows the DON concentration present in the samples at the respective time points. The FAF buffer mixture contains substances that can take on the role of externally added synthetic redox cofactors, as the DON concentration continuously decreases in the absence of the external synthetic redox cofactor. These substances originate from the piglet feed, since, as previously shown, no DON transformation could be measured in buffer without an external synthetic redox cofactor. In the presence of the external synthetic redox cofactor, the transformation of DON in the FAF buffer mixture proceeds comparatively faster.

[0076] In the mixture of FAF and saliva, alcohol dehydrogenase also showed activity independent of the presence of the external synthetic redox cofactor. In the transformation mixtures containing the external synthetic redox cofactor, a faster reduction of DON occurred.

[0077] Surprisingly, the alcohol dehydrogenase of SEQ ID No. 1 is also active in the FAF-gastric juice mixture without the addition of the external synthetic redox cofactor. The addition of FAF to the gastric juice increased the pH of the gastric juice and dissolved naturally occurring redox cofactors from the FAF, which can replace the external synthetic redox cofactor. Alcohol dehydrogenase activity was only detected in the intestinal contents when an external synthetic redox cofactor was added to the transformation mixture. Table 10: DON [ppm] sample 0 h 3 h 5 h 24 h FAF in buffer negative control 21 20 20 20 0 mM PFC(III) 20 10 9 5 positive control 20 mM PFC(III) 21 0 0 0 FAF in saliva negative control 20 20 20 20 0 mM PFC(III) 20 12 9 8 positive control 20 mM PFC (III) 21 1 0,8 0,5 FAF in gastric juice negative control 21 21 20 20 0 mM PFC(III) 20 7 5 2 positive control 20 mM PFC(III) 20 5 0,7 0 FAF in intestinal contents negative control 21 20 20 20 0 mM PFC(III) 21 20 18 16 positive control 20 mM PFC(III) 20 5 3 0,7

Claims

1. Use of an alcohol dehydrogenase of SEQ ID no. 1 containing metal ions and a quinone cofactor, or in addition, a functional variant exhibiting a sequence identity of at least 80%, preferably 86%, especially preferred at least 89%, and at least one redox cofactor for the transformation of at least one trichothecene exhibiting a hydroxyl group on the C-3 atom.

2. Use according to Claim 1 characterised in that the amino acid sequence of the functional variant is selected from the group of sequence ID numbers 2 to 4.

3. Use according to Claim 1 or 2 characterised in that the quinone cofactor is selected from the group PCC, TTC, TPC, LTC, and CTC, preferably PCC.

4. Use according to Claim 1, 2, or 3 characterised in that the quinone cofactor is bound to the alcohol dehydrogenase by at least one metal ion selected from the group Li+, Na+, K+, Mg2+, Ca2+, Zn2+, Zn3+, Mn2+, Mn3+, Fe2+, Fe3+, Cu2+, Cu3+, Co2+ and Co3, preferably Ca2+ and Mg2+.

5. Use according to one of the Claims 1 to 4 characterised in that at least one redox cofactor is selected from the group PMS, PMS derivatives, potassium hexacyanoferrate (III), sodium hexacyanoferrate (III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue, and TMPD, preferably PMS, coenzyme Q1 and coenzyme Q10.

6. Use according to one of the Claims 1 to 5 characterised in that the transformation of trichothecenes exhibiting a hydroxyl group on the C-3 atom in food and feed, especially for swine, poultry, cattle, horses, fish, aquaculture, and domestic animals, and in plant-based raw materials used for the production or processing of food and feed, is carried out.

7. Procedure for the enzymatic transformation of trichothecenes, characterised in that at least one trichothecene exhibiting a hydroxyl group on the C-3 atom is brought into contact with an alcohol dehydrogenase of sequence ID no. 1 containing metal ions and a quinone cofactor, or with a functional variant additionally exhibiting a sequence identity of at least 80%, preferably at least 86%, especially preferred at least 89% with at least one redox cofactor and water, and if necessary at least one excipient.

8. Procedure according to Claim 7 characterised in that the amino acid sequence of the functional variant is selected from the group of sequence ID numbers 2 to 4.

