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

Abstract

A polypeptide hydrolytically cleaving zearalenone and / or at least one zearalenone derivative is a hydrolytic enzyme having an amino acid sequence selected from the group consisting of 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 an additive comprising the polypeptide; and an isolated polynucleotide encoding the polypeptide; and a method for hydrolytically cleaving zearalenone and / or at least one zearalenone derivative with the polypeptide.

Description

[0001] This application is a divisional application of the invention patent application filed on August 27, 2014, with application number 201480055221.7, entitled "for hydrolyzing polypeptides of zearalenone and / or zearalenone derivatives, isolating their polynucleotides, and additives containing polypeptides, their uses and methods".

[0002] This invention relates to polypeptides for hydrolyzing zearalenone and / or at least one zearalenone derivative, isolated polynucleotides encoding such polypeptides, and additives containing such polypeptides, as well as the use of such polypeptides and methods for hydrolyzing zearalenone and / or at least one zearalenone derivative.

[0003] Mycotoxins are secondary metabolites produced by filamentous fungi. A prominent example is zearalenone (ZEN) (formerly known as F-2 toxin), which is produced by numerous Fusarium fungi. These fungi particularly infect cultivated plants, such as a wide variety of cereals, where infection typically occurs before harvest, while fungal growth or mycotoxin production can occur before harvest or, in cases of improper storage, after harvest. The FAO estimates that 25% of agricultural products worldwide are contaminated with mycotoxins, resulting in significant economic losses. In a recent worldwide study analyzing a total of 23,781 samples from January 2009 to December 2011, 81% tested positive for at least one mycotoxin and 45% 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 (dried distillers' grains and solubles), and in prepared feed mixtures, at a frequency of up to 100%.

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

[0005]

[0006] The IUPAC name is “(2E,11S)-15,17-dihydroxy-11–methyl-12-oxabicyclo[12.4.0]octadec-1(18),2,14,16-tetraene-7,13-dione”.

[0007] However, many ZEN derivatives exist in nature, formed through enzymatic or chemical modification of ZEN. Examples include glycosidic or sulfate-containing ZEN conjugates formed by the metabolism of fungi, plants, or mammals; and ZEN metabolites, which are formed particularly in human or animal organisms. In the following text, ZEN derivatives refer to ZEN conjugates or ZEN metabolites that exist in nature or are prepared through chemical or biochemical synthesis, but specifically to α-zearalenol (α-ZEL;(2E,7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadec-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]octadec-1(18),2,14,16-tetraen-13-one), α-zearalenol (α-ZAL;(7R,11S)-7,15,17-trihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadec-1(18),14,16-trien-13-one), β-zearalenol (β-ZAL;(7S,11S)-7,15,17-trihydroxy-1 1-Methyl-12-oxabicyclo[12.4.0]octadec-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]octadec-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]octadec-1(18),2,14,16-tetraene-7,13-dione) and zearalenone (ZAN;(11S)-15,17-dihydroxy-11-methyl-12-oxabicyclo[12.4.0]octadec-1(18),14,16-triene-7,13-dione).

[0008] ZEN, as well as 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 imbalances. It is immediately absorbed after oral ingestion and converted by mammals into two stereoisomers, α-ZEL and β-ZEL. Here, α-ZEL, as well as α-ZAL or ZAN, has a much stronger estrogenic effect than ZEN. Conjugated ZEN derivatives sometimes have lower estrogenic activity than ZEN; however, ZEN can be re-released from these ZEN derivatives into the digestive tract if possible.

[0010] Although ZEN has relatively low acute toxicity and an oral LD50 up to 20,000 mg / kg body weight, subacute and / or subchronic toxic effects, such as teratogenic, carcinogenic, estrus-inducing, and immunosuppressive effects, have been observed in animals or humans with prolonged exposure. ZEN-contaminated feed causes developmental disorders in mammals, with pigs, particularly piglets, being extremely susceptible. Concentrations of ZEN in feed exceeding 0.5 ppm cause developmental disorders, with concentrations exceeding, for example, 1.5 ppm can lead to super-estrogenic activity in pigs, and concentrations of 12 ppm ZEN are considered responsible for abortion in cattle. Because zearalenone is rapidly absorbed through mucous membranes, particularly the gastric mucosa, but also through the oral mucosa, immediate and especially quantitative inactivation is necessary. ZENs can already be detected in the blood within 30 minutes after oral administration. In this case, the use of isolated enzymes targeting the microorganism has advantages, such as higher specific activity or faster action. Due to the harmful effects of ZEN, there are mandatory ZEN limits in the EU for food and recommendations for ZEN limits in animal feed (EC NO:1881 / 2006).

[0011] The initial strategy for reducing ZEN contamination in food or feed is to limit fungal growth, for example, by following "good agricultural practices." This specifically includes ensuring seeds are free from pests and fungal infestation, or timely removal of agricultural waste from the field. Additionally, fungal growth in the field can be reduced by using fungicides. After harvest, the harvest should be stored at a residual moisture content below 15% and at low temperatures to inhibit fungal growth. Similarly, contaminated material due to fungal infection should be removed before reprocessing. Despite these measures, I. Rodriges 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 tested maize samples, respectively, were contaminated with ZEN between 2009 and 2011.

[0012] Other possibilities for removing ZEN from feed or food include the adsorption or conversion of mycotoxins. For this to be effective, the binding of the mycotoxin to the adsorbent must be strong and specific over a wide pH range and remain stable throughout digestion in the gastrointestinal tract. While several non-biosorbents, such as activated charcoal, silicates, or synthetic polymers like cholestyramine, can be used effectively for aflatoxins, their use for other mycotoxins is limited. A major drawback of adsorbents is the non-specific binding to other molecules that are sometimes essential for nutrient delivery. Biosorbents, such as yeast or yeast extracts, are also described in the literature; however, biosorbents, like non-biosorbents, have similar limitations.

[0013] Detoxification of ZEN through physical and chemical treatments is also limited. Heat treatment is ineffective at passivating ZEN, but extrusion and treatment with oxidants, such as treatment with a 10% hydrogen peroxide solution at 80°C for 16 hours, can reduce the ZEN content by 83.9%. In feed and food preparation, the use of extrusion methods and oxidants such as ozone or hydrogen peroxide is limited due to high cost, quality loss, sometimes low efficiency, and low specificity.

[0014] Biotransformation of ZEN using microorganisms such as *Trichosporon mycotoxinivorans*, *Gliocladium roseum*, or *Bacillus subtilis* strains, or enzymes isolated from them such as hydrolases or peroxidases, has been described, for example, in E. Vekiru et al., *Appl. and Environ. Microb.*, 2010, 76, 7, 2353-2359.

[0015] ZEN degradation properties of bacteria in the genera *Rhodococcus* and *Nocardia*, particularly *Rhodococcus globerulus*, *Rhodococcus erythropolis*, and *Nocardia globerula*, were learned from EP 0 938 575 B1.

[0016] The ZEN degradation of enzymes isolated from *Pseudomonas pulmonarius* is known from WO 02 / 076205, particularly α / β-hydrolases, zearalenone hydrolase 1 (ZHD1), which catalyzes the degradation of ZEN by means of a catalytic ternary structure.

[0017] Recombinant Zonase, or ZEN degrading enzyme, is known from WO 2012 / 113827. It remains stable in the gastrointestinal tract and specifically describes microorganisms such as *Thermobifidia fusca*, *Streptomyces exfoliatus*, *Acidovorans delafieldii*, and *Streptomyces* sp.

[0018] A polypeptide or enzyme capable of hydrolyzing ZEN and / or at least one ZEN derivative can also be called a Zonase.

[0019] The terms used below are derived from technical terms and are always used in their conventional sense unless otherwise specified. Therefore, the term "polynucleotide" refers 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 their fragments. The name "polypeptide" includes proteins, such as enzymes and antibodies, as well as polypeptides having up to 500 amino acids, such as peptide inhibitors, protein domains, and short polypeptides with small sequence lengths, such as fewer than 10 amino acids, such as receptors, ligands, peptide hormones, tags, etc. The name "position in a polynucleotide or polypeptide" refers to a single specific base or amino acid within the sequence of said polynucleotide or polypeptide.

[0020] The present invention aims to provide a polypeptide for use in which the rapid and reliable conversion of ZEN and / or at least one ZEN derivative into hydrolyzed ZEN and / or hydrolyzed ZEN derivatives can be successfully achieved. To achieve this objective, the present invention is characterized primarily 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 invention, the term "sequence identity" refers 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 performed within sequence segments, wherein a continuous sequence of the reference sequence is understood as a segment, and preferably includes conserved regions of that sequence.

[0022] In the current context, sequence identity is determined using the NCBI BLAST (Basic Local Alignment Search Tool) program, specifically BLASTP for peptides and BLASTN for polynucleotides, which is available on the National Center for Biotechnology Information homepage (NCBI; http: / / www.ncbi.nlm.nih.gov / ). Therefore, it is possible to compare two or more sequences against each other using the algorithm described by Altschul et al., 1997 (NucleicAcids Res., 25:3389-3402). For the purposes of this invention, the version of the program from May 15, 2013, is used. As part of the program setup, consider the basic settings, but specifically, for amino acid sequence comparisons: "max target sequence" = 100; "expected threshold" = 10; "word size" = 3; "matrix" = BLOSOM62; "gap costs" = "Existence: 11; Extension: 1"; "computational adjustment" = "Conditional compositional score matrix adjustment"; and for nucleotide sequence comparisons: word size: 11; expected value: 10; gap costs: existence = 5, extension = 2; filter = low complexity activated; match / mismatch scores: 2, -3; filter string: L; m.

[0023] The term "functional peptide variant" or "functional variant" refers, on the one hand, to an "allelic variant" and a "functional fragment" of a peptide, and on the other hand, to a "modification" of a peptide in which its enzymatic function is substantially unchanged. The name "allelic variant" refers to a peptide produced by a random mutation of its nucleotide sequence occurring in nature, resulting in a change in its amino acid sequence, in which its enzymatic function is unaffected. "Modification" can be, for example, a fusion with the C-terminus or N-terminus of a peptide or a mutated peptide, where the mutation can be obtained by substitution, insertion, or deletion of at least one amino acid, particularly by site-specific mutagenesis or random mutagenesis, recombination, and / or any other protein engineering method. The terms "substitution," "insertion," and "deletion" are used in their usual and familiar meaning in genetic technology. The term "functional fragment" refers to a portion or partial sequence of a peptide or a functional variant thereof, in which its enzymatic function is substantially preserved. When the enzymatic reaction mechanism remains unchanged—that is, the fungal toxin is hydrolyzed at the same location—and the specific residual activity of the "functional variant" relative to the original polypeptide is at least 5%, preferably at least 10%, and particularly at least 50%, then the enzymatic function is substantially preserved. The polypeptide having the amino acid sequences of SEQ ID No. 1-15 is a functional allelic variant of another or the same enzyme, wherein said sequences are derived from different microorganisms. This is clearly evident from the close mutual kinship measured by the percentage of sequence identity, and the fact that all polypeptides act on ZEN and ZEN derivatives through the same degradation mechanism.

[0024] Due to the similarity between the amino acid sequences of the polypeptides having SEQ ID No. 1-15, 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, a surprisingly rapid and complete hydrolysis of ZEN and / or at least one ZEN derivative was identified.

[0026] As in a preferred further development of the invention, the polypeptide has an amino acid sequence comprising 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 the sequence having SEQ ID No. 1, i.e., +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, and +1 to +328. The presence of at least one such conserved amino acid sequence segment enables the successful provision of polypeptides for use that, in addition to rapid and complete hydrolysis of ZEN and / or at least one ZEN derivative, possesses a particularly high activity value compared to ZEN-degrading polypeptides known to date.

[0027] When the functional variant has at least one amino acid modification selected from one or more amino acid substitutions, deletions, and insertions, as in a further modification of the invention, good results can still be obtained.

[0028] By further modifying the invention in this way, the polypeptide is obtained to have 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 to have a maximum of 50 μM, preferably at most 3.5 μM, particularly at most 0.5 μM of K+ cleaved by hydrolysis of ZEN. M Value, and / or having at least 0.05s -1 Preferably at least 0.6s -1 Especially at least 5 seconds -1 The hydrolytic cracking of ZEN k cat Value, and / or having at least 0.00001 μM -1 s -1 Preferably at least 0.0001 μM -1 s -1 Specifically, at least 0.001 μM -1 s -1 The hydrolytic cracking of ZEN v max ZEN and / or ZEN derivatives can be hydrolyzed particularly rapidly and completely, especially for detoxification.

[0029] As in a preferred further development of the 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. This further development ensures that the polypeptide will also cleave or detoxify zearalenone and / or at least one zearalenone derivative in acidic media, and thus, for example, in the case of a mammalian stomach. Here, the pH stability of the polypeptide is defined as the percentage of residual activity of the polypeptide at pH 5.0, relative to its activity at its respective optimum pH.

[0030] As in a preferred further development of the 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 exhibits the highest enzymatic activity in a temperature range between 30°C and 75°C, preferably between 38°C and 55°C, and particularly preferably between 38°C and 52°C. This further development of the invention ensures that zearalenone and / or at least one zearalenone derivative are hydrolyzed or detoxified by the polypeptide at mesophilic temperatures, particularly at the body temperature of humans and useful animals. The temperature at which the polypeptide exhibits the highest enzymatic activity is defined as the optimal temperature of the polypeptide.

[0031] As in a preferred further development of the 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 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 (e.g., this could be during container transport or during feed pelleting). The temperature stability of a polypeptide is defined as a temperature at which the polypeptide retains 50% of its residual activity after a 15-minute pre-incubation, compared to its activity at its respective optimum temperature.

[0032] Therefore, the polypeptide can be selected such that it is a suitable α / β-hydrolase for hydrolyzing the ester groups of zearalenone and / or ZEN derivatives in an oxygen-independent and cofactor-free manner, having an amino acid ternary structure that catalyzes the hydrolytic cleavage, the ternary structure consisting of serine, an acidic amino acid selected from glutamic acid and aspartic acid, particularly aspartic acid, and histidine, and the catalytic ternary structure being, for example, S128, D264, and H303, wherein the location relative to SEQ ID No. 1 is described.

[0033] The hydrolysis of ZEN and ZEN derivatives at the ester group of zearalenone or its derivatives can be successfully achieved using each of the polypeptides in 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 polypeptide according to the invention, particularly the α / β-hydrolase. Further decarboxylation of HZEN to decarboxylated hydrolyzed ZEN (DHZEN) or decarboxylated hydrolyzed ZEN derivatives usually occurs spontaneously.

[0036] In particular, the complete hydrolysis of ZEN and ZEN derivatives can be successfully achieved by means of the catalytic ternary structure mentioned above, and the degradation reaction has good pH stability, especially at pH values ​​in the acidic range.

[0037] Surprisingly, it has been confirmed that using a polypeptide consisting of a sequence segment consisting of three amino acids before and three amino acids after serine in the aforementioned catalytic ternary form, containing at least one polar amino acid selected from Y, Q, N, T, K, R, E, D and at least one nonpolar amino acid selected from F, M, L, I, V, A, G, P, can successfully achieve good results and further improve at least one enzyme kinetic parameter.

