Polypeptide for hydrolytic cleavage of zearalenone and / or zearalenone derivatives, isolated polynu

专利摘要:
A polypeptide for the hydrolytic cleavage of zearalenone and / or zearalenone derivatives contains an amino acid sequence which has a degree of identity to amino acid sequences +24 to +50, and / or +52 to +77, and / or +79 to +87, and / or +89 to +145, and / or +150 to +171, and / or +177 to +193, and / or +223 to +228, and / or +230 to +237, and / or +239 to + 247, and / or +249 to +255, and / or +257 to +261, and / or +263 to +270, and / or +272 to +279, and / or +297 to +301, and / or +303 to +313, and / or +24 to 328, and / or +1 to +328 of a sequence SEQ. ID. NO: 1 of at least 70%, or a variant thereof, or a functional fragment thereof, or a variant of having functional fragment, and an additive containing the polypeptide and a polynucleotide.
公开号:AT514775A1
申请号:T667/2013
申请日:2013-08-28
公开日:2015-03-15
发明作者:
申请人:Erber Ag；
IPC主号:

专利说明:

The present invention relates to a polypeptide for the hydrolytic cleavage of zearalenone and / or zearalenone derivatives, an isolated polynucleotide encoding such a polypeptide, and an additive containing such a polypeptide.
Mycotoxins are secondary metabolites produced by filamentous fungi. An important member of the same is the world wide spread zearalenone (ZEN), formerly known as F-2 toxin produced by a variety of Fusar / en fungi. These fungi infect among other crop plants, such as different types of cereals, with the fungus infestation usually occurring before harvest, whereby the fungus growth or the mycotoxin production can take place before and / or if not stored properly even after the harvest. The Food and Agriculture Organization (FAO) estimates that 25% of agrarian products worldwide are contaminated with mycotoxins, leading to significant economic losses. In a more recent world-wide study by I. Rodrigues and K. Naehrer, Toxins, 2012, 4, 663-675, a total of 23,781 samples were analyzed over the period from January 2009 to December 2011, with 81% positive for at least one mycotoxin and 45% positive for ZEN were tested. ZEN could be found in all regions of the world as well as in all cereal and feed classes tested, such as maize, soybean meal, wheat, wheat bran, DDGS and in ready meal mixtures with a frequency of up to 100%. ZEN is a non-steroid, estrogen, macrocyclic lactone with the following structural formula:
ZEN is synthesized via the polyketide pathway and, following IUPAC, as (2E, 11S) -15,17-dihydroxy-11-methyl-12-oxabicyclo [12.4.0] octadeca-1 (18), 2,14,16- tetraene-7,13-dione.
In nature, however, also occur a variety of ZEN derivatives, which can be caused by enzymatic or chemical modifications of ZEN. Examples of these are glycosidic or sulphate-containing ZEN conjugates which can be formed by fungi, plants or the mammalian metabolism, as well as ZEN metabolites, which are formed inter alia in the human or animal organism. In the following, ZEN derivatives are considered to be naturally occurring or produced by chemical or biochemical synthesis, ZEN conjugates or ZEN metabolites, but especially a-zearalenol (α-ZEL; (2E, 7R, 11S) -7,15,17-trihydroxy-11 methyl 12-oxabicyclo [12.4.0] octadeca-1 (18), 2,14,16-tetraen-13-one), β-zearalenol (β-ZEL; (2E, 7S, 11S) -7, 15,17-trihydroxy-11-methyl-12-oxabicyclo [12.4.0] octadeca-1 (18), 2,14,16-tetraen-13-one), α-zearalanol (a-ZAL; (7R, 11 S) -7,15,17-trihydroxy-11-methyl-12-oxabicyclo [12.4.0] octadeca1 (18), 14,1,16-trien-13-one), β-zearalanol (β-ZAL; (7S, 11S) -7,15,17-trihydroxy-11-methyl-12-oxabicyclo [12.4.0] octadeca-1 (14), 15,17-trien-13-one), zearalenone-14-sulfate (Z14S; (2 E, 11 S) -15-hydroxy-11-methyl-7,13-dioxo-12-oxabicyclo [12.4.0] octadeca-1 (18), 2,14,16-tetraen-17-yl] -hydrogen sulfate ), Zearalenone-14-glycoside (Z14G; (2E, 11S) -15-hydroxy-11-methyl-17 - [(3R, 4S, 5S, 6R) -3,4,5-trihydroxy-6- (hy- droxymethyl) tetrahydropyran-2-yl] oxy-12-oxabicyclo [12.4.0] octadeca-1 (18) 2.14 , 16-tetraene-7,13-dione), as well as zearalanone (ZAN; (11S) -15,17-dihydroxy-11-methyl-12-oxa-bicyclo [12.4.0] octadeca-1 (18), 14,16-triene-7,13-dione). Due to its high chemical and physical stability, ZEN can also be used in processed foodstuffs or feedstuffs such as e.g. Bread or beer can be detected. However, ZEN derivatives, in particular α-ZEL, β-ZEL, Z14S, α-ZAL, β-ZAL, Z14G and ZAN, could also be detected in a large number of tested feed and food samples in addition to ZEN. ZEN binds to the estrogen receptor and can cause hormonal imbalances. ZEN is absorbed immediately after oral intake and converted by mammals into the two stereoisomeric metabolites α-ZEL and β-ZEL, with α-ZEL, but also α-ZAL or ZAN, having a much stronger estrogenic activity than ZEN. Conjugated ZEN derivatives sometimes have a lower estrogenicity than ZEN, but may possibly be released from these ZEN in the digestive tract again.
Although ZEN has relatively low acute toxicity and an oral LD50 of up to 20,000 mg / kg of body weight, prolonged ingestion may result in subacute and / or subchronic toxic effects such as teratogenic, carcinogenic, estrogenic and immunosuppressive effects in animals or humans. ZEN-contaminated feedstuff leads to developmental disorders in mammals, with pigs, and especially juveniles, being extremely sensitive to ZEN. ZEN concentrations in feed greater than 0.5 ppm lead to developmental disorders, e.g. Concentrations of over 1.5 ppm can lead to hyper-estrogenicity in pigs and concentrations of 12 ppm ZEN have been blamed for cattle birth defects. Since zearalenone is rapidly absorbed via the mucous membranes, in particular via the stomach but also via the oral mucosa, an immediate and above all quantitative deactivation is necessary. Just 30 minutes after oral administration of ZEN, this can be detected in the blood. Here, the use of isolated enzymes to microorganisms has advantages such as a higher specific activity or a faster effect. Due to the demonstrated harmful effects of ZEN, binding ZEN food limits and recommendations for ZEN caps in feed have been agreed in the European Union (EC NO: 1881/2006).
The primary strategy to reduce ZEN contamination of food or feed is to limit fungal growth, for example, by complying with "Good Agricultural Practice". This includes, among other things, that seed is free from SchädlĤgen and fungal infestation, or that agricultural waste products are removed in time from the field. Furthermore, the use of fungicides can reduce fungal growth on the field. After harvesting, the crop should be stored at a residual moisture level below 15% and low temperature to prevent fungal growth. Similarly, contaminated material should be removed prior to further processing by fungal infestation. Despite these measures, I. Rodriges and K. Naehrer (2012) reported that even in regions with the highest agricultural standards, such as the US and Central Europe, between 2009 and 2011, 29% and 39% of the tested corn samples contaminated with ZEN were.
Further possibilities for removing ZEN from contaminated feed or foodstuffs are the adsorption or transformation of the mycotoxin. For this, it is necessary that the binding of the mycotoxin to the adsorbent be strong and specific over a wide pH range and remain stable throughout the digestive process in the gastrointestinal region. Although some non-biological adsorbents, such as e.g. Activated charcoal, silicates or synthetic polymers, such as cholestryamine, can be used efficiently for aflatoxins, their use is limited to other mycotoxins. The major disadvantage of the adsorbents is the non-specific binding of other molecules that are in part essential for nutrition. Biological adsorbents, such as e.g. Yeast or yeast extracts are likewise described in the literature, but have similar limitations as non-biological adsorption agents.
The detoxification of ZEN by physical and chemical treatments is also limited. By thermal treatment, ZEN can not be effectively deactivated, however, the ZEN content can be reduced by 83.9% by extrusion and treatment with oxidizing agents, for example for 16 hours at 80 ° C with 10% hydrogen peroxide solution. The use of extrusion and oxidation agents, such as ozone or hydrogen peroxide in feed and food production is limited mainly because of the high costs incurred, the quality loss, the sometimes low efficiency and low specificity.
Also, the biotransformation of ZEN by microorganisms, e.g. Trichosporon mycotoxinivorans, Gliocladium roseum or Bacillus subtilis strains or enzymes isolated therefrom, such as hydrolases or peroxidases, are described, for example, in E. Vekiru et al., In Appl. andEnviron. Microb., 2010, 76, 7, 2353-2359.
ZEN degrading properties of bacteria of the genus Rhodococcus and Nocardia, in particular R. globerulus, R. erythropolis and N. globerulabekannt have become known from EP 0 938 575 B1.
WO 02/076205 discloses the ZEN-degrading action of enzymes isolated from Gliocladium roseum, inter alia the α / β-hydrolase, zearalenone hydrolase 1 (ZHD1), which catalyze the degradation of ZEN by means of a catalytic triad.
WO 2012/113827 discloses recombinant zonases, namely ZEN-degrading enzymes, which remain stable in the gastrointestinal tract, in particular microorganisms such as Thermobifidia fusca, Streptomyces exfoliatus, Acidovorans delafieldii and Streptomyces sp , described.
The terms used in the following are taken from the technical language and are each, unless otherwise stated, used in the conventional meanings. Thus, the term "polynucleotide" refers to any type of genetic material, all lengths and all sequences, such as e.g. Single-stranded and double-stranded DNA and RNA molecules, including regulatory elements, structural elements, groups of genes, plasmids, entire genomes and fragments thereof. The term "polypeptide" includes proteins such as enzymes, antibodies and polypeptides of up to 500 amino acids, such as peptide inhibitors, domains of proteins, but also short polypeptides of small sequence lengths, e.g. less than 10 amino acids, such as receptors, ligands, peptide hormones, tags and the like. The term "position" in a polynucleotide or polypeptide refers to single, specific bases or amino acids in the sequence of the polynucleotide or polypeptide.
The present invention now aims to provide a polypeptide with which it is possible to transform ZEN, and / or ZEN derivatives quickly and reliably into hydrolyzed ZEN and / or into hydrolyzed ZEN derivatives.
