Use of alcohol dehydrogenase for converting trichothecenes, method for converting trichothecenes and additive for converting trichothecenes

Abstract

Use of an alcohol dehydrogenase of SEQ ID No. 1 containing a metal ion and a quinone cofactor, or a functional variant thereof showing at least 80 %, preferably at least 86 %, especially preferably at least 89 % sequence identity and at least one redox cofactor, for the conversion of at least one trichothecene compound presenting a hydroxyl group at the C-3 atom, as well as methods for the enzymatic conversion of trichothecene compounds and trichothecene conversion additives.

Description

[0001] This invention relates to the use of trichothecene-transforming alcohol dehydrogenase, methods for transforming trichothecene compounds, and additives for transforming trichothecene.

[0002] Trichothecene compounds represent a frequently occurring class of fungal toxins, including in particular deoxyfusarium oleoresin (DON, CAS No. 51481-10-8), T-2 toxin (CAS No. 21259-20-1), HT-2 toxin (CAS No. 26934-87-2), fuseranon-X (CAS No. 23282-20-4), and funeranon-X (CAS No. 21481-10-8). 23255-69-8), purstilbene triol, 15-acetoxypurstilbene alcohol (CAS No. 2623-22-5), 4,15-diacetoxypurstilbene alcohol (CAS No. 2270-40-8), Trichoderma alcohol (CAS No. 2198-93-8), Verrucosporin A (CAS No. 3148-09-2), Verrucosporin J (CAS No. 4643-58-7) ), isotrichol (CAS No. 91423-90-4), hydroxyisotrichol (CAS No. 344781-02-8), calonectrin (CAS No. 38818-51-8), T-2 tetraol (CAS No. 34114-99-3), deacetylated neotrichol (CAS No. 74833-39-9), neotrichol (CAS No. 36519-25-2), acetylated neotrichol (CAS No. 65041-92-1), sporotrichiol (CAS No. 101401-89-2), trichotriol (CAS No. 109890-37-1), elderberry fumigin (CAS No. 90044-33-0), and fumigin (CAS No. 18374-83-9). Trichothecene compounds, particularly DON, also known as vomitoxin, can be produced by various Fusarium fungi, especially *F. graminearum* and *F. culmorum*. These fungi infect crops such as maize, various grains such as wheat, oats, or barley. Fungal infection usually occurs before harvest, but fungal growth or mycotoxin formation can also occur before harvest or after harvest if improperly stored.

[0003] The Food and Agriculture Organization (FAO) estimates that 25% of global agricultural products are contaminated with mycotoxins, resulting in significant economic losses. In a more recent worldwide 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. Of these, 81% of the tests were positive for at least one mycotoxin, and 59% were positive for trichothecenes, particularly DON. Trichothecenes, especially DON, can be found with a frequency of up to 100% in all regions of the world, and in all tested cereals and feeds such as maize, soybean meal, wheat, wheat bran, DDGS (distilled grains with solubles), and in prepared feed mixtures. In addition to basic, unprocessed ingredients, evidence of trichothecene compounds has also been found in processed foods such as flour, breakfast cereals, pasta products, bread, pasta, and wheat-based children's and baby foods.

[0004] Trichothecene compounds have the following structural formulas:

[0005]

[0006] The different substitution residues R1 to R5 vary depending on the type of trichothecene. It is known that, in addition to the epoxy group, the intact α-hydroxyl group at the C-3 atom of trichothecene compounds is responsible for their toxic effects. Types of trichothecenes with a hydroxyl group at the C-3 atom include deoxycucurbitacin, T-2 toxin, HT-2 toxin, cucurbitacin, fuseranon-X, 15-acetoxycucurbitacinol, 4,15-diacetoxycucurbitacinol, trichothecinol, T-2 tetraol, deacetylated neosolacritinol, acetylated neosolacritinol, sportrichiol, trichotriol, elderberry fusarium, and fusarium macrosporum.

[0007] Deoxycucurbitacin (DON) has a characteristic carbonyl group at C-8 and the following structural formula:

[0008]

[0009] And the IUPAC name (3α,7α)-3,7,15-trihydroxy-12,13-epoxytrichosporon-9-en-8-one. In nature, several toxic DON isoforms also exist with a hydroxyl group at the C-3 atom. Examples of these are acetylated DON (e.g., 15-acyl DON), glycosylated DON, sulfonic acid DON (e.g., DONS-1, DONS-2), or sulfated DON (DON sulfate 15). These DON isoforms also belong to the trichosporine type with a hydroxyl group or a substituted hydroxyl group at the C-3 atom.

[0010] Due to the toxicity of DON, food and feed authorities have established limits or maximum levels. Therefore, the European Union has regulated the DON content in food (EC No. 1881 / 2006, EC No. 1126 / 2007) and has recommended maximum levels in feed (2006 / 576 / EC). In the United States, the FDA has published maximum levels.

[0011] Diseases caused by the ingestion of fungal toxins in humans or animals are called fungal poisoning. In the case of trichothecene compounds or types of trichothecene, these diseases are also called "trichothecene poisoning," more specifically "poisoning caused by trichothecene compounds exhibiting a hydroxyl group at the C-3 atom," or even more specifically "DON fungal poisoning." It is a well-known fact that the toxic effects of trichothecene compounds on animals and humans are based on several factors. These factors include inhibition of protein biosynthesis, possible interaction between serotonin and dopamine receptors, and upregulation of pro-inflammatory cytokines (EFSA Journal 2004, 73, 1-41). In addition, DON fungal poisoning causes changes in biomarkers, such as those diagnosed by: increased blood IgA concentrations, increased SOCS3 concentrations in the liver, or decreased plasma IGFALS levels (Pestka et al., 2004, Toxicol. Lett. 153, 61-73), and decreased concentrations of tight junction protein (claudin) in the intestine (Pinton et al., 2009, Tox. Appl. Pharmacol. 237, 41-48).

[0012] For example, trichothecene poisoning in pigs manifests as reduced feed intake, decreased growth, vomiting and diarrhea, as well as impaired intestinal immune function and impaired nutrient absorption. In poultry, trichothecene poisoning causes, in particular, deterioration of feed intake, less weight gain, morbidity of diarrhea, and reduced eggshell weight. In ruminants, reduced feed intake and reduced milk production have been described. In aquaculture, trichothecene poisoning causes, in particular, deterioration of feed intake and growth rate in fish (such as salmon, catfish, or trout) and shrimp (Binder et al., Guide to Mykotoxins; ISBN 978-0-9573721-0-8). Toxic effects have also been described in dogs and cats (EFSA Journal 2004, 73, 1-41). In humans, trichothecene poisoning can cause, in particular, nausea, vomiting, diarrhea, abdominal pain, headache, or fever (Sobrova et al., Interdisc. Toxicol. 2010, 3(3), 94-99).

[0013] The primary strategy for reducing trichothecene or DON contamination in food or feed is, for example, to limit fungal invasion by complying with Good Agricultural Practices (GAP) guidelines. This includes using seeds free of parasites and fungi, or deep plowing with crop residues. Additionally, fungal growth in the field can be reduced through the proper use of fungicides. After harvest, crops should be stored at residual moisture levels below 15% and at low temperatures to prevent fungal growth. Similarly, crops contaminated with fungi should be removed before any further treatment. Despite these measures, I. Rodriges and K. Naehrer reported (2012) that even in regions with the highest agricultural standards, such as the USA and Central Europe, DON contamination was found in 79% or 72% of all maize samples tested between 2009 and 2011.