[0038] In a preferred further modification of the invention, the polypeptide has at least one mutation in 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 sequence identity and are particularly active. By altering the polypeptide having SEQ ID No. 1 at at least one of these positions, i.e., by employing amino acid variants of SEQ ID Nos. 2-6 at that position, it has been successfully demonstrated that these positions have a significant effect on the enzyme kinetic parameters of the polypeptide, and furthermore, the combination of SEQ ID No. 1 with SEQ ID Nos. 2-6, which have a high degree of sequence identity, results in 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 of 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, L 132V, A141S, I146V, I146L, A148G, A149V, A154P, P163T, A164T, Y165C, Y165H, V169I, L170R, A172G, A176M, A176V, Y180F, D182T, F 183Y, I190V, G191S, K194T, K194E, F196Y, V197C, V197R, E198R, E198S, K201D, K201G, P204S, P204A, A205S, K206P, A207M, M208A, Q 209R, 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 hydrolyzing 1 μM mol ZEN / min in 50 mM Tris-HCl buffer (pH 8.2) at 32 °C. “Catalytic activity” refers to the enzymatic conversion of the substrate under defined reaction conditions, and “specific activity” refers to the ratio of catalytic activity to peptide mass concentration (mass / volume unit).

[0040] The polypeptide, having a specific activity of at least 7.00 U / mg, preferably at least 8.00 U / mg, can be successfully provided by generating the polypeptide in such a manner that it comprises at least one amino acid motif having the sequence of SEQ ID No. 32-50. Surprisingly, it has been shown that the enzymatic activity of the polypeptide is further increased, for example, relative to a motif comprising 7 amino acids, when at least one amino acid motif has the sequence of SEQ ID No. 51-58 is included. An even higher specific activity is achieved when at least one amino acid motif has the sequence of SEQ ID No. 59-69 is included.

[0041] According to a further modification of the invention, the polypeptide comprises at least one conserved amino acid substitution at at least one position, wherein the conserved 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 "conserved amino acid substitution" refers to a substitution of an amino acid by another amino acid that is considered conserved by a person skilled in the art (i.e., has similar specific properties). These special properties include, for example, the size, polarity, hydrophobicity, charge, or pK value of an amino acid. Conservative mutations refer to, for example, the substitution of one acidic amino acid for another, the substitution of one basic amino acid for another, or the substitution of one polar amino acid for another.

[0042] By using such conserved amino acid substitutions, functional peptide variants can be successfully prepared, with specific activity approximately as strong as that of the parent peptide, but preferably increased by at least 0.1 U / mg.

[0043] Furthermore, the present invention aims to provide isolated polynucleotides for use, from which polypeptides can be successfully prepared for rapid and reliable hydrolytic cleavage of ZEN and / or at least one ZEN-derived compound.

[0044] To address 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 any one of claims 1 to 11; and / or the nucleotide sequence has a 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 under moderately stringent conditions with at least one nucleotide sequence selected from SEQ ID No. 16-31, and / or hybridizes with a portion thereof having at least 200 nucleotides, particularly at least 100 nucleotides, and / or hybridizes with the complementary strand of the aforementioned nucleotide sequence or a portion thereof.

[0045] The nucleotide sequence to be expressed, especially its triples (codons), typically varies with the host cell, thus optimizing codon bias. This results in polynucleotides with sequence identity levels well below 80%, and some even below 70% or 60%, encoding the same polypeptide. Sequence comparisons used to determine sequence identity levels must also be performed within sequence regions, where a region refers to a contiguous section of a reference sequence. For nucleotide sequences, the length of sequence regions is typically 15 to 600 units.

[0046] Nucleic acid probes, typically at least 15, 30, or 40 nucleotides in length, can be successfully generated using existing isolated nucleotide sequences or sequence segments. These probes (which are usually further supplemented, for example, with...) 3 H, 32 P, 35 Labeled with S, biotin, or avidin, nucleotide sequences encoding polypeptides that degrade ZEN and / or ZEN derivatives can be identified using standard methods. Starting materials for identifying such sequences may include, for example, DNA, RNA, or cDNA from a single microorganism, a genomic DNA library, or a cDNA library.

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

[0048] For nucleotide sequences or probes with lengths from 15 to 100 nucleotides, moderately stringent conditions were 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 solution, 1 mM sodium pyrophosphate, 1 mM sodium dihydrogen phosphate, 0.1 mM ATP, and 0.2 mg / ml yeast RNA, performed at a temperature 5 to 10 °C lower than the calculated melting temperature (Tm), which was determined according to calculations performed by Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA, 48:1390). The assay was then performed under standard Southern blotting conditions (J. Sambrook, EFFritsch and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor, New York). Finally, the carrier material was washed once with 6×SCC buffer containing 0.1% SDS for 15 minutes; and twice with 6×SSC buffer at a temperature 5 to 10°C lower than the calculated Tm.

[0049] Furthermore, the present invention aims to provide an additive for use in which the rapid and reliable hydrolytic cleavage of ZEN and / or at least one ZEN derivative can be successfully achieved in defined or complex matrices, such as feed or food.

[0050] To address this task, an additive is provided for use that hydrolyzes zearalenone and / or at least one zearalenone derivative, 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 an adjuvant.

[0051] With such an additive, the biochemical conversion of ZEN and / or at least one ZEN derivative to hydrolyzed ZEN and / or hydrolyzed ZEN derivatives 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 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 fungal toxins. By using such an additive, it can be ensured, for example in feed or food, that the amount of ZEN and / or ZEN derivatives optionally contained is safely and reliably hydrolyzed, particularly detoxified, to such an extent that there are no harmful effects on the bodies of subjects ingesting such feed or food.

[0053] In this case, the polypeptide according to the invention may also be present in an enzyme preparation, which, in addition to containing at least one polypeptide according to the invention, contains at least one enzyme, such as an enzyme involved in the degradation of proteins, such as proteases, or an enzyme involved in the metabolism of starch or fiber or fat or glycogen, such as amylase, cellulase or glucanase, and such as hydrolases, lipases, mannosidases, oxidases, oxidoreductases, phytases, xylanases and / or combinations thereof.

[0054] Other applications of the present invention include enzyme preparations that, in addition to containing at least one polypeptide according to the present invention, also contain at least one component for detoxifying mycotoxins, such as mycotoxin-degrading enzymes, such as aflatoxin oxidase, ergotamine hydrolase, ergotamine amidase, zearalenone esterase, zearalenone lactone esterase, ochratoxin amidase, fusarium carboxylesterase, fusarium aminotransferase, amino polyol amino oxidase, deoxyfusarium alcohol 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 a microbial cell wall or an inorganic material such as bentonite.

[0055] According to a particularly preferred further modification of the invention, the polypeptide is contained in the additive at a concentration of up to 10,000 U / g, preferably up to 1,000 U / g, more preferably up to 100 U / g, and most preferably up to 10 U / g, thereby successfully achieving the rapid, and particularly prior, conversion of ZEN and / or ZEN derivatives into non-toxic or low-toxic metabolites, especially HZEN and DHZEN, before their absorption by subjects (especially mammals) who have consumed contaminated feed or food.

[0056] According to a further modification of the invention, the polypeptide is present in an encapsulated or coated form, wherein standard methods, such as those described in WO 92 / 12645, can be considered for the encapsulation or coating process. Through this encapsulation or coating process, the polypeptide can be successfully transported to its site of use without alteration, particularly without degradation or damage, so that the polypeptide only begins to function after the protective shell dissolves (e.g., in the digestive tract of an animal). This allows for more targeted, rapid, and complete degradation of ZEN and / or ZEN derivatives, even in acidic, protease-rich, and anaerobic media. Furthermore, the temperature stability of the polypeptide in the additive can be successfully improved through the encapsulation or coating process.

[0057] Furthermore, the present invention also aims to utilize the additive for the hydrolytic cleavage of zearalenone and / or at least one zearalenone derivative in feed (particularly for pig, poultry, and aquaculture), in food, or in distillers' grains. By using the additive according to the present invention, the hydrolysis or detoxification of ZEN and / or ZEN derivatives contained in food or feed or in distillers' grains can be successfully achieved, with such detoxification already successfully achieved at a peptide concentration of approximately 1 U / g in contaminated feed or food.

[0058] Furthermore, the present invention aims to provide a method for use that enables rapid and reliable hydrolytic cleavage of ZEN and / or at least one ZEN derivative.

[0059] To solve this task, the method is performed 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 invention, the method is carried out in such a way that the polypeptide is used in an additive corresponding to the invention.

[0061] According to a preferred further development, the method is carried out such that the polypeptide or additive is mixed with feed or food contaminated with zearalenone and / or at least one zearalenone derivative, so that the contaminated feed or food comes 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. In the case of moist feed or food, such as malt syrup or paste, the hydrolysis of zearalenone and / or at least one zearalenone derivative occurs in the moist feed or food before oral ingestion. This method ensures that the harmful effects of zearalenone and zearalenone derivatives on humans and animals are minimized. Here, moisture refers to the presence of water or an aqueous liquid, including, for example, saliva or other liquids present in the digestive tract. The digestive tract is defined as the oral cavity, pharynx (throat), esophagus, and gastrointestinal tract, or equivalents thereof, although different names may exist in animals or individual components may not be present in the animal's digestive tract.

[0062] The method of the present invention can also be carried out in such a way as to granulate the feed or food before oral ingestion.

[0063] According to a further modification 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 are hydrolyzed. This prevents subacute and / or subchronic toxic effects in animals or humans, such as teratogenic, carcinogenic, estrus-inducing, and immunosuppressive effects.

[0064] The present invention will now be described in more detail with reference to the embodiments and accompanying drawings. Wherein:

[0065] Figure 1 The study showed the degradation of ZEN over time via the peptide having SEQ ID No. 1, and the increase in the metabolites HZEN and DHZEN, wherein... Figure 1 In A, the polypeptide is unlabeled. Figure 1 In B, the polypeptide has a C-terminal 6×His tag, and in Figure 1 In C, the polypeptide has an N-terminal 6×His tag.

[0066] Figure 2 The Michaelis-Menten kinetics of the peptide having SEQ ID No. 1 are shown.

[0067] Figure 3 shows the purified product with SEQ ID No. 1 ( Figure 3A), SEQ ID No.2 Figure 3B ), SEQ ID No. 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 The peptides used to detect the degradation of ZEN over time and the increase of its metabolites HZEN and DHZEN, all of which have a C-terminal 6×His tag.

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

[0069] According to the instructions, using the "Quick-change Site-directed Mutagenesis Kits" (Stratagene), amino acid substitutions, insertions, or deletions are performed by PCR through mutation of the nucleotide sequence. Alternatively, complete nucleotide sequences (GeneArt) are also available for this purpose. The nucleotide sequences generated by PCR mutagenesis or purchased from GeneArt may optionally also contain a C-terminal or N-terminal 6×His tag at the amino acid level, and are integrated into expression vectors for expression in *E. coli* or *Pichia pastoris* using standard methods, transformed into *E. coli* or *Pichia pastoris*, and expressed in *E. coli* or *Pichia pastoris* (JMCregg, 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 may 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. Specifically, the term encompasses DNA constructs adapted to transfer a nucleotide sequence encoding the polypeptide into a host cell for integration into the genome or free existence in extrachromosomal space, and to express the nucleotide sequence encoding the polypeptide within the cell and optionally extract the polypeptide from the cell.

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

[0072] To determine the catalytic properties of peptides, soluble cell lysates should be considered in the case of *E. coli*, or culture supernatant in the case of *Pichia pastoris*. To determine K... M value, v max k cat The specific activity of the polypeptide was determined by selective enrichment of the polypeptide using a nickel-Sepharose column in a chromatographic manner using standard methods. Protein concentration was determined by standard methods, either using the BCA method (Pierce BCA Protein Assay Kit Prod#23225), but preferably by spectrophotometry using the specific extinction coefficient of the respective protein, calculated using the online program “ProtParam” available 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 percentage of sequence identity between polypeptides having amino acid sequences of SEQ ID No. 1-15 over their total polypeptide length (Table 1) can be determined using the BLAST (Basic Local Alignment Search Tool) program, specifically BLASTP, which is available on the homepage of the National Center for Biotechnology Information (NCBI; http: / / www.ncbi.nlm.nih.gov / ). Therefore, it is possible to compare two or more sequences against each other using the algorithm described by Altschul et al., 1997 (Nucleic Acids Res. (1997) 25:3389-3402). As program settings, the basic settings are considered, but specifically: "Maximum Target Sequence" = 100; "Expected Threshold" = 10; "Word Length" = 3; "Matrix" = BLOSOM62; "Gap Cost" = "Presence: 11; Extension: 1"; "Calculation Adjustment" = "Conditional Composition Score Matrix Adjustment".

[0075] To determine conserved amino acid sequence segments, the software COBALT (JS Papadopoulos and R. Agarwala, 2007, COBALT: constraint-based alignment tool for multiple protein sequences, Bioinformatics 23:1073-79) was used to adjust the alignment of polypeptides with at least 70% sequence identity between each other, considering standard parameters, particularly the following parameters: ("gap penalty": -11, -1; "terminal 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": standard; "Homology conservatism setting": on. (setting): 3 bits). The results of this analysis depicted conserved amino acids. Conserved amino acid sequence segments were defined as the following regions having at least 5 consecutive conserved amino acids: i.e., those relating to SEQ ID The sequence No.1 consists of the following segments: A (position +24 to position +50), B (position +52 to position +77), C (position +79 to position +87), D (position +89 to position +145), E (position +150 to position +171), F (position +177 to position +193), G (position +223 to position +228), H (position +230 to position +237), I (position +239 to position +247), J (position +249 to position +255), K (position +257 to position +261), L (position +263 to position +270), M (position +272 to position +279), N (position +297 to position +301), and O (position +303 to position +313).

[0076] As described above, the percentage of sequence identity between peptides and between individual peptides was determined relative to the conserved amino acid sequence of the sequence having SEQ ID No. 1. The results are presented in Tables 1 and 2.

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

[0078]

[0079]

[0080]

[0081]

[0082] Table 2: Percentage of sequence identity of conserved amino acid sequence regions A to O.

[0083]

[0084]

[0085]

[0086]

[0087] Example 3: Hydrolysis of ZEN by peptides in cell lysates

[0088] To determine its ability to degrade ZEN into non-toxic or low-toxic metabolites HZEN and DHZEN, a polypeptide having the nucleotide sequence of SEQ ID No. 1 encoded by the nucleotide sequence of SEQ ID No. 17 was prepared in *E. coli* as described in Example 1, the polypeptide being prepared with a 6×His tag having a C-terminus or N-terminus. A polypeptide having the amino acid sequence of SEQ ID No. 2-15 encoded by the nucleotide sequence of SEQ ID No. 18-31 was tagged with a 6×His tag only at the C-terminus. 100 ml of *E. coli* cultures with an optical density (OD600 nm) of 2.0-2.5 were harvested by centrifugation at 4°C and resuspended in 20 ml of Brunner mineral medium (DSMZ Microbial Culture Medium No. 462, 2012). The cell suspension was lysed by treating three times with a Freund's crusher at 20,000 psi. The cell lysates thus obtained were used at dilutions of 1:10, 1:100, or 1:1,000, 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 diluted cell lysates, and 31 μl of ZEN substrate stock solution were used. In summary, the cell lysates were thus diluted at 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 (Romer Labs biopure standard, trade number 001109, purity at least 98%) was weighed, bottled, and dissolved accordingly. Each degradation batch was performed in 25 ml glass vials and incubated for a total of 120 hours at 25°C with shaking at 100 rpm. At time points 0, 0.5, 1, 2, 5, 24, 47, 72, and 120 hours, 1 ml of sample was taken, the peptide was inactivated by heating at 99°C for 10 minutes, and stored at -20°C. After thawing, insoluble components were separated by centrifugation. ZEN, HZEN, and DHZEN were analyzed by LC / MS / MS. For this purpose, metabolites were separated by chromatography using a Phenomenex Luna C18(2) column with dimensions of 250 mm × 3 mm and a particle size of 5 μm. An acetonitrile-water mixture with 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 using QTrap / LC / MS / MS (triple quadrupole, Applied Biosystems) in "enhanced mode." After at least 24 hours, no large amounts of ZEN were detected in the batch. The majority (over 80%) of ZEN was converted to HZEN or DHZEN.