To achieve this object, the present invention is essentially characterized in that the polypeptide has an amino acid sequence which has a degree of sequence identity to amino acid sequences +24 to +50, and / or +52 to +77, and / or + 79 to + 87, and / or +89 to +145, and / or +150 to +171, and / or +177 to +193, and / or +223 to +228, and / or +230 to +237, and and / or +239 to +247, and / or +249 to +255, and / or +257 to +261, and / or +263 to +270, and / or +272 to +279, and / or +297 to + 301, and / or +303 to +313, and / or +24 to 328, and / or +1 to +328 of a sequence having the SEQ ID NO: 1 of at least 70%, preferably at least 84%, most preferably we¬ at least 92%, and more preferably at least 98%, or a variant thereof, or a functional fragment thereof, or a variant of the functional fragment.
The term "sequence identity" according to the present invention refers to a percent sequence identity. For amino acid sequences, the identity visu¬ell, but preferably be calculated using a computer program. As a reference sequence, the amino acid sequence is defined by SEQ ID NO: 1. The sequence comparison is also carried out within sequence sections, wherein a section is to be understood as a continuous sequence of the reference sequence and preferably encompasses a conserved region of the sequence.
The terms "polypeptide variant" or "variant" as used herein refer, on the one hand, to "allelic variants" of the polypeptide and, on the other hand, to "modification" of the parental polypeptide, the enzymatic function being essentially unchanged. The term "allelic variant" refers to a polypeptide which has been formed by naturally occurring mutation (s) of the nucleotide sequence and causes a change in the amino acid sequence, the enzymatic function of which is unaffected thereby. "Modifications" may be exemplified by C or N-terminal fusions to polypeptides or mutated polypeptides, where mutations are obtained by substitution, insertion or deletion of at least one amino acid, in particular by site-directed mutagenesis, recombination and / or any other protein engineering method be able to. The terms substitution, insertion and deletion are used here in genetic engineering and mean familiar to the person skilled in the art.
The term "functional fragment" as used herein refers to a part or a partial sequence of a polypeptide or a part or a partial sequence of a variant thereof, wherein the enzymatic function is substantially retained.
By choosing such an amino acid sequence, a functional fragment thereof, a variant thereof or a functional fragment of a variant, a surprisingly rapid and complete hydrolysis of ZEN and / or ZEN derivatives was found, with particularly high activity values in comparison to previously known ZEN degrading polypeptides could be detected.
Consistently good results could be achieved if, as is the case with a further development of the invention, the polypeptide variant has at least one amino acid modification selected from the group substitution, deletion and insertion of one or more amino acids.
By further developing the invention such that the polypeptide has a specific activity of at least 0.01 U / mg, preferably at least 0.1 U / mg, especially at least 1 U / mg; and / or a KM value of the hydrolytic cleavage of ZEN of at most 50 μΜ, preferably at most 3.5 μΜ, in particular at most 0.5 μΜ; and / or a kcar value of the hydrolytic cleavage of ZEN of at least 0.05 s -1, preferably at least 0.6 s -1, in particular at least 5 s -1; and / or has a vmax value of the hydrolytic cleavage of ZEN of at least 0.00001 μΜ.sup.-1, preferably at least 0.0001 μΜ.s -1, preferably at least 0.001 μΜ.s -1, ZEN and / or ZEN Derivatives particularly quickly and completely hydrolyzed, in particular detoxified.
According to one embodiment of the invention, the polypeptide is selected such that it is an α / β-hydrolase suitable for oxygen-independent and cofactor-free hydrolytic cleavage of the ester grouping of zearalenone and / or the ZEN derivatives, that it contains an amino acid triad catalyzing the hydrolytic cleavage, consisting of serine, an acidic amino acid selected from glutamic acid and aspartic acid, in particular aspartic acid and histidine, and in that the catalytic triad is S128, D264 and H303, wherein the positioning relative to the sequence is represented by SEQ ID NO: 1.
Hydrolysis of ZEN and / or ZEN derivatives occurs on the ester group of the zearalenone or its derivatives according to the following reaction mechanism:
The hydrolysis of ZEN to non-toxic hydrolysed zearalenone (HZEN) or hydrolyzed ZEN derivatives is effected by the polypeptides according to the invention, in particular the α / β-hydroals. The further decarboxylation of HZEN to decarboxylated, hydrolyzed ZEN (DHZEN) or decarboxylated, hydrolyzed ZEN derivatives is usually spontaneous.
In particular, it is possible by means of the catalytic triad to completely hydrolyze ZEN and / or ZEN derivatives, the degradation reaction having a good stability with respect to the pH, in particular pH values in the acidic range. Surprisingly, it has been found that it is possible, as is the case for a further development of the invention, with a polypeptide which consists of a sequence segment consisting of 3 amino acids before and 3 amino acids after the serine of the catalytic triad, 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 contains consistently good results and moreover at least one enzyme kinetic parameter to improve.
In a preferred embodiment of the invention, the polypeptide has at least one mutation of the amino acid sequence with respect to SEQ ID NO: 1 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 on. These positions result from the sequence differences of the polypeptide of SEQ ID NO: 1 and the high level of identity and particularly active polypeptides having that sequence with the sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 SEQ ID NO: 5 and SEQ ID NO: 6. By altering the polypeptide of SEQ ID NO: 1 at at least one of these positions such that the amino acid variants of the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO 4, SEQ ID NO: 5 and SEQ ID NO: 6, it is possible to show that these positions have a significant influence on the enzyme kinetic parameters of the polypeptide and, furthermore, that combinations of the sequence with SEQ ID NO: 1 with the sequences having a high degree of sequence identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 lead to higher activities.
According to one embodiment of the invention, the polypeptide has at least one mutation of the amino acid sequence with respect to SEQ ID NO: 1 selected from: 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, 1118V, K119R, V123I, L132V, A141S, 1146V, I146L, A148G, A149V, A154P, P163T, A164T, Y165C, Y165H, V169I, L170R, A172G, A176M, A176V, Y180F, D182T, F183Y, 1190V, G191S, K194T, K194E, F196Y, V197C, V197R, E198R, E198S, K201D, K201G, P204S, P204A, A205S, K206P, A207M, M208A, Q209R, L210A, L210S, ΔΡ212, 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 it is possible to completely hydrolyze ZEN within a short time, in particular to detoxify it, the specific activity of the polypeptide being at least 6.00 U / mg, preferably at least 7.00 U / mg, in particular at least 8.00 U / mg. The unit "U" or "unit" is a measure of the absolute catalytic activity and is defined by the hydrolysis of 1 μΜΜΙ ZEN per minute at 32 ° C in 50 mM Tris-HCl buffer (pH 8.2), wherein - "Catalytic activity" is understood to mean the enzymatic conversion of a substrate under defined reaction conditions and "specific activity" is understood to mean the ratio of the catalytic activity and the polypeptide mass concentration (mass per volume unit).
In that the polypeptide is designed such that at least one of the following amino acid motifs having the sequence having the SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO : 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, successful polypeptides having a specific activity of at least 7, 00U / mg, preferably at least 8.00 U / mg. Surprisingly, it has been found that when at least one of the following amino acid motifs having the sequence having SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, the enzymatic activity of the polypeptide, for example, compared to a motif containing 7 amino acids is further increased. An even higher specific activity is achieved if at least one of the following amino acid motifs having the sequence having the SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67.
According to one development of the invention, the polypeptide contains at least one amino acid sequence selected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 or at least one ZEN and / or a ZEN derivatives hydrolyzing variant thereof or at least one functional fragment thereof. Not only do these polypeptides show high specific activity in the hydrolysis, particularly detoxification of ZEN and / or ZEN derivatives, but they are also capable of activity in a wide range of pH, e.g. pH 4 to 9 and temperature range between 15 and 65 ° C to maintain. Surprisingly, it has been found that the polypeptides having the sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 to each other to a high degree of degrees Have sequence identity and have at least as high specific Aktivi¬ tty as the polypeptide having SEQ ID NO: 1.
According to a development of the invention, the polypeptide contains at least one conservative amino acid substitution in at least one position, and the conservative amino acid substitution is selected from substitutions of 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, Μ, 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 to 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 Czu S, T, A; or N to D, Q, H, S; or Q to E, N, Η, K, R. Where the term conservative amino acid substitution is to be understood as meaning the substitution of amino acids by other amino acids which are considered conservative by one skilled in the art, that is, possessing similar specific properties , Such specific properties are, for example, their size, polarity, hydrophobicity, charge or pKs. Therefore, a conservative mutation is understood as meaning, for example, a substitution of one acidic amino acid for another acidic amino acid, one basic amino acid for another basic amino acid, one polar for another polar amino acid, and the like.
With such conservative amino acid substitutions it is possible to prepare polypeptide variants whose specific activity is approximately the same in comparison to the parent polypeptide, but can preferably be increased by at least 0.1 U / mg.
The present invention further aims to provide an isolated polynucleotide with which it is possible to produce a polypeptide for the rapid and reliable hydrolysis of ZEN and / or ZEN derivatives.
To achieve this object, the invention is characterized in that the nucleotide sequence codes for a polypeptide having a zearalenone and / or zearalenone derivative hydrolyzing property and encodes at least one polypeptide according to any one of claims 1 to 10 and / or a degree of sequence identity to select at least one nucleotide sequence from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 has at least 70% and / or under moderate stringency conditions with at least one nucleotide sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO : 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 and / or with a partial sequence of m at least 200 nucleotides, in particular of at least 100 nucleotides thereof and / or hybridized with a complementary strand of the nucleotide sequence or partial sequence thereof.
Nucleotide sequences to be expressed, in particular their triplets, are generally changed, in particular optimized, according to the host cell. This results in the fact that also polynucleotides with a degree of sequence identity well below 80%, but also below 70% or below 60% can encode one and the same polypeptide. The sequence comparison for the determination of the degree of identity must also be carried out within sequence sections, a section being understood as a continuous sequence of the reference sequence. The length of the sequence sections for nucleotide sequences is normally 15 to 600.
With the aid of the stated nucleotide sequences or sequence sections it is possible to generate nucleic acid probes which have a length of generally at least 15, 30 or 40 nucleotides. With the help of such probes, which are typically additionally marked, e.g. by means of 3H, 32P, 35S, biotin or avidin, nucleotide sequences coding for polypeptides having ZEN and / or ZEN derivatives degrading action can be identified using standard methods. As starting material for the identification of such sequences, for example, DNA, RNA or cDNA of single microorganisms, genomic DNA libraries or cDNA libraries may be used.
The present invention further aims to provide an additive capable of rapid and reliable hydrolytic cleavage of ZEN and / or ZEN derivatives in a defined or complex matrix, such as in feed or food.