[0014] Other options for reducing mycotoxin contamination in food or feed include adsorption or conversion. For adsorption, it is essential that the binding of the mycotoxin to the adsorbent is strong and specific over a wide pH range, and that it remains stable in the gastrointestinal tract throughout digestion. While some non-biosorbents, such as activated carbon, silicates, or esters, or synthetic polymers such as cholestyramine, are effective against aflatoxins, they are ineffective against other mycotoxins, particularly trichothecenes. Biosorbents, such as yeast or yeast extracts, are also described in the literature, but have similar limitations to non-biosorbents. A significant drawback of adsorbents is that they may non-specifically bind to other molecules that may be nutritionally necessary.

[0015] Similarly, the conversion of trichothecene through physical and chemical treatment, especially detoxification, is limited because DON is very stable and remains stable even under heat treatment up to 350°C.

[0016] Possible microbial transformation of DON is described in EP-B 1 042 449, where, according to the aforementioned literature, microorganism BBSH 797 (DSM 11798) is used for the detoxification of DON. This detoxification is based on the opening of the epoxide rings on the C-12 and C-13 atoms of DON. US2012 / 0263827 A describes the bioconversion of DON to 3-epi-DON by a microorganism with International Canadian Registry Number 040408-1. However, for many technical feed or food processing methods, mixtures of microorganisms or adsorbents are not feasible or legally permitted, making the transformation or detoxification of trichothecene compounds such as DON or DON subtypes impossible.

[0017] Trichothecene compounds, such as DON and its subtypes, are rapidly absorbed into the gastrointestinal tract of humans or animals, which is why rapid and targeted detoxification is important.

[0018] JP-A 2003 / 159079 first describes the alcohol dehydrogenase of SEQ ID No. 1 for the production of 2-ketogulonic acid. WO 2009 / 133464 describes, particularly in the baking industry, a method for slowing down the aging processes of bread by oxidizing sugars with the enzyme of SEQ ID No. 1 used in food and feed for the oxidation of starch. Here, the alcohol dehydrogenase is used for the oxidation of the hydroxyl groups of carbohydrates.

[0019] Alcohol dehydrogenases with SEQ ID numbers 2 and 3 were identified during genome sequencing of microorganisms of the genus *Devosia* sp. and are stored online on the servers of the National Center for Biotechnology Information (NCBI) under identification numbers GI: 737041022 and GI: 630002266. More precise characterization of the alcohol dehydrogenases with SEQ ID numbers 2 and 3 is not provided in this work.

[0020] Because of the various toxic effects and frequency of trichothecene compounds, there is a need for substances or groups of substances-like enzymes that can be used for the specific, safe, and permissible transformation (especially detoxification) of trichothecene compounds.

[0021] The object of the present invention is to convert at least one trichothecene with a hydroxyl group at C-3 atom into a less toxic product using specific alcohol dehydrogenases and their variants.

[0022] To address the aforementioned task, it has been surprisingly demonstrated that using the alcohol dehydrogenase of SEQ ID No. 1 containing metal ions and quinone cofactors, or other functional variants exhibiting at least 80%, preferably 86%, particularly preferably at least 89% sequence identity and at least one redox cofactor for the conversion of at least one trichothecene with a hydroxyl group at C-3 atom, can specifically and reliably convert trichothecene compounds with a hydroxyl group at C-3 atom, such as DON, T-2 toxin, or Fusarium quinacrine.

[0023] Transformation is understood to occur when the structure of a toxin changes, preferably transforming it into a non-toxic or less toxic metabolite. In the present case, the structural change occurs, particularly at the C-3 atom of trichothecenes exhibiting a hydroxyl group, due to the catalytic conversion of the C-3 hydroxyl group to a ketone group. Surprisingly, according to the invention, using alcohol dehydrogenases, the transformation of trichothecene compounds, especially DON, exhibiting a hydroxyl group at the C-3 atom is achieved in the most diverse chemical and biological environments (e.g., in buffer solutions, feed mash, saliva, or feed-containing gastric juice or feed-containing intestinal contents). This is remarkable because important parameters, such as pH, protease concentration, ionic strength, or substrate, vary considerably in different environments where enzyme activity occurs. As a result, enzyme activity can be maintained from the addition of water to food and feed, to oral ingestion, and in the oral cavity and gastrointestinal tract. Surprisingly, for some environments, the external addition of redox factors can be omitted; this is particularly applicable to feed mixtures, saliva, and gastric juice.

[0024] The alcohol dehydrogenase in SEQ ID No. 1 is a quinone cofactor-dependent alcohol dehydrogenase. To produce an active holoenzyme or an active alcohol dehydrogenase, a metal ion (preferably calcium ion (Ca) is present. 2+ In the presence of [a specific ingredient], a quinone cofactor (preferably pyrroloquinoline quinone (PCC)) can bind to the enzyme. Therefore, the activated alcohol dehydrogenase comprises a quinone cofactor and a metal ion, wherein the molar ratio of enzyme to quinone cofactor is 1:1. Furthermore, the catalytic activity of the alcohol dehydrogenase also requires a redox cofactor, wherein the redox cofactor is used in the form of a synthetically produced redox factor in addition to activating the alcohol dehydrogenase, or redox factors also present in food or feed and in animal or human secretions may also be used. For example, these naturally occurring redox cofactors can be formed in the mouth and gastrointestinal tract of humans or animals during the provision, processing, or digestion of food or feed, and these naturally occurring redox cofactors can be extracted from food or feed if desired. Examples of human or animal secretions containing such naturally occurring redox cofactors are digestive secretions such as saliva, gastric juice, intestinal juice, pancreatic juice, bile, or rumen fluid.

[0025] The term "peptide variant" or "variant" refers to a functional polypeptide with at least amino acid substitutions compared to SEQ ID No. 1, wherein the enzyme's function is retained. Transformation, particularly the oxidation of the hydroxyl group at the C-3 atom of a trichothecene compound to a ketone group, is understood as enzyme function. Furthermore, a "peptide variant" may also have amino acid insertions or deletions, particularly sequences that are extended or shortened at the C or N terminus relative to the polypeptide sequence of SEQ ID No. 1. The enzyme function is "substantially retained" if the enzymatic reaction mechanism remains unchanged; that is, the trichothecene is oxidized at the same position, and the variant's enzyme activity is at least 10%, preferably at least 50%, more preferably at least 90%, and especially >100%, based on the original, parental polypeptide of SEQ ID No. 1.

[0026] The term "sequence identity" refers to the percentage of sequence identity. For amino acid and nucleotide sequences, sequence identity can be visually determined, but is preferably calculated by a computer program. The amino acid sequence of SEQ ID No. 1 is defined as the reference sequence. Sequence comparisons are also performed within sequence segments, in which case the segment is understood as a contiguous sequence of the reference sequence. The length of a peptide sequence segment is typically 3 to 200 amino acids, preferably 15 to 65, and most preferably 30 to 50 amino acids. Many commercially available or free bioinformatics programs exist for determining homology and are being further refined. Examples include the GCG Wisconsin BestFit package (Devereux et al., 1984), BLAST (Altschul et al., 1990), or BLAST 2 (Tatusova and Madden 1999). Because of the different setting options for these algorithms, it is possible to obtain different results for the same input sequence. Therefore, the search algorithm and associated settings must be defined. In the current context, the NCBI BLAST (Basic Local Alignment Search Tool) program, specifically BLASTP for peptides (available from the National Center for Biotechnology Information homepage (NCBI, HTTP: / / www.ncbi.nlm.nih.gov / )), is used to calculate sequence identity. In this way, it is possible to compare two or more sequences according to the algorithm of Altschul et al., 1997 (Nucleic Acids Res., 25: 3389-3402). Here, the version of the program from August 12, 2014, is used. Basic settings are used for program settings, especially for amino acid comparison: "Maximum target sequence" = 100; "Expected threshold" = 10; "Word length" = 3; "Matrix" = BLOSOM62; "Gap costs" = "Existence: 11; Extension: 1"; "Calculation adjustment" = "Conditional component score matrix adjustment".