[0089] exist Figure 1 As seen in the example, regarding cell lysate solutions diluted 1:10,000, for unlabeled ( Figure 1 A) and for those with a C-terminal 6×His tag ( Figure 1 B) and those with an N-terminal 6×His tag ( Figure 1 C) The peptide having SEQ ID No. 1, the degradation of ZEN over time and the increase of HZEN and DHZEN over time. It is clearly evident from this that: 1. The conversion of ZEN occurs immediately and completely, as ZEN is already almost undetectable in the first sample taken immediately after the start of the experiment (0 hours); and 2. No noteworthy loss of activity was observed by attaching C-terminal or N-terminal tags.

[0090] Example 4: Hydrolysis of ZEN derivatives by peptides in cell lysates

[0091] To determine the ability of the peptide to convert ZEN derivatives, in addition to ZEN, into non-toxic or low-toxic metabolites, peptides having SEQ ID No. 1-15 were prepared with a C-terminal His tag as described in Example 3, and synthetic nucleotide sequences having SEQ ID No. 17-31 were considered as cell lysates in “Degradation 15”.

[0092] Degradation assays were performed as described in Example 3, wherein each peptide was tested with each ZEN derivative selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S, and ZAN. Cell lysates were used at a total dilution of 1:10,000. An equimolar (i.e., 2.08 mM) solution of a ZEN derivative was used instead of a 2.08 mM ZEN solution (40 vol% ACN + 60 vol% H2O) as a substrate stock solution. α-ZEL, β-ZEL, α-ZAL, β-ZAL, and ZAN were purchased from Sigma and used as standards for this analysis. Z14G and Z14S were prepared with a purity of at least 90% according to methods described in 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 analysis. Another difference from Example 3 is that only one sample was taken, i.e., after 24 hours. The decrease in ZEN derivative concentration during the degradation assay 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, in all degradation experiments, only 0% to a maximum of 13% of the initial amount of ZEN derivatives were present after 24 hours of incubation.

[0093] Example 5: Specific activity and enzyme kinetic parameters of peptides and their variants

[0094] The specific activity of the peptides and their variants was determined by spectrophotometry, wherein all peptides used had a C-terminal 6×His tag. The preparation, enrichment, and purification of the peptides or their variants were performed as described in Example 1. 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 Lμmol -1 cm -1The extinction coefficient is strongly pH-dependent and therefore must always be measured at the exact same pH, preferably even in the same matrix. Measurements were performed at 32°C in a UV-VIS photometer (Hitachi U-2001), in a wavelength range of 200 to 2500 nm, in a quartz cuvette, in a 50 mM Tris-HCl (pH = 8.2) buffer solution.

[0095] 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 (Romer Labs biopure standard, trade number 001109, purity at least 98%) was weighed, bottled, and dissolved accordingly. ZEN substrate dilutions (0.79 μM, 1.57 μM, 2.36 μM, 3.14 μM, 4.71 μM, 6.28 μM, 7.85 μM, 9.42 μM, 10.99 μM, 12.56 μM, 14.13 μM, 15.71 μM, 17.28 μM, 18.85 μM) were prepared using 50 mM Tris-HCl (pH 8.2). Dilute the peptide solution with 50 mM Tris-HCl buffer (pH 8.2) to a final concentration of approximately 70 ng / ml. Preheat the ZEN substrate dilution to 32°C in a water bath.

[0096] Add 0.2 μl of peptide solution to 100 μl of each ZEN substrate diluent and measure absorbance for 5 minutes, with each "peptide solution-ZEN substrate diluent" combination measured at least twice.

[0097] By taking into account the extinction coefficients of ZEN and HZEN, the reaction rate with respect to each substrate concentration was calculated via the slope of absorbance over time.

[0098] Name "K" M The "value" or "Miemann constant" refers to a parameter used to describe enzyme affinity, which has units of [μM] or [mM] and is calculated according to H. Bisswang (2002, Enzyme Kinetics, ISBN 3-527-30343-X, p. 19) using a linear Hanes plot, where for this purpose the function "Enzyme Kinetics, Single Substrate" in the program SigmaPlot 12.0 is preferably used. The name "catalytic constant of enzyme reaction" or "k" is also used. cat The "value" refers to a parameter used to describe the conversion rate of a polypeptide or enzyme, expressed in [s]. -1 The value is given, and preferably calculated using the function "Enzyme Kinetics, Single Substrate" in the SigmaPlot 12.0 program. "Maximum enzyme rate" or "v" is also given. maxThe value is given in units of [μM / s] or [mM / s], and is similar to K. M The value is calculated using a linear Hanes plot, preferably using the function "Enzyme Kinetics, Single Substrate" in the SigmaPlot 12.0 program.

[0099] With the help of v max The specific activity is calculated using the following formula, based on the concentration of the enzyme used.

[0100]

[0101] One of the units is defined as 1 μmol ZEN per minute of hydrolysis in 50 mM Tris-HCl buffer solution (pH = 8.2) at 32 °C.

[0102] Below, for example, for a polypeptide having SEQ ID NO:1, examples are given regarding the enzyme parameter K. M v max k cat And the raw data for the specific activity determination. Table 3 shows the reaction rates at various ZEN substrate concentrations. Figure 2 The respective Mi-Mann diagrams are shown, and the corresponding enzyme kinetic parameters are given in Table 4. The enzyme solutions used had a concentration of 68 ng / L.

[0103] Table 3: Reaction rates of the polypeptide with SEQ ID NO:1 at different ZEN concentrations.

[0104]

[0105] Table 4: Enzyme kinetic parameters of the polypeptide with SEQ ID No. 1.

[0106]

[0107] The specific activities of the studied peptides 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.

[0108] The specific activities of the studied peptide variants are listed in Tables 5 and 6.

[0109] Table 5: Specific activity of functional variants of the polypeptide having SEQ ID No. 1; conserved amino acid sequence segment where the mutation is located; and sequence identity of the functional variants relative to the parent sequence having SEQ ID No. 1. The location of the mutation relative to the amino acid sequence having SEQ ID No. 1 is given. Sequence identity was measured by means of BLAST as described in Example 2.

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] Table 6: Specific activities of functional variants of the polypeptide having SEQ ID No. 2. The mutation position is relative to the amino acid sequence having SEQ ID No. 2. Sequence identity was determined by BLAST as described in Example 2.

[0118]

[0119] Example 6: Degradation of ZEN and ZEN derivatives in contaminated corn

[0120] To determine the ability of peptides to degrade naturally occurring ZEN and ZEN derivatives in complex matrices and at low pH, different concentrations of one of the peptides with SEQ ID No. 1-6 were incorporated into contaminated corn each time, and the degradation of ZEN and ZEN derivatives was tracked.

[0121] Contaminated corn was milled and used in the degradation assay, with each batch consisting of 1 g of milled contaminated corn, 8.9 ml of 100 mM acetate buffer (pH 4.0), and 0.1 ml of peptide solution. Enriched and purified peptide solutions were prepared as described in Example 5, diluted to concentrations of 10 mU / ml, 100 mU / ml, or 1,000 mU / ml. Therefore, 1 mU (= 1 mU / g corn), 10 mU (= 10 mU / g corn), or 100 mU (= 100 mU / g corn) was used in the batch in an absolute manner. Each degradation batch was prepared in 25 ml portions and incubated at 37°C with shaking at 100 rpm. Before enzyme addition and after 1 hour of incubation, 1 ml samples were taken, the peptides were inactivated by heating at 99°C for 10 minutes, and the samples were stored at -20°C. After thawing, 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 contents of ZEN and ZEN derivatives in this maize were: 238 ppb for ZEN; 15 ppb for α-ZEL; 23 ppb for β-ZEL; 32 ppb for Z14G; and 81 ppb for Z14S. The percentage decrease in ZEN and ZEN derivative contents during the degradation experiment is presented in Table 7.

[0122] Table 7: Reduction of ZEN and ZEN derivatives relative to the starting content, expressed as a percentage, in degradation tests of different peptides and peptide amounts.

[0123]

[0124] Example 7: Additive containing peptides for hydrolytic cleavage of ZEN and / or ZEN derivatives

[0125] To prepare additives for the hydrolytic cleavage of ZEN, fermentation supernatants containing peptides expressed by *Pichia pastoris* with SEQ ID Nos. 1, 2, 6, and 13 were purified under standard conditions by microfiltration and ultrafiltration (exclusion limit: 10 kDa) and concentrated to approximately 9% by weight of dry matter. The peptide-containing solution was then further processed into dry powders under standard conditions using a spray dryer (Büchi Mini B290). These four powders were subsequently named Z1, Z2, Z6, and Z13. Furthermore, Z1, Z2, Z6, or Z13 were mixed with bentonite of an average particle size of approximately 1 μm in an upward-moving shaker at a ratio of 1% by weight of additive Z1, Z2, Z6, or Z13 to 99% by weight of bentonite. The resulting additives were named additives Z1.B, Z2.B, Z6.B, and Z13.B. Furthermore, Z1, Z2, Z6, and Z13 were mixed with bentonite and a vitamin-trace element concentrate in an upward shaking apparatus at 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 additives Z1.BVS, Z2.BVS, Z6.BVS, and Z13.BVS. 100g of additives Z1.BVS, Z2.BVS, Z6.BVS and Z13.BVS contain 200mg ferric sulfate, 50mg copper sulfate, 130mg zinc oxide, 130mg manganese oxide, 2.55mg calcium carbonate, 160mg vitamin E, 6.5mg vitamin K3, 6.5mg vitamin B1, 14mg vitamin B2, 15mg vitamin B6, 0.15mg vitamin B12, 150mg niacin, 30mg pantothenic acid and 5.3mg folic acid.

[0126] The additive was extracted in 50 mM Tris-HCl buffer (pH = 8.2) for 30 minutes and further diluted in the same buffer to bring the final concentration of the peptide to approximately 70 ng / ml.

[0127] Subsequently, as described in Example 5, the effects of these solutions on the degradation of zearalenone were determined. 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.

[0128] 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 ZEN derivatives α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S, and ZAN was exercised as described in Example 4, but 100 μl of a peptide solution with a peptide concentration of approximately 70 ng / ml was used instead of 100 μl of cell lysate. After 6 hours of incubation, only a maximum of 15% of the initial amount remained as unhydrolyzed ZEN derivatives.

[0129] Example 8: Optimal Temperature

[0130] To determine the optimal temperature for the peptides having SEQ ID Nos. 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 tests, for each peptide, the concentration at which complete conversion of ZEN could be ensured after a 3-hour test duration under the test conditions (Teorell and Stenhagen buffer (Teorell and Stenhagen, Ein Universal puffer für den pH-Bereich 2.0 bis 12.0. Biochem Ztschrft, 1938, 299: pp. 416-419), pH 7.5, with 0.1 mg / ml BSA, at 30°C). The preparations were used at the calculated concentration in the degradation batch used to determine the optimal temperature. The experiment was performed in a PCR cycler (Eppendorf) using a temperature gradient function at 20°C ± 10°C, 40°C ± 10°C, and, if necessary, 60°C ± 10°C (10 temperatures within their respective ranges; predetermined PCR cycler temperatures). For batches, Teorell-Stenhagen buffer was infused with the appropriate enzyme concentration, along with 0.1 mg / ml BSA and 5 ppm ZEN, at their respective optimal pH. Batches with 0.1 mg / ml BSA and 5 ppm ZEN without enzyme addition served as negative controls. After incubation times of 0, 0.5, 1, 2, and 3 hours, one sample was taken for each incubation temperature, inactivated by heating 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 using HPLC-DAD. For this purpose, metabolites were separated by chromatography 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 containing 5 mM ammonium acetate was used as the mobile phase. The UV signal was recorded at 274 nm. Quantification of metabolites was performed by including a series of standards. The optimal temperature was determined by calculating the slope of the degradation curve, where the temperature at which the slope was maximized was defined as the optimal temperature. The optimal temperatures are shown in Table 8.

[0131] Table 8: Optimal temperature for peptides.

[0132]

[0133] Example 9: Temperature Stability

[0134] To determine the temperature stability of the peptides having SEQ ID Nos. 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. They were incubated in a PCR cycler with a gradient function at their respective optimal temperatures + / - 10 °C. One sample was taken from each batch and at each temperature after 0, 15, 30, and 60 minutes. Subsequently, in degradation assays, these pre-incubated samples were used in batches in Teorell-Stenhagen buffer at their respective optimal pH values ​​containing 0.1 mg / ml BSA and 5 ppm ZEN. In preliminary assays, for each peptide, the concentration at which complete ZEN conversion was ensured after a 3-hour assay duration under the test conditions (Teorell-Stenhagen buffer, pH 7.5, with 0.1 mg / ml BSA, at 30 °C). The calculated enzyme concentrations were used in each batch. The degradation batch was incubated at 30°C. Samples were taken after incubation times of 0, 0.5, 1, 2, and 3 hours. Subsequently, the peptides were inactivated by heating 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 using HPLC-DAD as described in Example 8.

[0135] Temperature stability is defined as the temperature at which the peptide retains 50% of its residual activity after a 15-minute pre-incubation, compared to the optimum temperature. The slope of the degradation curve is considered as a measure of activity. Temperature stability is shown in Table 9.

[0136] Table 9: Temperature stability of peptides (50% residual activity after 15 minutes of pre-incubation).

[0137]

[0138] Example 10: Optimal pH

[0139] To determine the optimal pH for the peptides having SEQ ID Nos. 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 peptide, the concentration at which complete ZEN conversion could be ensured after a 3-hour test duration under the test conditions (Teorell Stenhagen buffer, pH 7.5, with 0.1 mg / ml BSA, at 30°C). The respective enzyme concentrations were used in the batches. 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, degradation batches containing 0.1 mg / ml BSA and 5 ppm ZEN were incubated at 30°C. Batches containing 0.1 mg / ml BSA and 5 ppm ZEN in Teorell-Stenhagen buffer at pH 3.0, pH 7.0, and pH 12.0 were used as negative controls. Samples were taken after incubation times of 0 h, 0.5 h, 1 h, 2 h, and 3 h. Subsequently, the peptide was inactivated by heating at 99°C for 10 min, and the samples were stored at -20°C. After thawing, the samples were transferred to HPLC vials and analyzed using HPLC-DAD as described in Example 8. The optimal pH was determined by calculating the slope of the degradation curve, where the pH at which the slope was maximized was defined as the optimal pH. The optimal pH is shown in Table 10.

[0140] Table 10: Optimal pH of peptides.