To achieve this object, a polypeptide according to the invention capable of hydrolytic cleavage of zearalenone and / or zearalenone derivatives is used. With such an additive, the biochemical conversion of ZEN and / or ZEN derivatives to hydrolyzed ZEN and / or hydrolyzed ZEN derivatives is successful, and this can be used, for example, for the stereoselective hydrolysis of ZEN and / or ZEN derivatives in the industry.
In a preferred embodiment of the invention, the additive is formed such that additionally at least one inert carrier and optionally further constituents, such as vitamins and / or minerals and / or enzymes and / or further components for detoxification of mycotoxins are contained. By using such an additive it is possible, for example, to ensure in feedstuffs or foods that the amounts of ZEN and / or ZEN derivatives optionally retained are hydrolyzed, in particular detoxified, to the extent necessary, which has a detrimental effect the organism of the subject receiving this food or feed is absent.
In this case, a polypeptide according to the invention can also be present in an enzyme preparation which, in addition to at least one polypeptide according to the invention, additionally contains at least one enzyme which is involved, for example, in the degradation of proteins, e.g. Proteases, or that is involved in the metabolism of starch or fiber or fat or glycogen, e.g. Amy¬lase, cellulase or glucanase, and for example, hydrolases, lipolytic enzymes, man-nosidases, oxidases, oxidoreductases, phytases, xylanases and / or combinations thereof.
Further areas of use of the invention are enzyme preparations which, in addition to at least one polypeptide according to the invention, additionally contain at least one component for detoxifying mycotoxins, such as an enzyme which reduces mycotoxin, such as alfa toxin oxidase, ergotamine hydrolases, ergotamine amidases, zearalenone esterases, zearalenone lactonases, Ochratoxin amidases, fumonisin carboxylesterases, fumonisin aminotransferases, aminopolyol amine oxidases, deoxynivalenol epoxide hydrolases; and / or at least one mycotoxin-degrading microorganism such as Bacillus subtilis; and / or at least one mycotoxin-binding component, for example microbial cell walls or inorganic materials, such as bentonites.
According to a particularly preferred development of the invention, the polypeptide in the additive is present in a concentration of at most 10,000 U / g, preferably at most 1000 U / g, particularly preferably at most 100 U / g and in particular preferably at most 10 U / g, whereby it is possible to ZEN and / or ZEN derivatives rapidly and in particular before absorption by the body of the subject eating this food or food, in non-or less toxic metabolites, in particular HZEN and DHZEN umzuw-.
According to a development of the invention, the polypeptide is present in encapsulated or coated form, standard methods for encapsulation or coating, such as e.g. in WO92 / 12645, can be used. By encapsulating or coating, it is desirable to transport the polypeptide to its site of application without alteration, in particular without degradation and damage, so that the polypeptide begins to act only after dissolution of the protective envelope, for example in the digestive tract of animals an even more targeted, faster and complete degradation of ZEN and / or ZEN derivatives can also be achieved in an acidic, high-yielding and anaerobic environment.
The invention will be explained below with reference to exemplary embodiments and drawings closer. In these shows:
FIG. 1 shows the degradation of ZEN over time and the increase in the metabolites HZEN and DHZEN by the polypeptide of SEQ ID NO: 1, wherein in FIG. 1A the polypeptide is not tagged, in FIG. 1B, the polypeptide has a C-terminal 6xHis tag and in Fig. 1C an N-terminal 6xHisTag.
FIG. 2 shows the Michaelis-Menten kinetics of the polypeptide with SEQ ID NO: 1. FIG.
Example 1: Modification, Cloning and Expression of Polynucleotides Coding for Poly Peptides with ZEN and / or ZEN Derivatives Hydrolyzing Effect
Amino acid substitutions, insertions or deletions were performed by mutation of the nucleotide sequences by PCR using the "Quick-change Site-directed Mutagenesis Kit" (Stratagene) as instructed. Alternatively, complete nucleotide sequences were also obtained (GeneArt). The nucleotide sequences generated by PCR mutagenesis or by gene species optionally additionally contained a C- or N-terminal 6xHis tag at the amino acid level and were integrated by standard methods into expression vectors for expression in Escherichia coli or Pichia pastoris, in E. coli or P. .pastoris, as well as expressed in E. coli or P. pastoris (JM Cregg, Pichia Protocols, Second Edition, ISBN 10: 1588294293, 2007, J. Sambrook et al., 2012, Molecular Cloning, A La Boratory Manual 4th Edition , Cold Spring Harbor), which can be used for this task, any other suitable host cell.
The term "expression vector" refers to a DNA construct that is capable of expressing a gene in vivo or in vitro. In particular, these are understood to mean DNA constructs which are suitable for transferring the nucleotide sequence coding for the polypeptide into the host cell in order to integrate into the genome or to exist freely in the extrachromosomal space and to express the nucleotide sequence coding for the polypeptide intracellularly and optionally from the To remove cells.
The term "host cell" refers to all cells which either contain a nucleotide sequence to be expressed or an expression vector and are capable of producing an enzyme according to the invention. In particular, these are understood as meaning prokaryotic and / or eukaryotic cells, preferably Pichia pastoris, Eschichia coli, Bacillus subtilis, Streptomyces, Hansenula, Trichoderma, Lactobacillus, Aspergillus, plant cells and / or spores of Bacillus, Trichoderma or Aspergillus.
The soluble cell lysate in the case of E. coli or the culture supernatant in the case of P. pastoris were used to determine the catalytic properties of the polypeptides. To determine the KM value, of vmax, kcat and the specific activity, the polypeptides were enriched by standard methods by chromatography over nickel-Sepharoseseulen selek¬tiv. The protein concentration was determined by standard methods, either by the BCA method (Pierce BCA Protein Assay Kit # 23225), but preferably photometrically with the specific extinction coefficients for the respective proteins, using the "ProtParam" program, available online at http: //web.expasv .org / DrotDaram (Gasteiger E. et al., Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press, 2005, pp. 571-607).
Example 2: Determination of Sequence Identity and Conserved Regions
To determine the conserved regions of the polypeptides having SEQ ID NO: 1SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, these sequences have sequence identity of at least 70% to each other using the software COBALT (JS Papadopoulos and R. Agarwala, 2007, COBALT: constraint-based alignment tool for multiple protein sequences, Bioinformatics 23: 1073-79), using the standard parameters, in particular the parameters ("gap penalties ": -11, -1;" End-GapPenalties ": -5, -1;" Use RPS BLAST ": on;" Blast E-value ": 0,003;" Find Conserved columns andRecompute ": on;" use query clusters ": On;" word size ": 4;" may cluster distance ": 0.8;" alphabet ": regular;" Homology conversation setting ": 3 bits). The result of this analysis is the conserved amino acids. As conserved regions, the following regions were defined of at least 5 consecutive conserved amino acids, namely, the sequence having SEQ ID NO: 1 region A from +24 to +50, B from +52 to + 77, C from + 79 to +87, D from +89 to +145, E from +150 to +171, F from +177 to +193, G from +223 to +228, H from +230 to +237, I from +239 to +247, J from +249 to +255, K from +257 to +261, L from +263 to +270, M from +272 to +279, N from +297 to +301, and O from + 303 to +313.
The determination of the percent sequence identity over the entire polypeptide relative to SEQ ID NO: 1 (Table 2, column 2) was made using the program BLAST (Basic Local Alignment Search Tool), in particular BLASTP, which is available on the homepage of the National Center for Biotechnology Information "(NCBI; http://www.ncbi.nlm.nih.gov/) can be used. The basic settings were used as program settings, but in particular: "max target sequence" = 100; "Expected treshold" = 10; "Word size" = 3; "matrix" = BLOSOM62; "Gap costs" = "Existence: 11; Extention: 1 "; "Computational adjustment" = "Conditional compositional score matrix adjustment
The determination of the percent sequence identity of the conserved regions of the individual sequences relative to the conserved region of the sequence having SEQ ID NO: 1 (Table 1 Column 3 and Column 4) was carried out, as described above, by means of the COBALT program.
Table 1: Percent sequence identity over the entire polypeptide length and from the two selected conserved regions D and G.
Example 3: Hydrolysis of ZEN by polypeptides in cell lysates
In order to determine their ability to degrade ZEN into the non-or less toxic metabolites, HZEN and DHZEN, the polypeptide of SEQ ID NO: 1 encoded by the nucleotide sequence of SEQ ID NO: 15, as such, and of C and N, respectively, was determined terminal 6xHis tag in E. coli as described in Example 1. In each case 100 ml of an E. coli culture 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 microorganisms medium number 462.2012). The cell suspension was lysed by treating FrenshPress 3 times at 20,000 psi. The cell lysate thus obtained was used in a 1:10, a 1: 100 or a 1: 1000 dilution prepared in Brunner mineral medium including 0.1 mg / ml BSA (bovine serum albumin). For a ZEN degradation experiment, 9.9 ml of mineral mineral medium including 0.1 mg / ml BSA, 0.1 ml diluted cell lysate and 31 μl ZEN substrate strand solution were used. In total, the cell lysates were thus diluted 1: 1000, 1: 10,000 and 1: 100,000, respectively. The ZEN substrate stock solution was a 2.08 mM ZEN solution (40% by volume ACN + 60% by volume H 2 O). For the preparation of this solution, ZEN was weighed and dissolved in crystalline form (Biopu¬re Standard from Römer Labs, Art. No. 001109, purity at least 98%). Each digestion batch was carried out in 25 ml glass vials and incubated at 25 ° C. with shaking at 100 rpm for a total of 120 hours. At the times 0; 0.5; 1; 2; 5; 24; 47; A sample of 1 ml was taken at 72 and 120 h, heat-inactivated at 99 ° C. for 10 min and stored at -20 ° C. After thawing the sample, the non-soluble components were separated by centrifugation. ZEN, HZEN and DHZEN were analyzed by LC / MS / MS. For this purpose, the metabolites were separated by chromatography using a Phenomenex Luna C18 (2) column with the dimensions 250 mm x 3 mm and a particle size of 5 pm. The eluent was an acetonitrile-water mixture with a formic acid concentration of 1 ml / l. The UV signal at 270 nm was recorded. The ionization source used was electro-spray ionization (ESI). ZEN, HZEN and DHZEN were quantified using QTrap / LC / MS / MS (triple quadrupole, Applied Biosystems) in "enhanced mode".
In Figure 1, the degradation of ZEN and the increase in HZEN and DHZEN are exemplified for a 1: 10,000 diluted cell lysate solution for ungagged (Figure 1A) as well as 6xHis tagged C (Figure 1B) and 6xHis N-terminal (Figure 1C) to see polypeptide having SEQ ID NO: 1. It is clear from this that, first, the conversion of ZEN is immediate and complete, since already in the first sample (0 h) pulled immediately after the start of the experiment, almost no ZEN could be detected any more, and secondarily, by adding a tag, C or N-terminal, no significant Akti¬vitätsverlusten came.