[0027] By using an alcohol dehydrogenase containing a metal ion and a quinone cofactor or a functional variant thereof according to the invention, at least 20%, preferably at least 50%, particularly at least 90% of at least one trichothecene presenting a hydroxyl group at the C-3 atom, especially DON, can be converted, wherein sufficiently, the alcohol dehydrogenase containing a metal ion and a quinone cofactor or a functional variant thereof is contacted with at least one trichothecene presenting a hydroxyl group at the C-3 atom for at least one minute, preferably at least 5 minutes, particularly at least 60 minutes.

[0028] According to a further embodiment of the invention, an amino acid sequence of a functional variant selected from the group consisting of SEQ ID Nos. 2 to 4 is used. Employing these functional variants having at least 86% sequence identity with the alcohol dehydrogenase of SEQ ID No. 1, it is possible to convert trichothecene compounds, particularly DON, which exhibit consistently good effects, by presenting a hydroxyl group at the C-3 atom.

[0029] According to a further embodiment of the invention, a quinone cofactor selected from the group consisting of PCC, TTC, TPC, LTC, and CTC is used, preferably PCC. By using one of the quinone cofactors—pyrroloquinoline quinone (PCC, CAS No. 72909-34-3), tryptophan tryptophan quinone (TTQ, CAS No. 134645-25-3), topaquinone (TPC, CAS No. 64192-68-3), lysine tyrosine quinone (LTQ, CAS No. 178989-72-5), or cysteine ​​tryptophan quinone (CTC, CAS No. 400616-72-0)—on an alcohol dehydrogenase, it is possible to convert trichothecene compounds (e.g., DON) exhibiting a hydroxyl atom at C-3 into derivatives that are non-toxic or harmless from a toxicological perspective.

[0030] Through 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+ With Co 3+ At least one of the metal ions in the group, preferably Ca 2+ and Mg 2+ The combination enables a particularly rapid and complete binding of the quinone cofactor to the alcohol dehydrogenase.

[0031] Also by using at least one redox cofactor selected from the group consisting of phenazine methosulphate (PMS), PMS derivatives, potassium hexacyanoferrate(III), sodium hexacyanoferrate(III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), preferably phenazine methosulphate (PMS, CAS No.: 299-11-6), coenzyme Q1, and coenzyme Q10, complete and rapid conversion of the trichothecene compounds may be carried out only in the presence of moisture, to ensure, for example, during feed production and in any case before use in animals, the conversion of trichothecene compounds contained in feed components into non-toxic derivatives. Examples of PMS derivatives are: 1-hydroxyphenazine, 2-(pentapentadienyloxy)dihydrophenazine, 5,10-dihydro-9-dimethylallylphenazine-1-carboxylic acid, 5,10-dihydrophenazine-1-carboxylic acid, 5-methylphenazine sulfate, 6-acetylphenazine-1-carboxylic acid, bethophoenin, chlorofazimine, dihydromethanophenazine, esmeraldic acid, esmeraldin B, izumiphenazine AC, Janus Green B cation, methanophenazine pelagiomicin A, phenazine, phenazine-1,6-dicarboxylic acid, phenazine-1-carboxamide, phenosafranine, pyocyanin, saphenamycin, or methyl saphenic acid. Due to the transformation of trichothecene compounds exhibiting a hydroxyl group at the C-3 atom in food and feed (particularly for pigs, poultry, cattle, horses, fish, aquaculture, and livestock feed) and in plant-based raw materials used in the production or processing of food and feed, it is possible to prevent harm to animal and human health through the use according to the invention.

[0032] Furthermore, the present invention aims to provide a method for safely and reliably converting trichothecene compounds (especially trichothecene compounds with a hydroxyl group at the C-3 atom) into products with lower toxicity, regardless of whether the agricultural products containing trichothecene compounds have been treated or not.

[0033] To address this problem, the essential feature of the method for the enzymatic conversion of trichothecene compounds according to the present invention is that at least one trichothecene exhibiting a hydroxyl group at the C-3 atom is contacted with an alcohol dehydrogenase of SEQ ID No. 1 containing a metal ion and a quinone cofactor, or additionally with a functional variant having at least 80%, preferably at least 86%, particularly preferably at least 89% sequence identity, contacted with at least one redox cofactor and water, and, if necessary, with at least one excipient. By contacting the trichothecene exhibiting a hydroxyl group at the C-3 atom with the alcohol dehydrogenase of SEQ ID No. 1 containing a metal ion and a quinone cofactor, and further with at least one redox cofactor and water, it is possible to oxidize the hydroxyl group present at the C-3 atom of the trichothecene compound to a ketone, in which case the trichothecene is thus detoxified and converted into a non-toxic or low-toxic compound.

[0034] By continuously using functional variants of the amino acid sequence selected from the group consisting of SEQ ID No. 2 to 4, instead of the amino acid sequence of SEQ ID No. 1, the same advantages achieved by using the alcohol dehydrogenase of SEQ ID No. 1 can be realized, and the conversion of trichothecene compounds contained in food and feed can be achieved particularly rapidly and reliably, regardless of their treatment status, i.e., whether they are already processed agricultural products or not.

[0035] The particularly rapid and complete conversion of trichothecenes exhibiting a hydroxyl group at C-3 atom is achieved at temperatures between 5°C and 55°C, preferably between 10°C and 50°C, and particularly preferably between 28°C and 45°C, using the method according to the invention. Because the method according to the invention can be performed over such a wide temperature range, the alcohol dehydrogenase of SEQ ID No. 1 or a functional variant thereof exhibiting at least 80% of the sequence of SEQ ID No. 1 can be used for a variety of different applications, such as aquaculture or also for technological processes at elevated temperatures. Examples of technological processes where the conversion of trichothecene compounds at elevated temperatures is important are processes for processing feed, producing pasta and other corn products such as corn porridge, popcorn, corn flakes, tortillas, or unfermented tortillas, as well as intensive liquefaction processes, saccharification processes, or fermentation processes, such as crushing or fermentation processes, particularly bioethanol production. It is important here to ensure that the food or feed produced by these methods does not contain any harmful amounts of trichothecene compounds exhibiting a hydroxyl group at C-3 atom.

[0036] A further embodiment of the method according to the invention is carried out by contacting at least one trichothecene exhibiting a hydroxyl group at C-3 atom with an alcohol dehydrogenase or at least its functional variant containing a metal ion and a quinone cofactor, a redox factor, water, and, if necessary, an excipient, for at least one minute, preferably at least five minutes, particularly preferably at least sixty minutes. Because a contact time of one minute to more than sixty minutes is sufficient to fully convert trichothecene compounds into non-toxic or low-toxic derivatives, the method according to the invention can be used, for example, for treating basic agricultural materials used in food or feed. Alternatively, it can be applied by farmers immediately before feeding, for example, by adding water to the feed and allowing it to stand at a temperature between 5°C and 55°C for one minute to up to about one hour, which will initiate the conversion of trichothecene compounds into non-toxic products.