[0141]

[0142] Example 11: pH stability at pH 5.0

[0143] To determine pH stability, peptides from Example 10 were incubated for 1 hour at 25°C in Teroell-Stenhagen buffer at pH 5.0 and their respective optimum pH. In degradation assays, these pre-incubated samples were used in batches at the same concentrations of their respective peptides used for determining the optimum pH in 100 mM Tris-HCl buffer at their respective optimum pH with 0.1 mg / ml BSA and 5 ppm ZEN. Samples were taken after incubation times of 0, 0.5, 1, 2, and 3 hours. Subsequently, the peptides were inactivated by heating 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 using HPLC-DAD as described in Example 8. pH stability was defined as the percentage of residual activity of the peptide at pH 5.0, relative to its activity at its respective optimum pH. pH stability at pH 5.0 is shown in Table 11.

[0144] Table 11: pH stability of peptides at pH 5.0.

[0145]

[0146] Example 12: ZEN Degradation Test

[0147] ZEN to HZEN and DHZEN degradation were performed, exemplarily for peptides having SEQ ID Nos. 1, 2, 5, 6, 7, 9, 11, 12, and 15. The degradation batches were prepared in Teorell Stenhagen buffer (pH 7.5) containing 0.1 mg / ml BSA and 5 ppm ZEN. The degradation batches were incubated at 30°C. Samples were taken after incubation times of 0 h, 0.5 h, 1 h, 2 h, and 3 h. Subsequently, the peptides were inactivated by heating at 99°C for 10 min, 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. The peptide concentration was thus selected so that complete degradation was achieved in approximately 3 hours. The degradation kinetics are depicted in Figure 3, where the y-axis shows the concentrations of ZEN, HZEN, and DHZEN in micromoles per liter (μmol / L), and the x-axis shows the incubation time in hours (h).

[0148] *μM represents the microvolume molar concentration, and corresponds to the unit μmol / l.

[0149] Some embodiments of the present invention are as follows:

[0150] 1. A polypeptide that hydrolyzes zearalenone and / or at least one zearalenone derivative, 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%.

[0151] 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 having SEQ ID No. 1, i.e., +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.

[0152] 3. The polypeptide according to embodiment 1 or 2, characterized in that the functional variant has an amino acid modification selected from the following: substitution, deletion and insertion of one or more amino acids respectively.

[0153] 4. A 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+ cleavage concentration of zearalenone by hydrolysis at a maximum of 50 μM, preferably at a maximum of 3.5 μM, particularly at a maximum of 0.5 μM. M Value, and / or having at least 0.05s -1 Preferably at least 0.6s -1 Especially at least 5 seconds -1 The hydrolytic cleavage of zearalenone by k cat Value, and / or having at least 0.00001 μM -1 s -1 Preferably at least 0.0001 μM -1 s -1 Specifically, at least 0.001 μM -1 s -1 The hydrolytic cleavage of zearalenone v max value.

[0154] 5. A polypeptide according to any 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.

[0155] 6. A polypeptide according to any 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 a temperature range between 30°C and 75°C, preferably between 38°C and 55°C, particularly preferably between 38°C and 52°C.

[0156] 7. A polypeptide according to any 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 90°C, preferably 75°C, and particularly preferably 60°C.

[0157] 8. A polypeptide according to any one of embodiments 1 to 7, characterized in that the polypeptide has at least one mutation in 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.

[0158] 9. The polypeptide according to embodiment 8, characterized in that the polypeptide has at least one mutation selected from the amino acid sequence of 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, F183 Y, I190V, G191S, K194T, K194E, F196Y, V197C, V197R, E198R, E198S, K201D, K201G, P204S, P204A, A205S, K206P, A207M, M208A, Q20 9R, L210A, L210S, ΔP212, T213V, P214A, E216T, E216G, A217I, N220H, L221M, K222R, K222Q, G229A, A231V, F233W, F233Y, F233H, A2 38G, H240N, H240S, D244E, R245Q, M246L, S248T, S248N, S248G, Q249R, K251N, I254V, I256L, A260M, T262D, T262G, I263T, E266D, E 269H, 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.

[0159] 10. A 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 No. 32-69.

[0160] 11. The polypeptide according to embodiment 9, characterized in that the polypeptide comprises at least one conserved amino acid substitution at at least one position, and the conserved 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.

[0161] 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 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 under moderately stringent conditions with at least one nucleotide sequence selected from SEQ ID No. 16-31, and / or hybridizes with a portion thereof having at least 200 nucleotides, particularly at least 100 nucleotides, and / or hybridizes with the complementary strand of the aforementioned nucleotide sequence or a portion thereof.

[0162] 13. An additive for hydrolyzing 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.

[0163] 14. The additive according to embodiment 13, characterized in that it comprises at least one polypeptide according to one of embodiments 1 to 11.

[0164] 15. An 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 fungal toxins.

[0165] 16. An additive according to any one of embodiments 13, 14 or 15, characterized in that the additive contains at least one polypeptide according to any one of embodiments 1 to 11 at a concentration of up to 10,000 U / g, preferably up to 1,000 U / g, more preferably up to 100 U / g, and most preferably up to 10 U / g.

[0166] 17. An additive according to any one of embodiments 13 to 16, characterized in that the additive is present in an encapsulated or coated form.

[0167] 18. The use of an additive according to any one of embodiments 13 to 17 for hydrolytically cleaving zearalenone and / or at least one zearalenone derivative in feed, food, or elsewhere, said feed being particularly for pig, poultry, and aquaculture.

[0168] 19. A method for hydrolyzing 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%.

[0169] 20. The method according to embodiment 19, characterized in that the polypeptide is used in an additive according to any one of embodiments 14 to 17.

[0170] 21. The method according to embodiment 20, characterized in that the polypeptide or additive is mixed 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 is hydrolyzed to zearalenone and / or at least one zearalenone derivative contained in the contaminated feed or food.