Example 4: Hydrolysis of ZEN derivatives by polypeptides in cell lysates
To determine the ability of the polypeptides in addition to ZEN also ZEN derivatives in nichtbzw. To transform less toxic metabolites, the polypeptides having the SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO. 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, as in Example 3, prepared and used as cell lysates in Abbaubauuch. For the preparation of the polypeptides having the sequences having the SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively, were the respective synthetic nucleotide sequences having the sequences of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, respectively.
The degradation experiments were carried out as described in Example 3, each polypeptide being tested with each ZEN derivative, α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN. The cell lysates were used at a total dilution of 1: 10,000. As a substrate stock solution, instead of a 2.08 mM ZEN solution (40% v / v ACN + 60 v / v% H 2 O), equimolar, i. 2.08 mM solutions of ZEN derivatives used. α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were purchased from Sigma, Z14G and Z14S were at least 90% pure by the methods described in P. Krenn et al., 2007, Mycotoxin Research , 23, 4, 180-184 and M. Sulyok et al., Anal. Bioanal. Chem., 2007, 289, 1505-1523. Another difference to Example 3 is that only one sample, after 24 h, genom¬men was. The reduction of the concentration of ZEN derivatives during the degradation experiment was quantified by LC / MS / MS. α-ZEL, β-ZEL, Z14G and Z14S were prepared by the method of M. Sulyok et al. (2010, Food Chemistry, 119, 408-416); α-ZAL, β-ZAL and ZAN were synthesized according to the method of P. Songsermaskul et al. (2011, J. of Animal Physiol, and Animal Nutr., 97, 155-161). Surprisingly, it has been found that after 6 h Inkuba¬¬ tion, only 0 to a maximum of 13% of the starting amounts of ZEN derivatives were present.
Example 5 Specific activity and enzyme kinetic parameters of the polypeptides and variants thereof
The determination of the specific activity of the polypeptides and variants thereof was carried out photometrically, with all the polypeptides used suspending a C-terminal 6 × his day. The preparation, enrichment and purification of the polypeptides or variants thereof were successful as described in Example 1. The degradation of ZEN to HZEN was measured by the decrease in adsorption at the wavelength of 315 nm. The molar extinction coefficients [ε] of ZEN and HZEN were determined experimentally to be 0.0078895 L pmol'1 cm'1 and 0.0030857 L pmol'1 cm'1. The extinction coefficients are strongly pH-dependent and therefore the activity measurement must always be carried out at exactly the same pH and preferably also in the same matrix. The measurements were carried out in a 50 mM Tris-HCl pH = 8.2 buffer solution in quartz cuvettes with a wavelength range of 200 to 2500 nm in a UV-VIS photometer (Hitachi U-2001) at 32 ° C.
The ZEN substrate stock solution was a 2.08 mM ZEN solution (40% by volume ACN + 60% H 2 O). To prepare this solution, ZEN was weighed and dissolved in crystalline form (Biopure Standard from Römer Labs, Art. No. 001109, purity at least 98%). The ZEN substrate dilutions (0.79 μΜ, 1.57 μΜ, 2.36 μΜ, 3.14 μΜ, 4.71 μΜ, 6.28 μΜ, 7.85 μΜ, 9.42 μΜ, 10.99 μΜ, 12.56 μΜ, 14.13 μΜ, 15.71 μΜ, 17.28 μΜ, 18.85 μΜ) were prepared with 50 mM Tris-HCl pH = 8.2. The polypeptide solution was diluted with 50 mM Tris-HCl buffer pH = 8.2 to a final concentration of approximately 70 ng / ml. The ZEN substrate dilutions were preheated to 32 ° C in a water bath. 0.2 μΙ of enzyme solution was added to 100 μΙ of the respective ZEN substrate dilution and the absorbance was measured for 5 min, with each combination of enzyme solution -ZEN substrate dilution being measured at least twice.
Taking into account the extinction coefficients of ZEN and HZEN, the rate of absorption for each substrate concentration was calculated by increasing the absorbance.
The terms "KM value" or "Michaelis-Menten constant" refer to a parameter for description of the enzymatic affinity having the units [μΜΙ] or [mMI] and using the linear Hanes plot of H. Bisswang (2002, Enzymes Kinetics, ISBN 3-527-30343-X, page 19) is calculated, for which purpose preferably the function "Enzymki¬netik, single substrate" of the program SigmaPlot 12.0 is used. The terms "catalytic enzyme reaction constant" or "kcar value" refer to a parameter for describing the turnover rate of an enzyme indicated in [s] " 1 and preferably using the enzyme enzyme single substrate function of SigmaPlot 12.0 program The "maximum enzyme rate" or the "vmax value" is given in the units [μΜ / s] or [mM / s] and determined analogously to the KM value by means of the linear Hanes plot, with the function " Enzyme Kinetics, Single Substrate "SigmaPlot 12.0 program is used.
By means of vmax and the enzyme concentration used, the specific activity was according to the formula. ..... , r, .., vmax [μΜ / s] x 60 [sfmin] specific activity [U / mg] == ----. .- =
Enzyme Konzentratton [mg / l] calculated, with one unit as hydrolysis of 1 μπιοΙ ZEN per minute at 32 ° C in 50 mM Tris-HCl buffer solution, pH = 8.2 is defined.
The raw data for the determination of the enzyme parameters KM, vmax, k ^ t and the specific activity are given below by way of example for the polypeptide having SEQ ID NO: 1. Table 2 shows the reaction rates at the respective ZEN substrate concentrations, FIG associated Michaelis-Menton graphics and in Table 3 the corre- sponding enzyme kinetic parameters are given. The enzyme solution used had a concentration of 68 ng / l.
Table 2: Reaction rates of the polypeptide of SEQ ID NO: 1 at different ZEN concentrations.
Table 3: Enzyme kinetic parameters of the polypeptide having SEQ ID NO: 1.
The specific activity of the investigated polypeptides for SEQ ID NO: 1 is 8.25 U / mg, for SEQ ID NO: 2 10.56 U / mg, for SEQ ID NO: 3 8.36 U / mg, for SEQ ID NO: 4 8.33U / mg, for SEQ ID NO: 5 8.56 U / mg, for SEQ ID NO: 6 9.95 U / mg, for SEQ ID NO: 7 3.83 U / mg, for SEQ ID NO : 8 2.57 U / mg, for SEQ ID NO: 9 4.87 U / mg, for SEQ ID NO: 10 5.12 U / mg, for SEQ ID NO: 11 3.88 U / mg, for SEQ ID NO: 12 2.78 U / mg, for SEQ ID NO: 13 6.43 U / mg, for SEQ ID NO: 14 3.33 U / mg.
The specific activities of the tested polypeptide variants are listed in Table 4 and Table 5.
Table 4: Specific activity of variants of the polypeptide of SEQ ID NO: 1, conserved region (s) in which the mutation (s) lie and sequence identity of the variant to the parental sequence of SEQ ID NO: 1. Numbering of the mutation (s ) is relative to the amino acid sequence of SEQ ID NO: 1. Sequence identity was determined by BLAST as described in Example 2.
Table 5: Specific activity of variants of the polypeptide of SEQ ID NO: 2. The numbering of the mutation (s) is relative to the amino acid sequence of SEQ ID NO: 2. Sequence identity was determined by BLAST as described in Example 2.
Example 6: Degradation of ZEN and ZEN derivatives in contaminated maize
In order to determine the ability of the polypeptides to degrade naturally occurring ZEN and ZEN derivatives in a complex matrix and at low pH, contaminating maize with different concentrations of the polypeptides having SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and the degradation of ZEN and ZEN derivatives.
The contaminated corn was milled and used in the digestion experiment, the batch consisting of 1 g of ground, contaminated corn, 8.9 ml of 100 mM acetate buffer pH = 4.0, and 1 ml of polypeptide solution. Enriched and purified polypeptide solutions, described in Example 5, were prepared, diluted to a concentration of 10 mU / ml, 00 mU / ml and 1,000 mU / ml, respectively. In the approach were thus absolutely 0.1 mU, 1 mlzw. 10 mU used. Each digestion batch was carried out in 25 ml glass vials and incubated at 37 ° C. with shaking at 100 rpm. Before the addition of enzyme or after 1 hour incubation in each case a sample of 1 ml were removed, heat inactivated at 99 ° C for 10 min are stored at -20 ° C. After thawing the sample, the non-soluble components were separated by centrifugation. Concentrations of ZEN as well as ZEN derivatives were determined with sl-LC / MS / MS as described in M. Sulyok et al. (2007, Anal., Bioanal Chem., 289, 1505-1523). The content of ZEN and ZEN derivatives in this maize was 238ipb for ZEN, 15 ppb for a-ZEL, 23 ppb for β-ZEL, 32 ppb for Z14G and 81 ppb for Z14S. Table 8 shows the percentage decrease in ZEN content and ZEN derivatives in the degradation study.
Table 6: Reduction of ZEN and ZEN derivatives in percent relative to the starting content of the degradation assay of different polypeptides and polypeptide amounts.
Example 7: Polypeptide-containing additives for hydrolytic cleavage of ZENjnd / or ZEN derivatives
For preparation of additives for the hydrolytic cleavage of ZEN, fermentation supernatants of P. pastoris-expressed polypeptides having the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 13 by microfiltration and ultrasound were used. rafiltration (cut off limit: 10 kDa) under standard conditions and concentrated to a bulk solids concentration of about 9% by weight. Subsequently, these olypetide-containing solutions were further processed into dry powders under standard conditions using a spray drier (Büchi Mini B290) also under standard conditions. These four powders are referred to in more detail as additives Z1, Z2, Z6 and Z13. Z1, Z2, Z6 and Z13 were further compounded with bentonite having an average grain size of about 1 pm, in a ratio of 13 wt% additive Z1, Z2, Z6 and Z13 and 99 wt% bentonite in an overhead shaker, respectively , The additives thus contained are referred to as additive Z1.B, Z2.B, Z6.B and Z13.B. Furthermore, Z1, Z2, Z6 and Z13 with bentonite and a vitamin-trace element concentrate in a ratio of 0.1 wt .-% additive Z1, Z2, Z6 or Z13, 0.9Gew .-% vitamin trace elements Concentrate and 99 wt .-% bentonite in a overhead shaker, mixed. The additives thus contained are referred to as additive Z1.BVS, Z2.BVS, Z6.BVS and Z13.BVS. 100 g of the additives Z1.BVS, Z2.BVS, Z6.BVS and Z13.BVS contained 200 mg of iron sulfate, 50 mg of copper sulfate, 130 mg of tin oxide, 130 mg of manganese oxide, 2.55 mg of calcium carbonate, 160 mg of vitamin E, 6, 5 mg vitamin K3, 6.5 mg vitamin B1, 14 mg vitamin B2, 15 mg vitamin B6, 0.15 mg vitamin B12, 150 mg nicotinic acid, 30 mg pantothenic acid and 5.3 mg folic acid.