[0037] If the quinone cofactor is selected from the group consisting of PCC, TTC, PTC, LTC, and CTC, preferably PCC, then particularly rapid and complete conversion is possible, as this corresponds to another embodiment of the method according to the invention. Such a quinone cofactor allows alcohol dehydrogenases to rapidly and reliably attack the hydroxyl group on the C-3 atom of trichothecene compounds and convert it into a non-toxic derivative containing a ketone group.

[0038] If the cofactor in the method according to the invention is 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+ With Co 3+ At least one of the metal ions in the group, preferably Ca 2+ and Mg 2+ By binding to alcohol dehydrogenase, further completion of the reaction, and particularly its acceleration, becomes possible. Performing this method in this manner not only generates a strong binding of the quinone cofactor to the alcohol dehydrogenase but also enables rapid and reliable conversion of trichothecene compounds.

[0039] To further improve the conversion of trichothecenes, and particularly for the completion of the conversion reaction, the method according to the invention is continued, thereby using a redox factor selected from the group consisting of PMS, PMS derivatives, potassium hexaferrate(III), sodium hexaferrate(III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue, and TMPD, preferably a redox factor selected from PMS, coenzyme Q1, and coenzyme Q10. By adding such a redox cofactor, the conversion of trichothecene compounds exhibiting a hydroxyl group at C-3 atom in an aqueous medium can be performed, for example, in a redox-free feed slurry or feed administered to aquaculture animals. This redox factor can be obtained, for example, from saliva, gastric juice, or intestinal juice that must be added or present, or in a feed slurry or feed administered to animals that have already ingested the feed slurry or feed, in which case the absorption of trichothecene compounds by the ingesting animals can be prevented.

[0040] Finally, the object of the present invention is to provide a trichothecene conversion additive, which can be used to safely and reliably convert trichothecene compounds into non-toxic derivatives in feed or food.

[0041] To address this issue, the essential characteristic of the additive according to the invention is that it comprises an alcohol dehydrogenase of SEQ ID No. 1 containing a metal ion and a quinone cofactor, or further comprises a functional variant exhibiting at least 80%, preferably at least 86%, particularly preferably at least 89% sequence identity, and, if necessary, at least one additional component selected from the group consisting of a synthetic redox cofactor and at least one excipient. Such an additive can be mixed with conventional feed at low concentrations, for example, about 10 grams to 1 kilogram per ton of feed, and at such low concentrations, allows trichothecene compounds exhibiting a hydroxyl group at C-3 atom to be converted into non-toxic derivatives, thereby improving, for example, the health and behavioral performance of animals fed with this feed, thus reducing not only failure rates but also improving feed utilization.

[0042] Consistently good results can be achieved by using the additive according to the invention (containing a functional variant of the alcohol dehydrogenase selected from SEQ ID No. 2 to 4 of SEQ ID No. 1, but not containing the alcohol dehydrogenase of SEQ ID No. 1).

[0043] In order to substantially completely convert the hydroxyl groups present at the C-3 atom of trichothecene compounds by means of the additive according to the invention, the additive further embodies the presence of a quinone cofactor selected from the group consisting of PCC, TTC, TPC, LTC, and CTC, and a cofactor 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+ With Co 3+ The group consists of metal ions. With such a further implementation, on the one hand, it is possible to safely and reliably bind quinone cofactors to alcohol dehydrogenases, and on the other hand, using alcohol dehydrogenases containing such supplements, it is possible to achieve the complete conversion of trichothecene compounds, such as deoxycucurbitacinol, which exhibit a hydroxyl group at the C-3 atom of the molecule.

[0044] In order to carry out such a reaction in the absence of redox cofactors (such as those naturally present in saliva, gastric juice, or intestinal juice, etc.), the additive according to the invention is further characterized in that, in addition to being a further redox cofactor, the synthetic redox cofactor is selected from 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.

[0045] According to a further embodiment of the invention, an additive has been developed such that the excipients are selected from inert carriers, vitamins, minerals, plant-derived substances, enzymes, and additional components for the detoxification of mycotoxins, such as enzymes that degrade mycotoxins, especially aflatoxin oxidase, ergotamine hydrolase, ergotamine amidase, zearalenone esterase, zearalenone lactone lactonease, zearalenone hydrolase, ochratoxin amidase, fumonisin aminotransferase, fumonisin carboxyltransferase, aminopolyolamine oxidase, deoxyfuricinol epoxide hydrolase, deoxyfuricinol dehydrogenase, deoxyfuricinol oxidase, trichothecene dehydrogenase, trichothecene oxidase; and microorganisms that transform mycotoxins, such as DSM 11798; and mycotoxin binding substances such as microbial cell walls or inorganic materials, such as bentonite or montmorillonite. For example, the use of such additives can ensure that any amount of trichothecene compounds with hydroxyl groups at the C-3 atom that may be included in feed or food, as well as any additional mycotoxins such as Fusarium toxin, ergotamine, ochratoxin, are definitively detoxified to the point that no mycotoxins have any harmful effects on the individual organisms that ingest such feed or food.

[0046] A further application of the present invention is that, in addition to at least one alcohol dehydrogenase according to the present invention, it contains an additive containing at least one enzyme involved in protein breakdown (e.g., protease) or an enzyme involved in starch or fiber or fat or glycogen metabolism (e.g., amylase, cellulase or glucanase, and, for example, hydrolases, lipases, mannosidases, oxidases, oxidoreductases, phytases or xylanases).

[0047] It goes without saying that additives can certainly exist in encapsulated or coated forms, in which case standard methods, such as those described in WO 92 / 12645, can be used. Encapsulation or coating allows the additive to be delivered to its place of use without modification, and particularly without any degradation or damage, so that the polypeptide only begins to function after the outer shell dissolves, for example, in the animal's digestive tract. This allows for even more targeted, faster, and more complete destruction of trichothecene compounds exhibiting a hydroxyl group at the C-3 atom, even in acidic, protease-rich, and anaerobic environments. Furthermore, encapsulation or coating can also improve the temperature stability of alcohol dehydrogenases in the additive, thus improving its use, for example, in feed pelleting processes.

[0048] The additives according to the invention can be used in a wide variety of applications, such as in the production of compounds, to prevent and / or treat trichothecene fungal poisoning, preferably fungal poisoning caused by trichothecene compounds exhibiting a hydroxyl group at the C-3 atom, particularly fungal poisoning such as deoxycucurbitacin. Such fungal poisoning has serious consequences for humans and animals. By using the additives as described in the invention, in the preventive case, even with oral ingestion of trichothecene compounds (especially trichothecene compounds exhibiting a hydroxyl group at the C-3 atom, particularly deoxycucurbitacin), it is possible to maintain the health status of humans and animals at substantially the same level as that obtained with no or reduced toxins. In the case of treatment of fungal poisoning, it is possible to alleviate the symptoms of this disease, particularly by normalizing SOCS3 concentrations in the liver or IGFALS levels in plasma and claudin concentrations in the intestine.

[0049] Furthermore, it is possible to improve livestock productivity, particularly feed utilization and weight gain, and reduce mortality through this use of the invention.

[0050] The invention is explained below based on the implementation scheme and accompanying drawings. Here:

[0051] Figure 1 The time-series transformation of deoxy-fusarium alcohol against activated SEQ ID No. 1 and as a comparative test (CTR) is shown, and

[0052] Figure 2 The diagram shows the temporal sequence of conversions of activated alcohol dehydrogenases using SEQ ID No. 1 to 4 and DON as a comparative test (CTR).