[0171] 22. The method according to any 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 are hydrolyzed. sequence list <110> DSM AUSTRIA GMBH <120> Polypeptides, isolated polynucleotides thereof, and additives containing polypeptides, their uses and methods. <130> P05341PCT <160> 69 <170> PatentIn version 3.5 <210> 1 <211> 328 <212> PRT <213> Rhodococcus erythropolis <400> 1 Met Ala Glu Glu Gly Thr Arg Ser Glu Ala Ala Asp Ala Ala Thr Gln 1 5 10 15 Ala Arg Gln Leu Pro Asp Ser Arg Asn Ile Phe Val Ser His Arg Phe 20 25 30 Pro Glu Arg Gln Val Asp Leu Gly Glu Val Val Met Asn Phe Ala Glu 35 40 45 Ala Gly Ser Pro Asp Asn Pro Ala Leu Leu Leu Leu Pro Glu Gln Thr 50 55 60 Gly Ser Trp Trp Ser Tyr Glu Pro Val Met Gly Leu Leu Ala Glu Asn 65 70 75 80 Phe His Val Phe Ala Val Asp Ile Arg Gly Gln Gly Arg Ser Thr Trp 85 90 95 Thr Pro Arg Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 100 105 110 Phe Ile Ala Leu Val Ile Lys Arg Pro Val Val Val Ala Gly Asn Ser 115 120 125 Ser Gly Gly Leu Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro Gly 130 135 140 Gln Ile Arg Ala Ala Leu Cys Glu Asp Ala Pro Phe Phe Ala Ser Glu 145 150 155 160 Leu Val Pro Ala Tyr Gly His Ser Val Leu Gln Ala Ala Gly Pro Ala 165 170 175 Phe Glu Leu Tyr Arg Asp Phe Leu Gly Asp Gln Trp Ser Ile Gly Asp 180 185 190 Trp Lys Gly Phe Val Glu Ala Ala Lys Ala Ser Pro Ala Lys Ala Met 195 200 205 Gln Leu Phe Pro Thr Pro Asp Glu Ala Pro Gln Asn Leu Lys Glu Tyr 210 215 220 Asp Pro Glu Trp Gly Arg Ala Phe Phe Glu Gly Thr Val Ala Leu His 225 230 235 240 Cys Pro His Asp Arg Met Leu Ser Gln Val Lys Thr Pro Ile Leu Ile 245 250 255 Thr His His Ala Arg Thr Ile Asp Pro Glu Thr Gly Glu Leu Leu Gly 260 265 270 Ala Leu Ser Asp Leu Gln Ala Glu His Ala Gln Asp Ile Ile Arg Ser 275 280 285 Ala Gly Val Arg Val Asp Tyr Gln Ser His Pro Asp Ala Leu His Met 290 295 300 Met His Leu Phe Asp Pro Ala Arg Tyr Ala Glu Ile Leu Thr Ser Trp 305 310 315 320 Ser Ala Thr Leu Pro Ala Asn Asp 325 <210> 2 <211> 308 <212> PRT <213> Streptomyces violaceusniger <400> 2 Met Ala Asp Pro Ala Gln Arg Asp Val Tyr Val Pro His Ala Tyr Pro 1 5 10 15 Glu Lys Gln Ala Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu Ala 20 25 30 Gly Glu Pro Asp Met Pro Ala Val Leu Leu Ile Pro Glu Gln Thr Gly 35 40 45 Ser Trp Trp Gly Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu Asn Phe 50 55 60 His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp Ala 65 70 75 80 Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg Phe ​​Ile Ala Leu Val Val Lys Arg Pro Val Ile Val Ala Gly Asn Ser Ser 100 105 110 Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly Gln 115 120 125 Val Arg Gly Ala Leu Cys Glu Asp Ala Pro Phe Phe Ala Ser Glu Leu 130 135 140 Val Thr Thr Cys Gly His Ser Ile Arg Gln Ala Ala Gly Pro Met Phe 145 150 155 160 Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp Trp 165 170 175 Thr Gly Tyr Cys Arg Ala Ala Asp Ala Ser Ser Ser Pro Met Ala Arg 180 185 190 Tyr Phe Val Ala Asp Glu Ile Pro Gln His Met Arg Glu Tyr Asp Pro 195 200 205 Glu Trp Ala Arg Ala Phe Trp Glu Gly Thr Val Ala Leu His Cys Pro 210 215 220 His Glu Gln Leu Leu Thr Gln Val Lys Thr Pro Val Leu Leu Thr His 225 230 235 240 His Met Arg Asp Ile Asp Pro Asp Thr Gly His Leu Val Gly Ala Leu 245 250 255 Ser Asp Glu Gln Ala Ala Arg Ala Arg Leu Leu Met Glu Ser Ala Gly 260 265 270 Val Lys Val Asp Tyr Ala Ser Val Pro Asp Ala Leu His Met Met His 275 280 285 Gln Phe Asp Pro Pro Arg Tyr Val Glu Ile Phe Thr Gln Trp Ala Ala 290 295 300 Thr Leu Ala Ala 305 <210> 3 <211> 309 <212> PRT <213> Streptomyces coelicolor <400> 3 Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr 1 5 10 15 Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu 20 25 30 Ala Gly Asp Pro Gly Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr 35 40 45 Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His 50 55 60 Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp 65 70 75 80 Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 85 90 95 Phe Ile Ala Leu Val Val Arg Arg Pro Val Val Val Ala Gly Asn Ser 100 105 110 Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly 115 120 125 Gln Ile Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu 130 135 140 Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val 145 150 155 160 Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp 165 170 175 Trp Glu Gly Phe Arg Ser Ala Ala Asp Ala Ser Ala Ser Pro Met Ala 180 185 190 Arg Ser Phe Val Ala Asp Thr Ile Pro Gln His Leu Lys Glu Tyr Asp 195 200 205 Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Gly Leu Asn Cys 210 215 220 Pro His Glu Arg Met Leu Asn Arg Val Asn Thr Pro Val Leu Leu Thr 225 230 235 240 His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala 245 250 255 Leu Ser Asp Glu Gln Ala Ala Gln Val Arg Arg Leu Met Glu Ser Ala 260 265 270 Gly Val Lys Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met 275 280 285 His Gln Ser Asp Pro Ala Arg Tyr Ala Glu Ile Leu Thr Pro Trp Thr 290 295 300 Ala Ala Leu Ala Pro 305 <210> 4 <211> 309 <212> PRT <213> Streptomyces rapamycinicus <400> 4 Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr 1 5 10 15 Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu 20 25 30 Ala Gly Asp Pro Asp Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr 35 40 45 Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His 50 55 60 Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp 65 70 75 80 Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 85 90 95 Phe Ile Ala Leu Val Val Lys Arg Pro Val Val Val Ala Gly Asn Ser 100 105 110 Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly 115 120 125 Gln Leu Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu 130 135 140 Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val 145 150 155 160 Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Ser Asp 165 170 175 Trp Glu Gly Phe Cys Arg Ala Ala Gly Ala Ser Ala Ser Pro Met Ala 180 185 190 Arg Ser Phe Val Ala Asp Gly Ile Pro Gln His Leu Lys Glu Tyr Asp 195 200 205 Pro Glu Trp Ala Arg Ala Phe His Glu Gly Thr Val Gly Leu Asn Cys 210 215 220 Pro His Glu Arg Met Leu Gly Arg Val Asn Thr Pro Val Leu Leu Thr 225 230 235 240 His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala 245 250 255 Leu Ser Asp Glu Gln Ala Ala Gln Ala Arg Leu Leu Met Glu Ser Ala 260 265 270 Gly Val Arg Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met 275 280 285 His Gln Ser Asp Pro Ala Arg Tyr Ala Glu Ile Phe Thr Arg Trp Ala 290 295 300 Ala Ala Leo Ala Pro 305 <210> 5 <211> 309 <212> PRT <213> Streptomyces lividans <400> 5 Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr 1 5 10 15 Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu 20 25 30 Ala Gly Asp Pro Gly Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr 35 40 45 Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His 50 55 60 Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp 65 70 75 80 Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 85 90 95 Phe Met Ala Leu Val Val Arg Arg Pro Val Val Val Ala Gly Asn Ser 100 105 110 Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly 115 120 125 Gln Ile Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu 130 135 140 Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val 145 150 155 160 Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp 165 170 175 Trp Glu Gly Phe Arg Ser Ala Ala Gly Ala Ser Ala Ser Pro Met Ala 180 185 190 Arg Ser Phe Val Ala Asp Thr Ile Pro Gln His Leu Lys Glu Tyr Asp 195 200 205 Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Gly Leu Asn Cys 210 215 220 Pro His Glu Arg Met Leu Asn Arg Val Asn Thr Pro Val Leu Leu Thr 225 230 235 240 His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala 245 250 255 Leu Ser Asp Glu Gln Ala Ala Gln Ala Arg Arg Leu Met Glu Ser Ala 260 265 270 Gly Val Lys Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met 275 280 285 His Gln Ser Asp Pro Ala Arg Tyr Ala Glu Ile Leu Thr Pro Trp Ala 290 295 300 Ala Ala Leu Ala Pro 305 <210> 6 <211> 309 <212> PRT <213> Streptomyces coelicoflavus <400> 6 Met Val Thr Ser Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr 1 5 10 15 Pro Glu Gln Gln Val Asp Leu Gly Glu Ile Thr Met Asn Tyr Ala Glu 20 25 30 Ala Gly Asp Pro Asp Arg Pro Ala Val Leu Leu Ile Pro Glu Gln Thr 35 40 45 Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ser Glu His 50 55 60 Phe His Val Tyr Ala Val Asp Leu Arg Gly Gln Gly Arg Ser Ser Trp 65 70 75 80 Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg 85 90 95 Phe Ile Ala Leu Val Val Lys Arg Pro Val Val Val Ala Gly Asn Ser 100 105 110 Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly 115 120 125 Gln Leu Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu 130 135 140 Leu Val Pro Ala His Gly His Ser Val Arg Gln Gly Ala Gly Pro Val 145 150 155 160 Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gln Trp Ser Val Gly Asp 165 170 175 Trp Glu Gly Phe Cys Arg Ala Ala Gly Ala Ser Ala Ser Pro Met Ala 180 185 190 Arg Ser Phe Val Ala Asp Gly Ile Pro Gln His Leu Gln Glu Tyr Asp 195 200 205 Pro Glu Trp Ala Arg Val Phe Tyr Glu Gly Thr Val Gly Leu Ser Cys 210 215 220 Pro His Glu Arg Met Leu Gly Gln Val Lys Thr Pro Val Leu Leu Thr 225 230 235 240 His His Met Arg Gly Ile Asp Pro Glu Thr Gly Asn Leu Leu Gly Ala 245 250 255 Leu Ser Asp Glu Gln Ala Leu Arg Ala Arg Arg Leu Met Asp Ser Ala 260 265 270 Gly Val Thr Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met 275 280 285 His Gln Ser Ala Pro Ala Arg Tyr Val Glu Ile Phe Thr Arg Trp Ala 290 295 300 Ala Ala Leu Ala Pro 305 <210> 7 <211> 300 <212> PRT <213> Rhodococcus triatome <400> 7 Met Pro His Asp Tyr Glu Glu Lys Leu Val Asp Leu Gly Glu Ile Asp 1 5 10 15 Leu Asn Tyr Ala Glu Ala Gly Ser Pro Asp Lys Pro Ala Leu Leu Leu 20 25 30 Ile Pro Ser Gln Ser Glu Ser Trp Trp Gly Tyr Glu Glu Ala Met Gly 35 40 45 Leu Leu Ala Glu Asp Tyr His Val Phe Ala Val Asp Met Arg Gly Gln 50 55 60 Gly Arg Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly 65 70 75 80 Asn Asp Leu Val Arg Phe Ile Asp Leu Val Ile Gly Arg Thr Val Ile 85 90 95 Val Ser Gly Asn Ser Ser Gly Gly Val Val Ala Ala Trp Leu Ala Ala 100 105 110 Phe Ser Leu Pro Gly Gln Val Arg Ala Ala Leu Ala Glu Asp Ala Pro 115 120 125 Phe Phe Ala Ser Glu Leu Asp Pro Lys Val Gly His Thr Ile Arg Gln 130 135 140 Ala Ala Gly His Ile Phe Val Asn Trp Arg Asp Tyr Leu Gly Asp Gln 145 150 155 160 Trp Ser Val Gly Asp Tyr Ala Gly Phe Leu Lys Ala Met Lys Ser Ser 165 170 175 Glu Val Pro Met Leu Arg Gln Val Pro Leu Pro Glu Thr Ala Pro Gln 180 185 190 Asn Leu Leu Glu Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly 195 200 205 Thr Val Ala Gln Thr Cys Pro His Asp Tyr Met Leu Ser Gln Val Lys 210 215 220 Val Pro Met Leu Val Thr His His Ala Arg Met Ile Asp Glu Ala Thr 225 230 235 240 Ser Gly Leu Val Gly Ala Met Ser Asp Leu Gln Val Gln Lys Ala Ala 245 250 255 Glu Ile Ile Arg Gly Thr Gly Val Gln Val Asp Val Val Asp Leu Pro 260 265 270 Glu Ala Pro His Ile Leu His Gln Leu Ala Pro Lys Glu Tyr Val Glu 275 280 285 Ile Leu Asn Asn Trp Val Glu Lys Leu Pro Pro Val 290 295 300 <210> 8 <211> 307 <212> PRT <213> Hirschia baltica <400> 8 Met Ile Gln Asn Asn Lys Thr Ala Pro Tyr Lys Tyr Lys Glu Lys Leu 1 5 10 15 Val Asp Leu Gly Glu Ile Lys Met Asn Tyr Ile Val Ala Gly Ala Asp 20 25 30 Val Ser Pro Ala Leu Leu Leu Ile Pro Gly Gln Thr Glu Ser Trp Trp 35 40 45 Gly Phe Glu Ala Ala Ile Glu Lys Leu Glu Ser Asn Phe Gln Val Phe 50 55 60 Ala Ile Asp Leu Arg Gly Gln Gly Lys Ser Thr Gln Thr Pro Gly Arg 65 70 75 80 Tyr Ser Leu Asn Leu Met Gly Asn Asp Leu Val Arg Phe Ile Ser Leu 85 90 95 Val Ile Lys Arg Pro Val Ile Val Ser Gly Asn Ser Ser Gly Gly Leu 100 105 110 Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro Asn Gln Ile Arg Ala 115 120 125 Ile His Cys Glu Asp Ala Pro Phe Phe Thr Ala Glu Lys Ala Pro Leu 130 135 140 Tyr Gly His Ala Ile Gln Gln Ala Ala Gly Pro Ile Phe Ser Leu Met 145 150 155 160 Ser Lys Phe Leu Gly Asp Gln Trp Ser Ile Asn Asn Trp Glu Gly Leu 165 170 175 Lys Ala Ala Gln Ala Lys Asp Thr His Pro Ala Asn Lys Met Ile Ser 180 185 190 Gln Val Glu Gln Pro Pro Gln His Leu Lys Glu Tyr Asp Pro Glu Trp 195 200 205 Gly Arg Ala Phe Ile Glu Gly Lys Phe Asn Leu Asn Ser Pro His His 210 215 220 Thr Leu Leu Ser Asp Ile Lys Thr Pro Met Leu Tyr Thr His His Met 225 230 235 240 Arg Phe Glu Asp Pro Gln Thr Gly Leu Leu Ile Gly Ala Thr Ser Asp 245 250 255 Phe Gln Ala Ser Lys Ile Lys Glu Ile Ala Leu Lys Thr Gly Asn Ser 260 265 270 Phe Glu Leu Ile Asp Ala Pro Asp Ala Phe His Ser Met His Glu Ala 275 280 285 Asp Pro Gln Arg Phe Val Asp Ile Leu Thr Ser Trp Ile Glu Arg Leu 290 295 300 Asn Leu Gln 305 <210> 9 <211> 321 <212> PRT <213> Nocardia brasiliensis <400> 9 Met Gly Ile Ser Glu Ala Ala Asp Arg Ala Asp Thr Phe Val Ala His 1 5 10 15 Lys Phe Glu Glu Gln Leu Val Asp Leu Gly Glu Ile Arg Met Asn Tyr 20 25 30 Val Ala Ala Gly Asp Pro Thr Ser Pro Ala Leu Leu Leu Ile Pro Ala 35 40 45 Gln Gly Glu Ser Trp Trp Gly Tyr Glu Asn Ala Ile Thr Leu Leu Ala 50 55 60 Asn Asp Phe Arg Val Phe Ala Ile Asp Leu Arg Gly Gln Gly Arg Ser 65 70 75 80 Thr Trp Thr Pro Gly Arg Tyr Asn Leu Asn Thr Trp Gly Asn Asp Val 85 90 95 Glu Arg Phe Ile Asp Leu Val Ile Gly Arg Pro Thr Leu Val Ala Gly 100 105 110 Asn Ser Ser Gly Gly Val Ile Ala Ala Trp Leu Ala Ala Tyr Ala Lys 115 120 125 Pro Gly Gln Ile Arg Gly Ala Met Leu Glu Asp Pro Pro Leu Phe Ala 130 135 140 Ser Gln Ala Ala Pro Pro Tyr Gly Pro Gly Ile Met Gln Thr Leu Gly 145 150 155 160 Pro Ile Phe Val Leu Trp Ala Lys Trp Leu Gly Pro Gln Trp Ser Val 165 170 175 Gly Asp Trp Asp Gly Met Val Ala Ala Ala Pro Arg Glu Leu Pro Glu 180 185 190 Phe Leu His Pro Gly Ile Ala Phe Leu Phe Gly Asp Gly Thr Gly Glu 195 200 205 Gly Ala Ala Ala Thr Pro Pro Gln His Leu Lys Glu Tyr Asp Pro Glu 210 215 220 Trp Ala Gln Ala Trp Ala Thr Asp Val Ala Asn Ala Gly Cys Asp His 225 230 235 240 Ala Thr Met Leu Ala Gln Asn Arg Val Pro Val Leu Leu Thr His His 245 250 255 Phe His Leu Thr Asp Pro Asp Thr Gly Gln Leu Met Gly Ala Met Thr 260 265 270 Asp Ile Gln Ala Gln Gln Ala Arg Arg Leu Leu Ala Ala Thr Gly Gln 275 280 285 Pro Val Thr Phe Thr Ala Leu Asp Ala Pro His Thr Met His Asp Pro 290 295 300 Glu Pro Glu Arg Tyr Phe Glu Val Leu Thr Glu Trp Ala Ser Ala Leu 305 310 315 320 Asp <210> 10 <211> 319 <212> PRT <213> Mycobacterium vaccae <400> 10 Met Gly Arg Tyr Ala Gly Val Phe Gly Pro His Ala Pro Glu Ser Thr 1 5 10 15 Tyr Val Gly His Ala Tyr Pro Glu Gln Leu Phe Asp Thr Gly Glu Val 20 25 30 Arg Leu Asn Tyr Ala Val Ala Gly Asp Ala Ser Ala Ser Pro Leu Leu 35 40 45 Leu Ile Pro Gly Gln Thr Glu Ser Trp Trp Gly Tyr Glu Pro Ala Met 50 55 60 Gly Leu Leu Ala Glu His Phe His Val His Ala Val Asp Leu Arg Gly 65 70 75 80 Gln Gly Arg Ser Thr Arg Thr Pro Arg Arg Tyr Thr Leu Asp Asn Ile 85 90 95 Gly Asn Asp Leu Val Arg Phe Leu Asp Gly Val Ile Gly Arg Pro Ala 100 105 110 Phe Val Ser Gly Leu Ser Ser Gly Gly Leu Leu Ser Ala Trp Leu Ser 115 120 125 Ala Phe Ala Glu Pro Gly Gln Val Leu Ala Ala Cys Tyr Glu Asp Pro 130 135 140 Pro Phe Phe Ser Ser Glu Leu Asp Pro Val Ile Gly Pro Gly Leu Met 145 150 155 160 Ser Thr Val Gly Pro Leu Phe Ala Leu Tyr Val Lys Tyr Leu Gly Asp 165 170 175 Gln Trp Ser Ile Gly Asp Trp Asp Gly Phe Val Ala Gly Ala Pro Gln 180 185 190 Glu Leu Ala Gly Trp Gln Ala His Val Ala Leu Ala Gly Gly Thr Ala 195 200 205 Glu Pro Pro Gln His Leu Lys Glu Tyr Asp Pro Glu Trp Gly Arg Ala 210 215 220 Phe Val Gly Gly Thr Phe Thr Thr Gly Cys Pro His Gln Val Met Leu 225 230 235 240 Ser Gln Val Lys Val Pro Val Leu Phe Thr His His Phe Arg Met Leu 245 250 255 Asp Asp Glu Ser Gly Ser Leu Ile Gly Ala Ala Thr Asp Asp Gln Ala 260 265 270 Ala Arg Val Val Glu Leu Val Glu Asn Ser Gly Ala Pro Leu Thr Tyr 275 280 285 Arg Ser Phe Pro Met Met Gly His Ser Met His Ala Gln Asp Pro Ala 290 295 300 Leu Phe Ala Gly Thr Leu Val Asp Trp Phe Thr Ala Ala Arg Ser 305 310 315 <210> 11 <211> 319 <212> PRT <213> Mycobacterium gilvum <400> 11 Met Gly Arg Tyr Ala Gly Val Phe Gly Pro His Ala Pro Glu Ala Thr 1 5 10 15 Tyr Val Glu His Gly Tyr Pro Glu Arg Leu Phe Asp Thr Gly Glu Val 20 25 30 Gln Leu Asn Tyr Val Val Ala Gly Asp Ala Ala Ala Pro Pro Leu Leu 35 40 45 Leu Ile Pro Gly Gln Ser Glu Ser Trp Trp Gly Tyr Glu Ala