Polypeptides, isolated polynucleotides thereof, and additives comprising polypeptides, uses and methods thereof

Abstract

A polypeptide that hydrolytically cleaves zearalenone and / or at least one zearalenone derivative, which is a hydrolase having an amino acid sequence selected from SEQ ID No.1-15 or a functional variant thereof wherein the sequence identity between said functional variant and at least one of said amino acid sequences is at least 40%; and an additive comprising the polypeptide; and isolated polynucleotides encoding the polypeptides; and a method for hydrolytically cleaving zearalenone and / or at least one zearalenone derivative with said polypeptide.

Description

[0001] This application is a divisional application of the patent application for invention titled "Polypeptides for Hydrolytic Cleavage of Zearalenone and / or Zearalenone Derivatives, Isolated Polynucleotides Thereof, and Additives Containing the Polypeptides, Their Uses and Methods", with the application date of August 27, 2014, application number 201480055221.7.

[0002] The present invention relates to polypeptides for hydrolytically cleaving zearalenone and / or at least one zearalenone derivative, isolated polynucleotides encoding such polypeptides, and additives containing such polypeptides, and also relates to the uses of such polypeptides and to methods for hydrolytically cleaving zearalenone and / or at least one zearalenone derivative.

[0003] Mycotoxins are secondary metabolites produced by filamentous fungi. An important representative thereof is zearalenone (ZEN) (formerly known as F-2 toxin), which is produced by many Fusarium fungi. These fungi particularly infect cultivated plants, such as various cereals, where fungal infection usually occurs before harvest, where fungal growth or mycotoxin production can occur before harvest, or can also occur after harvest in the case of improper storage. The FAO estimates that 25% of agricultural products worldwide are contaminated with mycotoxins, which results in huge economic losses. In a recent worldwide study, a total of 23,781 samples were analyzed from January 2009 to December 2011, of which 81% were tested positive for at least one mycotoxin and 45% were positive for ZEN. ZEN can be found in all regions of the world, equally in all tested cereal and feed categories, such as corn, soybean meal, wheat, wheat bran, DDGS (distillers dried grains with solubles), and in pre-mixed feed mixtures, with frequencies up to 100%.

[0004] ZEN is a non-steroidal, estrogenic, macrocyclic lactone synthesized via the polyketide pathway, and has the following structural formula:

[0005]

[0006] and the IUPAC nomenclature name "(2E,11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraene-7,13-dione".

[0007] However, there are also many ZEN derivatives in nature, which are formed by enzymatic or chemical modification of ZEN. Examples of this are glycosidic or sulfated ZEN conjugates, which are formed by fungal, plant or mammalian metabolism; and ZEN metabolites, which are formed especially in the human or animal body. Hereinafter, ZEN derivatives refer to ZEN conjugates or ZEN metabolites that exist in nature or are prepared by chemical or biochemical synthesis, but particularly refer to α-zearalenol (α-ZEL; (2E,7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-13-one), β-zearalenol (β-ZEL; (2E,7S,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-13-one), α-zearalanol (α-ZAL; (7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),14,16-trien-13-one), β-zearalanol (β-ZAL; (7S,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(14),15,17-trien-13-one), zearalenone-14-sulfate (Z14S; [(2E,11S)-15-hydroxy-11-methyl-7,13-dioxo-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraen-17-yl] hydrogen sulfate), zearalenone-14-glycoside (Z14G; (2E,11S)-15-hydroxy-11-methyl-17-[(3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-12-oxabicyclo[12.4.0]octadeca-1(18),2,14,16-tetraene-7,13-dione) and zearalanone (ZAN; (11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadeca-1(18),14,16-trien-7,13-dione).

[0008] ZEN, and likewise ZEN derivatives, especially α-ZEL, β-ZEL, Z14S, α-ZAL, β-ZAL, Z14G and ZAN, can also be detected in processed foods or feeds such as bread or beer due to their high chemical and physical stability.

[0009] ZEN binds to estrogen receptors and can cause hormonal disorders. It is immediately absorbed after oral ingestion and is converted by mammals into two stereoisomeric metabolites, namely α-ZEL and β-ZEL. Here, for example, α-ZEL, as well as α-ZAL or ZAN, have much stronger estrogenic effects than ZEN. Conjugated ZEN derivatives sometimes have lower estrogenic activity than ZEN. However, if possible, ZEN can be released back into the digestive tract from these ZEN derivatives.

[0010] Although ZEN has relatively low acute toxicity and an oral LD50 value up to 20,000 mg / kg body weight, subacute and / or subchronic toxic effects such as teratogenic, carcinogenic, estrogenic, and immunosuppressive effects occur in animals or humans in the case of longer-term intake. ZEN-contaminated feed causes developmental disorders in mammals, and pigs, especially piglets, are extremely sensitive to ZEN. A ZEN concentration in feed above 0.5 ppm causes developmental disorders. For example, a concentration above 1.5 ppm can cause hyperestrogenic activity in pigs, and a concentration of 12 ppm of ZEN is considered responsible for abortions in cattle. Since zearalenone is rapidly absorbed through mucous membranes, especially through the gastric mucosa but also through the oral mucosa, immediate and especially quantitative inactivation is necessary. They can be detected in the blood 30 minutes after oral administration of ZEN. In this case, using isolated enzymes against microorganisms has advantages such as higher specific activity or faster action. Due to the harmful effects of ZEN, there are thus mandatory ZEN limits in foods and recommendations for ZEN limits in feeds in the European Union (EC NO: 1881 / 2006).

[0011] The initial strategy to reduce ZEN contamination of food or feed is to limit fungal growth, for example by following "good agricultural practice". This especially includes that seeds are free from pest and fungal infestation, or agricultural waste is removed from the fields in a timely manner. In addition, fungal growth on fields can also be reduced by using fungicides. After harvest, the harvested product should be stored at a residual moisture content below 15% and a low temperature to prevent fungal growth. Similarly, materials contaminated by fungal infestation should be removed before reprocessing. Despite this series of measures, I. Rodrigez and K. Naehrer (2012) still reported that even in regions with the highest agricultural standards, such as the USA and Central Europe, 29% and 39% of the tested maize samples were contaminated with ZEN during 2009 - 2011, respectively.

[0012] Other possibilities for removing ZEN from feed or food are the adsorption or transformation of mycotoxins. For this to be necessary, the binding of mycotoxins to the adsorbent must be strong and specific over a wide pH range and remain stable throughout the digestion process in the gastrointestinal region. Although several abiotic adsorbents, such as activated carbon, silicates or synthetic polymers such as cholestyramine, can be effectively used for aflatoxins, their use for other mycotoxins is limited. The main disadvantage of adsorbents is the non-specific binding of other molecules that are sometimes necessary for nutrient supply. Biotic adsorbents, such as yeast or yeast extracts, have also been described in the literature, however, biotic adsorbents, like abiotic adsorbents, also have similar limitations.

[0013] Detoxification of ZEN by physical and chemical treatments is also limited. By heat treatment, ZEN cannot be effectively inactivated, but the ZEN content can be reduced by 83.9% by extrusion and treatment with oxidants, such as treatment with a 10% hydrogen peroxide solution at 80 °C for 16 hours. The use of extrusion methods and oxidants such as ozone or hydrogen peroxide in the preparation of feed and food is limited due to high costs, quality losses, sometimes low efficiency and low specificity.

[0014] The biotransformation of ZEN by means of microorganisms such as Trichosporon mycotoxinivorans, Gliocladium roseum or Bacillus subtilis strains or enzymes isolated therefrom, such as hydrolases or peroxidases, has also been described, for example, in E. Vekiru et al., Appl. and Environ. Microb., 2010, 76, 7, 2353-2359.

[0015] The ZEN degradation properties of bacteria of the genus Rhodococcus and Nocardia, in particular Rhodococcus globerulus, Rhodococcus erythropolis and Nocardia globerula, are known from EP 0 938 575 B1.

[0016] The ZEN degradation by enzymes isolated from Gliocladium roseum, in particular α / β-hydrolases, zearalenone hydrolase 1 (ZHD1), which catalyzes the degradation of ZEN by means of a catalytic triad, is known from WO 02 / 076205.

[0017] Recombinant Zonase, i.e., ZEN-degrading enzyme, is known from WO 2012 / 113827, which is stable in the gastrointestinal tract, and in particular microorganisms such as Thermobifidia fusca, Streptomyces exfoliatus, Acidovorans delafieldii, and Streptomyces sp. are described therein.

[0018] A polypeptide or enzyme capable of hydrolyzing ZEN and / or at least one ZEN derivative may also be referred to as Zonase.

[0019] The terms used below are taken from the technical jargon and are always used in their traditional sense, unless otherwise indicated. Thus, the term "polynucleotide" relates to every type of genetic material having all lengths and sequences, such as single-stranded and double-stranded DNA and RNA molecules, including regulatory elements, structural elements, gene clusters, plasmids, whole genomes, and fragments thereof. The name "polypeptide" includes proteins, such as enzymes, antibodies, and polypeptides having up to 500 amino acids, such as peptide inhibitors, domains of proteins, and short polypeptides having a small sequence length, such as less than 10 amino acids, such as receptors, ligands, peptide hormones, tags, etc. The name "position" in a polynucleotide or polypeptide relates to a single specific base or amino acid in the sequence of the polynucleotide or polypeptide.

[0020] Now, the present invention aims to provide such polypeptides for use, with which the rapid and reliable conversion of ZEN and / or at least one ZEN derivative into hydrolyzed ZEN and / or hydrolyzed ZEN derivative can be successfully achieved. To solve this task, the present invention is mainly characterized in that the polypeptide is a hydrolase having an amino acid sequence selected from SEQ ID No. 1-15 or a functional variant thereof, wherein the sequence identity between the functional variant and at least one of the amino acid sequences is at least 40%.

[0021] According to the present invention, the name "sequence identity" relates to the percentage of sequence identity. For amino acid sequences and nucleotide sequences, sequence identity can be determined visually, but is preferably calculated using a computer program. Sequence comparisons are also made within sequence segments, where a continuous sequence of the reference sequence is understood as a segment and preferably includes the conserved regions of the sequence.

[0022] In the current situation, sequence identity is determined with the aid of the program NCBI BLAST (Basic Local Alignment Search Tool), in particular for polypeptides, with BLASTP, and for polynucleotides, with BLASTN, which is made available for use on the homepage of the "National Center for Biotechnology Information" (NCBI; http: / / www.ncbi.nlm.nih.gov / ). Thus, it is possible to compare two or more sequences with each other according to the algorithm of Altschul et al., 1997 (Nucleic Acids Res., 25: 3389-3402). For the purposes of the present invention, the version of the program of May 15, 2013 is used. As program settings, the basic settings are considered, but in particular, for 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"; and for nucleotide sequence comparison: word length: 11; Expect value: 10; gap costs: Existence = 5, Extention = 2; Filter = low complexity activated; Match / Mismatch Scores: 2, -3; Filter String: L; m.