[0053] Example 1: Cloning and purification of the gene for alcohol dehydrogenase

[0054] The codon-optimized nucleotide sequences of the alcohol dehydrogenases SEQ ID numbers 1 to 4 for each host cell were derived from DNA 2.0 and contain restriction sites at the 5' and 3' ends of the sequence at the nucleic acid level, and also contain a C- or N-terminal 6xHis tag at the amino acid level. These nucleotide sequences were integrated into expression vectors for expression in *Escherichia coli* or *Komagataella pastoris* using standard methods, transformed into *E. coli* or *Komagataella* yeast, and expressed in *E. coli* or *Komagataella* yeast (JMCregg, Pichia Protocols, 2nd Edition, ISBN-10: 1588294293, 2007; J. Sambrook et al., 2012, *Molecular Cloning: A Laboratory Manual 4th Edition*, Cold Spring Harbor).

[0055] Alcohol dehydrogenases having SEQ ID Nos. 1 to 4 were selectively enhanced by chromatography via standard methods using a nickel agarose column, in the case of expression in *E. coli* and intercellular expression in *K. yeast*, or in the case of extracellular expression in *K. yeast*, from cell lysates. The selectively enhanced eluent was incubated and activated in the presence of a metal ion and a quinone cofactor, where “activation” refers to the alcohol dehydrogenase exhibiting both the bound metal ion and the quinone cofactor. These activated alcohol dehydrogenases were used to determine the enzymatic properties of the alcohol dehydrogenases having SEQ ID Nos. 1 to 4 in Examples 3 to 7 below. Total protein concentrations were determined by photometry using Bradford reagent (Sigma#B6916), and in this case, absorbance was measured at a wavelength of 595 nm in a microplate photometer (plate reader, Baxter, Synergy HT). Protein concentrations were determined based on calibration curves. The calibration curves were established using the Bradford assay by measuring bovine serum albumin (BSA, Sigma#A4919) solutions with concentrations up to 1500 μg / mL.

[0056] Example 2: Determination of sequence identity

[0057] Using the BLAST program (Basic Local Alignment Search Tool), particularly BLASTP, the percentage of sequence identity relative to the full-length amino acid sequences of alcohol dehydrogenases having SEQ ID numbers 1-4 was determined. The BLAST program (Basic Local Alignment Search Tool), particularly BLASTP, is available from the homepage of the National Center for Biotechnology Information (NCBI; http: / / www.ncbi.nlm.nih.gov / ). The BLAST program (Basic Local Alignment Search Tool), particularly BLASTP, is capable of comparing two or more sequences according to the algorithm of Altschul et al. 1997 (Nucleic Acids Res. (1997) 25: 3389-3402). Basic settings were used as program settings, specifically: "Maximum Target Sequence" = 100; "Expected Threshold" = 10; "Word Length" = 3; "Matrix" = BLOSOM62; "Gap Cost" = "Presence: 11; Extension: 1"; "Calculation Adjustment" = "Conditional Component Score Matrix Adjustment". The percentage of amino acid sequence identity between them is shown in Table 1:

[0058] Table 1:

[0059] 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％

[0060] Example 3: Transformation of trichothecene with a hydroxyl group at C-3 atom

[0061] To determine whether alcohol dehydrogenases with SEQ ID numbers 1-4 are suitable for converting trichothecene compounds (especially DON, cucurbitacin, and T-2 toxin) with a hydroxyl group at the C-3 atom, alcohol dehydrogenases with SEQ ID numbers 1-4 were prepared in Escherichia coli with a C-terminal 6xHis tag, as described in Example 1.

[0062] Transformation occurs when the amount of trichothecene with a hydroxyl group at C-3 is reduced by contacting an activated alcohol dehydrogenase (i.e., an alcohol dehydrogenase containing a metal ion and a quinone cofactor) with trichothecene that has a hydroxyl group at C-3.

[0063] In each case, 100 mL of *E. coli* culture with an optical density (OD) of 2.0–2.5 (600 nm) was harvested by centrifugation at 4 °C and resuspended in 20 mL of potassium phosphate buffer. The cell suspension was lysed three times using a French press at 20,000 psi. The cell lysates were separated into soluble and insoluble fractions by centrifugation. The supernatant was sterilely filtered and enhanced with alcohol dehydrogenase via a standard method using a nickel agarose column. Buffer exchange was then performed by dialysis using a specific tube with a cutoff of 10 kDa. The resulting total protein concentration was determined using the Bradford assay.

[0064] In an aqueous solution, quinone cofactors and metal ions are incubated to bind to alcohol dehydrogenase. Here, quinone cofactors, such as pyrroloquinolinequinone (PCC, CAS No. 72909-34-3), tryptophan tryptophan quinone (TTC, CAS No. 134645-25-3), topaquinone (TPC, CAS No. 64192-68-3), lysine tyrosine quinone (LTC, CAS No. 178989-72-5), and cysteine ​​tryptophan quinone (CTC, CAS No. 400616-72-0), are added as an aqueous solution in approximately a 20-fold molar excess to the existing total protein concentration. (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+ With Co 3+ The metal ions are used in the form of their salts in aqueous solution. Unless otherwise stated, alcohol dehydrogenases are generally used with PCC (Sigma-Aldrich #D7783) and Ca. 2+ Together, it was activated to a 5 mM CaCl2 solution. The enzyme purified and activated in this manner was used for the in vitro conversion assay of trichothecenes exhibiting a hydroxyl group at C-3. Unless otherwise stated, the term "enzyme" or "alcohol dehydrogenase" is always understood to refer to a properly activated alcohol dehydrogenase containing a metal ion and a quinone cofactor.

[0065] The conversion assay was performed in an aqueous solution containing the following components: 100 mM Tris-HCl pH 7.5 or 10% Teorell Stenhagen pH 7.5; a synthetic redox cofactor selected from the following: 1 mM phenazine methylsulfate 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 sodium hexacyanoferrate (III) PFC(III) (Fluka #60300); 10 ppm to a maximum of 100 ppm of trichothecene with a hydroxyl group at C-3 atom, obtained by adding the desired amount of toxin substrate stock solution; and 10 nM to 100 nM, up to 300 nM of activated alcohol dehydrogenase containing metal ions and quinone cofactors of SEQ ID No. 1, 2, 3, or 4. Unless otherwise stated, Tris-HCl buffer, redox cofactor PMS, DON, and the alcohol dehydrogenase of SEQ ID No. 1 are typically used. Each conversion assay is performed in a 1.5 mL brown Eppendorf reaction vessel. The reaction mixture is incubated at 30 °C in a thermoblock for up to 120 min, with a minimum of 40 min. After 0, 10, 20, 30, and 40 min, 0.1 mL of sample is taken in each case, mixed with 0.1 mL of methanol, and stored at -20 °C, or alternatively, the sample is analyzed immediately by LC-MS / MS or HPLC.

[0066] A sterile filtered DON solution containing 2000 ppm water was used as the DON substrate stock solution. To produce this solution, crystalline DON (from Romer Labs' Biopure Standard, item number 001050, purity at least 98%) was weighed and dissolved.

[0067] To quantify trichothecenes exhibiting a hydroxyl group at C-3 and their metabolites, high-performance liquid chromatography (HPLC) analysis was performed, wherein the substances were separated by chromatography using a Phenomenex C18Gemini NX column with dimensions of 150 mm × 4.6 mm and a particle size of 5 μm. A methanol / water mixture with a concentration of 5 mM ammonium acetate was used as the eluent. UV signals were recorded and evaluated at 220 nm. For quantification by means of LC-MS / MS analysis, the substances were separated by chromatography using a Zorbax eclipse C8 column with dimensions of 150 mm × 4.6 mm and a particle size of 5 μm. A methanol / water mixture with a concentration of 5 mM ammonium acetate was used as the eluent. UV signals were recorded at 220 nm. Electrospray ionization (ESI) was used as the ionization source. Trichothecene compounds exhibiting hydroxyl groups at C-3 atom were quantified by QTrap / LC / MS / MS (triple quadrupole, applied biosystems) in "enhanced mode".