The adjuncts were extracted in a 50mM Tris-HCl buffer pH = 8.2 for 30 minutes and further diluted in the same buffer to give the final concentration of polypeptide. 70 ng / ml.
Subsequently, the zearalenone degrading effect of these solutions, as described in Example 5, determined. The corresponding activities were for Z1 8,230 U / g, for Z29,310 U / g, for Z6 9,214 U / g, for Z1.B 83 U / g, for Z2.B 92 U / g, for Z2.C 90 U / g, for Z13.B 57 U / g, for Z1 .BVS 8 U / g, for Z2.BVS 9 U / g, for Z6.BVS 9 U / g and for Z13.BVS 6 U / g.
The ability to degrade the ZEN derivatives α-ZEL, β-ZEL, α-ZAL, β-ZAL, Z14G, Z14S and ZAN by the additives Z1, Z2, Z6, Z13, Z1.B, Z2.B, Z6.B , Z13.B, Z1.BVS, Z2.BVS, Z6.BVS and Z13.BVS, was carried out as described in Example 4, but instead of 100 μl of a cell lysate, 100 μl of a polypeptide solution having a polypeptide concentration of approx. 70 ng / ml used. After incubation for 6 hours, only a maximum of 15% of the starting amount was present as unhydrolyzed ZEN derivative. SEQUENCE LISTING &lt; 110 &gt; Erber Aktiengesellschaft &lt; 120 &gt; Polypeptide for hydrolytic cleavage of zearalenone and / or zearalenone derivatives <130>. P05244 &lt; 160 &gt; 67 &lt; 170 &gt; Patent version 3.5 &lt; 210 &gt; 1 &lt; 211 &gt; 328
&Lt; 212 &gt; PRT &lt; 213 &gt; Rhodococcus erythropolis &lt; 400 &gt; 1
Met Ala Glu Glu Gly Thr Argus Glu Ala Ala Asp Ala Ala Thr Gin 15 10 15
Ala Arg Gin Leu Pro Asp Ser Arg Asn Ile Phe Val ser His Arg Phe 20 25 30 per Glu Arg Gin Val Asp Leu Gly Glu Val Val Met Asn Phe Ala Glu35 40 45
Ala Gly Ser Pro Asp Asn pro Ala Leu Leu Leu Leu Pro Glu Gin Thr50 55 60
Glv 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 Gin 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 Ser115 120 125 ser Gly Gly Leu Leu Ala Ala Trp Leu ser Ala Tyr Ala Met Pro Gly130 135 140
Gin Ile Arg Ala Ala Leu Cys Glu Asp Ala Pro Phe Phe Ala Ser Glu 145 150 155 160 I pij val Pro Ala Tyr Gly His Ser Val Leu Gin Ala Ala Gly Pro Ala eu 165 170 175 phe Glu Leu Tyr Arg Asp Phe Leu Gly Asp Gin Trp Ser Ile Gly Asp 180 185 190
Trp Lys Gly Phe Val Glu Ala Ala Lys Ala Ser pro Ala Lys Ala Met195 200 205
Gin Leu Phe Pro Thr Pro Asp Glu Ala Pro Gin Asn Leu Lys Glu Tyr210 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 Gin 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 Gly260 265 270
Ala Leu Ser Asp Leu Gin Ala Glu His Ala Gin Asp Ile Ile Arg Ser275 280 285
Ala Gly val Arg Val Asp Tyr Gin Ser His Pro Asp Ala Leu His Met290 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 Asp325 &lt; 210 &gt; 2 &lt; 211 &gt; 308
&Lt; 212 &gt; PRT &lt; 213 &gt; Streptomyces violaceous &lt; 400 &gt; 2
Met Ala Asp Pro Ala Gin Arg Asp Val Tyr val Pro His Ala Tyr Pro15 10 15
Glu Lys Gin Ala Asp Leu Gly Glu Thr Thr Asn Tyr Ala Glu Ala20 25 30
Gly Glu Pro Asp Met Pro Ala Val Leu Leu Ile Pro Glu Gin Thr Gly35 40 45
Ser Trp Trp Gly Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu Asn Phe50 55 60
His Val Tyr Ala Val Asp Leu Arg Gly Gin 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 85 90 95
Ile Ala Leu Val val Lys Arg Pro Val Ile Val Ala Gly Asn Ser Ser P05244_ST25.txt 100 105 110
Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr ser Met Pro Gly Gin115 120 125
Val Arg Gly Ala Leu Cys Glu Asp Ala Pro Phe Phe Ala Ser Glu Leu130 135 140 Thr Thr Thrys Gly His Ser Ile Arg Gin Ala Ala Gly Pro Met Phe 145 150 155 160
Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gin Trp ser Val Gly Asp Trp 165 170 175
Thr Gly Tyr cys Arg Ala Ala Asp Ala Ser Ser pro Met Ala Arg 180 185 190
Tyr Phe Val Ala Asp Glu Pro Gin 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 Pro210 215 220
His Glu Gin Leu Leu Thr Gin val Lys Thr Pro val Leu Leu Thr His 225 230 235 240
His Met Arg Asp was Asp Asp Asp Gly His Leu val Gly Ala Leu 245 250 255
Ser Asp Glu Gin Ala Ala Arg Ala Arg Leu Leu Met Glu Ser Ala Gly260 265 270
Val Lys Val Asp Tyr Ala ser val Pro Asp Ala Leu His Met Met His275 280 285
Gin Phe Asp Pro Pro Arg Tyr val Glu lie Phe Thr Gin Trp Ala Ala290 295 300
Thr Leu Ala Ala305 &lt; 210 &gt; 3 &lt; 211 &gt; 309
&Lt; 212 &gt; PRT &lt; 213 &gt; Streptomyces coelicolor &lt; 400 &gt; 3
Met Val Thr Ser Pro Ala Leu Arg Asp val His val Pro His Ala Tvr 1 5 10 15
Pro Glu Gin Gin Val Asp Leu Gly Glu Thr Thr Asn Tyr Ala Glu 20 25 30
Page 3
Ala Gly Asp Pro Gly Arg Pro Ala Val Leu Leu Le Pro Glu Gin Thr35 40 45
Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His50 55 60
Phe His val Tyr Ala Val Asp Leu Arg Gly Gin 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 ala Gly Asn ser100 105 110
Ser Gly Gly Val Leu Ala Ala Trp Leu Ser Ala Tyr Ser Met Pro Gly115 120 125
Gin Ile Arg Gly val Leu Cys Glu Asp Pro Pro Phe Phe Ala Ser Glu130 135 140
Leu Val Pro Ala His Gly His Ser Arg Arg Gly Gly Ala Gly Pro val 145 150 155 160
Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gin Trp Ser val Gly Asp 165 170 175
Trp Glu Gly Phe Arg Ser Ala Ala Asp Ala Ser Ala Ser Pro Met Ala180 185 190
Argus Phe val Ala Asp Thr lie Pro Gin His Leu Lys Glu Tyr Asp195 200 205
Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Gly Leu Asn Cys210 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 Gin Ala Ala Gin val Arg Arg Leu Met Glu Ser Ala260 265 270
Gly Val Lys val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met275 280 285
His Gin Ser Asp Pro Ala Arg Tyr Ala Glu Le Le Thr Pro Trp Thr290 295 300
Ala Ala Leu Ala Pro305 &lt; 210 &gt; 4 &lt; 211 &gt; 309
&Lt; 212 &gt; PRJ. &Lt; 213 &gt; Streptomyces rapamycinicus &lt; 400 &gt; 4
Met Val Thr Ser Pro Ala Leu Arg Asp val His Val Pro His Ala Tyr15 10 15
Pro Glu Gin Gin Val Asp Leu Gly Glu Thr Thr Asn Tyr Ala Glu20 25 30
Ala Gly Asp Pro Asp Arg Pro Ala Val Leu Leu Le Pro Glu Gin Thr35 40 45
Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His50 55 60
Phe His Val Tyr Ala Val Asp Leu Arg Gly Gin Gly Arg Ser Ser Trp 65 70 75 80
Thr Pro Lys Arg Tyr ser Le Asp Asn Phe Gly Asn Asp Leu val Arg 85 90 95 phe lie Ala Leu val Val Lys Arg Pro Val Val Val Ala Gly Asn Ser100 105 110 ser Gly Gly val Leu Ala Ala Trp Leu ser Ala Tyr Ser Met Pro Gly115 120 125
Gin 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 Gin Gly Ala Gly Pro val 145 150 155 160
Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gin Trp Ser val Ser Asp 165 170 175
Trp Glu Gly Phe cys Arg Ala Ala Gly Ala Ser Ala ser Pro Met Ala180 185 190
Arq ser Phe Val Ala Asp Gly ile Pro Gin His Leu Lys Glu Tyr Asp195 200 205 per Glu Trp Ala Arg Ala Phe His Glu Gly Thr Val Gly Leu Asn cys210 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 Gin Ala Ala Gin Ala Arg Leu Leu Met Glu ser Ala260 265 270
Gly Val Arg val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met275 280 285
His Gin Ser Asp Pro Ala Arg Tyr Ala Glu Ile Phe Thr Arg Trp Ala290 295 300
Ala Ala Leu Ala Pro 305 &lt; 210 &gt; 5 &lt; 211 &gt; 309
&Lt; 212 &gt; PRT &lt; 213 &gt; Streptomyces lividans &lt; 400 &gt; 5
Met val Thrers Pro Ala Leu Arg Asp Val His Val Pro His Ala Tyr15 10 15 per Glu Gin Gin val Asp Leu Gly Glu Thr Thr Asn Tyr Ala Glu20 25 30
Ala Gly Asp Pro Gly Arg Pro Ala val Leu Leu lie Pro Glu Gin Thr35 40 45
Gly ser Trp Trp ser Tyr Glu Glu Ala Met Gly Leu Leu Ala Glu His50 55 60
Phe His Val Tyr Ala Val Asp Leu Arg Gly Gin 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 ser100 105 110
Ser Gly Gly Val Leu Ala Ala Trp Leu ser Ala Tyr ser Met Pro Gly115 120 125
Gin Ile Arg Gly Val Leu Cys Glu Asp pro Pro Phe Phe Ala ser Glu130 135 140 P05244_ST25.txt
Leu Val Pro Ala His Gly His Ser Val Ara Gin Gly Ala Gly Pro Val 145 150 155 160
Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gin Trp Ser val Gly Asp 165 170 175
Trp Glu Gly Phe Arg Ser Ala Ala Gly Ala Ser Ala Ser Pro Met Ala180 185 190