[0023] The term "functional polypeptide variant" or "functional variant" relates on the one hand to "allelic variants" of a polypeptide and "functional fragments" of a polypeptide, and on the other hand to "modifications" of a polypeptide, in which the enzymatic function is essentially unchanged. The name "allelic variant" relates to such a polypeptide which is produced by a nucleotide sequence mutation occurring randomly in nature and causes an alteration of the amino acid sequence, in which its enzymatic function is not affected. A "modification" can be, for example, a fusion with the C-terminus or N-terminus of a polypeptide or a mutated polypeptide, where the mutation can be obtained by substitution, insertion or deletion of at least one amino acid, which is carried out in particular by site-directed mutagenesis or random mutagenesis, recombination and / or any other protein engineering method. The terms "substitution", "insertion" and "deletion" are used in the meanings customary and familiar to the person skilled in the art in genetic engineering. The term "functional fragment" relates to a part or partial sequence of a polypeptide or a part or partial sequence of its functional variant, in which the enzymatic function is essentially retained. The enzymatic function is essentially retained when the enzymatic reaction mechanism remains unchanged, i.e., the mycotoxin is hydrolyzed at the same position, and the specific residual activity of the "functional variant" relative to the original polypeptide is at least 5%, preferably at least 10%, in particular at least 50%. The polypeptides having the amino acid sequences of SEQ ID No. 1-15 are functional allelic variants of another or the same enzyme, where the sequences are each derived from different microorganisms. This is clearly evident from the following: the close phylogenetic relationship measured by means of the percentage of sequence identity, and the fact that all polypeptides act on ZEN and ZEN derivatives by the same degradation mechanism.

[0024] Due to the similarity of the amino acid sequences of the polypeptides having SEQ ID No. 1-15 to each other, it is possible that a functional variant of one of these polypeptides has at least 40% sequence identity with more than one of the claimed polypeptides having SEQ ID No. 1-15.

[0025] By selecting such amino acid sequences or their functional variants, surprisingly rapid and complete hydrolysis of ZEN and / or at least one ZEN derivative was found.

[0026] In a preferred further development corresponding to the present invention, the polypeptide has an amino acid sequence which comprises at least one conserved amino acid sequence segment or a functional variant thereof, wherein the functional variant of the amino acid sequence segment has at least 70%, preferably at least 84%, more preferably at least 92%, and most preferably at least 98% sequence identity, and the at least one conserved amino acid sequence segment is selected from the amino acid sequences of +24 to +50, +52 to +77, +79 to +87, +89 to +145, +150 to +171, +177 to +193, +223 to +228, +230 to +237, +239 to +247, +249 to +255, +257 to +261, +263 to +270, +272 to +279, +297 to +301, +303 to +313, +24 to 328, +1 to +328 of the sequence of SEQ ID No.1. By the presence of at least one such conserved amino acid sequence segment, it is possible to successfully provide such a polypeptide for use, which, in addition to the rapid and complete hydrolysis of ZEN and / or at least one ZEN derivative, also has a particularly high activity value compared to the hitherto known ZEN-degrading polypeptides.

[0027] Good results can still be achieved when, as in a further modification corresponding to the present invention, the functional variant has at least one amino acid modification selected from substitutions, deletions and insertions of one or more amino acids.

[0028] By further modifying the present invention in this way, such that the polypeptide has a specific activity of at least 0.01 U / mg, preferably at least 0.1 U / mg, particularly at least 1 U / mg, and / or has a K M value for hydrolytic cleavage of ZEN of at most 50 μΜ, preferably at most 3.5 μΜ, particularly at most 0.5 μΜ, and / or has a k -1 value for hydrolytic cleavage of ZEN of at least 0.05 s -1 , preferably at least 0.6 s -1 , particularly at least 5 s cat , and / or has a v -1 value for hydrolytic cleavage of ZEN of at least 0.00001 μΜ -1 s -1 , preferably at least 0.0001 μΜ -1 s -1 , particularly at least 0.001 μΜ -1 s max , ZEN and / or ZEN derivatives can be hydrolyzed particularly rapidly and completely, in particular detoxified.

[0029] According to a preferred further development corresponding to the present invention, the polypeptide comprises an amino acid sequence selected from SEQ ID No. 2, 5 - 7, 9, 11, 12, and 15 or a functional variant thereof, wherein the functional variant has at least 40% sequence identity with at least one of the amino acid sequences, and the polypeptide has a pH stability of at least 15%, preferably 50%, and particularly preferably at least 90% at pH 5.0. By such a further development, it can be ensured that the polypeptide cleaves or detoxifies zearalenone and / or at least one zearalenone derivative in an acidic medium, thus for example in the case of the stomach of a mammal. Here, the pH stability of the polypeptide is defined as the percentage of the residual activity of the polypeptide at pH 5.0 relative to the activity at the respective optimal pH.

[0030] According to a preferred further development corresponding to the present invention, the polypeptide comprises an amino acid sequence selected from SEQ ID No. 1, 2, 5 - 7, 9, 11, and 15 or a functional variant thereof, wherein the functional variant has at least 40% sequence identity with at least one of the amino acid sequences, and the polypeptide has the highest enzymatic activity in the temperature range between 30 °C and 75 °C, preferably between 38 °C and 55 °C, and particularly preferably between 38 °C and 52 °C. By such a further development of the present invention, it is ensured that zearalenone and / or at least one zearalenone derivative are also hydrolyzed or detoxified by the polypeptide at mesophilic temperatures, especially at the body temperature of humans and useful animals. The temperature at which the polypeptide has the highest enzymatic activity is defined as the optimal temperature of the polypeptide.

[0031] According to a preferred further development corresponding to the present invention, the polypeptide comprises an amino acid sequence selected from SEQ ID No. 1, 5, 6, 9, 11, 12, and 15 or a functional variant thereof, wherein the functional variant has at least 40% sequence identity with at least one of the amino acid sequences, and the polypeptide is temperature - stable up to a temperature of 90 °C, preferably 75 °C, and particularly preferably 60 °C. This ensures that the polypeptide and its enzymatic function remain substantially intact even under increased temperature loads (for example, this can be the case during transportation in a container or during feed granulation). The temperature stability of the polypeptide is defined as the temperature at which the polypeptide has 50% residual activity after a 15 - minute pre - incubation compared to the activity at the respective optimal temperature.

[0032] Thus, the polypeptide can be selected such that it is an α / β-hydrolase suitable for hydrolytically cleaving the ester group of zearalenone and / or ZEN derivatives in an oxygen-independent and cofactor-free manner, which has an amino acid triad that catalyzes the hydrolytic cleavage, the triad consisting of serine, an acidic amino acid selected from glutamic acid and aspartic acid, in particular aspartic acid, and histidine, and the catalytic triad being, for example, S128, D264, and H303, where the positions relative to SEQ ID No.1 are described.

[0033] Hydrolysis of ZEN and ZEN derivatives at the ester group of zearalenone or its derivatives can be successfully achieved with each of the polypeptides of SEQ ID No.1-15 according to the following reaction mechanism:

[0034]

[0035] Hydrolysis of ZEN to non-toxic hydrolyzed zearalenone (HZEN) or hydrolyzed ZEN derivatives is carried out by the polypeptides according to the invention, in particular the α / β-hydrolase. Further decarboxylation of HZEN to decarboxylated hydrolyzed ZEN (DHZEN) or decarboxylated hydrolyzed ZEN derivatives usually occurs spontaneously.

[0036] In particular, with the aid of the above-mentioned catalytic triad, complete hydrolysis of ZEN and ZEN derivatives can be successfully achieved, and the degradation reaction has good pH stability, especially at pH values in the acidic range.

[0037] Surprisingly, it has been confirmed that polypeptides containing at least one polar amino acid selected from Y, Q, N, T, K, R, E, D and at least one non-polar amino acid selected from F, M, L, I, V, A, G, P in the sequence segment consisting of the 3 amino acids before and the 3 amino acids after the serine of the above-mentioned catalytic triad can successfully achieve still good results and furthermore improve at least one enzyme kinetic parameter.

[0038] In a preferred further modification of the present invention, the polypeptide has at least one mutation with respect to the amino acid sequence of SEQ ID No. 1 at at least one of the following positions: 22, 23, 25, 26, 27, 29, 31, 32, 35, 37, 42, 43, 46, 51, 53, 54, 57, 60, 69, 72, 73, 78, 80, 84, 88, 95, 97, 99, 114, 118, 119, 123, 132, 141, 146, 148, 149, 154, 163, 164, 165, 169, 170, 172, 176, 180, 182, 183, 190, 191, 194, 196, 197, 198, 201, 204, 205, 206, 207, 208, 209, 210, 212, 213, 214, 216, 217, 220, 221, 222, 229, 231, 233, 238, 240, 244, 245, 246, 248, 249, 251, 254, 256, 260, 262, 263, 266, 269, 271, 277, 280, 281, 282, 283, 284, 285, 286, 287, 292, 296, 298, 302, 307, 308, 309, 311, 314, 317, 319, 321, 323, 325 and 326. These positions are derived from the sequence differences between the polypeptide having SEQ ID No. 1 and the polypeptides having SEQ ID Nos. 2-6 which have a high degree of identity with this sequence and are particularly active. By changing the polypeptide having SEQ ID No. 1 in this way at at least one of these positions, i.e., by adopting the amino acid variants of SEQ ID Nos. 2-6 at this position, it can be successfully shown that these positions have a significant influence on the enzyme kinetic parameters of the polypeptide, and furthermore, the combination of SEQ ID No. 1 with SEQ ID Nos. 2-6 having a high degree of sequence identity leads to higher activity.

[0039] According to a further modification of the present invention, the polypeptide has at least one mutation selected from the following in the amino acid sequence with respect to SEQ ID No. 1: D22A, S23Q, S23L, N25D, I26V, F27Y, F27H, S29P, R31A, F32Y, R35K, R35Q, V37A, V42I, V43T, F46Y, S51E, S51D, D53G, N54M, N54R, L57V, L60I, S69G, P72E, V73A, A78S, N80H, F84Y, I88L, T95S, T97A, R99K, I114M, I118V, K119R, V123I, L132V, A141S, I146V, I146L, A148G, A149V, A154P, P163T, A164T, Y165C, Y165H, V169I, L170R, A172G, A176M, A176V, Y180F, D182T, F183Y, I190V, G191S, K194T, K194E, F196Y, V197C, V197R, E198R, E198S, K201D, K201G, P204S, P204A, A205S, K206P, A207M, M208A, Q209R, L210A, L210S, ΔP212, T213V, P214A, E216T, E216G, A217I, N220H, L221M, K222R, K222Q, G229A, A231V, F233W, F233Y, F233H, A238G, H240N, H240S, D244E, R245Q, M246L, S248T, S248N, S248G, Q249R, K251N, I254V, I256L, A260M, T262D, T262G, I263T, E266D, E269H, E269N, L271V, L277E, E280A, E280L, H281R, H281Q, A282V, Q283R, D284L, D284R, I285L, I286M, R287E, R287D, R292K, R292T, Q296A, Q296E, H298V, L302S, L307Q, F308S, D309A, A311P, A314V, L317F, S319Q, S319P, S319R, S321A, S321T, T323A, P325A, A326P. With such a polypeptide, complete hydrolysis of ZEN can be successfully achieved in a short time, especially its detoxification, wherein the specific activity of the polypeptide is at least 6.00 U / mg, preferably at least 7.00 U / mg, and particularly at least 8.00 U / mg.The unit "U" or "Unit" is a measure of absolute catalytic activity and is defined by the hydrolysis of 1 μMol of ZEN per minute at 32 °C in 50 mM Tris-HCl buffer (pH 8.2), where "catalytic activity" refers to the enzymatic conversion of a substrate under defined reaction conditions, and "specific activity" refers to the ratio of catalytic activity to the polypeptide mass concentration (mass / volume unit).

[0040] By producing the polypeptide such that it contains at least one of the following amino acid motifs having SEQ ID No. 32 - 50, a polypeptide can be successfully provided for use that has a specific activity of at least 7.00 U / mg, preferably at least 8.00 U / mg. It has surprisingly been shown that the enzymatic activity of the polypeptide is further increased when it contains at least one of the following amino acid motifs having a sequence of SEQ ID No. 51 - 58, for example relative to a motif containing 7 amino acids. An even higher specific activity is achieved when it contains at least one of the following amino acid motifs having a sequence of SEQ ID No. 59 - 69.