[0068] The negative slope of the conversion line (i.e., the decrease in toxin concentration over time) within the linear range is used as a standard for the activity of alcohol dehydrogenases. To determine residual activity, measured activities relative to the baseline activity, measured under standard conditions (particularly 30°C and pH 7.5), are applied and are typically expressed as a percentage. Figure 1 This illustrates the chronological conversion of DON against the activated alcohol dehydrogenase of SEQ ID No. 1, and Figure 2 The activated alcohol dehydrogenases of SEQ ID numbers 2-4 are shown. It is evident from the illustrations that DON conversion occurs because the concentration of DON decreases based on real-time.

[0069] Figure 1 The DON conversion using the alcohol dehydrogenase of SEQ ID No. 1 is shown in the presence of 50 ppm DON and 1 mM PMS in 100 mM Tris HCl at pH 7.5. Measurements were obtained by LC-MS / MS analysis, and the DON conversion using alcohol dehydrogenases of SEQ ID Nos. 1-4 is illustrated. Figure 2 The results were obtained by HPLC analysis. CTR was used as a negative test in the test, containing all components of the conversion assay, compared with alcohol dehydrogenases of SEQ ID numbers 1-4.

[0070] To compare the efficiency of quinone cofactors, in the conversion assay, quinone cofactors PCC, TTC, TPC, LTC, and CTC-activated 10 nM of SEQ ID No. 1 alcohol dehydrogenase, 10 ppm DON, and 1 mM synthetic redox factor PMS were each mixed in 100 mM Tris-HCl pH 7.5 and incubated at 30 °C. The DON concentration was determined by LC-MS / MS after 30 minutes. The results are shown in Table 2.

[0071] To compare the efficiency of redox cofactors, in the conversion assay, 10 nM of the activating enzyme (ethanol dehydrogenase of SEQ ID No. 1), 10 ppm DON, and 1 mM or 20 mM of the synthesized redox cofactor to be tested were mixed in 100 nM Tris-HCl pH 7.5 and incubated at 30 °C. The DON concentration was determined by LC-MS / MS after 30 minutes. The results are shown in Table 2.

[0072] Table 2:

[0073] Quinone cofactor DON[ppm] Redox cofactor DON[ppm] PCC 1.94 1mM PMS 1.95 TTQ 2.32 20mM PFC(III) 2.11 TPQ 2.41 1mM Coenzyme Q1 8.58 LTQ 2.04 1mM Methylene Blue 6.88

[0074] To test the effect of metal ions on transformation in activated enzymes, alcohol dehydrogenase and PCC of SEQ ID No. 1 were activated, but with different metal ions used in each case, namely, Mg. 2+ Ca 2+ Zn 2+ Mn 2+ Fe 2+ and Cu 2+ The conversion assay consisted of 10 nM activated alcohol dehydrogenase, 10 ppm DON, and 1 mM PMS, respectively, incubated at 30 °C. The DON concentration was determined by LC-MS / MS after 30 minutes. The results are shown in Table 3.

[0075] Table 3:

[0076] metal ions DON[ppm] metal ions DON[ppm] <![CDATA[Mg 2+ ]]> 1.90 <![CDATA[Mn 2+ ]]> 2.57 <![CDATA[Ca 2+ ]]> 1.98 <![CDATA[Fe 2+ ]]> 2.17 <![CDATA[Zn 2+ ]]> 2.46 <![CDATA[Cu 2+ ]]> 2.61

[0077] Similar to the DON conversion assay described above, other trichothecene compounds exhibiting a hydroxyl group at the C-3 atom were used for the conversion assay. In these assays, instead of 50 ppm DON, 50 ppm T-2 toxin or 50 ppm Fusarium quinolone was used. All four alcohol dehydrogenases of SEQ ID Nos. 1 to 4, containing metal ions and quinone cofactors, can also convert T-2 toxin and Fusarium quinolone; in this case, more than half of the initially used toxin was converted within 30 minutes.

[0078] Example 4: Measurement of active regions

[0079] To determine the ability of the alcohol dehydrogenases of SEQ ID Nos. 1-4 to convert DON under different conditions, the alcohol dehydrogenase of SEQ ID No. 1 was used as an example.

[0080] The alcohol dehydrogenase of SEQ ID No. 1 was prepared and activated with calcium ions and PCC as described in Example 3. To determine the enzyme activity in the temperature range of 10°C to 50°C and in the pH range of 3.0 to 9.0, 10% Teorell Stenhagen buffer was used instead of 100 mM Tris-HCl pH 7.5 buffer.

[0081] A conversion assay to determine activity at different temperatures was performed in an aqueous solution containing the following components: 10% Teorell Stenhagen pH 7.5, 1 mM of the synthetic redox cofactor PMS, 50 ppm of DON, and 10 nM of activated alcohol dehydrogenase (SEQ ID No. 1). The conversion assay was incubated for up to 60 minutes in a thermal cycler (Eppendorf) with a temperature gradient from 10°C to 50°C. After 0, 10, 20, 30, 40, and 60 minutes, 0.05 mL of sample was taken in each case, and the sample was mixed with 0.05 mL of methanol to terminate the reaction and stored at -20°C. The sample was prepared for LC-MS / MS as described in Example 3 and analyzed by LC-MS / MS. The DON reduction process and activity were determined for each temperature as described in Example 3. The slope of the linear range of the conversion line at 30°C was used as a reference to calculate the residual activity at other temperatures. Table 4 shows the temperatures in °C and the associated residual activities as a percentage. Surprisingly, it has been shown that the alcohol dehydrogenase of SEQ ID No. 1 is active over a wide temperature range. At 10 °C, 48% residual activity was measured, and at approximately 50 °C, 67% residual activity was measured.

[0082] Table 4:

[0083]

[0084]

[0085] The determination was carried out in an aqueous solution containing the following components. Conversion assay of activity within a pH range: 10% Teorell Stenhagen pH 4.0 to pH 10.0, 20 mM synthesized redox cofactor PFC, 100 ppm DON, and 20 nM activated alcohol dehydrogenase of SEQ ID No. 1. Conversion assay was performed by incubation in a thermal cycler at 30 °C for up to 60 min. After 0, 10, 20, 30, 40, and 60 min, 0.05 mL of sample was taken in each case and the sample was mixed with 0.05 mL of methanol to terminate the reaction and stored at -20 °C. The sample was diluted and analyzed by LC-MS / MS as described in Example 3. The DON reduction process and activity were determined at each pH and calculated as described in Example 3. The slope of the linear range of the conversion line at pH 7.5 was used as a reference to calculate the residual activity at other temperatures. Table 5 shows the pH values ​​and associated residual activities (DON reduction based on a reference pH of 7.5).