Ala Ile 50 55 60 Pro Leu Leu Ala Arg His Phe His Val His Ala Val Asp Leu Arg Gly 65 70 75 80 Gln Gly Arg Ser Thr Arg Thr Pro Gly Arg Tyr Thr Leu Asp Asn Val 85 90 95 Gly Asn Asp Leu Val Arg Phe Leu Asp Gly Val Ile Gly Arg Pro Ala 100 105 110 Phe Val Ser Gly Leu Ser Ser Gly Gly Leu Ala Ser Ala Trp Leu Ser 115 120 125 Ala Phe Ala Lys Pro Gly Gln Val Val Ala Ala Cys Trp Glu Asp Pro 130 135 140 Pro Phe Phe Ser Ser Glu Thr Ala Pro Ile Val Gly Pro Pro Ile Thr 145 150 155 160 Asp Ser Ile Gly Pro Leu Phe Gly Met Trp Ala Arg Tyr Leu Gly Asp 165 170 175 Gln Trp Ser Val Gly Asp Trp Asp Gly Phe Val Ala Ala Val Pro Thr 180 185 190 Glu Leu Ala Asp Trp Gln Ala His Val Ala Leu Val Val Gly Thr Ala 195 200 205 Asp Pro Pro Gln Asn Leu Arg Glu Tyr Asp Pro Glu Trp Gly Lys Ala 210 215 220 Phe Ile Thr Gly Thr Phe Ala Ala Ser Cys Pro His His Val Met Leu 225 230 235 240 Ser Lys Val Lys Val Pro Val Leu Tyr Thr His His Phe Arg Met Ile 245 250 255 Asp Glu Gly Ser Gly Gly Leu Ile Gly Ala Cys Ser Asp Ile Gln Ala 260 265 270 Gly Arg Val Thr Gln Leu Ala Lys Ser Gly Gly Arg Ser Val Thr Tyr 275 280 285 Arg Ser Phe Pro Met Met Ala His Ser Met His Gly Gln Asp Pro Ala 290 295 300 Leu Phe Ser Glu Thr Leu Val Glu Trp Phe Ser Arg Phe Thr Gly 305 310 315 <210> 12 <211> 322 <212> PRT <213> Gordonia effusa <400> 12 Met Pro Lys Ser Glu Ala Ala Asp Arg Ala Asp Ser Phe Val Ser His 1 5 10 15 Asp Phe Lys Glu Asn Ile Val Asp Leu Gly Glu Ile Arg Met Asn Tyr 20 25 30 Val Val Gln Gly Asn Lys Lys Ser Pro Ala Leu Leu Leu Ile Pro Ala 35 40 45 Gln Gly Glu Ser Trp Trp Gly Tyr Glu Ala Ala Ile Pro Leu Leu Ala 50 55 60 Lys His Phe Gln Val Phe Ala Ile Asp Leu Arg Gly Gln Gly Arg Thr 65 70 75 80 Thr Trp Thr Pro Gly Arg Tyr Thr Leu Asp Ile Phe Gly Asn Asp Val 85 90 95 Val Arg Phe Ile Asp Leu Val Ile Gly Arg Glu Thr Leu Ile Ala Gly 100 105 110 Asn Ser Ser Gly Gly Leu Ile Gly Ala Trp Leu Ala Ala Phe Ala Lys 115 120 125 Pro Gly Gln Val Arg Ala Val Met Leu Glu Asp Pro Pro Leu Phe Ala 130 135 140 Ser Glu Ile Arg Pro Pro Tyr Gly Pro Gly Ile Trp Gln Gly Leu Gly 145 150 155 160 Pro Met Phe Ala Ala Trp Ala Lys Trp Leu Gly Pro Gln Trp Ser Ile 165 170 175 Gly Asp Trp Asp Gly Met Val Lys Ala Leu Pro Asp Glu Leu Pro Glu 180 185 190 Asp Leu Leu Pro Gly Ile Gly Phe Met Leu Gly Asp Gly Glu Ser Asp 195 200 205 Gly Ala Ala Pro Thr Pro Pro Gln His Leu Lys Glu Tyr Asp Pro Glu 210 215 220 Trp Gly Ala Ser Trp Ala Ser Gly Phe Ala Asn Thr Gly Cys Glu His 225 230 235 240 Glu Ala Val Ile Ser Gln Val Arg Val Pro Val Leu Leu Thr His His 245 250 255 Phe Arg Gln Ile Asn Glu Glu Thr Gly His Leu Met Gly Ala Leu Ser 260 265 270 Asp Leu Gln Ala Ala Gln Val Arg His Ile Ile Glu Glu Val Ala Gly 275 280 285 Gln Glu Val Thr Tyr Val Ser Leu Asp Ala Pro His Thr Met His Glu 290 295 300 Pro Gln Pro Glu Arg Tyr Thr Asp Val Leu Leu Asp Trp Val Lys Lys 305 310 315 320 Leu Gly <210> 13 <211> 328 <212> PRT <213> Togninia minima <400> 13 Met Asn Tyr Ala Thr Ala Gly Ser Ser Asp Lys Pro Ala Leu Leu Leu 1 5 10 15 Val Pro Gly Gln Ser Glu Ser Trp Trp Gly Tyr Glu Met Ala Met Trp 20 25 30 Leu Leu Lys Asp Asp Tyr Gln Val Phe Ala Val Asp Met Arg Gly Gln 35 40 45 Gly Gln Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Thr Phe Gly 50 55 60 Asn Asp Leu Val Lys Phe Ile Asp Ile Val Ile Lys Arg Pro Val Val 65 70 75 80 Val Ser Gly Leu Ser Ser Gly Gly Val Val Ser Ala Trp Leu Ser Ala 85 90 95 Phe Ala Lys Pro Gly Gln Ile Arg Ala Ala Val Tyr Glu Asp Pro Pro 100 105 110 Leu Phe Ala Ser Gln Ser Lys Pro Ala Ile Gly Gln Ser Val Met Gln 115 120 125 Thr Val Ala Gly Pro Phe Phe Asn Leu Trp Tyr Lys Trp Leu Gly Ala 130 135 140 Gln Trp Thr Ile Gly Asp Gln Ala Gly Met Val Ala Ala Met Pro Lys 145 150 155 160 Glu Ile Pro Ala Trp Ile Leu Gln Tyr Leu Gly Asn Thr Thr Ser Gly 165 170 175 Pro Thr Gly Leu Asp Leu Thr Leu Asn Glu Tyr Asp Pro Glu Trp Gly 180 185 190 His Gly Phe Val Ser Gly Thr Val Asp Ala Thr Cys Asp His Glu Ala 195 200 205 Met Leu Thr His Val Lys Val Pro Val Leu Phe Thr His His Ser Arg 210 215 220 Ala Ile Asp Pro Tyr Thr Gly Asn Leu Ile Gly Ser Val Ser Asp Thr 225 230 235 240 Gln Val Ser Tyr Ala Gln Gly Leu Ile Thr Thr Asn Gly Asn Gln Ser 245 250 255 Phe Thr Leu Lys Asn Phe Pro Leu Ala Ser His Asp Met His Asn Ser<<<MASK>>> 260 265 270 Asp Pro Ala Thr Tyr Val Ser Ala Ile Thr Thr Trp Met Ala Ser Leu 275 280 285 Gly Ile Gly Ser Ala Val Ile Pro Gly Pro Val Lys Val Ala Ser Ala 290 295 300 Ser Ala Gln Val Ser Ala Ala Ser Thr Ala Pro Pro Ser Cys Thr Ser 305 310 315 320 Thr Ser Ala Pro Ser Thr Gly His 325 <210> 14 <211> 280 <212> PRT <213> Actinosynnema mirum <400> 14<<<MASK>>> Met Thr Val Val Asp Pro Pro Ala Pro Arg Asp Phe Pro Glu Leu Leu <<<MASK>>>1 5 10 15 Val Asp Leu Gly Glu Val Val Leu Asn His Ala Glu Ala Gly Ser Pro 20 25 30 Asp Arg Pro Ala Leu Val Pro Val Pro Glu Gln Gly Gly Ser Trp Trp 35 40 45 Ser Tyr Glu Arg Val Met Pro Leu Pro Ala Arg Asp Phe His Val Phe 50 55 60 Ala Val Asp Leu Arg Gly Arg Gly Arg Ser Thr Arg Thr Pro Arg Arg 65 70 75 80 Tyr Ser Leu Asp Asp Phe Gly Asn Asp Leu Val Arg Phe Leu Ala Leu 85 90 95 Val Val Arg Arg Pro Ala Val Val Ala Gly Asn Ser Ser Gly Gly Val 100 105 110 Leu Ala Ala Trp Ser Ser Ala Tyr Ala Met Pro Gly Gln Val Arg Ala 115 120 125 Val Leu Leu Glu Asp Pro Pro Leu Phe Ser Ser Glu Leu Thr Pro Val 130 135 140 Cys Gly Pro Gly Val Arg Gln Ala Ala Gly Pro Leu Phe Glu Leu Leu 145 150 155 160 Ser Thr His Leu Gly Asp Gln Trp Gly Gly Gly Arg Pro Gly Arg Val 165 170 175 His Gly Gly Val Pro Arg Leu Gly Leu Ala Ala Ala Ala Ala Val Arg 180 185 190 Val Ala Arg Arg Ala Ala Ala Thr Asp Ala Arg Gly Arg Pro Gly Ala 195 200 205 Ala Arg Gly Arg Pro Ala Gly Val Gly Gly Ala Ala Arg Arg Gly Arg 210 215 220 Gly Gly Arg Glu Arg Thr Gly Thr Thr Thr Val Leu Ser Gly Leu Thr 225 230 235 240 Gly Ser Arg Thr Ser Gly Thr Gly Arg Cys Arg Lys Pro Phe Arg Leu 245 250 255 Arg Gln Trp Trp Ala Gly Gly Ala Arg Gly Pro Pro Pro Pro Arg Gln 260 265 270 Ile Arg Ala Asp Val Arg Thr Arg 275 280 <210> 15 <211> 326 <212> PRT <213> Kutzneria albida <400> 15 Met Ser Val Pro Val Thr Pro Ser Ala Arg Asn Val Phe Val Pro His 1 5 10 15 Ala Phe Pro Glu Lys Gln Ile Asp Leu Gly Glu Val Val Leu Asn Tyr 20 25 30 Ala Glu Ala Gly Thr Pro Asp Lys Pro Ala Leu Leu Leu Leu Pro Glu 35 40 45 Gln Thr Gly Ser Trp Trp Ser Tyr Glu Pro Ala Met Gly Leu Leu Ala 50 55 60 Glu His Phe His Val Phe Ala Val Asp Leu Arg Gly Gln Gly Arg Ser 65 70 75 80 Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu 85 90 95 Val Arg Phe Ile Ala Leu Ala Ile Arg Arg Pro Val Val Val Ala Gly 100 105 110 Cys Ser Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ala Leu 115 120 125 Pro Gly Gln Ile Arg Gly Ala Leu Cys Glu Asp Ala Pro Leu Phe Ala 130 135 140 Ser Glu Leu Thr Pro Ala His Gly His Gly Val Arg Gln Gly Ala Gly 145 150 155 160 Pro Val Phe Glu Leu Tyr Arg Asp Tyr Leu Gly Asp Gln Trp Ser Val 165 170 175 Gly Asp Trp Ala Gly Leu Val Ala Ala Ala Gln Ala Ser Pro Ala Lys 180 185 190 Met Met Ser Leu Phe Lys Met Pro Gly Glu Pro Pro Gln Asn Leu Arg 195 200 205 Glu Tyr Asp Pro Glu Trp Ala Arg Val Phe Phe Glu Gly Thr Val Gly 210 215 220 Leu His Cys Pro His Asp Arg Met Leu Ser Gln Val Lys Thr Pro Val 225 230 235 240 Leu Ile Thr His His Ala Arg Thr Thr Asp Pro Glu Thr Gly Glu Phe 245 250 255 Leu Gly Ala Leu Ser Glu Leu Gln Ala Glu Arg Ala Gln Ala Ile Ile 260 265 270 Arg Ala Ala Gly Val Pro Val Asp Tyr Gln Ser Phe Pro Asp Ala Ala 275 280 285 His Ala Met His Thr Thr Glu Pro Ala Arg Tyr Ala Ala Val Leu Thr 290 295 300 Ala Trp Ala Ala Lys Leu Pro Pro Val Ala Asp Thr Ser Pro Ser Ala 305 310 315 320 Ala Ala Ser Ala His Val 325 <210> 16 <211> 987 <212> DNA <213> Rhodococcus erythropolis <220> <221> misc_feature Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 1 <400> 16 atggccgaag aaggaactag gtccgaagca gcggatgctg ccacacaagc gagacagcta 60 cccgattcgc ggaacatctt tgtctcgcac cgatttccgg aaaggcaggt cgatctcggt 120 gaagtggtga tgaacttcgc ggaggcgggc tctccggaca acccggcact gctcctcctc 180 cccgagcaga ccgggtcgtg gtggagttac gagccagtga tgggtcttct ggcagagaac 240 tttcatgtct ttgccgtcga tatccgtggg caaggtcgca gtacctggac gccacggcga 300 tacagcctgg acaacttcgg caatgatctg gtgcgtttca tcgctctggt catcaagcgc 360 cctgtcgtcg tggcagggaa ctcctcgggg gggctgctgg ccgcctggct ctcggcgtac 420 gcgatgcccg gccagatccg tgcagcattg tgtgaggacg caccgttctt tgcgtcggag 480 ttggtccccg catacggtca ctcggttctg caggcggcgg gtccggcatt cgagttgtac 540 cgggacttcc tcggggacca gtggtcgatt ggggactgga aagggttcgt tgaggcagcc 600 aaagcgtcgc cggcaaaggc tatgcaatta tttccgaccc cggatgaggc gccgcagaat 660 ctcaaggaat acgacccgga atgggggcgc gcattcttcg aagggactgt ggcactgcac 720 tgcccacacg acaggatgct ctcgcaagtc aagacaccaa ttctcatcac tcaccacgcg 780 cggacgatcg accccgagac gggcgagctg ttgggcgcgc tctccgacct tcaggcagag 840 catgcgcagg acatcattcg gtctgcgggc gttcgggtgg actatcagtc gcaccccgac 900 gcgcttcaca tgatgcatct gttcgatccc gctcgttacg cggagatctt gacatcctgg 960 tccgcaacac tgcctgcgaa cgactag 987 <210> 17 <211> 987 <212> DNA <213> Artificial Sequence <220> <223> The ORF is codon-optimized and thus different from the naturally occurring DNA sequence <400> 17 atggcagaag aaggcacccg tagcgaagca gcagatgcag caacccaggc acgtcagctg 60 ccggatagcc gtaacatttt tgttagccat cgttttccgg aacgtcaggt tgatctgggt 120 gaagttgtta tgaattttgc agaagcaggt agtccggata atccggcatt actgctgctg 180 ccggaacaga ccggtagttg gtggtcttat gaaccggtta tgggtctgct ggcagaaaac 240 tttcatgttt ttgcagttga tattcgtggt cagggtcgta gcacctggac accgcgtcgt 300 tatagcctgg ataattttgg taatgatctg gtgcgtttta ttgccctggt tattaaacgt 360 ccggttgttg ttgcaggtaa tagcagcggt ggcctgctgg ctgcatggct gagcgcctat 420 gcaatgcctg gtcagattcg tgcagcactg tgtgaagatg caccgttttt tgcaagcgaa 480 ctggttcctg cctatggtca tagcgttctg caggcagcag gtccggcatt tgaactgtat 540 cgtgattttc tgggtgatca gtggtcaatt ggtgattgga aaggttttgt tgaagcagca 600 aaagcaagtc cggctaaagc aatgcagctg tttccgacac cggatgaagc accgcagaat 660 ctgaaagaat atgatccgga atggggtcgt gcattttttg aaggcaccgt tgcactgcat 720 tgtccgcatg atcgtatgct gagccaggtt aaaaccccga ttctgattac ccatcatgca 780 cgtaccatcg atccggaaac cggtgaactg ctgggtgcac tgagtgatct gcaggccgaa 840 catgcacagg atattattcg tagtgccggt gttcgtgttg attatcagag ccatcctgat 900 gcactgcaca tgatgcacct gtttgatccg gcacgttatg cagaaattct gaccagttgg 960 agcgcaaccc tgcctgcaaa tgattaa 987 <210> 18 <211> 927 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 2 <400> 18 atggcagatc cggcacagcg tgatgtttat gttccgcatg catatccgga aaaacaggca 60 gatctgggtg aaattaccat gaattatgcc gaagccggtg aacctgatat gcctgcagtt 120 ctgctgattc cggaacagac cggtagttgg tggggttatg aagaagcaat gggtctgctg 180 gcagaaaact ttcatgttta tgcagttgat ctgcgtggtc agggtcgtag cagctgggca 240 ccgaaacgtt atagcctgga taattttggt aatgatctgg tgcgttttat tgccctggtt 300 gttaaacgtc cggttatattgt tgcaggtaat agcagcggtg gtgttctggc agcatggctg 360 agcgcatata gcatgcctgg tcaggttcgt ggtgcactgt gtgaagatgc accgtttttt 420 gcaagcgaac tggttaccac ctgtggtcat agcattcgtc aggcagcagg tccgatgttt 480 gaactgtttc gtacctatct gggcgatcag tggtcagttg gtgattggac cggctattgt 540 cgtgcagcag atgcaagcag cagcccgatg gcacgttattttgttgcaga tgaaattccg 600 cagcacatgc gtgaatatga tccggaatgg gcacgtgcat tttgggaagg caccgttgca 660 ctgcattgtc cgcatgaaca gctgctgacc caggttaaaa caccggtgct gctgacacat 720 cacatgcgcg atattgatcc tgataccggt catctggttg gtgccctgag tgatgaacag 780 gcagcccgtg cacgtctgct gatggaaagt gccggtgtta aagttgatta tgcaagcgtt 840 ccggatgcac tgcacatgat gcaccagttt gatccgcctc gttatgttga aatctttacc 900 cagtgggcag caaccctggc agcataa 927 <210> 19 <211> 930 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 3 <400> 19 atggttacca gtccggcact gcgtgatgtt catgttccgc atgcatatcc ggaacagcag 60 gttgatctgg gtgaaattac catgaattat gccgaagccg gtgatccggg tcgtccggca 120 gttctgctga tcccggaaca gaccggtagt tggtggtctt atgaagaagc aatgggtctg 180 ctggcagaac attttcatgt ttatgcagtt gatctgcgtg gtcagggtcg tagcagctgg 240 accccgaaac gttatagcct ggataatttt ggtaatgatc tggtgcgttt tattgccctg 300 gttgttcgtc gtccggttgt tgttgcaggt aatagcagcg gtggtgttct ggcagcatgg 360 ctgagcgcat atagcatgcc tggtcagatt cgtggtgtgc tgtgtgaaga tccgcctttt 420 tttgcaagcg aactggttcc ggcacatggt catagcgttc gtcagggtgc aggtccggtt 480 tttgaactgt ttcgtaccta tctgggcgat cagtggtcag ttggtgattg ggaaggtttt 540 cgtagcgcag cagatgcaag cgcaagcccg atggcacgta gctttgttgc agataccatt 600 ccgcagcatc tgaaagaata tgatccggaa tgggcacgtg cattttatga aggcaccgtt 660 ggtctgaatt gtccgcatga acgtatgctg aatcgtgtta atacaccggt gctgctgacc 720 catcacatgc gtggcaccga tccggaaacc ggtaatctgc tgggtgcact gagtgatgaa 780 caggcagcac aggtgcgtcg tctgatggaa agtgccggtg ttaaagttga ttatgaaagc 840 gttccggatg caagccacat gatgcaccag agcgatccgg cacgttatgc agaaattctg 900 accccgtgga ccgcagcact ggcaccgtaa 930 <210> 20 <211> 930 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 4 <400> 20 atggttacca gtccggcact gcgtgatgtt catgttccgc atgcatatcc ggaacagcag 60 gttgatctgg gtgaaattac catgaattat gccgaagccg gtgatcctga tcgtccggca 120 gttctgctga tcccggaaca gaccggtagt tggtggtcat atgaagaagc aatgggtctg 180 ctggcagaac attttcatgt ttatgcagtt gatctgcgtg gtcagggtcg tagcagctgg 240 accccgaaac gttatagcct ggataatttt ggtaatgatc tggtgcgttt tattgccctg 300 gttgttaaac gtccggttgt tgttgcaggt aatagcagcg gtggtgttct ggcagcatgg 360 ctgagcgcat atagcatgcc tggtcagctg cgtggtgtgc tgtgtgaaga tccgcctttt 420 tttgcaagcg aactggttcc ggcacatggt catagcgttc gtcagggtgc aggtccggtt 480 tttgaactgt ttcgtaccta tctgggcgat cagtggtcag ttagcgattg ggaaggtttt 540 tgtcgtgcag ccggtgcaag cgcaagcccg atggcacgta gctttgttgc agatggtatt 600 ccgcagcatc tgaaagaata tgatccggaa tgggcacgtg catttcatga aggcaccgtt 660 ggtctgaatt gtccgcatga acgtatgctg ggtcgtgtta atacaccggt gctgctgacc 720 catcatatgc gtggcaccga tccggaaacc ggtaatctgc tgggtgcact gagtgatgaa 780 caggcagcac aggcacgtct gctgatggaa agtgccggtg ttcgtgttga ttatgaaagc 840 gttccggatg caagccatat gatgcaccag agcgatccgg cacgttatgc agaaatcttt 900 acccgttggg cagcagccct ggcaccgtaa 930 <210> 21 <211> 930 <212> DNA <213> Artificial sequence <220> <223> The ORF is codon-optimized and thus different from the naturally occurring DNA sequence <220> <221> misc_feature Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 5 <400> 21 atggttacca gtccggcact gcgtgatgtt catgttccgc atgcatatcc ggaacagcag 60 gttgatctgg gtgaaattac catgaattat gccgaagccg gtgatccggg tcgtccggca 120 gttctgctga tcccggaaca gaccggtagt tggtggtctt atgaagaagc aatgggtctg 180 ctggcagaac attttcatgt ttatgcagtt gatctgcgtg gtcagggtcg tagcagctgg 240 accccgaaac gttatagcct ggataatttt ggtaatgatc tggtgcgttt tatggcactg 300 gttgttcgtc gtccggttgt tgttgcaggt aatagcagcg gtggtgttct ggcagcatgg 360 ctgagcgcat atagcatgcc tggtcagatt cgtggtgtgc tgtgtgaaga tccgcctttt 420 tttgcaagcg aactggttcc ggcacatggt catagcgttc gtcagggtgc aggtccggtt 480 tttgaactgt ttcgtaccta tctgggcgat cagtggtcag ttggtgattg ggaaggtttt 540 cgtagcgcag ccggtgcaag cgcaagcccg atggcacgta gctttgttgc agataccatt 600 ccgcagcatc tgaaagaata tgatccggaa tgggcacgtg cattttatga aggcaccgtt 660 ggtctgaatt gtccgcatga acgtatgctg aatcgtgtta atacaccggt gctgctgacc 720 catcacatgc gtggcaccga tccggaaacc ggtaatctgc tgggtgcact gagtgatgaa 780 caggcagcac aggcacgtcg tctgatggaa agtgccggtg ttaaagttga ttatgaaagc 840 gttccggatg caagccacat gatgcaccag agcgatccgg cacgttatgc agaaattctg 900 accccgtggg cagcagccct ggcaccgtaa 930 <210> twenty two <211> 930 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 6 <400> twenty two atggttacca gtccggcact gcgtgatgtt catgttccgc atgcatatcc ggaacagcag 60 gttgatctgg gtgaaattac catgaattat gccgaagccg gtgatcctga tcgtccggca 120 gttctgctga tcccggaaca gaccggtagt tggtggtctt atgaagaagc aatgggtctg 180 ctgagcgaac attttcatgt ttatgcagtt gatctgcgtg gtcagggtcg tagcagctgg 240 accccgaaac gttatagcct ggataatttt ggtaatgatc tggtgcgttt tattgccctg 300 gttgttaaac gtccggttgt tgttgcaggt aatagcagcg gtggtgttct ggcagcatgg 360 ctgagcgcat atagcatgcc tggtcagctg cgtggtgtgc tgtgtgaaga tccgcctttt 420 tttgcaagcg aactggttcc ggcacatggt catagcgttc gtcagggtgc aggtccggtt 480 tttgaactgt ttcgtaccta tctgggcgat cagtggtcag ttggtgattg ggaaggtttt 540 tgtcgtgcag ccggtgcaag cgcaagcccg atggcacgta gctttgttgc agatggtatt 600 ccgcagcatc tgcaagaata tgatccggaa tgggcacgtg ttttttatga aggcaccgtt 660 ggtctgagct gtccgcatga acgtatgctg ggtcaggtta aaacaccggt gctgctgacc 720 catcacatgc gtggtatcga tccggaaacc ggtaatctgc tgggtgcact gagtgatgaa 780 caggccctgc gtgcacgtcg