[0041] According to a further modification of the invention, the polypeptide contains at least one conservative amino acid substitution at at least one position, where the conservative amino acid substitution is selected from: G to A; or A to G, S; or V to I, L, A, T, S; or I to V, L, M; or L to I, M, V; or M to L, I, V; or P to A, S, N; or F to Y, W, H; or Y to F, W, H; or W to Y, F, H; or R to K, E, D; or K to R, E, D; or H to Q, N, S; or D or N, E, K, R, Q; or E to Q, D, K, R, N; or S to T, A; or T to S, V, A; or C to S, T, A; or N to D, Q, H, S; or Q to E, N, H, K, R. The term "conservative amino acid substitution" refers to the substitution of an amino acid by another amino acid that is regarded by a person skilled in the art as conservative (i.e., having similar specific properties). Such specific properties are, for example, the size, polarity, hydrophobicity, charge, or pK value of the amino acid. A conservative mutation refers to, for example, substituting one acidic amino acid with another acidic amino acid, substituting one basic amino acid with another basic amino acid, or substituting one polar amino acid with another polar amino acid.

[0042] With such conservative amino acid substitutions, functional polypeptide variants can be successfully prepared that have a specific activity that is approximately as strong as that of the parent polypeptide, yet preferably increased by at least 0.1 U / mg.

[0043] Furthermore, the present invention also aims to provide isolated polynucleotides for use in successfully preparing polypeptides capable of hydrolytically cleaving ZEN and / or at least one ZEN-derivative rapidly and reliably.

[0044] To solve this task, the present invention is characterized in that the isolated polynucleotide has a nucleotide sequence encoding a polypeptide, wherein the polypeptide has the property of hydrolyzing zearalenone and / or at least one zearalenone derivative; and the nucleotide sequence encodes at least one polypeptide according to one of claims 1 to 11; and / or the nucleotide sequence has a degree of sequence identity with at least one nucleotide sequence selected from SEQ ID No. 16 - 31, wherein the selected nucleotide sequence is at least 40%; and / or the nucleotide sequence hybridizes with at least one nucleotide sequence selected from SEQ ID No. 16 - 31 under medium stringency conditions, and / or hybridizes with a partial sequence thereof having at least 200 nucleotides, particularly at least 100 nucleotides, and / or hybridizes with the complementary strand of the above nucleotide sequence or its partial sequence.

[0045] The nucleotide sequence to be expressed, particularly its triplets (codons), usually varies with the host cell, so that the codon bias is optimized with the host cell. This results in that polynucleotides with a degree of sequence identity far lower than 80%, and also lower than 70% or lower than 60% can also encode the same polypeptide. The sequence comparison for determining the degree of sequence identity must also be carried out within a sequence segment, where the segment refers to a continuous sequence of the reference sequence. For nucleotide sequences, the length of the sequence segment is usually 15 to 600.

[0046] With the existing isolated nucleotide sequences or sequence segments, nucleic acid probes with a length usually of at least 15, 30 or 40 nucleotides can be successfully produced. With such probes (which are usually additionally labeled, for example, with 3 H, 32 P, 35 S, biotin or avidin), nucleotide sequences encoding polypeptides having the effect of degrading ZEN and / or ZEN derivatives can be identified by using standard methods. As starting materials for identifying such sequences, for example, the DNA, RNA or cDNA of a single microorganism, a genomic DNA library, or a cDNA library can be considered.

[0047] For nucleotide sequences or nucleotide probes having a length of at least 100 nucleotides, moderate stringency conditions are defined as prehybridization and hybridization in a Na-EDTA buffer (SSPE, 0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA) with 5× NaCl containing 0.3% sodium dodecyl sulfate (SDS), 200 μg / ml sheared and denatured salmon sperm DNA, and 35% formamide at 42°C, followed by standard Southern blotting conditions, where the carrier material is finally washed three times with 2× sodium chloride-citrate buffer (SSC, 300 mM NaCl and 30 mM trisodium citrate, 0.2% SDS) at 55°C for 15 minutes.

[0048] For nucleotide sequences or nucleotide probes having a length of 15 to 100 nucleotides, moderate stringency conditions are defined as prehybridization and hybridization in a buffer consisting of 0.9 M NaCl, 0.09 M Tris-HCl pH = 7.6, 6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium dihydrogen phosphate, 0.1 mM ATP, and 0.2 mg / ml yeast RNA, where prehybridization and hybridization are carried out at a temperature 5 to 10°C lower than the calculated melting temperature (Tm), where Tm is determined by the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA, 48:1390). Subsequently, the assay is continued under standard Southern blotting conditions (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor, New York). Finally, the carrier material is washed once with 6× SCC buffer containing 0.1% SDS for 15 minutes; and washed twice with 6× SSC buffer at a temperature 5 to 10°C lower than the calculated Tm for 15 minutes each.

[0049] Furthermore, the present invention also aims to provide additives for use, with which it is possible to successfully achieve the hydrolytic cleavage of ZEN and / or at least one ZEN derivative in a defined or complex matrix, such as in feed or food, quickly and reliably.

[0050] To solve this task, an additive for cleaving zearalenone and / or at least one zearalenone derivative by hydrolysis is provided for use, wherein the additive comprises at least one polypeptide having an amino acid sequence selected from SEQ ID No. 1 - 15 or a functional variant thereof, wherein the sequence identity between the functional variant and at least one of the amino acid sequences is at least 40%; and optionally comprises adjuvants.

[0051] With such an additive, the biochemical conversion of ZEN and / or at least one ZEN derivative into hydrolyzed ZEN and / or hydrolyzed ZEN derivative can be successfully achieved. For example, this additive can also be considered for the stereoselective hydrolysis of ZEN and / or ZEN derivatives in industrial processes.

[0052] In a preferred further modification of the present invention, the additive is produced such that the adjuvant is selected from at least one inert carrier, and optionally, other components such as vitamins and / or minerals and / or enzymes and / or other components for detoxifying mycotoxins. By using such an additive, it can be ensured, for example, in feed or food, that the amount of optionally contained ZEN and / or ZEN derivative is safely and reliably hydrolyzed, especially detoxified to such an extent that there is no harmful effect on the organism of the subject ingesting these feeds or foods.

[0053] In this case, the polypeptide according to the present invention can also be present in an enzyme preparation, which in addition to comprising at least one polypeptide according to the present invention, further comprises at least one enzyme, which, for example, participates in the degradation of proteins, such as proteases, or the enzyme participates in the metabolism of starch or fiber or fat or glycogen, such as amylase, cellulase or dextranase, and for example, hydrolases, lipases, mannosidases, oxidases, oxidoreductases, phytases, xylanases and / or their combinations.

[0054] Other fields of use of the present invention are enzyme preparations, which in addition to comprising at least one polypeptide according to the present invention, further comprise at least one component for detoxifying mycotoxins, such as mycotoxin-degrading enzymes, such as aflatoxin oxidase, ergotamine hydrolase, ergotamine amide hydrolase, zearalenone esterase, zearalenone lactonase, ochratoxin amide hydrolase, moniliformin carboxylesterase, moniliformin aminotransferase, aminopolyol amino oxidase, deoxynivalenol epoxide hydrolase; and / or at least one microorganism for degrading mycotoxins, such as Bacillus subtilis; and / or at least one component for binding mycotoxins, such as microbial cell walls or inorganic materials such as bentonite.

[0055] According to a particularly preferred further modification of the present invention, in the additive, the polypeptide is contained therein at a concentration of at most 10,000 U / g, preferably at most 1,000 U / g, more preferably at most 100 U / g, and most preferably at most 10 U / g, whereby it is possible to successfully achieve the rapid conversion of ZEN and / or ZEN derivatives, and in particular before their absorption by the body of a subject (especially a mammal) that has consumed contaminated feed or food, into non-toxic or low-toxic metabolites, especially HZEN and DHZEN.

[0056] According to a further modification of the present invention, the polypeptide is present in a encapsulated or coated form, and for the encapsulation or coating process, standard methods can be considered, such as those described in WO 92 / 12645. Through the encapsulation or coating process, it is possible to successfully transport the polypeptide to its site of use without change, especially without degradation or impairment, so that the polypeptide only starts to act after the protective shell dissolves (for example, in the digestive tract of an animal), whereby a more targeted, rapid, and complete degradation of ZEN and / or ZEN derivatives can also be achieved in an acidic, protease-rich, and hypoxic medium. In addition, through the encapsulation or coating process, the temperature stability of the polypeptide in the additive can also be successfully improved.

[0057] Furthermore, the present invention also aims at the use of the additive for the hydrolytic cleavage of zearalenone and / or at least one zearalenone derivative in feed (which is especially used for pigs, poultry, and aquaculture), in food, or in dried distillers grains. By using the additive according to the present invention, it is possible to successfully hydrolyze or detoxify the ZEN and / or ZEN derivatives contained in food or feed or in dried distillers grains, and such detoxification has been successfully achieved at a polypeptide concentration of approximately 1 U / g of contaminated feed or food.

[0058] Furthermore, the present invention also aims to provide such a method for use, by which a rapid and reliable hydrolytic cleavage of ZEN and / or at least one ZEN derivative is made possible.

[0059] To solve this task, the method is carried out such that zearalenone and / or at least one zearalenone derivative is hydrolyzed by a polypeptide having an amino acid sequence selected from SEQ ID No. 1-15 or a functional variant thereof, wherein the sequence identity between the functional variant and at least one of the amino acid sequences is at least 40%.

[0060] According to a further modification of the present invention, the method is carried out such that the polypeptide is used in an additive corresponding to the present invention.

[0061] According to a preferred further development, the method is carried out such that the polypeptide or additive is admixed with feed or food contaminated with zearalenone and / or at least one zearalenone derivative, the contaminated feed or food is brought into contact with moisture, and the polypeptide or additive hydrolyzes the zearalenone and / or at least one zearalenone derivative comprised in the contaminated feed or food. In the case of moist feed or food such as mash or gruel, the hydrolysis of zearalenone and / or at least one zearalenone derivative takes place in the moist feed or food before oral ingestion. By this method, it can be ensured that the harmful effects of zearalenone and zearalenone derivatives on humans and animals are eliminated to the greatest extent possible. Herein, moisture refers to the presence of water or an aqueous liquid, where for example saliva or other liquids present in the digestive tract also fall within this category. The digestive tract is defined as the oral cavity, pharynx (throat), esophagus and gastrointestinal tract or their equivalents, where in animals there may be different names or individual components may not be present in the digestive tract of the animal.

[0062] The method of the invention can also be carried out such that the feed or food is granulated before oral ingestion.

[0063] According to a further refinement of the invention, the method is carried out such that at least 70%, preferably at least 80%, particularly preferably at least 90% of the zearalenone and / or at least one zearalenone derivative is hydrolyzed. Thereby, subacute and / or subchronic toxic effects in animals or humans, such as teratogenic, carcinogenic, estrogenic and immunosuppressive effects, can be prevented.

[0064] The invention will be explained in more detail below on the basis of examples and the figures. Among them:

[0065] Figure 1 Shows the time-dependent degradation of ZEN by the polypeptide having SEQ ID No.1 and the increase in the metabolites HZEN and DHZEN, where in Figure 1 A, the polypeptide is untagged, in Figure 1 B, the polypeptide has a C-terminal 6×His tag, and in Figure 1 C, the polypeptide has an N-terminal 6×His tag.

[0066] Figure 2 Shows the Michaelis-Menten kinetics of the polypeptide having SEQ ID No.1.