Arg Ser Phe Val Ala Asp Thr Ile Pro Gin His Leu Lys Glu Tyr Asp195 200 205
Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly Thr Val Gly Leu Asn Cys210 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 Gin Ala Ala Gin Ala Arg Arg Leu Met Glu Ser Ala260 265 270
Gly val Lys Val Asp Tyr Glu Ser Val Pro Asp Ala Ser His Met Met275 280 285
His Gin Ser Asp Pro Ala Arg Tyr Ala Gluile Leu Thr Pro Trp Ala290 295 300
Ala Ala Leu Ala Pro305 &lt; 210 &gt; & lt 6; 211 &gt; 309
&Lt; 212 &gt; PRT &lt; 213 &gt; streptomyces coelicoflavus &lt; 400 &gt; 6
Met val Thr Ser Pro Ala Leu Arg Asp val His val pro His Ala Tyr15 10 15
Pro Glu Gin Gin val Asp Leu Gly Glu Thr Thr Asn Tyr Ala Glu20 25 30
Ala Gly Asp Pro Asp Arg Pro Ala Val Leu Leu Ile Pro Glu Gin Thr35 40 45
Gly Ser Trp Trp Ser Tyr Glu Glu Ala Met Gly Leu Leu Ser Glu His50 55 60
Phe His Val Tyr Ala val Asp Leu Arg Gly Gin Gly Arg Ser Ser Trp
Page 7 65 70 75 80
Thr Pro Lys Arg Tyr Ser Leu Asp Asn Phe Gly Asn Asp Leu Val Arg85 90 95
Phe Ile Ala Leu Val val Lys Arg Pro val Val Val Ala Gly Asn ser100 105 110 ser Gly Gly val Leu Ala Ala Trp Leu Ser Ala Tyr ser Met Pro Gly115 120 125
Gin Leu Arg Gly Val Leu Cys Glu Asp Pro Pro Phe Phe Ala ser Glu130 135 140
Leu val Pro Ala His Gly His Ser Arg Arg Gly Gly Ala Gly Pro val 145 150 155 160
Phe Glu Leu Phe Arg Thr Tyr Leu Gly Asp Gin Trp ser val Gly Asp 165 170 175
Trp Glu Gly Phe Cys Arg Ala Ala Gly Ala Ser Ala water Pro Met Ala180 185 190
Arg Ser Phe val Ala Asp Gly Ile Pro Gin His Leu Gin Glu Tyr Asp195 200 205
Pro Glu Trp Ala Arg val Phe Tyr Glu Gly Thr val Gly Leu Ser cys210 215 220
Pro His Glu Arg Met Leu Gly Gin 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 Asp Glu Gin Ala Leu Arg Ala Arg Arg Leu Met Asp Ser Ala260 265 270
Gly val Thr val Asp Tyr Glu ser val Pro Asp Ala Ser His Met Met275 280 285
His Gin Ser Ala Pro Ala Arg Tyr val Glu Ile Phe Thr Arg Trp Ala290 295 300
Ala Ala Leu Ala Pro305 &lt; 210 &gt; 7 &lt; 211 &gt; 300
&Lt; 212 &gt; PRT &lt; 213 &gt; Rhodococcus triatome P05244_ST25.txt &lt; 400 &gt; 7
Met Pro His Asp Tyr Glu Glu Lys Leu Val Asp Leu Gly Glu lie Asp 1 5 10 15
Leu Asn Tyr Ala Glu Ala Glyser Pro Asp Lys Pro Ala Leu Leu Leu 20 25 30 Lp Pro Gin ser Glu Ser Trp Trp Gly Tyr Glu Glu Ala Met Gly35 40 45
Leu Leu Ala Glu Asp Tyr His val Phe Ala Val Asp Arg Arg Gly Gin50 55 60
Gly Arg Thr Thr Thr Pro Gly Arg Tyr ser Leu Asp Asn Phe Gly 65 70 75 80
Asn Asp Leu val Arg phe le Asp Leu val Ile Gly Arg Thr valval 85 90 95
Gly ser Val Gly Gly Gly val Val Ala Ala Trp Leu Ala Ala 100 105 110
Phe Ser Leu Pro Gly Gin 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 Gin130 135 140
Ala Ala Gly His Ile Phe val Asn Trp Arg Asp Tyr Leu Gly Asp Gin 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 Gin val Pro Leu pro Glu Thr Ala Pro Gin 180 185 190
Asn Leu Leu Glu Tyr Asp Pro Glu Trp Ala Arg Ala Phe Tyr Glu Gly195 200 205
Thr val Ala Gin Thr cys Pro His Asp Tyr Met Leu ser Gin val Lys210 215 220
Val Pro Met Leu Val Thr His His Ala Arg Met lie Asp Glu Ala Thr 225 230 235 240
Ser Gly Leu Val Gly Ala Met Ser Asp Leu Gin Val Gin Lys Ala Ala 245 250 255
Gly Arg Arg Gly Gly Gly Gin Val Asp Val Val Asp Leu Pro260 265 270
Glu Ala Pro His Ile Leu His Gin Leu Ala Pro Lys Glu Tyr val Glu275 280 285
Ile Leu Asn Asn Trp Val Glu Lys Leu Pro Pro Val290 295 300 &lt; 210 &gt; & lt 8; 211 &gt; 307
&Lt; 212 &gt; PRT &lt; 213 &gt; Hirschia baltica &lt; 400 &gt; 8th
Met Ile Gin Asn Asn Lys Thr Ala Pro Tyr Lys Tyr Lys Glu Lys Leu15 10 15 val Asp Leu Gly Glu Ile Lys Met Asn Tyr Ile Val Ala Gly Ala Asp20 25 30
Val Ser Pro Ala Leu Leu Lei Ile Pro Gly Gin Thr Glu Ser Trp Trp35 40 45
Gly Phe Glu Ala Ala Ile Glu Lys Leu Glu Ser Asn Phe Gin val Phe50 55 60
Ala Ile Asp Leu Arg Gly Gin Gly Lys Ser Thr Gin Thr Pro Gly Arg 65 70 75 80
Tvr 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 Leu100 105 110
Leu Ala Ala Trp Leu Ser Ala Tyr Ala Met Pro Asn Gin Ile Arg Ala115 120 125
Ile His cys Glu Asp Ala Pro Phe Phe Thr Ala Glu Lys Ala Pro Leu130 135 140
Tvr Gly His Ala Ile Gin Gin Ala Ala Gly Pro Ile Phe Ser Leu Met 145 150 155 160
Ser Lys Phe Leu Gly Asp Gin Trp ser Ile Asn Trn Trp Glu Gly Leu 165 170 175
Lys Ala Ala Gin Ala Lys Asp Thr His Pro Ala Asn Lys Met Ile Ser 180 185 190
Gin Val Glu Gin Pro Pro Gin His Leu Lys Glu Tyr Asp Pro Glu Trp195 200 205
Gly Arg Ala Phe He Glu Gly Lys Phe Asn Leu Asn Ser Pro His His210 215 220
Thr Leu Leu Ser Asp lie Lys Thr Per Met Leu Tyr Thr His His Met 225 230 235 240
Arg Phe Glu Asp Pro Gin Thr Gly Leu Leu Gly Ala Thr Ser Asp 245 250 255
Phe Gin Ala Ser Lys Ile Lys Glu Ile Ala Leu Lys Thr Gly Asn Ser260 265 270 phe Glu Leu l Asp Ala Pro Asp Ala Phe His Ser Met His Glu Ala275 280 285
Asp Pro Gin Arg Phe val Asp Ile Leu Thr Ser Trp Ile Glu Arg Leu290 295 300
Asn Leu Gin305 &lt; 210 &gt; 9 &lt; 211 &gt; 321
&Lt; 212 &gt; PRT &lt; 213 &gt; Nocardia brasiliensis &lt; 400 &gt; 9
Met Gly lie Ser Glu Ala Ala Asp Arg Ala Asp Thr Phe val Ala His15 10 15
Lys Phe Glu Glu Gin Leu val Asp Leu Gly Glu Ile Arg Met Asn Tyr20 25 30 val Ala Ala Gly Asp Pro Thr Ser Pro Ala Leu Leu Leu lie Pro Ala35 40 45
Gin Gly Glu Ser Trp Trp Gly Tyr Glu Asn Ala Ile Thr Leu Leu Ala50 55 60
Asn Asp Phe Arg val Phe Ala He Asp Leu Arg Gly Gin 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 lie Asp Leu val lie Gly Arg Pro Thr Leu val Ala Gly100 105 110
Ser Ser Gly Gly val He Ala Ala Trp Leu Ala Ala Tyr Ala Lys115 120 125
Gly Gin Ile Arg Gly Ala Met Leu Glu Asp Pro Leu Phe Ala130 135 140 gin Ala Ala Pro Pro Tyr Gly Pro Gly Met Gin Thr Leu Gly 145 150 155 160 per Ile Phe val Leu Trp Ala Lys Trp Leu Gly Pro Gin 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 Glu195 200 205
Gly Ala Ala Ala Thr Pro Pro Gin His Leu Lys Glu Tyr Asp Pro Glu210 215 220
Trp Ala Gin Ala Trp Ala Thr Asp Val Ala Asn Ala Gly Cys Asp His 225 230 235 240
Ala Thr Met Leu Ala Gin Asn Arg val Pro Val Leu Leu Thr His His 245 250 255
Phe His Leu Thr Asp Pro Asp Thr Gly Gin Leu Met Gly Ala Met Thr260 265 270
Asp Ile Gin Ala Gin Gin Ala Arg Arg Leu Leu Ala Ala Thr Gly Gin275 280 285
Pro Val Thr Phe Thr Ala Leu Asp Ala Pro His Thr Met His Asp Pro290 295 300
Glu Pro Glu Arg Tyr Phe Glu val Leu Thr Glu Trp Ala ser Ala Leu 305 310 315 320
Asp &lt; 210 &gt; 10 &lt; 211 &gt; 319
&Lt; 212 &gt; PRT &lt; 213 &gt; Mycobacterium vaccae &lt; 400 &gt; 10
Met Gly Arg Tyr Ala Gly Val Phe Gly Pro His Ala Pro Glu ser Thr15 10 15
Tyr val Gly His Ala Tyr Pro Glu Gin Leu Phe Asp Thr Gly Glu val20 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 Gin Thr Glu ser Trp Trp Gly Tyr Glu Pro Ala Met50 55 60
Gly Leu Leu Ala Glu His Phe His Val His Ala Val Asp Leu Arg Gly65 70 75 80
Gin Gly Arg Ser Thr Arg Thr Pro Arg Arg Tyr Thr Leu Asp Asn Ile85 90 95
Gly Asn Asp Leu val Arg Phe Leu Asp Gly val Ile Gly Arg Pro Ala100 105 HO
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 Gin 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
Gin Trp Ser Ile Gly Asp Trp Asp Gly Phe val Ala Gly Ala Pro Gin 180 185 190
Glu Leu Ala Gly Trp Gin Ala His val Ala Leu Ala Gly Gly Thr Ala 195 200 205
Glu Pro Pro Gin 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 Gin val Met Leu 225 230 235 240 ser Gin 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 Gin Ala 260 265 270
Ala Ara val Val Glu Leu val Glu Asn ser Gly Ala Pro Leu Thr Tyr a 275 280 285
Ara Ser Phe Pro Met Met Gly His Ser His His Ala Gin Asp Pro Ala 290 295 300
Leu Phe Ala Gly Thr Leu val Asp Trp phe Thr Ala Ala Arg ser P05244_ST25.txt 305 310 315 &lt; 210 &gt; & lt 11; 211 &gt; 319
&Lt; 212 &gt; PRT &lt; 213 &gt; Mycobacterium gilvum &lt; 400 &gt; 11
Met Gly Arg Tyr Ala Gly val Phe Gly Pro His Ala Pro Glu Ala Thr15 10 15
Tyr val Glu His Gly Tyr Pro Glu Arg Leu Phe Asp Thr Gly Glu val20 25 30