[0086] Table 5:

[0087] 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％

[0088] Example 5: Determination of temperature stability

[0089] The temperature stability of the alcohol dehydrogenase of SEQ ID No. 1 was determined within the range of 30°C to 55°C. To do this, the activated alcohol dehydrogenase was incubated in 100 nM Tris-HCl buffer, pH 7.5, at a specific temperature for up to 60 minutes in a thermal cycler (Eppendorf). After 0, 5, 10, 15, 20, 30, 40, and 60 minutes, aliquots of the alcohol dehydrogenase were taken and their activity was determined in a DON conversion assay as described in Example 3. The conversion assay comprised 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 incubation reaction was performed as described in Example 3, and samples were taken after 0, 10, 20, 30, 40, and 60 minutes to determine activity. The DON reduction process was determined for each temperature and each incubation time. The slope of the linear range of the DON conversion line was calculated to determine temperature stability. The slope of the linear range of the DON conversion line at t=0 minutes for each temperature was used as a reference for calculating residual activity. Table 6 shows the temperature in °C, incubation time in minutes, and associated residual activity as a percentage. The alcohol dehydrogenase of SEQ ID No. 1 was most stable when stored at 30 °C and 37 °C for one hour. In contrast, the alcohol dehydrogenase still had 73% residual activity at 40 °C after one hour of storage. 50% residual activity was measured after storage at 45 °C for 30 minutes. Surprisingly, 84% residual activity was detected after storage at 50 °C for 5 minutes.

[0090] Table 6:

[0091]

[0092] Example 6: Determining pH stability

[0093] The pH stability of the activated alcohol dehydrogenase of SEQ ID No. 1 was determined within the pH range of 4.0 to 10.0. To do this, a tenfold concentration of the activated alcohol dehydrogenase (100 nM) was stored in 10% Teorell-Stenhagen buffer at pH 4.0 to 10.0 for up to 120 minutes at 30°C. After 0, 60, and 120 minutes, aliquots of the alcohol dehydrogenase were taken and their activity in a conversion assay was determined, as described in Example 3, at 30°C with the following components: 100 mM Tris-HCl, pH 7.5, 1 mM PMS, 50 ppm DON, and 10 nM activated SEQ ID No. 1 alcohol dehydrogenase. Sampling for activity determination was performed after 0, 10, 20, 30, and 40 minutes. The DON reduction process was measured each time for each pH value. To determine stability, the slope of the linear range of the DON conversion line was calculated for each pH value at each time point. The slope of the linear range of the DON conversion line at each pH value at t=0 minutes was used as a reference for the activity calculations for the following incubation times. Table 7 shows the pH values, pH incubation times in minutes, and the associated residual activity as a percentage. The alcohol dehydrogenase of SEQ ID No. 1 was stable at pH 5.0 to pH 9.0 after 60 minutes of incubation. Surprisingly, the alcohol dehydrogenase exhibited particularly good stability in acidic environments (no loss of activity at pH 5.0) and heavily alkaline environments (no loss of activity after 120 minutes of incubation at pH 9.0).

[0094] Table 7:

[0095]

[0096] Example 7: Conversion of DON in a composite matrix

[0097] To determine the ability of activated alcohol dehydrogenase to convert trichothecene compounds in a composite matrix without the addition of external synthetic redox cofactors, activated alcohol dehydrogenase of SEQ ID No. 1 was prepared as described in Example 3, and the DON conversion assay was performed in the composite matrix. Here, the composite matrix is ​​specifically defined as bovine rumen fluid, porcine jejunal intestinal contents, porcine gastric juice, human and porcine saliva, pelleted piglet feed, and pelleted piglet feed mixed with saliva, rumen fluid, or intestinal contents. For comparison of buffer systems, tests were performed using Tris-HCl as described in Example 3. Standard feeds based on corn, soybean, and barley were used as piglet feed.

[0098] To determine the alcohol dehydrogenase activity in rumen fluid (pH 5.9), 1 ml of sterile rumen fluid filtrate was added to 100, 200, and 300 nM of activated alcohol dehydrogenase of SEQ ID No. 1 and 50 ppm DON in each case. Control batches were tested in aqueous solution as described in Example 3. The conversion assay was performed by incubation at 30°C in a hot block for up to 24 hours. Samples were taken at 0, 0.5, 1.0, 5.0, and 24.0 hours, in which case 0.1 ml of sample was taken each time, and the reaction was stopped with 0.1 ml of methanol. Samples were stored at -20°C, thawed, and centrifuged at 13,000 rpm for 10 minutes using an Eppendorf benchtop centrifuge and aseptically filtered through a 0.2 μm Spartan filter. For LC-MS / MS, samples were diluted as described in Example 3 and analyzed by LC-MS / MS. The concentration of DON at t = 0 h was used as a reference value (100%) for the following values. Table 8 shows the percentage of DON concentration measured at a relative t=0h. The presence of an externally added synthetic redox cofactor is necessary for the activity of the Tris-HCl buffer because DON conversion occurs slowly and is only detectable after 24 hours with 300 nM alcohol dehydrogenase. Surprisingly, it has been confirmed that DON is converted in rumen fluid filtrate at pH 5.9 without the addition of an externally added synthetic redox cofactor. This clearly demonstrates the presence of a natural redox cofactor in the rumen fluid. With 300 nM, only 42% of the initial DON amount was contained in the formulation after 5 hours of incubation. After 24 hours of incubation, only low amounts of DON were detectable with alcohol dehydrogenase concentrations greater than 200 nM.

[0099] Table 8:

[0100]

[0101] To determine the activity of alcohol dehydrogenase in porcine gastric fluid without a pH of approximately 3, in porcine intestinal contents with a pH of approximately 6, and in porcine and human saliva, in each case, 300 nM of activated alcohol dehydrogenase SEQ ID No. 1, approximately 20 ppm DON, was mixed with 1 ml of gastric fluid (sterilely filtered), 1 ml of pasty intestinal contents, or 1 ml of saliva. As a negative test, a assay containing only digestive fluid with 20 ppm DON was introduced, and as a positive test, a conversion assay containing all components, including 20 mM of synthetic redox cofactor PFC(III), was used. Samples were taken at 0, 3.0, 5.0, and 24.0 hours, in which case 0.1 ml of sample was taken each time, and the reaction was stopped with 0.1 ml of methanol. Samples were stored at 20°C, thawed, and centrifuged at 13,000 rpm for 10 minutes using an Eppendorf benchtop centrifuge, and sterilely filtered (0.2 μM Spartan filter). For LC-MS / MS, samples were diluted 1:10 in eluent (see Example 3) and analyzed by LC-MS / MS as in Example 3. Table 9 shows the DON concentrations measured at sampling time. Surprisingly, a reduction in DON in saliva occurred without the external addition of synthetic redox cofactors (regardless of breed). This clearly demonstrates that human and swine salivary secretions contain substances suitable as natural redox cofactors for the conversion of DON using alcohol dehydrogenase SEQ ID No. 1. No significant reduction in DON concentration was measured in pure gastric juice without mush. The reduction in DON concentration in intestinal contents was achieved solely by the addition of synthetic redox cofactors.

[0102] Table 9:

[0103]

[0104]

[0105] To determine the activity of alcohol dehydrogenase in piglet feed, 100 mg of piglet feed was mixed with 400 μl of 100 mM Tris-HCl buffer (pH 7.5), 400 μl of pig saliva, 400 μl of sterile pig gastric juice, or 400 μl of pig intestinal contents, respectively. These piglet feed suspensions were stored overnight at 4°C. Afterward, approximately 20 ppm DON, and / or 300 nM of activated SEQ ID No. 1 alcohol dehydrogenase, and / or 20 mM of synthetic redox cofactor PFC(III) were added to all samples. Preparations without alcohol dehydrogenase and without externally synthesized redox cofactor were used as negative tests. Preparations with added alcohol dehydrogenase and synthetic redox cofactor were used as positive tests. Samples were collected at 0, 3, 5, and 24 hours. One complete sample was used each time. For each sample, 500 μl of methanol was added, followed by homogenization at 300 rpm on a shaker for 30 minutes. Following this, the sample was centrifuged for 15 minutes (Eppendorf benchtop centrifuge, 13,000 rpm), and the supernatant was filtered through a 0.2 μM Spartan filter using a syringe. The supernatant was stored at -20°C, thawed, and diluted 1:10 in eluent for LC-MS / MS analysis as described in Example 3.