[0067] Figure 3 shows by the purified polypeptide having SEQ ID No.1 ( Figure 3A)、SEQ ID No.2( Figure 3B )、SEQ IDNo.5( Figure 3C )、SEQ ID No.6( Figure 3D )、SEQ ID No.7( Figure 3E )、SEQ ID No.9( Figure 3F )、SEQ ID No.11( Figure 3G )、SEQ ID No.12( Figure 3H ) and SEQ ID No.15( Figure 3I ) for the time-dependent degradation of ZEN and the increase in metabolites HZEN and DHZEN by means of polypeptides, where all sequences have a C-terminal 6×His tag.

[0068] Example 1: Modification, cloning and expression of polynucleotides encoding polypeptides capable of hydrolytically cleaving ZEN and / or at least one ZEN derivative

[0069] According to the instructions, using "Quick-change Site-directed Mutagenesis Kits" (Stratagene), amino acid substitutions, insertions or deletions are carried out by mutation of the nucleotide sequence with the aid of PCR. Alternatively, the complete nucleotide sequence (GeneArt) was also purchased for this purpose. The nucleotide sequences generated by means of PCR mutagenesis or purchased from GeneArt optionally additionally contain a C-terminal or N-terminal 6×His tag at the amino acid level and are integrated into an expression vector for expression in Escherichia coli (E. coli) or Pichia pastoris (P. pastoris) by means of standard methods, transformed into Escherichia coli or Pichia pastoris, and expressed in Escherichia coli or Pichia pastoris (J.M. Cregg, Pichia Protocols, 2nd edition, ISBN-10: 1588294293, 2007; J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, 4th edition, Cold Spring Harbor), where any other suitable host cell can also be considered for this task.

[0070] The term "expression vector" refers to a DNA construct capable of expressing a gene in vivo or in vitro. In particular, this term encompasses such DNA constructs that are suitable for transferring the nucleotide sequence encoding the polypeptide into a host cell in order to integrate it into the genome there or to exist extrachromosomally in the cell, and to express the nucleotide sequence encoding the polypeptide intracellularly and optionally extract the polypeptide from the cell.

[0071] The term "host cell" refers to all such cells that either contain the nucleotide sequence to be expressed or an expression vector and are capable of producing the polypeptide according to the present invention. In particular, the term encompasses prokaryotic and / or eukaryotic cells, preferably Pichia pastoris, Escherichia coli, Bacillus subtilis, Streptomyces, Hansenula, Trichoderma, Lactobacillus, Aspergillus, plant cells, and / or spores of the genus Bacillus, Trichoderma or Aspergillus.

[0072] To determine the catalytic properties of the polypeptide, in the case of Escherichia coli, the soluble cell lysate is considered, or in the case of Pichia pastoris, the culture supernatant is considered. To determine the K M value, v max , k cat and specific activity, the polypeptide is selectively enriched chromatographically by means of a nickel-Sepharose column using standard methods. The determination of the protein concentration is carried out using standard methods, either by the BCA method (Pierce BCA Protein Assay Kit Prod#23225), however preferably by photometry using the specific extinction coefficient of the respective protein, which is calculated using the program "ProtParam" available online at http: / / web.expasy.org / protparam (Gasteiger E. et al., Protein Identification and Analysis Tools on the ExPASy Server, John M. Walker (ed.): The Proteomics Protocols Handbook, Humana Press, 2005, pp. 571-607).

[0073] Example 2: Determination of sequence identity and conserved amino acid sequence segments

[0074] The determination of the percentage of sequence identity between the total polypeptide lengths of polypeptides having the amino acid sequences of SEQ ID No. 1-15 (Table 1) can be carried out with the aid of the program BLAST (Basic Local Alignment Search Tool), in particular with BLASTP which can be used on the homepage of the "National Center for Biotechnology Information" (NCBI; http: / / www.ncbi.nlm.nih.gov / ). Thus, it is possible to compare two or more sequences with each other according to the algorithm of Altschul et al., 1997 (Nucleic Acids Res. (1997) 25: 3389-3402). As program settings, the basic settings are considered, but in particular: "Maximum target sequences" = 100; "Expect threshold" = 10; "Word size" = 3; "Matrix" = BLOSOM62; "Gap costs" = "Existence: 11; Extension: 1"; "Calculation adjustments" = "Conditional compositional score matrix adjustment".

[0075] To identify conserved amino acid sequence segments, the polypeptides with SEQ ID No. 1-6 that have at least 70% sequence identity to each other were aligned using the software COBALT (J.S. Papadopoulos and R.Agarwala, 2007, COBALT: constraint-based alignment tool for multiple protein sequences, Bioinformatics 23: 1073-79), considering standard parameters, in particular the following parameters: ("gap penalty": -11, -1; "end gap penalty": -5, -1; "Use RPS BLAST": on; "Blast E-value": 0.003; "Find Conserved columns and Recompute": on; "use query clusters": on; "word length": 4; "may cluster distance": 0.8; "alphabet": regular; "Homology conversation setting": 3 bits). The result of this analysis delineated the conserved amino acids. The conserved amino acid sequence segments were defined as the following regions having at least 5 consecutive conserved amino acids: for the sequence with SEQ ID No. 1, segment A from position +24 to position +50, segment B from position +52 to position +77, segment C from position +79 to position +87, segment D from position +89 to position +145, segment E from position +150 to position +171, segment F from position +177 to position +193, segment G from position +223 to position +228, segment H from position +230 to position +237, segment I from position +239 to position +247, segment J from position +249 to position +255, segment K from position +257 to position +261, segment L from position +263 to position +270, segment M from position +272 to position +279, segment N from position +297 to position +301, and segment O from position +303 to position +313.

[0076] As described above, the determination of the percentage of sequence identity of the conserved amino acid sequence segments among the polypeptides and of the individual polypeptides relative to the conserved amino acid sequence segments of the sequence with SEQ ID No. 1 was performed. The results are presented in Tables 1 and 2.

[0077] Table 1: Percentage of sequence identity among the polypeptides.

[0078]

[0079]

[0080]

[0081] Table 2: Percentage sequence identity of the conserved amino acid sequence segments A to O.

[0082]

[0083]

[0084] Example 3: Hydrolysis of ZEN by the polypeptide in the cell lysate

[0085] To determine its ability to degrade ZEN into non-toxic or low-toxic metabolites HZEN and DHZEN, as described in Example 1, a polypeptide having SEQ ID No. 1 encoded by the nucleotide sequence having SEQ ID No. 17 was prepared in Escherichia coli, and the polypeptide was thus prepared with a C-terminal or N-terminal 6×His tag. Polypeptides having amino acid sequences of SEQ ID Nos. 2-15 encoded by the nucleotide sequences having SEQ ID Nos. 18-31 were labeled only with 6×His at the C-terminus. Each 100 ml of Escherichia coli culture having an optical density (OD600 nm) of 2.0-2.5 was harvested by centrifugation at 4°C and resuspended in 20 ml of Brunner mineral medium (DSMZ microbial medium No. 462, 2012). The cell suspension was lysed by treatment with a French press at 20,000 psi three times. The cell lysate thus obtained was used at dilutions of 1:10, 1:100 or 1:1,000, which were prepared in Brunner mineral medium containing 0.1 mg / ml BSA (bovine serum albumin). For the ZEN degradation assay, 9.9 ml of Brunner mineral medium containing 0.1 mg / ml BSA, 0.1 ml of the diluted cell lysate and 31 μl of the ZEN substrate stock solution were used. In total, the cell lysate was thus diluted 1:1,000, 1:10,000 or 1:100,000. A 2.08 mM ZEN solution (40 vol% ACN + 60 vol% H2O) was used as the ZEN substrate stock solution. To prepare this solution, ZEN in crystalline form (biopure standard from Romer Labs, product number 001109, purity at least 98%) was weighed accordingly and bottled and dissolved. Each degradation batch was carried out in a 25 ml glass vial and incubated at 25°C with shaking at 100 rpm for a total of 120 hours. At time points 0, 0.5, 1, 2, 5, 24, 47, 72 and 120 hours, 1 ml of the sample was taken, the polypeptide was heat inactivated at 99°C for 10 minutes and stored at -20°C. After thawing the sample, the insoluble components were separated by centrifugation. ZEN, HZEN and DHZEN were analyzed by LC / MS / MS. For this purpose, the metabolites were separated chromatographically by means of a Phenomenex Luna C18(2) column with dimensions of 250 mm × 3 mm and a particle size of 5 μm. An acetonitrile-water mixture having a formic acid concentration of 1 ml / l was used as the mobile phase. The UV-signal was recorded at 270 nm. Electrospray ionization (ESI) was used as the ionization source.ZEN, HZEN, and DHZEN were quantified in “enhanced mode” by QTrap / LC / MS / MS (triple quadrupole, Applied Biosystems). No significant amount of ZEN was detected in the batch after 24 h at the latest. Most (more than 80%) of the ZEN was converted to HZEN or DHZEN.

[0086] As seen Figure 1 in, exemplarily for a 1:10,000 diluted cell lysate solution, for the untagged ( Figure 1 A) and for the C-terminally 6×His-tagged ( Figure 1 B) and N-terminally 6×His-tagged ( Figure 1 C) polypeptide with SEQ ID No.1, the degradation of ZEN over time and the increase of HZEN and DHZEN over time. It is clearly evident therefrom that: 1. The conversion of ZEN proceeds immediately and completely, since in the first sample taken immediately after the start of the experiment (0 h), ZEN could hardly be detected anymore; and 2. By attaching a C-terminal or N-terminal tag, no notable loss of activity occurred.

[0087] Example 4: Hydrolysis of ZEN derivatives by polypeptides in cell lysates

[0088] To determine the ability of the polypeptides to convert ZEN derivatives, in addition to ZEN, into non-toxic or less toxic metabolites, as described in Example 3, polypeptides with SEQ ID No.1 - 15 were prepared with a C-terminal His tag and, as cell lysates in “Degradation 15”, the respective synthetic nucleotide sequences with the sequences SEQ ID No.17 - 31 were considered.

[0089] The degradation test was carried out as described in Example 3, in which each polypeptide was tested with each ZEN derivative selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S, and ZAN. The cell lysate was used at a total dilution of 1:10,000. As the substrate stock solution, an equimolar (i.e., 2.08 mM) solution of the ZEN derivative was used instead of the 2.08 mM ZEN solution (40 vol% ACN + 60 vol% H2O). α-ZEL, β-ZEL, α-ZAL, β-ZAL, and ZAN were purchased from Sigma and used as standards for this assay. Z14G and Z14S were prepared with at least 90% purity according to the methods described in, for example, P. Krenn et al., 2007 (Mykotoxin Research, 23, 4, 180 - 184) and M. Sulyok et al., 2007 (Anal. Bioanal. Chem. 289, 1505 - 1523), and used as standards for this assay. Another difference from Example 3 was that only one sample was taken, i.e., after 24 hours. The decrease in the concentration of the ZEN derivative during the degradation test was quantified by LC / MS / MS. α-ZEL, β-ZEL, Z14G, and Z14S were measured according to the method of M. Sulyok et al. (2010, Food Chemistry, 119, 408 - 416); α-ZAL, β-ZAL, and ZAN were measured according to the method of P. Songsermaskul et al. (2011, J. of Animal Physiol. and Animal Nutr., 97, 155 - 161). Surprisingly, it was shown that in all degradation tests, after 24 hours of incubation, only 0% to a maximum of 13% of the starting amount of the ZEN derivative remained.