Gin Leu Asn Tyr val val Ala Gly Asp Ala Ala Ala Pro pro Leu Leu35 40 45
Leu Pro Gly Gin Ser Glu Ser Trp Trp Gly Tyr Glu Ala Ala lie50 55 60
Pro Leu Leu Ala Arg His Phe His Val His Ala Val Asp Leu Arg Gly 65 70 75 80
Gin 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 Ala100 105 110
Phe Val Ser Gly Leu ser Ser Gly Gly Leu Ala Ser Ala Trp Leu ser115 120 125
Ala Phe Ala Lys Pro Gly Gin val val Ala Ala Cys Trp Glu Asp Pro 130 135 140
Per Phe Phe ser Ser Glu Thr Ala Pro Val Gly Pro Pro lie Thr 145 150 155 160
Asp Ser Ile Gly Pro Leu Phe Gly Met Trp Ala Arg Tyr Leu Gly Asp 165 170 175
Gin Trp Ser val Gly Asp Trp Asp Gly Phe val Ala Ala val Pro Thr 180 185 190
Glu Leu Ala Asp Trp Gin Ala His val Ala Leu Val Val Gly Thr Ala195 200 205
Asp Pro Pro Gin Asn Leu Arg Glu Tyr Asp Pro Glu Trp Gly Lys Ala210 215 220
Phe lie Thr Gly Thr Phe Ala Ala ser cys Pro His His Met Met Leu 225 230 235 240
Ser Lys Val Lys Val Pro val Leu Tyr Thr His His phe Arg Met Ile245 250 255
Asp Glu Gly Ser Gly Gly Leu Ile Gly Ala Cys Ser Asp Ile Gin Ala260 265 270
Gly Arg Val Thr Gin Leu Ala Lys Ser Gly Gly Arg Ser Val Thr Tyr275 280 285
Arg Ser Phe Pro Met Met Ala His Ser Met His Gly Gin Asp Pro Ala290 295 300
Leu Phe Ser Glu Thr Leu val Glu Trp Phe Ser Arg Phe Thr Gly 305 310 315 &lt; 210 &gt; & lt 12; 211 &gt; 322
&Lt; 212 &gt; PRT &lt; 213 &gt; Gordonia effusa &lt; 400 &gt; 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 Gin Gly Asn Lys Lys Ser Pro Ala Leu Leu Leu Ile Pro Ala 35 40 45
Gin Gly Glu Ser Trp Trp Gly Tyr Glu Ala Ala Ile Pro Leu Leu Ala50 55 60 i vs His Phe Gin val Phe Ala Ile Asp Leu Arg Gly Gin Gly Arg Thr 6¾ 70 75 80
Thr Tro Thr Pro Gly Arg Tyr Thr Leu Asp Ile Phe Gly Asn Asp val μ 85 90 95 val Ara Phe Ile Asp Leu Val Ile Gly Arg Glu Thr Leu Ile Ala Gly y 100 105 110
Asn Ser Ser Gly Gly Leu Ile Gly Ala Trp Leu Ala Ala Phe Ala Lys 115 120 125 per Glv Gin val Arg Ala val Met Leu Glu Asp Pro Pro Leu Phe Ala130 135 140 ser Glu Ile Arg Pro Pro Tyr Gly Pro Gly trp Gin Gly Leu Gly 145 150 155 160 per Met Phe Ala Ala Trp Ala Lys Trp Leu Gly Pro Gin Trp ser lie 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 Gly Gly Gly Methe Le Gly Asp Gly Glu Asp195 200 205
Glv Ala Ala Pro Thr Pro Pro Gin His Leu Lys Glu Tyr Asp Pro Glu 210 210 220
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 Gin val Arg val Pro Val Leu Leu Thr His His 245 250 255 phe Arg Gin lie As Glu Glu Thr Gly His Leu Met Gly Ala Leu ser260 265 270
Asp Leu Gin Ala Ala Gin Val Arg His lie Glu Glu Val Ala Gly275 280 285
Gin Glu Val Thr Tyr Val Ser Leu Asp Ala Pro His Thr Met His Glu290 295 300
Pro Gin Pro Glu Arg Tyr Thr Asp Val Leu Leu Asp Trp val Lys Lys 305 310 315 320
Leu Gly &lt; 210 &gt; 13
&Lt; 211 &gt; & lt 328; 212 &gt; PRT &lt; 213 &gt; Togninia minima &lt; 400 &gt; 13
Met Asn Tyr Ala Thr Ala Gly Ser Asp Lys Pro Ala Leu Leu Leu 1 5 10 15 val Pro Gly Gin Ser Glu Ser Trp Trp Gly Tyr Glu Met Ala Met Trp 20 25 30
Leu Leu Lys Asp Asp Tyr Gin val Phe Ala Val Asp Arg Arg Gly Gin35 40 45
Gly Gin Ser Thr Trp Thr Pro Gly Arg Tyr Ser Leu Asp Thr Phe Gly50 55 60
Asn Asp Leu val Lys Phe lie Asp Ile val Ile Lys Arg Pro val val 65 70 75 80 val Ser Gly Leu Ser Ser Gly Gly val Val Ala Trp Leu ser Ala .85 90 95
Phe Ala Lys Pro Gly Gin Ile Arg Ala Ala Val Tyr Glu Asp Pro Pro 100 105 110
Leu Phe Ala Ser Gin ser Lys Pro Ala lie Gly Gin ser Val Met Gin115 120 125
Thr Val Ala Gly Pro Phe Phe Asn Leu Trp Tyr Lys Trp Leu Gly Ala130 135 140
Gin Trp Thr lie Gly Asp Gin Ala Gly Met Val Ala Ala Met Pro Lys 145 150 155 160
Glu Ile Pro Ala Trp Ile Leu Gin Tyr Leu Gly Asn Thr Thr ser Gly 165 170 175
Per Thr Gly Leu Asp Leu Thr Leu Asn Glu Tyr Asp Pro Glu Trp Gly180 185 190
His Gly Phe val ser Gly Thr val Asp Ala Thr Cys Asp His Glu Ala195 200 205
Met Leu Thr His val Lys Val Pro val Leu Phe Thr His His Arg Arg 210 215 220
Ala lie Asp Pro Tyr Thr Gly Asn Leu Gly Ser val Ser Asp Thr 225 230 235 240
Gin Val Ser Tyr Ala Gin Gly Leu Thr Thr Asn Gly Asn Gin Ser 245 250 255
Phe Thr Leu Lys Asn Phe Pro Leu Ala Ser His Asp Met His Asn Ser260 265 270
Asp Pro Ala Thr Tyr val ser Ala Thr Thr Trp Met Ala Ser Leu275 280 285
Gly al Gly Ala Val lie Pro Gly Pro val Lys val Ala ser Ala290 295 300 ser Ala Gin val ser Ala Ala ser Thr Ala Pro Pro Cys Thr ser 305 310 315 320
Threads Ala Proser Thr Gly His325 &lt; 210 &gt; 14
&Lt; 211 &gt; & lt 280; 212 &gt; PRT &lt; 213 &gt; Actinosynnema mi &lt; 400 &gt; 14
Met Thr Val Val Pro Pro Ala Pro Arg Asp Phe Pro Glu Leu Leu 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 Gin Gly Gly Trp Trp 35 40 45
Ser Tyr Glu Arq 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 HO
Leu Ala Ala Trp ser Ser Ala Tyr Ala Met Pro Gly Gin val Arg Ala 115 120 125 val Leu Leu Glu Asp Pro Leu Phe Ser Ser Glu Leu Thr Pro val 130 135 140 CVS Gly Pro Gly val Arg Gin Ala Ala Gly Pro Leu Phe Glu Leu Leu 145 150 155 160 ser Thr His Leu Gly Asp Gin 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 180 185 100 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 Thrers Gly Thr Gly Arg cys Arg Lys Pro Phe Arg Leu 245 250 255
Arg Gin Trp Trp Ala Gly Gly Ala Arg Gly Pro Pro Pro Arg Gin260 265 270
Ile Arg Ala Asp val Arg Thr Arg275 280 &lt; 210 &gt; 15 &lt; 211 &gt; 987
&Lt; 212 &gt; DNA &lt; 213 &gt; Rhodococcus erythropolis &lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encoding polypeptide having SEQ ID NO: 1 &lt; 400 &gt; 15 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 acatcat tcg gtctgcgggc gttcgggtgg actatcagtc gcaccccgac 900 gcgcttcaca tgatgcatct gttcgatccc gctcgttacg cggagatctt gacatcctgg 960 tccgcaacac tgcctgcgaa cgactag 987 &lt; 210 &gt; & lt 16; 211 &gt; 987
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 400 &gt; 16 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 atattat tcg tagtgccggt gttcgtgttg attatcagag ccatcctgat 900 gcactgcaca tgatgcacct gtttgatccg gcacgttatg cagaaattct gaccagttgg 960 agcgcaaccc tgcctgcaaa tgattaa 987 &lt; 210 &gt; 17 &lt; 211 &gt; 927
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encoding polypeptide having SEQ ID NO: 2 &lt; 400 &gt; 17 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 cggttattgt 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 gcacgttatt ttgttgcaga 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 tgcacat gat gcaccagttt gatccgcctc gttatgttga aatctttacc 900 cagtgggcag caaccctggc agcataa 927 &lt; 210 &gt; & lt 18; 211 &gt; 930
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encoding polypeptide having SEQ ID NO: 3 &lt; 400 &gt; 18 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 caagcca cat gatgcaccag agcgatccgg cacgttatgc agaaattctg 900 accccgtgga ccgcagcact ggcaccgtaa 930 &lt; 210 &gt; 19 &lt; 211 &gt; 930
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 22B &gt; Artificial DNA sequence encoding polypeptide having SEQ ID NO: 4 &lt; 400 &gt; 19 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 ttegtaecta 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 caagcca did gatgcaccag agcgatccgg cacgttatgc agaaatcttt 900 acccgttggg cagcagccct ggcaccgtaa 930 &lt; 210 &gt; & lt 20; 211 &gt; 930
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; orf was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encoding polypeptide having SEQ ID NO: 5 &lt; 400 &gt; 20 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 caagcca cat gatgcaccag agcgatccgg cacgttatgc agaaattctg 900 accccgtggg cagcagccct ggcaccgtaa 930 &lt; 210 &gt; & lt 21; 211 &gt; 930
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; orf was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encodes polypeptide with SEQ ID NO: 6 &lt; 400 &gt; 21 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 caagcca cat gatgcaccag agcgcaccgg cacgttatgt tgaaatcttt 900 acccgttggg cagcagccct ggcaccgtaa 930 &lt; 210 &gt; & lt 22; 211 &gt; 903