tctgatggat agtgccggtg ttaccgttga ttatgaaagc 840 gttccggatg caagccacat gatgcaccag agcgcaccgg cacgttatgt tgaaatcttt 900 acccgttggg cagcagccct ggcaccgtaa 930 <210> 23 <211> 903 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 7 <400> twenty three atgccgcacg attatgaaga aaaactggtt gatctgggcg aaatcgatct gaattatgca 60 gaagcaggta gtccggataa accggcactg ctgctgattc cgagccagag cgaaagttgg 120 tggggctatg aagaagcaat gggtctgctg gccgaagatt atcatgtttt tgcagttgat 180 atgcgtggtc agggtcgtag cacctggaca ccgggtcgtt atagcctgga taattttggt 240 aatgatctgg tgcgctttat cgatctggtt attggtcgta ccgttatttgt tagcggtaat 300 agcagcggtg gtgttgttgc agcatggctg gcagcattta gcctgcctgg tcaggttcgt 360 gcagcactgg cagaagatgc accgtttttt gcaagcgaac tggacccgaa agtgggtcat 420 accattcgtc aggcagcagg tcatattttt gttaactggc gtgattatct gggtgatcag 480 tggtcagttg gtgattatgc aggttttctg aaagcaatga aaagcagcga agttccgatg 540 ctgcgtcagg ttccgctgcc ggaaaccgca ccgcagaatc tgctggaata tgatccggaa 600 tgggcacgtg cattttatga aggcaccgtt gcacagacct gtccgcatga ttatatgctg 660 agccaggtta aagtgcctat gctggttacc catcatgcac gtatgattga tgaagcaacc 720 agcggtctgg ttggtgcaat gagcgatctg caggttcaga aagcagcaga aattattcgt 780 ggcaccggtg ttcaggttga tgttgttgat ctgccggaag caccgcatat tctgcatcag 840 ctggcaccga aagaatatgt ggaaattctg aataactggg tggaaaaact gcctccggtt 900 taa 903 <210> twenty four <211> 924 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 8 <400> twenty four atgatccaga acaataaaac cgcaccgtat aaatacaaag aaaaactggt tgatctgggc 60 gaaatcaaaa tgaactatat tgttgccggt gcagatgtta gtccggcact gctgctgatt 120 ccgggtcaga ccgaaagttg gtggggtttt ttgagaaact ggaaagcaac tttcaggtgt ttgcaattga tctgcgtggt cagggtaaaa gcacccagac accgggtcgt 240 tatagcctga atctgatggg taatgatctg gttcgtttta ttagcctggt tattaaacgt ccggttattg ttagcggtaa tagcagcggt ggtctgctgg cagcatggct gagcgcctat 360 gcaatgccga atcagattcg tgcaattcat tgtgaagatg caccgttttt taccgcagaa 420 aaagcaccgc tgtatggtca tgcaattcag caggcagcag gtccgatttt tagcctgatg 480 agcaaatttc tgggtgatca gtggtcaatt aacaattggg aaggtctgaa agcagcacag gcaaaagata cccatccggc aaacaaaatg attagccagg ttgaacagcc tccgcagcat ctgaagaat atgatccgga atggggtcgt gcatttattg aaggcaaatt taacctgaac agtccgcatc ataccctgct gagcgacatt aaaaccccga tgctgtatac ccatcacatg cgttttgaag atccgcagac aggtctgctg attggtgcaa ccagcgattt tcaggcaagc 840. aaaatcaaag aaattgccct gaaaaccggc aatagcttcg aactgattga tgcaccggat gcatttcata gtatgcatga agccgatccg cagcgttttg ttgatattct gaccagctgg 900 attgaacgtc tgaatctgca gtaa 924 <210> 25 <211> 966 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 9 <400> 25 atgggtatta gcgaagcagc agatcgtgca gatacctttg ttgcacataa atttgaagaa 60 cagctggttg atctgggtga aattcgtatg aattatgttg cagccggtga tccgaccagt 120 ccggcactgc tgctgattcc ggcacagggt gaaagttggt ggggttatga aaatgcaatt 180 accctgctgg caaatgattt tcgtgttttt gcaattgatc tgcgtggtca gggtcgtagc 240 acctggacac cgggtcgtta taatctgaat acctggggta atgatgtgga acgctttatt 300 gatctggtta ttggtcgtcc gaccctggtt gcaggtaata gcagcggtgg tgttattgca 360 gcatggctgg cagcctatgc aaaaccgggt cagattcgtg gtgcaatgct ggaagatccg 420 cctctgtttg caagccaggc agcaccgcct tatggtccgg gtattatgca gaccctgggt 480 ccgatttttg ttctgtgggc aaaatggctg ggtccgcagt ggtcagttgg tgattggggat 540 ggtatggttg cagcggcacc gcgtgaactg ccggaatttc tgcatccggg tatcgcattt 600 ctgtttggtg atggcaccgg tgaaggtgca gcagcaaccc ctccgcagca tctgaaagaa 660 tatgatccgg aatgggcaca ggcatgggca accgatgttg caaatgcagg ttgtgatcat 720 gcaaccatgc tggcacagaa tcgtgttccg gttctgctga cccatcattt tcatctgacc 780 gatccggata caggccagct gatgggtgca atgaccgata ttcaggcaca gcaggcacgt 840 cgtctgctgg cagcaaccgg tcagccggtt acctttaccg cactggatgc accgcatacc 900 atgcatgatc ctgaacctga acgttatatttt gaagttctga ccgaatgggc aagtgcactg 960 gattaa 966 <210> 26 <211> 960 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 10 <400> 26 atgggtcgtt atgccggtgt ttttggtccg catgcaccgg aaagcaccta tgttggtcat 60 gcatatccgg aacaactgtt tgataccggt gaagttcgtc tgaattatgc agttgccggt 120 gatgcaagcg caagtccgct gctgctgatt ccgggtcaga ccgaaagttg gtggggttat 180 gaaccggcaa tgggtctgct ggcagaacat tttcatgttc atgcagttga tctgcgtggt 240 cagggtcgta gcacccgtac accgcgtcgt tataccctgg ataatattgg taatgatctg 300 gtgcgttttc tggatggtgt tattggtcgt ccggcatttg ttagcggtct gagcagcggt 360 ggtctgctga gcgcatggct gagcgcctt gcagaaccgg gtcaggttct ggcagcatgt 420 tatgaagatc cgcctttttt tagcagcgaa ctggacccgg tgattggtcc gggtctgatg 480 agcaccgttg gtccgctgtt tgcactgtat gttaaatatc tgggtgatca gtggtcaatt 540 ggtgattggg atggttttgt tgcaggcgca ccgcaagaac tggcaggttg gcaggcacat 600 gttgcactgg caggcggtac agcagaaccg cctcagcatc tgaaagaata tgatccggaa 660 tggggtcgtg catttgttgg tggcaccttt accaccggtt gtccgcatca ggttatgctg 720 agccaggtta aagttccggt tctgtttacc catcattttc gtatgctgga tgatgaaagc 780 ggtagcctga ttggtgcagc aaccgatgat caggcagcac gtgttgttga actggttgaa 840 aatagtggtg caccgctgac ctatcgtagc tttccgatga tgggtcatag tatgcatgca 900 caagatccgg cactgtttgc aggcaccctg gttgattggt ttaccgcagc acgtagctaa 960 <210> 27 <211> 960 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 11 <400> 27 atgggtcgtt atgccggtgt ttttggtccg catgcaccgg aagcaaccta tgttgaacat 60 ggttatccgg aacgtctgtt tgataccggt gaagtgcagc tgaattatgt tgttgccggt 120 gatgcagcag caccgcctct gctgctgatt ccgggtcaga gcgaaagttg gtggggttat 180 gaagcagcaa ttccgctgct ggcacgtcat tttcatgttc atgcagttga tctgcgtggt 240 cagggtcgta gcaccgtac accgggtcgc tatacccctgg ataatgttgg taatgatctg 300 gtgcgttttc tggatggtgt tattggtcgt ccggcatttg ttagcggtct gagcagcggt 360 ggtctggcaa gcgcatggct gagcgcattt gcaaaaccgg gtcaggttgt tgcagcatgt 420 tgggaagatc cgcctttttt tagcagcgaa accgcaccga ttgttggtcc gcctattacc 480 gatagcattg gtccgctgtt tggtatgtgg gcacgttatc tgggtgatca gtggtcagtt 540 ggtgattggg atggttttgt tgccgcagtt ccgaccgaac tggcagattg gcaggcacat 600 gttgcactgg ttgttggcac cgcagatcct ccgcagaatc tgcgtgaata tgatccggaa 660 tggggtaaag catttattac cggcaccttt gcagcaagct gtccgcatca tgttatgctg 720 agcaaagtta aagttccggt tctgtatacc catcactttc gcatgattga tgaaggtagt 780 ggtggtctga ttggtgcatg tagcgatatt caggcaggtc gtgttaccca gctggcaaaa 840 tcaggtggtc gtagcgttac ctatcgtagc tttccgatga tggcacatag catgcatggt 900 caagatccgg cactgtttag cgaaaccctg gttgaatggt ttagccgttt taccggttaa 960 <210> 28 <211> 969 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 12 <400> 28 atgccgaaaa gcgaagcagc agatcgtgca gatagctttg ttagccatga tttcaaagaa 60 aacattgtgg atctgggcga aatccgcatg aattatgttg ttcagggcaa caaaaaaagt 120 ccggcactgc tgctgattcc ggcacagggt gaaagttggt ggggttatga agcagcaatt 180 ccgctgctgg caaaacattt tcaggttttt gcaattgatc tgcgtggtca gggtcgtacc 240 acctggacac cgggtcgtta taccctggat atttttggta atgatgtggt gcgctttatc 300 gatctggtta ttggtcgtga aaccctgatt gcaggtaata gcagcggtgg tctgattggt 360 gcatggctgg cagcatttgc aaaaccgggt caggttcgtg cagttatgct ggaagatccg 420 cctctgtttg caagcgaaat tcgtccgcct tatggtccgg gtatttggca gggtctgggt 480 ccgatgtttg cagcatgggc aaaatggctg ggtccgcagt ggtcaattgg tgattggggat 540 ggtatggtta aagcactgcc ggatgaactg ccggaagatc tgctgcctgg tattggtttt 600 atgctgggtg atggtgaaag tgatggtgca gcaccgaccc ctccgcagca tctgaaagaa 660 tatgatccgg aatggggtgc aagctgggca agcggttttg ccaataccgg ttgtgaacat 720 gaagcagtta ttagccaggt gcgtgttccg gttctgctga cccatcattt tcgtcagatt 780 aatgaagaaa ccggtcatct gatgggtgca ctgagcgatc tgcaggcagc acaggttcgt 840 catatcattg aagaagttgc aggtcaagag gttacctatg ttagcctgga tgcaccgcat 900 accatgcatg aaccgcagcc ggaacgttat accgatgttc tgctggattg ggttaaaaaa 960 ctgggttaa 969 <210> 29 <211> 987 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 13 <400> 29 atgaattatg caaccgcagg tagcagcgat aaaccggcac tgctgctggt tccgggtcag 60 agcgaaagtt ggtggggtta tgaaatggca atgtggctgc tgaaagatga ttatcaggtt 120 tttgcagttg atatgcgtgg tcagggtcag agtacctgga caccgggtcg ttatagcctg 180 gatacctttg gtaatgatct ggtgaaattc atcgatatcg tgattaaacg tccggttgtt 240 gttagcggtc tgagcagcgg tggtgttgtg agcgcatggc tgagcgcatt tgcaaaacct 300 ggtcagattc gtgcagcagt ttatgaagat ccgcctctgt ttgcaagcca gagcaaaccg 360 gcaattggtc agagtgttat gcagaccgtt gcaggtccgt tttttaacct gtggtataaa 420 tggctgggtg cacagtggac cattggtgat caggcaggta tggttgcagc aatgccgaaa 480 gaaattccgg catggattct gcagtatctg ggtaatacca ccagtggtcc gaccggtctg 540 gatctgacac tgaatgaata tgatccggaa tggggtcatg gttttgttag tggcaccgtt 600 gatgcaacct gtgatcatga agcaatgctg acccatgtta aagttccggt tctgtttacc 660 catcatagcc gtgcaattga tccgtatacc ggtaatctga ttggtagcgt tagcgatacc 720 caggttagct atgcacaggg tctgattacc accaatggca atcagagctt taccctgaaa 780 aactttccgc tggcaagcca tgatatgcat aattctgatc cggcaaccta tgttagcgca 840 attaccacct ggatggcaag cctgggtatt ggtagtgcag ttatccggg tccggttaaa 900 gttgcaagcg caagcgcaca ggttagcgca gcaagcaccg caccgcctag ctgtaccagc 960 accagcgcac cgagcaccgg tcattaa 987 <210> 30 <211> 843 <212> DNA <213> Artificial sequence <220> <223> ORFs are codon-optimized and therefore differ from naturally occurring DNA sequences. <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 14 <400> 30 atgaccgttg ttgatccgcc tgcaccgcgt gattttccgg aactgctggt tgatctgggt 60 gaagttgttc tgaatcatgc agaagcaggt agtccggatc gtccggcact ggttccggtg 120 ccggaacagg gtggtagttg gtggtcttat gaacgtgtta tgccgctgcc tgcacgcgat 180 tttcatgttt ttgcagttga tctgcgtggt cgtggtcgta gcacccgtac accgcgtcgt 240 tatagcctgg atgattttgg taatgatctg gttcgttttc tggccctggt tgttcgccgt 300 ccggcagttg ttgcaggtaa tagcagcggt ggtgttctgg cagcatggtc aagcgcctat 360 gcaatgcctg gtcaggttcg tgcagttctg ctggaagatc cgcctctgtt tagcagcgaa 420 ctgacaccgg tttgtggtcc gggtgttcgt caggcagcag gtccgctgtt tgaactgctg 480 agcacccatc tgggcgatca gtggggtggt ggtcgtccgg gtcgtgttca tggtggcgtt 540 ccgcgtctgg gtctggcagc cgcagcagca gttcgtgttg cacgtcgtgc agcagcaacc 600 gatgcacgtg gtcgccctgg tgcagcacgt ggacgtcctg ccggtgttgg tggtgcagct 660 cgtcgcggtc gcggtggtcg tgaacgcacc ggtacaacca ccgttctgag cggtctgacc 720 ggtagccgta ccagcggcac cggtcgttgt cgtaaaccgt ttcgtctgcg tcagtggtgg 780 gcaggcggtg cccgtggtcc tcctccgcct cgtcagattc gcgcagatgt tcgtacccgt 840 taa 843 <210> 31 <211> 981 <212> DNA <213> Artificial Sequence <220> <221> misc_feature <223> Artificial DNA sequence encoding a polypeptide having SEQ ID NO: 15 <400> 31 atgagcgttc cggttacccc gagcgcacgt aatgtttttg ttccgcatgc atttccagag 60 aaacaaattg atctgggtga agtggttctg aattatgcag aagcaggtac accggataaa 120 ccggcattac tgctgctgcc ggaacagacc ggtagttggt ggtcttatga accggcaatg 180 ggtctgctgg cagaacattt tcatgttttt gcagttgatc tgcgtggtca gggtcgtagc 240 acctggacac cgggtcgtta tagcctggat aattttggta atgatctggt gcgttttatt 300 gcactggcaa ttcgtcgtcc ggttgttgtt gcaggttgta gcagcggtgg tgttctggca 360 gcatggctga gcgcctatgc actgcctggt cagattcgtg gtgcactgtg tgaagatgca 420 ccgctgtttg caagcgaact gacaccggca catggtcatg gtgttcgtca gggtgcaggt 480 ccggtttttg aactgtatcg tgattatctg ggcgatcagt ggtcagttgg tgattgggca 540 ggtctggttg cagcagcaca ggcaagtccg gcaaaaatga tgagcctgtt taaaatgcct 600 ggtgaaccgc ctcagaatct gcgtgaatat gatccggaat gggcacgtgt tttttttgaa 660 ggcaccgttg gtctgcattg tccgcatgat cgtatgctga gccaggttaa aacaccggtt 720 ctgattaccc atcatgcacg taccaccgat ccggaaaccg gtgaatttct gggtgcactg 780 agcgaactgc aggcagaacg tgcacaggcc attattcgtg cagccggtgt tccggttgat 840 tatcagagct ttccggatgc agcacatgca atgcatacca cagaaccggc acgttatgca 900 gcagttctga ccgcatgggc agcaaaactg cctccggttg cagataccag cccgtcagca 960 gcagcaagcg cacatgttta a 981 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid Motif <220> <221> PEPTIDE <222> (1)..(7) <400> 32 Ala Gly Asn Ser Ser Gly Gly 1 5 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid Motif <220> <221> PEPTIDE <222> (1)..(7) <400> 33 Arg Thr Ile Asp Pro Glu Thr 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 34 Asp Ala Leu His Met Met His 1 5 <210> 35 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 35 Ala Gly Asp Ser Ser Gly Gly 1 5 <210> 36 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 36 Ala Gly Asp Ser Ser Leu Gly 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 37 Ala Gly Gln Ser Ser Gly Gly 1 5 <210> 38 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 38 Ala Gly His Ser Ser Gly Gly 1 5 <210> 39 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 39 Ala Gly Ser Ser Ser Ser Gly Gly 1 5 <210> 40 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 40 Ser Gly Asn Ser Ser Gly Gly 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 41 Ser Gly Asp Ser Ser Gly Gly 1 5 <210> 42 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 42 Ser Gly Gln Ser Ser Gly Gly 1 5 <210> 43 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 43 Ser Gly His Ser Ser Gly Gly 1 5 <210> 44 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 44 Ser Gly Ser Ser Ser Ser Gly Gly 1 5 <210> 45 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 45 Arg Thr Ile Asp Pro Glu Thr 1 5 <210> 46 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 46 Arg Asp Ile Asp Pro Asp Thr 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 47 Arg Gly Thr Asp Pro Glu Thr 1 5 <210> 48 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 48 Arg Gly Ile Asp Pro Glu Thr 1 5 <210> 49 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 49 Asp Ala Leu His Met Met His 1 5 <210> 50 <211> 7 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(7) <400> 50 Asp Ala Ser His Met Met His 1 5 <210> 51 <211> 11 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(11) <400> 51 Val Val Ala Gly Asn Ser Ser Gly Gly Leu Leu 1 5 10 <210> 52 <211> 11 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(11) <400> 52 Ile Val Ala Gly Asn Ser Ser Gly Gly Val Leu 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(11) <400> 53 His Ala Arg Thr Ile Asp Pro Glu Thr Gly Glu 1 5 10 <210> 54 <211> 11 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(11) <400> 54 His Met Arg Asp Ile Asp Pro Asp Thr Gly His 1 5 10 <210> 55 <211> 11 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(11) <400> 55 His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn 1 5 10 <210> 56 <211> 11 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(11) <400> 56 His Pro Asp Ala Leu His Met Met His Leu Phe 1 5 10 <210> 57 <211> 11 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(11) <400> 57 Val Pro Asp Ala Leu His Met Met His Gln Phe 1 5 10 <210> 58 <211> 11 <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(11) <400> 58 Val Pro Asp Ala Ser His Met Met His Gln Ser 1 5 10 <210> 59 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 59 Ile Lys Arg Pro Val Val Val Ala Gly Asn Ser Ser Gly Gly Leu Leu 1 5 10 15 Ala Ala Trp Leu Ser 20 <210> 60 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 60 Val Lys Arg Pro Val Ile Val Ala Gly Asn Ser Ser Gly Gly Val Leu 1 5 10 15 Ala Ala Trp Leu Ser 20 <210> 61 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 61 Val Arg Arg Pro Val Val Val Ala Gly Asn Ser Ser Gly Gly Val Leu 1 5 10 15 Ala Ala Trp Leu Ser 20 <210> 62 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 62 Val Lys Arg Pro Val Val Val Ala Gly Asn Ser Ser Gly Gly Val Leu 1 5 10 15 Ala Ala Trp Leu Ser 20 <210> 63 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 63 Ile Leu Ile Thr His His Ala Arg Thr Ile Asp Pro Glu Thr Gly Glu 1 5 10 15 Leu Leu Gly Ala Leu 20 <210> 64 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 64 Val Leu Leu Thr His His Met Arg Asp Ile Asp Pro Asp Thr Gly His 1 5 10 15 Leu Val Gly Ala Leu 20 <210> 65 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 65 Val Leu Leu Thr His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn 1 5 10 15 Leu Leu Gly Ala Leu 20 <210> 66 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 66 Val Leu Leu Thr His His Pro Asp Ala Leu His Met Met His Leu Phe 1 5 10 15 Leu Leu Gly Ala Leu 20 <210> 67 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 67 Val Asp Tyr Gln Ser His Pro Asp Ala Leu His Met Met His Leu Phe 1 5 10 15 Asp Pro Ala Arg Tyr 20 <210> 68 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> amino acid motif <220> <221> PEPTIDE <222> (1)..(21) <400> 68 Val Asp Tyr Ala Ser Val Pro Asp Ala Leu His Met Met His Gln Phe 1 5 10 15 Asp Pro Pro Arg Tyr 20 <210> 69 <211> twenty one <212> PRT <213> Artificial sequence <220> <221> PEPTIDE <222> (1)..(21) <400> 69 Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met His Gln Ser 1 5 10 15 Ala Pro Ala Arg Tyr 20