[0090] Example 5: Specific Activity and Enzyme Kinetic Parameters of Polypeptides and Their Variants

[0091] The determination of the specific activity of the polypeptides and their variants was carried out photometrically, where all polypeptides used had a C-terminal 6×His tag. The preparation, enrichment, and purification of the polypeptide or its variant were carried out as described in Example 1. The degradation of ZEN to HZEN was measured by the decrease in absorbance at a wavelength of 315 nm. The molar extinction coefficients [ε] of ZEN and HZEN were determined experimentally and were 0.0078895 L μmol -1 cm -1 and 0.0030857 L μmol -1 cm -1。The extinction coefficient is strongly pH-dependent and therefore the activity measurements must always be carried out at exactly the same pH value, preferably even in the same matrix. Measurements are carried out in a quartz cuvette in a 50 mM Tris-HCl (pH = 8.2) buffer solution in the wavelength range from 200 to 2500 nm at 32 °C in a UV-VIS photometer (Hitachi U-2001).

[0092] A 2.08 mM ZEN solution (40 vol% ACN + 60 vol% H2O) is used as the ZEN substrate stock solution. To prepare this solution, ZEN in crystalline form (biopure standard from Romer Labs, article number 001109, purity at least 98%) is weighed accordingly and bottled and dissolved. ZEN substrate dilutions (0.79 μΜ, 1.57 μΜ, 2.36 μΜ, 3.14 μΜ, 4.71 μΜ, 6.28 μΜ, 7.85 μΜ, 9.42 μΜ, 10.99 μΜ, 12.56 μΜ, 14.13 μΜ, 15.71 μΜ, 17.28 μΜ, 18.85 μΜ) are prepared with 50 mM Tris-HCl (pH = 8.2). The polypeptide solution is diluted to a final concentration of approximately 70 ng / ml with 50 mM Tris-HCl buffer (pH = 8.2). The ZEN substrate dilutions are preheated to 32 °C in a water bath.

[0093] 0.2 μl of the polypeptide solution is added to 100 μl of each ZEN substrate dilution and the absorbance is measured for 5 minutes, where each "polypeptide solution - ZEN substrate dilution" combination is measured at least twice.

[0094] By taking into account the extinction coefficients of ZEN and HZEN, the reaction rate for each substrate concentration is calculated via the slope of the absorbance over time.

[0095] The name "K M value" or "Michaelis - Menten constant" refers to the parameter used to describe the enzyme affinity, which has the unit [μΜ] or [mM] and is calculated according to H. Bisswang (2002, Enzyme Kinetics, ISBN 3 - 527 - 30343 - X, page 19) with the help of a linear Hanes plot, where for this purpose the function "Enzyme kinetics, single substrate" of the program SigmaPlot 12.0 is preferably used. The name "catalytic constant of the enzyme reaction" or "k cat value" refers to the parameter used to describe the conversion rate of the polypeptide or enzyme, which is given in [s] -1 and is preferably calculated with the help of the function "Enzyme kinetics, single substrate" of the program SigmaPlot 12.0. "Maximum enzyme rate" or "v maxThe "value" is given in units of [μΜ / s] or [mM / s] and is similar to K M value, which is measured by means of a linear Hanes plot, for which the function "Enzyme Kinetics, Single Substrate" of the program SigmaPlot 12.0 is preferably used.

[0096] By means of v max and the enzyme concentration used, the specific activity is calculated according to the following formula

[0097]

[0098] where one unit is defined as hydrolyzing 1 μmοl of ZEN per minute at 32 °C in a 50 mM Tris-HCl buffer solution (pH = 8.2).

[0099] Below, for the polypeptide with SEQ ID NO:1 by way of example, the original data on the determination of the enzyme parameters K M , v max , k cat and the specific activity are listed. Table 3 shows the reaction rates at various ZEN substrate concentrations, Figure 2 shows the respective Michaelis-Menten plot, and the corresponding enzyme kinetic parameters are given in Table 4. The enzyme solution used had a concentration of 68 ng / l.

[0100] Table 3: Reaction rates of the polypeptide with SEQ ID NO:1 at different ZEN concentrations.

[0101]

[0102] Table 4: Enzyme kinetic parameters of the polypeptide with SEQ ID No.1.

[0103]

[0104] The specific activities of the polypeptides studied were: 8.25 U / mg for SEQ ID No.1; 10.56 U / mg for SEQ ID No.2; 8.36 U / mg for SEQ ID No.3; 8.33 U / mg for SEQ ID No.4; 8.56 U / mg for SEQ ID No.5; 9.95 U / mg for SEQ ID No.6; 3.83 U / mg for SEQ ID No.7; 2.57 U / mg for SEQ ID No.8; 4.87 U / mg for SEQ ID No.9; 5.12 U / mg for SEQ ID No.10; 3.88 U / mg for SEQ ID No.11; 2.78 U / mg for SEQ ID No.12; 6.43 U / mg for SEQ ID No.13; 3.33 U / mg for SEQ ID No.14; and 7.76 U / mg for SEQ ID No.15.

[0105] The specific activities of the polypeptide variants studied are listed in Tables 5 and 6.

[0106] Table 5: Specific activities of functional variants of the polypeptide having SEQ ID No.1; the conserved amino acid sequence segments in which the mutations are located; and the sequence identity of the functional variants relative to the parental sequence having SEQ ID No.1. The positions of the mutations are given relative to the amino acid sequence having SEQ ID No.1. As described in Example 2,

[0107] sequence identity was determined by BLAST.

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] Table 6: Specific activities of functional variants of the polypeptide having SEQ ID No.2. The positions of the mutations are relative to the amino acid sequence having SEQ ID No.2. As described in Example 2, sequence identity was determined by BLAST.

[0116]

[0117] Example 6: Degradation of ZEN and ZEN derivatives in contaminated maize

[0118] To determine the ability of the polypeptides to degrade naturally occurring ZEN and ZEN derivatives in a complex matrix and at low pH, different concentrations of one of the polypeptides having SEQ ID No. 1 - 6 were incorporated into contaminated maize each time, and the degradation of ZEN and ZEN derivatives was followed.

[0119] The contaminated maize was milled and used in the degradation assay, where the batch consisted of 1 g of milled contaminated maize, 8.9 ml of 100 mM acetate buffer (pH = 4.0), and 0.1 ml of polypeptide solution. The enriched and purified polypeptide solutions were prepared as described in Example 5, where they were diluted to concentrations of 10 mU / ml, 100 mU / ml, or 1,000 mU / ml. Thus, 1 mU (= 1 mU / g maize), 10 mU (= 10 mU / g maize), or 100 mU (= 100 mU / g maize) was used in absolute terms in the batch. Each degradation batch was carried out in a preparation of 25 ml and incubated at 37 °C with shaking at 100 rpm. Samples of 1 ml were taken before enzyme addition and after 1 h of incubation, the polypeptides were heat inactivated at 99 °C for 10 min, and the samples were stored at -20 °C. After thawing the samples, the insoluble components were separated by centrifugation. The concentrations of ZEN and ZEN derivatives were measured by LC / MS / MS as described in M. Sulyok et al. (2007, Anal. Bioanal. Chem., 289, 1505 - 1523). The content of ZEN and ZEN derivatives in this maize was: 238 ppb for ZEN; 15 ppb for α - ZEL; 23 ppb for β - ZEL; 32 ppb for Z14G; and 81 ppb for Z14S. The percentages of the decrease in the content of ZEN and ZEN derivatives in the degradation assay are presented in Table 7.

[0120] Table 7: Reduction of ZEN and ZEN derivatives in the degradation assay in percentage relative to the starting content for different polypeptides and amounts of polypeptide.

[0121]

[0122] Example 7: Additive containing polypeptide for hydrolytic cleavage of ZEN and / or ZEN derivatives

[0123] For the preparation of an additive for the hydrolytic cleavage of ZEN, the fermentation supernatants of the polypeptides with SEQ ID No. 1, 2, 6 and 13 expressed via Pichia pastoris were purified under standard conditions by microfiltration and ultrafiltration (exclusion limit: 10 kDa) and concentrated to a dry matter concentration of approximately 9 wt%. Subsequently, the polypeptide-containing solution was further processed into a dry powder in a spray dryer (Mini B290 from Büchi) also under standard conditions. These four powders were successively named Z1, Z2, Z6 and Z13. In addition, Z1, Z2, Z6 or Z13 was mixed with bentonite with an average particle size of approximately 1 µm in an up-shaker in a ratio of 1 wt% of the additive Z1, Z2, Z6 or Z13 and 99 wt% of bentonite. The additives thus obtained were named additive Z1.B, Z2.B, Z6.B and Z13.B. In addition, Z1, Z2, Z6 and Z13 were mixed with bentonite and a vitamin-trace element concentrate in an up-shaker in a ratio of 0.1 wt% of Z1, Z2, Z6 or Z13, 0.9 wt% of the vitamin-trace element concentrate, and 99 wt% of bentonite. The additives thus obtained were named additive Z1.BVS, Z2.BVS, Z6.BVS and Z13.BVS. 100 g of the additives Z1.BVS, Z2.BVS, Z6.BVS and Z13.BVS contain 200 mg of iron sulfate, 50 mg of copper sulfate, 130 mg of zinc oxide, 130 mg of manganese oxide, 2.55 mg of calcium carbonate, 160 mg of vitamin E, 6.5 mg of vitamin K3, 6.5 mg of vitamin B1, 14 mg of vitamin B2, 15 mg of vitamin B6, 0.15 mg of vitamin B12, 150 mg of niacin, 30 mg of pantothenic acid and 5.3 mg of folic acid.

[0124] The additives were extracted in 50 mM Tris-HCl buffer (pH = 8.2) for 30 minutes and further diluted in the same buffer such that the final concentration of the polypeptide was approximately 70 ng / ml.

[0125] Subsequently, the effect of these solutions on the degradation of zearalenone was determined as described in Example 5. The corresponding activities were: 8.230 U / g for Z1; 9.310 U / g for Z2; 9.214 U / g for Z6; 83 U / g for Z1.B; 92 U / g for Z2.B; 90 U / g for Z2.C; 57 U / g for Z13.B; 8 U / g for Z1.BVS; 9 U / g for Z2.BVS; 9 U / g for Z6.BVS; and 6 U / g for Z13.BVS.

[0126] The ability of additives Z1, Z2, Z6, Z13, Z1.B, Z2.B, Z6.B, Z13.B, Z1.BVS, Z2.BVS, Z6.BVS and Z13.BVS to degrade the ZEN derivatives α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN was carried out as described in Example 4, but using 100 μl of a polypeptide solution having a polypeptide concentration of approximately 70 ng / ml instead of 100 μl of the cell lysate. After 6 hours of incubation, only a maximum of 15% of the starting amount was present as the unhydrolyzed ZEN derivative.

[0127] Example 8: Optimal temperature

[0128] To determine the optimal temperature of the polypeptides with SEQ ID No. 1, 2, 5, 6, 7, 9, 11, 12, and 15, as described in Example 1, they were cloned with a C-terminal 6×His tag, expressed in E. coli, and purified. In preliminary experiments, for each polypeptide, that concentration was determined at which complete conversion of ZEN was ensured after a test duration of 3 hours under the test conditions (Teorell Stenhagen buffer (Teorell and Stenhagen, Ein Universalpuffer für den pH-Bereich 2.0 bis 12.0. Biochem Ztschrft, 1938, 299: pages 416 - 419), pH 7.5, with 0.1 mg / ml BSA, at 30 °C). The preparations were used in the degradation batches for determining the optimal temperature at the determined concentration. The test was carried out in a PCR cycler (Eppendorf) using a temperature gradient function at 20 °C ± 10 °C, at 40 °C ± 10 °C, and if required, at 60 °C ± 10 °C (10 temperatures in each range; temperatures of the pre-determined PCR cycler). For the batches, the corresponding enzyme concentration as well as 0.1 mg / ml BSA and 5 ppm ZEN were incorporated into the Teorell-Stenhagen buffer at the respective optimal pH. Batches with 0.1 mg / ml BSA and 5 ppm ZEN without enzyme addition were used as negative controls. After incubation times of 0 h, 0.5 h, 1 h, 2 h, and 3 h, one sample was taken for each incubation temperature, heat-inactivated at 99 °C for 10 minutes, and stored at -20 °C. After thawing, the samples were transferred to HPLC vials. ZEN, HZEN, and DHZEN were analyzed by means of HPLC-DAD. For this purpose, the metabolites were separated chromatographically using a Zorbax SB-Aq C18 column with dimensions of 4.6 mm × 150 mm and a particle size of 5 μm. A methanol-water mixture with 5 mM ammonium acetate was used as the mobile phase. The UV-signal was recorded at 274 nm. Quantification of the metabolites was carried out by including a series of carried standards. The optimal temperature was determined by the slope of the determined degradation curve, where the temperature at which the slope was maximum was defined as the optimal temperature. The optimal temperatures are shown in Table 8.