9. Procedure according to one of the Claims 7 or 8 characterised in that the trichothecene exhibiting a hydroxyl group on the C-3 atom is transformed at a temperature between 5°C and 55°C, preferably between 10°C and 50°C, especially preferred between 28°C and 45°C.

10. Procedure according to one of the Claims 7, 8, or 9 characterised in that at least one trichothecene exhibiting a hydroxyl group on the C-3 atom is brought into contact with the alcohol dehydrogenase containing metal ions and a quinone cofactor, or at least a functional variant thereof, with the redox factor, with water, and if necessary, with the excipient, for at least one minute, preferably at least 5 minutes, especially at least 60 minutes.

11. Procedure according to one of the Claims 7 to 10 characterised in that the quinone cofactor is selected from the group PCC, TTC, TPC, LTC, and CTC, preferably PCC.

12. Procedure according to one of the Claims 7 to 11 characterised in that the quinone cofactor is bound to the alcohol dehydrogenase by at least one metal ion selected from the group Li+, Na+, K+, Mg2+, Ca2+, Zn2+, Zn3+, Mn2+, Mn3+, Fe2+, Fe3+, Cu2+, Cu3+, Co2+ and Co3+, preferably Ca2+ and Mg2+.

13. Procedure according to one of the Claims 7 to 12 characterised in that at least one redox cofactor is selected from the group PMS, PMS derivatives, potassium hexacyanoferrate (III), sodium hexacyanoferrate (III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue, TMPD, preferably PMS, coenzyme Q1, and coenzyme Q10.

14. Trichothecene-transforming additive characterised in that the additive contains an alcohol dehydrogenase of sequence ID no. 1 containing metal ions and a quinone cofactor, or a functional variant additionally exhibiting a sequence identity of at least 80%, preferably at least 86%, especially preferred at least 89%, and if necessary, additionally at least one additional component selected from the group consisting of a redox cofactor and at least one excipient.

15. Additive according to Claim 14 characterised in that it contains a functional variant of the alcohol dehydrogenase of sequence ID no. 1 selected from the group of sequence ID numbers 2 to 4.

16. Additive according to Claim 14 or 15 characterised in that it contains a quinone cofactor selected from the group PCC, TTC, TPC, LTC, and CTC, preferably PCC.

17. Additive according to Claim 14, 15, or 16 characterised in that it contains the quinone cofactor bound to the alcohol dehydrogenase by at least one metal ion selected from the group Li+, Na+, K+, Mg2+, Ca2+, Zn2+, Zn3+, Mn2+, Mn3+, Fe2+, Fe3+, Cu2+, Cu3+, Co2+ and Co3+, preferably Ca2+ and Mg2+.

18. Additive according to one of the Claims 14 to 17 characterised in that a synthetic redox cofactor is selected from the group PMS, PMS derivatives, potassium hexacyanoferrate (III), sodium hexacyanoferrate (III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue, and TMPD, preferably PMS, coenzyme Q1, and coenzyme Q10 as the redox cofactor.

19. Additive according to one of the Claims 14 to 18 characterised in that the excipient is selected from a group of inert carriers, vitamins, mineral substances, phytogenetic substances, enzymes, and additional components for the detoxification of mycotoxins like mycotoxin-degrading enzymes, especially aflatoxin-oxidases, ergotamine hydrolases, ergotamine amidases, zearalenone esterases, zearalenone lactonases, zearalenone hydrolases, ochratoxin amidases, fumonisin aminotransferases, fumonisin carboxyltransferases, amino polyol amine oxidases, deoxynivalenol epoxide hydrolases, deoxynivalenol dehydrogenases, deoxynivalenol oxidases, trichothecene dehydrogenases, trichothecene oxidases; and mycotoxin-transforming microorganisms such as DSM 11798; and mycotoxin-binding substances such as microbial cell walls or inorganic materials like bentonite.

20. Additive according to one of the Claims 14 to 19 characterised in that it is present in an encapsulated or coated form.

21. Use of at least one additive according to one of the Claims 14 to 20 for the production of a compound for the prevention and / or treatment of trichothecene mycotoxicoses, preferably of mycotoxicoses caused by trichothecenes that exhibit a hydroxyl group on the C-3 atom, especially such as deoxynivalenol mycotoxicoses.