[0106] Table 10 shows the DON concentrations present in the samples at various time points. In the piglet feed buffer mixture, there are substances that can be assumed to have the effect of externally added synthetic redox cofactors, as the DON concentration continuously decreases in the absence of externally synthesized redox cofactors. These substances are derived from the piglet feed because, as previously shown, DON conversion cannot be measured in the buffer without externally synthesized redox cofactors. In the presence of externally synthesized redox cofactors, DON conversion in the piglet feed buffer mixture occurs relatively more rapidly.

[0107] In a mixture of piglet feed and saliva, alcohol dehydrogenase also showed activity independent of the presence of externally synthesized redox cofactors; and a faster reduction in DON occurred in conversion assays containing externally synthesized redox cofactors.

[0108] Surprisingly, the alcohol dehydrogenase of SEQ ID No. 1 was active in piglet feed mixtures without the addition of externally synthesized redox coenzymes. On the one hand, the addition of piglet feed to gastric juices increases the pH of the gastric juices; on the other hand, naturally occurring redox cofactors, which can replace externally synthesized redox cofactors, are released from the piglet feed. The activity of alcohol dehydrogenase in intestinal contents was determined only when externally synthesized redox cofactors were added to the conversion assay.

[0109] Table 10:

[0110]

Claims

1. Use of an alcohol dehydrogenase of SEQ ID No. 1 containing a metal ion and a quinone cofactor, or a functional variant of an amino acid sequence selected from SEQ ID NO: 2 to 4, and at least one redox cofactor in the transformation of at least one trichothecene exhibiting a hydroxyl group at C-3 atom, wherein the quinone cofactor is selected from PCC, TTC, TPC, LTC, and CTC, and the metal ion is 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+ At least one metal ion, and the at least one redox cofactor is selected from PMS, PMS derivatives, potassium hexacyanoferrate(III), sodium hexacyanoferrate(III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue and TMPD, wherein the trichothecene is selected from DON, cucurbitacinol and T-2 toxin.

2. The use according to claim 1, characterized in that, The quinone cofactor is PCC.

3. The use according to claim 1 or 2, characterized in that, The quinone cofactor is selected from Ca 2+ and Mg 2+ At least one metal ion binds to alcohol dehydrogenase.

4. The use according to claim 1 or 2, characterized in that, At least one redox cofactor is selected from PMS, coenzyme Q1 and coenzyme Q10.

5. The use according to claim 1 or 2, characterized in that, The conversion of trichothecene compounds with hydroxyl groups at the C-3 atom is carried out in food and feed, and in plant-based raw materials used in the production or processing of food and feed.

6. The use according to claim 5, characterized in that, The feed mentioned therein is feed for aquaculture or livestock.

7. The use according to claim 5, characterized in that, The feed mentioned therein is feed for pigs, poultry, cattle, horses or fish.

8. A method for the enzymatic transformation of trichothecene compounds, characterized in that, At least one trichothecene exhibiting a hydroxyl group at C-3 atom is contacted with an alcohol dehydrogenase of SEQ ID No. 1 or a functional variant of amino acid sequence selected from SEQ ID Nos. 2 to 4 containing a metal ion and a quinone cofactor, and then contacted with at least one redox cofactor and water, wherein the quinone cofactor is selected from PCC, TTC, TPC, LTC, and CTC, and the metal ion is 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+ At least one metal ion, and the at least one redox cofactor is selected from PMS, PMS derivatives, potassium hexacyanoferrate(III), sodium hexacyanoferrate(III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue and TMPD, wherein the trichothecene is selected from DON, cucurbitacinol and T-2 toxin.

9. The method according to claim 8, characterized in that, At least one trichothecene exhibiting a hydroxyl group at C-3 atom is contacted with an alcohol dehydrogenase of SEQ ID No. 1 or a functional variant of amino acid sequence selected from SEQ ID Nos. 2 to 4 containing a metal ion and a quinone cofactor, contacted with at least one redox cofactor and water, and contacted with at least one excipient, wherein the quinone cofactor is selected from PCC, TTC, TPC, LTC, and CTC, and the metal ion is 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+ At least one metal ion, and the at least one redox cofactor is selected from PMS, PMS derivatives, potassium hexacyanoferrate(III), sodium hexacyanoferrate(III), cytochrome C, coenzyme Q1, coenzyme Q10, methylene blue and TMPD, wherein the trichothecene is selected from DON, cucurbitacinol and T-2 toxin.

10. The method according to claim 8, characterized in that, The at least one trichothecene exhibiting a hydroxyl group at C-3 atom is converted at a temperature between 5°C and 55°C.

11. The method according to claim 10, characterized in that, The at least one trichothecene exhibiting a hydroxyl group at C-3 atom is converted at a temperature between 10°C and 50°C.

12. The method according to claim 10, characterized in that, At least one trichothecene exhibiting a hydroxyl group at C-3 atom is converted at a temperature between 28°C and 45°C.

13. The method according to any one of claims 8-12, characterized in that, The at least one trichothecene exhibiting a hydroxyl group at C-3 atom is contacted with the alcohol dehydrogenase or at least one of the functional variants containing a metal ion and a quinone cofactor, along with a redox cofactor, and with water, for at least one minute.

14. The method according to any one of claims 8-12, characterized in that, The at least one trichothecene exhibiting a hydroxyl group at C-3 atom is contacted with the alcohol dehydrogenase or at least one of the functional variants containing a metal ion and a quinone cofactor, with a redox cofactor, with water, and with an excipient for at least one minute.

15. The method according to claim 13, characterized in that, The at least one trichothecene exhibiting a hydroxyl group at C-3 atom is contacted with the alcohol dehydrogenase or at least one of the functional variants containing a metal ion and a quinone cofactor, along with a redox cofactor, and water, for at least 5 minutes.

16. The method according to claim 13, characterized in that, The at least one trichothecene exhibiting a hydroxyl group at C-3 atom is contacted with the alcohol dehydrogenase or at least one of the functional variants containing a metal ion and a quinone cofactor, with a redox cofactor, with water, and with an excipient for at least 5 minutes.

17. The method according to claim 13, characterized in that, The at least one trichothecene exhibiting a hydroxyl group at C-3 atom is contacted with the alcohol dehydrogenase or at least one of the functional variants containing a metal ion and a quinone cofactor, along with a redox cofactor, and with water, for at least 60 minutes.

18. The method according to claim 13, characterized in that, The at least one trichothecene exhibiting a hydroxyl group at C-3 atom is contacted with the alcohol dehydrogenase or at least one of the functional variants containing a metal ion and a quinone cofactor, with a redox cofactor, with water, and with an excipient for at least 60 minutes.

19. The method according to any one of claims 8-12, characterized in that, The quinone cofactor is PCC.

20. The method according to any one of claims 8-12, characterized in that, The quinone cofactor is selected from Ca 2+ and Mg 2+ At least one metal ion binds to alcohol dehydrogenase.

21. The method according to any one of claims 8-12, characterized in that, The at least one redox cofactor is selected from PMS, coenzyme Q1 and coenzyme Q10.

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