[0129] Table 8: Optimal temperatures of the polypeptides.

[0130]

[0131] Example 9: Temperature stability

[0132] To determine the temperature stability of the polypeptides having SEQ ID No. 1, 2, 5, 6, 7, 9, 11, 12 and 15, as described in Example 1, they were cloned with a C-terminal 6×His tag, expressed in E. coli and purified. They were incubated in a PCR cycler with a gradient function at their respective optimal temperatures ±10°C. After 0 minutes, 15 minutes, 30 minutes and 60 minutes, a sample was taken for each batch and each temperature. Subsequently, in the degradation experiment, these pre-incubated samples were used in the batch in Teorell-Stenhagen buffer at their respective optimal pH with 0.1 mg / ml BSA and 5 ppm ZEN. In a preliminary experiment, for each polypeptide, that concentration was determined at which complete conversion of ZEN could be ensured after a test duration of 3 hours under the test conditions (Teorell Stenhagen buffer, pH 7.5, with 0.1 mg / ml BSA, at 30°C). The respective determined enzyme concentration was used in the batch. The degradation batches were incubated at 30°C. Sampling was carried out after incubation times of 0 hours, 0.5 hours, 1 hour, 2 hours and 3 hours. Subsequently, the polypeptides were heat inactivated at 99°C for 10 minutes and the samples were stored at -20°C. After thawing, the samples were transferred to HPLC vials and analyzed by means of HPLC-DAD as described in Example 8.

[0133] The temperature stability was defined as the temperature at which the polypeptide had 50% residual activity after a 15-minute pre-incubation, compared to the optimal temperature. As a measure of activity, the slope of the degradation curve was considered. The temperature stability is shown in Table 9.

[0134] Table 9: Temperature stability of the polypeptides (50% residual activity after a 15-minute pre-incubation).

[0135]

[0136] Example 10: Optimal pH

[0137] To determine the optimal pH of the polypeptides having SEQ ID No. 1, 2, 5, 6, 7, 9, 11, 12 and 15, they were cloned with a C-terminal 6×His tag, expressed in E. coli and purified as described in Example 1. In preliminary experiments, for each polypeptide, that concentration was determined at which complete conversion of ZEN could be ensured after a test duration of 3 hours under the test conditions (Teorell - Stenhagen buffer, pH 7.5, with 0.1 mg / ml BSA, at 30 °C). The respective enzyme concentration was used in the batch. The degradation batches were prepared in Stenhagen buffer at pH values of 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11.0 and 12.0. For degradation, the degradation batches with 0.1 mg / ml BSA and 5 ppm ZEN were incubated at 30 °C. Batches in Teorell - Stenhagen buffer at pH 3.0, pH 7.0 and pH 12.0 with 0.1 mg / ml BSA and 5 ppm ZEN were used as negative controls. Sampling was carried out after incubation times of 0 h, 0.5 h, 1 h, 2 h and 3 h. Subsequently, the polypeptides were heat inactivated at 99 °C for 10 minutes and the samples were stored at -20 °C. After thawing, the samples were transferred to HPLC vials and analyzed by HPLC - DAD as described in Example 8. The determination of the optimal pH was carried out by the slope of the measured degradation curve, where the pH value at which the slope was maximum was defined as the optimal pH. The optimal pH is shown in Table 10.

[0138] Table 10: Optimal pH of the polypeptides.

[0139]

[0140] Example 11: pH stability at pH 5.0

[0141] To determine pH stability, the polypeptide from Example 10 was incubated at 25 °C for 1 hour in Teroell-Stenhagen buffer at pH 5.0 and at their respective optimal pH. In the degradation assay, these pre-incubated samples were used in batch in 100 mM Tris-HCl buffer at their respective optimal pH with 0.1 mg / ml BSA and 5 ppm ZEN at the same concentration of each polypeptide as used for the determination of the optimal pH. Sampling was performed after incubation times of 0 h, 0.5 h, 1 h, 2 h, and 3 h. Subsequently, the polypeptide was heat inactivated at 99 °C for 10 minutes and the samples were stored at -20 °C. After thawing, the samples were transferred to HPLC vials and analyzed by HPLC-DAD as described in Example 8. The pH stability was defined as the percentage of residual activity of the polypeptide at pH 5.0 relative to the activity at their respective optimal pH. The pH stability for pH 5.0 is shown in Table 11.

[0142] Table 11: pH stability of the polypeptide at pH 5.0.

[0143]

[0144] Example 12: ZEN degradation assay

[0145] Exemplarily, for the polypeptides having SEQ ID No. 1, 2, 5, 6, 7, 9, 11, 12, and 15, the degradation of ZEN to HZEN and DHZEN was performed. The degradation batch was prepared in Teorell-Stenhagen buffer (pH 7.5) with 0.1 mg / ml BSA and 5 ppm ZEN. The degradation batch was incubated at 30 °C. Sampling was performed after incubation times of 0 h, 0.5 h, 1 h, 2 h, and 3 h. Subsequently, the polypeptide was heat inactivated at 99 °C for 10 minutes and the samples were stored at -20 °C. After thawing, the samples were transferred to HPLC vials and analyzed by HPLC-DAD as described in Example 8. The polypeptide concentration was selected such that complete degradation was achieved after approximately 3 hours. The degradation kinetics are depicted in Figure 3, where the y-axis shows the concentration of ZEN, HZEN, and DHZEN in micromoles per liter (μmol / l), and the x-axis shows the incubation time in hours (h).

[0146] *μΜ represents microvolume molar concentration and corresponds to the unit μmol / l.

[0147] Some embodiments of the present invention are as follows:

[0148] 1. A polypeptide that cleaves zearalenone and / or at least one zearalenone derivative by hydrolysis, characterized in that the polypeptide is a hydrolase having an amino acid sequence selected from SEQ ID No. 1-15 or a functional variant thereof, wherein the sequence identity between the functional variant and at least one of the amino acid sequences is at least 40%.

[0149] 2. The polypeptide according to embodiment 1, characterized in that the polypeptide comprises at least one conserved amino acid sequence segment or a functional variant thereof, wherein the functional variant of the amino acid sequence segment has at least 70%, preferably at least 84%, more preferably at least 92%, and most preferably at least 98% sequence identity, and the at least one conserved amino acid sequence segment is selected from the amino acid sequences of SEQ ID No. 1: +24 to +50, +52 to +77, +79 to +87, +89 to +145, +150 to +171, +177 to +193, +223 to +228, +230 to +237, +239 to +247, +249 to +255, +257 to +261, +263 to +270, +272 to +279, +297 to +301, +303 to +313, +24 to 328, +1 to +328.

[0150] 3. The polypeptide according to embodiment 1 or 2, characterized in that the functional variant has amino acid modifications selected from: substitution, deletion and insertion of one or more amino acids each.

[0151] 4. The polypeptide according to any one of embodiments 1, 2 or 3, characterized in that the polypeptide has a specific activity of at least 0.01 U / mg, preferably at least 0.1 U / mg, particularly at least 1 U / mg, and / or has a K value for hydrolytic cleavage of zearalenone of at most 50 μM, preferably at most 3.5 μM, particularly at most 0.5 μM, and / or has a k value for hydrolytic cleavage of zearalenone of at least 0.05 s, preferably at least 0.6 s, particularly at least 5 s, and / or has a v value for hydrolytic cleavage of zearalenone of at least 0.00001 μM s, preferably at least 0.0001 μM s, particularly at least 0.001 μM s. M value, and / or has at least 0.05 s -1 preferably at least 0.6 s -1 particularly at least 5 s -1 for hydrolytic cleavage of zearalenone, and / or has a v cat value for hydrolytic cleavage of zearalenone of at least 0.00001 μM -1 s -1 preferably at least 0.0001 μM -1 s -1 particularly at least 0.001 μM -1 s -1 value. max

[0152] 5. A polypeptide according to one of embodiments 1 to 4, characterized in that the polypeptide comprises an amino acid sequence selected from SEQ ID No. 2, 5, 6, 7, 9, 11 and 15 or a functional variant thereof, wherein the functional variant has at least 40% sequence identity with at least one of the amino acid sequences, and the polypeptide has a pH stability of at least 15%, preferably 50%, and particularly preferably at least 90% at pH 5.0.

[0153] 6. A polypeptide according to one of embodiments 1 to 4, characterized in that the polypeptide comprises an amino acid sequence selected from SEQ ID No. 1, 2, 5, 6, 7, 9, 11, 15 or a functional variant thereof, wherein the functional variant has at least 40% sequence identity with at least one of the amino acid sequences, and the polypeptide has the highest enzymatic activity in the temperature range between 30 °C and 75 °C, preferably between 38 °C and 55 °C, particularly preferably between 38 °C and 52 °C.

[0154] 7. A polypeptide according to one of embodiments 1 to 4, characterized in that the polypeptide comprises an amino acid sequence selected from SEQ ID No. 1, 5, 6, 9, 11, 12 and 15 or a functional variant thereof, wherein the functional variant has at least 40% sequence identity with at least one of the amino acid sequences, and the polypeptide is temperature stable up to a temperature of 90 °C, preferably 75 °C, and particularly preferably 60 °C.

[0155] 8. A polypeptide according to one of embodiments 1 to 7, characterized in that the polypeptide has at least one mutation with respect to the amino acid sequence of SEQ ID No. 1 at at least one position selected from the following: 22, 23, 25, 26, 27, 29, 31, 32, 35, 37, 42, 43, 46, 51, 53, 54, 57, 60, 69, 72, 73, 78, 80, 84, 88, 95, 97, 99, 114, 118, 119, 123, 132, 141, 146, 148, 149, 154, 163, 164, 165, 169, 170, 172, 176, 180, 182, 183, 190, 191, 194, 196, 197, 198, 201, 204, 205, 206, 207, 208, 209, 210, 212, 213, 214, 216, 217, 220, 221, 222, 229, 231, 233, 238, 240, 244, 245, 246, 248, 249, 251, 254, 256, 260, 262, 263, 266, 269, 271, 277, 280, 281, 282, 283, 284, 285, 286, 287, 292, 296, 298, 302, 307, 308, 309, 311, 314, 317, 319, 321, 323, 325 and 326.