Claims

1. A polypeptide cleaved by hydrolysis of zearalenone and / or at least one zearalenone derivative, wherein the zearalenone derivative is selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S, and ZAN, characterized in that, The polypeptide is a hydrolase composed of the amino acid sequence of SEQ ID No.

13.

2. An isolated polynucleotide whose nucleotide sequence encodes a polypeptide, wherein the polypeptide has the property of hydrolyzing zearalenone and / or at least one zearalenone derivative, said zearalenone derivative being selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S, and ZAN, characterized in that, The nucleotide sequence encodes the polypeptide according to claim 1.

3. An additive for use in feed for pigs, poultry, or aquaculture, produced by hydrolyzing zearalenone and / or at least one zearalenone derivative, said additive for addition to food or dried distillers grains, said zearalenone derivative being selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S, and ZAN, characterized in that... The additive comprises the polypeptide according to claim 1.

4. The additive according to claim 3, characterized in that, The additive further includes auxiliary agents.

5. The additive according to claim 4, characterized in that, The auxiliary agent is at least one inert carrier.

6. The additive according to claim 4, characterized in that, The adjuvant is at least one inert carrier, and other components, wherein the other components are vitamins and / or minerals and / or enzymes and / or other components for detoxifying fungal toxins.

7. The additive according to claim 3, characterized in that, The additive contains the polypeptide according to claim 1 at a concentration of up to 10,000 U / g.

8. The additive according to claim 3, characterized in that, The additive contains the polypeptide according to claim 1 at a concentration of up to 1,000 U / g.

9. The additive according to claim 3, characterized in that, The additive contains the polypeptide according to claim 1 at a concentration of up to 100 U / g.

10. The additive according to claim 3, characterized in that, The additive contains the polypeptide according to claim 1 at a concentration of up to 10 U / g.

11. The additive according to claim 3 or 4, characterized in that, The additive is present in the form of encapsulation or coating.

12. Use of the polypeptide of claim 1 for hydrolyzing zearalenone and / or at least one zearalenone derivative in feed, food or dry distillers grains, said zearalenone derivative being selected from α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN.

13. The use according to claim 12, wherein the feed is for use in pig, poultry and aquaculture.

14. A method for hydrolyzing zearalenone and / or at least one zearalenone derivative, wherein the zearalenone derivative is 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 are hydrolyzed by the polypeptide according to claim 1.

15. The method according to claim 14, characterized in that, The polypeptide is used in an additive according to any one of claims 3 to 11.

16. The method according to claim 15, characterized in that, The polypeptide or additive is mixed 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 is hydrolyzed to contain zearalenone and / or at least one zearalenone derivative in the contaminated feed or food.

17. The method according to any one of claims 14 to 16, characterized in that, At least 70% of the zearalenone and / or at least one zearalenone derivative is hydrolyzed.

18. The method according to any one of claims 14 to 16, characterized in that, At least 80% of the zearalenone and / or at least one zearalenone derivative is hydrolyzed.

19. The method according to any one of claims 14 to 16, characterized in that, At least 90% of the zearalenone and / or at least one zearalenone derivative is hydrolyzed.

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