[0156] 9. The polypeptide according to embodiment 8, characterized in that the polypeptide has at least one mutation in the amino acid sequence of SEQ ID No. 1 selected from the following: D22A, S23Q, S23L, N25D, I26V, F27Y, F27H, S29P, R31A, F32Y, R35K, R35Q, V37A, V42I, V43T, F46Y, S51E, S51D, D53G, N54M, N54R, L57V, L60I, S69G, P72E, V73A, A78S, N80H, F84Y, I88L, T95S, T97A, R99K, I114M, I118V, K119R, V123I, L132V, A141S, I146V, I146L, A148G, A149V, A154P, P163T, A164T, Y165C, Y165H, V169I, L170R, A172G, A176M, A176V, Y180F, D182T, F183Y, I190V, G191S, K194T, K194E, F196Y, V197C, V197R, E198R, E198S, K201D, K201G, P204S, P204A, A205S, K206P, A207M, M208A, Q209R, L210A, L210S, ΔP212, T213V, P214A, E216T, E216G, A217I, N220H, L221M, K222R, K222Q, G229A, A231V, F233W, F233Y, F233H, A238G, H240N, H240S, D244E, R245Q, M246L, S248T, S248N, S248G, Q249R, K251N, I254V, I256L, A260M, T262D, T262G, I263T, E266D, E269H, E269N, L271V, L277E, E280A, E280L, H281R, H281Q, A282V, Q283R, D284L, D284R, I285L, I286M, R287E, R287D, R292K, R292T, Q296A, Q296E, H298V, L302S, L307Q, F308S, D309A, A311P, A314V, L317F, S319Q, S319P, S319R, S321A, S321T, T323A, P325A, A326P.

[0157] 10. The polypeptide according to any one of embodiments 1 to 8, characterized in that it comprises at least one of the following amino acid motifs selected from SEQ ID Nos. 32 - 69.

[0158] 11. The polypeptide according to embodiment 9, characterized in that the polypeptide comprises at least one conservative amino acid substitution at at least one position, and the conservative amino acid substitution is selected from: G to A; or A to G, S; or V to I, L, A, T, S; or I to V, L, M; or L to I, M, V; or M to L, I, V; or P to A, S, N; or F to Y, W, H; or Y to F, W, H; or W to Y, F, H; or R to K, E, D; or K to R, E, D; or H to Q, N, S; or D or N, E, K, R, Q; or E to Q, D, K, R, N; or S to T, A; or T to S, V, A; or C to S, T, A; or N to D, Q, H, S; or Q to E, N, H, K, R substitutions.

[0159] 12. An isolated polynucleotide having a nucleotide sequence encoding a polypeptide, wherein the polypeptide has the property of hydrolyzing zearalenone and / or at least one zearalenone derivative, characterized in that the nucleotide sequence encodes at least one polypeptide according to one of embodiments 1 to 11; and / or the nucleotide sequence has a degree of sequence identity of at least 40% with at least one nucleotide sequence selected from SEQ ID No. 16 - 31, wherein the selected nucleotide sequence is at least 40%; and / or the nucleotide sequence hybridizes with at least one nucleotide sequence selected from SEQ ID No. 16 - 31 under medium stringency conditions, and / or hybridizes with a partial sequence thereof having at least 200 nucleotides, particularly at least 100 nucleotides, and / or hybridizes with the complementary strand of the above nucleotide sequence or its partial sequence.

[0160] 13. An additive for hydrolytically cleaving zearalenone and / or at least one zearalenone derivative, characterized in that the additive comprises at least one polypeptide having an amino acid sequence selected from SEQ ID No. 1 - 15 or a functional variant thereof, wherein the sequence identity between the functional variant and at least one of the amino acid sequences is at least 40%; and optionally comprises an adjuvant.

[0161] 14. The additive according to embodiment 13, characterized in that it comprises at least one polypeptide according to one of embodiments 1 to 11.

[0162] 15. The additive according to one of embodiments 13 or 14, characterized in that the adjuvant is selected from: at least one inert carrier, and optionally, other components such as vitamins and / or minerals and / or enzymes and / or other components for detoxifying mycotoxins.

[0163] 16. An additive according to one of embodiments 13, 14 or 15, characterized in that in the additive, at least one polypeptide according to one of embodiments 1 to 11 is included at a concentration of at most 10,000 U / g, preferably at most 1,000 U / g, more preferably at most 100 U / g, and most preferably at most 10 U / g.

[0164] 17. An additive according to one of embodiments 13 to 16, characterized in that the additive is present in encapsulated or coated form.

[0165] 18. Use of an additive according to one of embodiments 13 to 17 for hydrolytic cleavage of zearalenone and / or at least one zearalenone derivative in feed, in food or in, said feed being in particular feed for pigs, poultry and aquaculture.

[0166] 19. A method for hydrolytic cleavage of zearalenone and / or at least one zearalenone derivative, characterized in that the zearalenone and / or at least one zearalenone derivative is hydrolyzed by a polypeptide having an amino acid sequence selected from SEQ ID No. 1 - 15 or a functional variant thereof, wherein the sequence identity between the functional variant and at least one of the amino acid sequences is at least 40%.

[0167] 20. A method according to embodiment 19, characterized in that the polypeptide is used in an additive according to one of embodiments 14 to 17.

[0168] 21. A method according to embodiment 20, characterized in that the polypeptide or additive is admixed with feed or food contaminated with zearalenone and / or with at least one zearalenone derivative, the contaminated feed or food is brought into contact with moisture, and the polypeptide or additive hydrolyzes the zearalenone and / or at least one zearalenone derivative comprised in the contaminated feed or food.

[0169] 22. A method according to one of embodiments 19 to 21, characterized in that at least 70%, preferably at least 80%, particularly preferably at least 90% of the zearalenone and / or at least one zearalenone derivative is hydrolyzed.

Claims

1. A method for hydrolytic cleavage of zearalenone and / or at least one zearalenone derivative selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN, characterized in that, The zearalenone and / or at least one zearalenone derivative is hydrolyzed by a polypeptide consisting of the amino acid sequence of SEQ ID No. 6 or a functional variant thereof, wherein the functional variant of the amino acid sequence of SEQ ID No. 6 has at least one mutation selected from the following with respect to the sequence of SEQ ID No. 1: D22A, S23Q, S23L, N25D, I26V, F27Y, F27H, S29P, R31A, F32Y, R35K, R35Q, V42I, V43T, F46Y, S51E, S51D, N54M, N54R, L57V, L60I, S69G, P72E, V73A, A78S, N80H, F84Y, I88L, T95S, R99K, I118V, L132V, A141S, I146V, I146L, A148G, A149V, A154P, Y165C, Y165H, L170R, A172G, A176M, A176V, Y180F, D182T, F183Y, I190V, K194T, K194E, V197C, V197R, E198R, E198S, K201D, K201G, P204S, P204A, A205S, K206P, A207M, M208A, Q209R, L210A, L210S, T213V, P214A, E216T, E216G, A217I, N220H, K222R, K222Q, G229A, A231V, F233W, F233Y, F233H, A238G, H240N, H240S, D244E, S248T, S248N, S248G, I254V, I256L, A260M, T262D, T262G, E269H, E269N, L277E, E280A, E280L, H281R, H281Q, Q283R, D284L, D284R, I285L, I286M, R287E, R287D, R292K, R292T, Q296A, Q296E, H298V, L302S, L307Q, F308S, D309A, A314V, L317F, S319Q, S319P, S319R, S321A, S321T, T323A, P325A and A326P.

2. The method according to claim 1, wherein The functional variants of the amino acid sequence of SEQ ID No.6 have mutations selected from the following with respect to the sequence of SEQ ID No.1: N25D, F27Y, F27H, R35K, R35Q, N25D / S29P / V42I / V43T, I26V / R31A / F32Y / F46Y, N25D / I26V / F27Y / S29P / R31A / F32Y / R35K / V37A / V42I / V43T / F46Y, N25D / I26V / F27H / S29P / R31A / F32Y / R35Q / V42I / V43T / F46Y, D53G, N54M, N54R, S69G, P72E, P72R, N54M / L57V / L60I / S69G / P72E / V73A, D53G / N54R / L57V / L60I / P72E / V73A, N54R / L57V / L60I / P72E / V73A, N54R / L58V / L59P / L60V / T64G / P72R / G75P / L77P, N80H, N80D, F84Y, N80H / F84Y, N80H / F84H, E79R / N80D, T95S, R99K, V123I, A125G, G126A, G130A, G130V, G131A, N127D, N127Q, A141S, F106W, I118V, I118V / V123L, I118V / K119R / L132V, W96Q / F106W / L116G / V122A, Q91R / N105D / K119G / A141S / M142K, T95S / T97A / R99K / I118V / V123I / L132V / A141S, T95S / R99K / I118V / K119R / L132V / A141S, T95S / R99K / I118V / L132V / A141S, T95S / R99K / I114M / I118V / K119R / L132V / A141S, R99G / A115D / K119G / P121T / V123I / A125S / L132V / L133V / S138A / Y140F / A141S / M142L, R93K / W96Q / R99G / D104N / N105L / F106M / A115S / V123I / A125S / G144N, R99G / S102N / D104N / N105T / F106W / L110V / V111E / A115D / K119G / V122T / V123L / L132V / L133I / S138A / M142K,W96R / S102T / F106I / I114L / A115S / L116G / K119G / V122A / V123F / A125S / A134S / Y140F / M142E, W96R / R99G / S102T / F106V / I114L / A115D / L116G / K119G / V122A / V123F / A125S / N127L / L133A / A134S / Y140F / M142K, S94T / R99G / S102T / N105I / L110V / A115D / K119G / P121E / V122T / V123L / V124I / L133I / A134G / S138A / Y140F / M142K, R93Q / R99G / N105T / R112K / A115D / L116I / A125S / N127L / L132V / L133V / A134S / Y140F / M142K, Q91R / W96R / N105D / I114L / I118V / K119R / V122A / L132V / L137S, Y165C, Y165H, P163T, A154P / Y165C, P163T / A164T / Y165C / V169I / L170R, A154P / Y165H / L170R, Y180F, D182T, D182K, Y180F / R181V / I190V, Y180F / D182T / F183Y / I190V / G191S, Y180F / D182T / F183Y / I190V, E178A / R181V / D182K / F183Y, T236K, V237F, E234G, F233W, F233Y, F233H, A231V / F233Y, F232W / F233A / E234T / G235D / L239A, H240N, H240S, D244E / R245Y, D244E / R245Q / M246L, H240N / D244E, H240S / D244E, L239Q / H240T / R245Y, Q249R, T252V, I254V, Q249R / K251N / I254V, T252V / I254M, T252V / I254V, A260M, A260F, A260S, E266Y, E266D, T262G, T262D / E266D / , T262G / I263T / , E266D / E269H, I263T / E269N, E269N, E266Y / E269N, L274M, L274C, L277E, L274M / A279V, L274T / L277F, L274C / L277I, H297L, H298V / L302S, H298V, H298L / P299DL307Q, F308S, L307Q / A311P, L307Q / F308S, L307Q / F308S / D309A, D53G / N54R / L57V / L60I / P72E / V73A / F233V / E234G / V237F, N80H / F84Y / T95S / R99K / I118V / K119R / L132V / A141S, T95S / T97A / R99K / I118V / V123I / L132V / A141S / A260M, T95S / T97A / R99K / I118V / V123I / L132V / A141S / L277E, A260M / H298V, A260M / T262D / E266D / E269H, A260M / T262G / I263T / E269N, Q296A / H298V / L307Q / A311P, Q296E / H298V / L302S / L307Q / F308S, N80H / F84Y / T95S / R99K / I118V / L132V / A141S / Q249R / K251N / I254V, D53G / N54R / L57V / L60I / P72E / V73A / T95S / R99K / I114M / I118V / K119R / L132V / A141S / A260M, I254V / I256L / A260M / T262G / I263T / E269N, I254V / I256L / A260M / T262D / E266D / E269H / L271V, E79R / N80D / D53G / N54R / L57V / L60I / P72E / V73A / W96R / R99G / S102T / F106V / I114L / A115D / L116G / K119G / V122A / V123F / A125S / N127L / L133A / A134S / Y140F / M142K / T252V / I254V, ΔP212, ΔG5 / ΔT6 / ΔR7 / ΔS8 / ΔE9 / ΔA10 / ΔA11 / ΔD12 / ΔA13 / ΔA14 / ΔT15 / ΔQ16 / ΔA17 / ΔR18 / ΔQ19, ΔN327 / ΔD328, N25D / I26V / F27Y / S29P / R31A / F32Y / R35K / V37A / V42I / V43T / F46Y / N54R / L58V / L59P / L60V / T64G / P72R / G75P / L77P / R99G / S102N / D104N / N105T / F106W / L110V / V111E / A115D / K119G / V122T / V123L / L132V / L133I / S138A / M142KΔG5 / ΔT6 / ΔR7 / ΔS8 / ΔE9 / ΔA10 / ΔA11 / ΔD12 / ΔA13 / ΔA14 / ΔT15 / ΔQ16 / ΔA17 / ΔR18 / ΔQ19 / ΔP212 / ΔN327 / ΔD328 / E79R / N80D / D53G / N54R / L57V / L60I / P72E / V73A / W96R / R99G / S102T / F106V / I114L / A115D / L116G / K119G / V122A / V123F / A125S / N127L / L133A / A134S / Y140F / M142K / T252V / I254V, ΔG5 / ΔT6 / ΔR7 / ΔS8 / ΔE9 / ΔA10 / ΔA11 / ΔD12 / ΔA13 / ΔA14 / ΔT15 / ΔQ16 / ΔA17 / ΔR18 / ΔQ19 / ΔP212 / ΔN327 / ΔD328 / N25D / I26V / F27Y / S29P / R31A / F32Y / R35K / V37A / V42I / V43T / F46Y / E79R / N80D / D53G / N54R / L57V / L60I / P72E / V73A / W96R / R99G / S102T / F106V / I114L / A115D / L116G / K119G / V122A / V123F / A125S / N127L / L133A / A134S / Y140F / M142K / T252V / I254V, and L302S.

3. The method according to claim 1 or 2, characterized in that, The polypeptide or an additive containing the polypeptide is admixed with feed or food contaminated with zearalenone and / or at least one zearalenone derivative, the contaminated feed or food is brought into contact with moisture, and the polypeptide or additive hydrolyzes the zearalenone and / or at least one zearalenone derivative contained in the contaminated feed or food.

4. A method for hydrolytic cleavage of zearalenone and / or at least one zearalenone derivative selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN, characterized in that, The zearalenone and / or at least one zearalenone derivative is hydrolyzed by a polypeptide consisting of the amino acid sequence of SEQ ID No.

6.

5. The method according to any one of claims 1-4, characterized in that At least 70% of the zearalenone and / or at least one zearalenone derivative is hydrolyzed.

6. The method according to any one of claims 1-4, characterized in that, At least 80% of the zearalenone and / or at least one zearalenone derivative is hydrolyzed.

7. The method according to any one of claims 1-4, characterized in that, At least 90% of the zearalenone and / or at least one zearalenone derivative is hydrolyzed.

8. Use of a polypeptide consisting of the amino acid sequence of SEQ ID No. 6 or a functional variant thereof for hydrolytic cleavage of zearalenone and / or at least one zearalenone derivative in feed, in food or in dried distillers grains, wherein the zearalenone derivative is selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN, The functional variants of the amino acid sequence of SEQ ID No.6 have at least one mutation selected from the following with respect to the sequence of SEQ ID No.1: D22A, S23Q, S23L, N25D, I26V, F27Y, F27H, S29P, R31A, F32Y, R35K, R35Q, V42I, V43T, F46Y, S51E, S51D, N54M, N54R, L57V, L60I, S69G, P72E, V73A, A78S, N80H, F84Y, I88L, T95S, R99K, I118V, L132V, A141S, I146V, I146L, A148G, A149V, A154P, Y165C, Y165H, L170R, A172G, A176M, A176V, Y180F, D182T, F183Y, I190V, K194T, K194E, V197C, V197R, E198R, E198S, K201D, K201G, P204S, P204A, A205S, K206P, A207M, M208A, Q209R, L210A, L210S, T213V, P214A, E216T, E216G, A217I, N220H, K222R, K222Q, G229A, A231V, F233W, F233Y, F233H, A238G, H240N, H240S, D244E, S248T, S248N, S248G, I254V, I256L, A260M, T262D, T262G, E269H, E269N, L277E, E280A, E280L, H281R, H281Q, Q283R, D284L, D284R, I285L, I286M, R287E, R287D, R292K, R292T, Q296A, Q296E, H298V, L302S, L307Q, F308S, D309A, A314V, L317F, S319Q, S319P, S319R, S321A, S321T, T323A, P325A and A326P.

9. Use according to claim 8, characterized in that, Functional variants of the amino acid sequence of SEQ ID No. 6 have mutations selected from the following with respect to the sequence of SEQ ID No. 1: N25D, F27Y, F27H, R35K, R35Q, N25D / S29P / V42I / V43T, I26V / R31A / F32Y / F46Y, N25D / I26V / F27Y / S29P / R31A / F32Y / R35K / V37A / V42I / V43T / F46Y, N25D / I26V / F27H / S29P / R31A / F32Y / R35Q / V42I / V43T / F46Y, D53G, N54M, N54R, S69G, P72E, P72R, N54M / L57V / L60I / S69G / P72E / V73A, D53G / N54R / L57V / L60I / P72E / V73A, N54R / L57V / L60I / P72E / V73A, N54R / L58V / L59P / L60V / T64G / P72R / G75P / L77P, N80H, N80D, F84Y, N80H / F84Y, N80H / F84H, E79R / N80D, T95S, R99K, V123I, A125G, G126A, G130A, G130V, G131A, N127D, N127Q, A141S, F106W, I118V, I118V / V123L, I118V / K119R / L132V, W96Q / F106W / L116G / V122A, Q91R / N105D / K119G / A141S / M142K, T95S / T97A / R99K / I118V / V123I / L132V / A141S, T95S / R99K / I118V / K119R / L132V / A141S, T95S / R99K / I118V / L132V / A141S, T95S / R99K / I114M / I118V / K119R / L132V / A141S, R99G / A115D / K119G / P121T / V123I / A125S / L132V / L133V / S138A / Y140F / A141S / M142L, R93K / W96Q / R99G / D104N / N105L / F106M / A115S / V123I / A125S / G144N, R99G / S102N / D104N / N105T / F106W / L110V / V111E / A115D / K119G / V122T / V123L / L132V / L133I / S138A / M142K,W96R / S102T / F106I / I114L / A115S / L116G / K119G / V122A / V123F / A125S / A134S / Y140F / M142E, W96R / R99G / S102T / F106V / I114L / A115D / L116G / K119G / V122A / V123F / A125S / N127L / L133A / A134S / Y140F / M142K, S94T / R99G / S102T / N105I / L110V / A115D / K119G / P121E / V122T / V123L / V124I / L133I / A134G / S138A / Y140F / M142K, R93Q / R99G / N105T / R112K / A115D / L116I / A125S / N127L / L132V / L133V / A134S / Y140F / M142K, Q91R / W96R / N105D / I114L / I118V / K119R / V122A / L132V / L137S, Y165C, Y165H, P163T, A154P / Y165C, P163T / A164T / Y165C / V169I / L170R, A154P / Y165H / L170R, Y180F, D182T, D182K, Y180F / R181V / I190V, Y180F / D182T / F183Y / I190V / G191S, Y180F / D182T / F183Y / I190V, E178A / R181V / D182K / F183Y, T236K, V237F, E234G, F233W, F233Y, F233H, A231V / F233Y, F232W / F233A / E234T / G235D / L239A, H240N, H240S, D244E / R245Y, D244E / R245Q / M246L, H240N / D244E, H240S / D244E, L239Q / H240T / R245Y, Q249R, T252V, I254V, Q249R / K251N / I254V, T252V / I254M, T252V / I254V, A260M, A260F, A260S, E266Y, E266D, T262G, T262D / E266D / , T262G / I263T / , E266D / E269H, I263T / E269N, E269N, E266Y / E269N, L274M, L274C, L277E, L274M / A279V, L274T / L277F, L274C / L277I, H297L, H298V / L302S, H298V, H298L / P299DL307Q, F308S, L307Q / A311P, L307Q / F308S, L307Q / F308S / D309A, D53G / N54R / L57V / L60I / P72E / V73A / F233V / E234G / V237F, N80H / F84Y / T95S / R99K / I118V / K119R / L132V / A141S, T95S / T97A / R99K / I118V / V123I / L132V / A141S / A260M, T95S / T97A / R99K / I118V / V123I / L132V / A141S / L277E, A260M / H298V, A260M / T262D / E266D / E269H, A260M / T262G / I263T / E269N, Q296A / H298V / L307Q / A311P, Q296E / H298V / L302S / L307Q / F308S, N80H / F84Y / T95S / R99K / I118V / L132V / A141S / Q249R / K251N / I254V, D53G / N54R / L57V / L60I / P72E / V73A / T95S / R99K / I114M / I118V / K119R / L132V / A141S / A260M, I254V / I256L / A260M / T262G / I263T / E269N, I254V / I256L / A260M / T262D / E266D / E269H / L271V, E79R / N80D / D53G / N54R / L57V / L60I / P72E / V73A / W96R / R99G / S102T / F106V / I114L / A115D / L116G / K119G / V122A / V123F / A125S / N127L / L133A / A134S / Y140F / M142K / T252V / I254V, ΔP212, ΔG5 / ΔT6 / ΔR7 / ΔS8 / ΔE9 / ΔA10 / ΔA11 / ΔD12 / ΔA13 / ΔA14 / ΔT15 / ΔQ16 / ΔA17 / ΔR18 / ΔQ19, ΔN327 / ΔD328, N25D / I26V / F27Y / S29P / R31A / F32Y / R35K / V37A / V42I / V43T / F46Y / N54R / L58V / L59P / L60V / T64G / P72R / G75P / L77P / R99G / S102N / D104N / N105T / F106W / L110V / V111E / A115D / K119G / V122T / V123L / L132V / L133I / S138A / M142KΔG5 / ΔT6 / ΔR7 / ΔS8 / ΔE9 / ΔA10 / ΔA11 / ΔD12 / ΔA13 / ΔA14 / ΔT15 / ΔQ16 / ΔA17 / ΔR18 / ΔQ19 / ΔP212 / ΔN327 / ΔD328 / E79R / N80D / D53G / N54R / L57V / L60I / P72E / V73A / W96R / R99G / S102T / F106V / I114L / A115D / L116G / K119G / V122A / V123F / A125S / N127L / L133A / A134S / Y140F / M142K / T252V / I254V, ΔG5 / ΔT6 / ΔR7 / ΔS8 / ΔE9 / ΔA10 / ΔA11 / ΔD12 / ΔA13 / ΔA14 / ΔT15 / ΔQ16 / ΔA17 / ΔR18 / ΔQ19 / ΔP212 / ΔN327 / ΔD328 / N25D / I26V / F27Y / S29P / R31A / F32Y / R35K / V37A / V42I / V43T / F46Y / E79R / N80D / D53G / N54R / L57V / L60I / P72E / V73A / W96R / R99G / S102T / F106V / I114L / A115D / L116G / K119G / V122A / V123F / A125S / N127L / L133A / A134S / Y140F / M142K / T252V / I254V and L302S., 10. Use of a polypeptide consisting of the amino acid sequence of SEQ ID No.6 for hydrolytically cleaving zearalenone and / or at least one zearalenone derivative in feed, in food or in dried distillers grains, said zearalenone derivatives being selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN.

11. Use according to any one of claims 8 - 10, characterized in that, The feed is feed for pigs, poultry and / or aquaculture.

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