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. , &Lt; 220 &gt; &Lt; 221 &gt; mi sc__feature &lt; 223 &gt; Artificial DNA sequence encodes polypeptide with SEQ ID NO: 7 &lt; 400 &gt; 22 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 ccgttattgt 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 aagaata tgt ggaaattctg aataactggg tggaaaaact gcctccggtt 900 taa 903 &lt; 210 &gt; 23 &lt; 211 &gt; 924
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encodes polypeptide with SEQ ID NO: 8 &lt; 400 &gt; 23 atgatccaga acaataaaac cgcaccgtat aaatacaaag aaaaactggt tgatctgggc 60 gaaatcaaaa tgaactatat tgttgccggt gcagatgtta gtccggcact gctgctgatt 120 ccgggtcaga ccgaaagttg gtggggtttt gaagcagcaa ttgagaaact ggaaagcaac 180 tttcaggtgt ttgcaattga tctgcgtggt cagggtaaaa gcacccagac accgggtcgt 240 tatagcctga atctgatggg taatgatctg gttcgtttta ttagcctggt tattaaacgt 300 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 540 gcaaaagata cccatccggc aaacaaaatg attagccagg ttgaacagcc tccgcagcat 600 ctgaaagaat atgatccgga atggggtcgt gcatttattg aaggcaaatt taacctgaac 660 agtccgcatc ataccctgct gagcgacatt aaaaccccga tgctgtatac ccatcacatg 720 cgttttgaag atccgcagac aggtctgctg attggtgcaa ccagcgattt tcaggcaagc 780 aaaatcaaag aaattgccct gaaaaccggc aatagcttcg aactgattga tgcaccggat 840 gcatttcata gtatgca tga agccgatccg cagcgttttg ttgatattct gaccagctgg 900 attgaacgtc tgaatctgca gtaa 924 &lt; 210 &gt; 24 &lt; 211 &gt; 966
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encoding polypeptide with seq id NO: 9 &lt; 400 &gt; 24 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 tgattgggat 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 atgaccgäta ttcaggcaca gcaggcacgt 840 cgtctgctgg cagcaa ccgg tcagccggtt acctttaccg cactggatgc accgcatacc 900 atgcatgatc ctgaacctga acgttatttt gaagttctga ccgaatgggc aagtgcactg 960 gattaa 966 &lt; 210 &gt; 25 &lt; 211 &gt; 960
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encode polypeptide having SEQ ID NO: 10 &lt; 400 &gt; 25 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 gagcgccttt 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 caccgct gac ctatcgtagc tttccgatga tgggtcatag tatgcatgca 900 caagatccgg cactgtttgc aggcaccctg gttgattggt ttaccgcagc acgtagctaa 960 &lt; 210 &gt; & lt 26; 211 &gt; 960
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encoding polypeptide having SEQ ID NO: 11 &lt; 400 &gt; 26 atgggtcgtt atgccggtgt ttttggtccg catgcaccgg aagcaaccta tgttgaacat 60 ggttatccgg.aacgtctgtt tgataccggt gaagt.gcagc tgaattatgt tgttgccggt .120 gatgcagcag caccgcctct gctgctgatt ccgggtcaga gcgaaagttg gtggggttat 180 gaagcagcaa ttccgctgct ggcacgtcat tttcatgttc atgcagttga tctgcgtggt 240 cagggtcgta gcacccgtac accgggtcgc tataccctgg 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 gtagc gttac ctatcgtagc tttccgatga tggcacatag catgcatggt 900 caagatccgg cactgtttag cgaaaccctg gttgaatggt ttagccgttt taccggttaa 960 &lt; 210 &gt; 27 &lt; 211 &gt; 969
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encoding polypeptide having SEQ ID NO: 12 &lt; 400 &gt; 27 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 tgattgggat 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 aagaagt tgc aggtcaagag gttacctatg ttagcctgga tgcaccgcat 900 accatgcatg aaccgcagcc ggaacgttat accgatgttc tgctggattg ggttaaaaaa 960 ctgggttaa 969 &lt; 210 &gt; & lt 28; 211 &gt; 987
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encoding polypeptide having SEQ ID NO: 13 &lt; 400 &gt; 28 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 attaccacct 840 ggatggc aag cctgggtatt ggtagtgcag ttattccggg tccggttaaa 900 gttgcaagcg caagcgcaca ggttagcgca gcaagcaccg caccgcctag ctgtaccagc 960 accagcgcac cgagcaccgg tcattaa 987 &lt; 210 &gt; 29 &lt; 211 &gt; 843
&Lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; ORF was codon optimized and thus different from natural occuring DNA sequence. &Lt; 220 &gt; &Lt; 221 &gt; misc_feature &lt; 223 &gt; Artificial DNA sequence encoding polypeptide having SEQ ID NO: 14 &lt; 400 &gt; 29 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 &lt; 210 &gt;; 30 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 30
Ala Gly Asn Ser Ser Gly Gly1 5 &lt; 210 &gt; 31 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 31
Arg Thr Asp Pro Glu Thr1 5 &lt; 210 &gt; 32 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 32
Asp Ala Leu His Met Met His1 5 &lt; 210 &gt; 33 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 33
Ala Gly Asp Ser Ser Gly Gly1 5 &lt; 210 &gt; 34 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; ¢ 1). (7) 400 &gt; 34
Ala Gly Asp Ser Ser Leu Gly 15 &lt; 210 &gt; 35 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 35
Ala Gly Gin Ser Ser Gly Gly1 5 &lt; 210 &gt; 36 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDES &lt; 22 2 &gt; (1) .. (7) &lt; 400 &gt; 36
Ala Gly His Ser Ser Gly Gly1 5 &lt; 210 &gt; 37 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 37
Ala Gly Ser Ser Ser Gly Gly1 5 &lt; 210 &gt; 38 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 38
Ser Gly Asn ser ser Gly Gly1 5 &lt; 210 &gt; 39 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 39 ser Gly Asp Ser Ser Gly Gly1 5 &lt; 210 &gt; 40 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 40
Ser Gly Gin Ser Ser Gly Gly1 5 &lt; 210 &gt; 41 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 41
Ser Gly His Ser Ser Gly Gly1 5 &lt; 210 &gt; 42 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 42
Ser Gly Ser Ser Ser Gly Gly1 5 &lt; 210 &gt; 43 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 43
Arg Thr Asp Pro Glu Thr1 5 &lt; 210 &gt; 44 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 44
Arg Asp is Asp Pro Asp Thr1 5 &lt; 210 &gt; 45 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; ¢ 1). (7) 400 &gt; 45
Arg Giy Thr Asp Pro Giu Thr1 5 &lt; 210 &gt; 46 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 46
Arg Gly is Asp Pro Glu Thr1 5 &lt; 210 &gt; 47 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 47
Asp Ala Leu His Met Met His1 5 &lt; 210 &gt; 48 &lt; 211 &gt; 7
&Lt; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1) .. (7) &lt; 400 &gt; 48
Asp Ala Ser His Met Met His 1 5 &lt; 210 &gt; 49
&Lt; 211 &gt; & lt 11; 212 &gt; PRT &lt; 21B &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (11) &lt; 400 &gt; 49
Val val Ala Gly Asn Ser Ser Gly Gly Leu Leu1 5 10 &lt; 210 &gt; 50
&Lt; 211 &gt; & lt 11; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (11) &lt; 400 &gt; 50
Ile Val Ala Gly Asn Ser Ser Gly Gly Val Leu1 5 10 &lt; 210 &gt; 51
&Lt; 211 &gt; & lt 11; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (11) &lt; 400 &gt; 51
His Ala Arg Thr was Asp Pro Glu Thr Gly Glu1 5 10 &lt; 210 &gt; 52
&Lt; 211 &gt; & lt 11; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDES &lt; 22 2 &gt; (1). (11) &lt; 400 &gt; 52
His Met Arg Asp was Asp Pro Asp Thr Gly His1 5 10 &lt; 210 &gt; 53
&Lt; 211 &gt; & lt 11; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (11) &lt; 400 &gt; 53
His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn15 10 &lt; 210 &gt; 54
&Lt; 211 &gt; & lt 11; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (11) &lt; 400 &gt; 54
His Pro Asp Ala Leu His Met Met His Leu Phe1 5 10 &lt; 210 &gt; 55
&Lt; 211 &gt; & lt 11; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (11) &lt; 400 &gt; 55
Val Pro Asp Ala Leu His Met Met His Gin Phe1 5 10 &lt; 210 &gt; 56
&Lt; 211 &gt; & lt 11; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (11) &lt; 400 &gt; 56 val Per Asp Ala Ser His Met Met His Gin Ser1 5 10 &lt; 210 &gt; 57
&Lt; 211 &gt; & lt 21; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 57 Lys Arq Pro val Val val Ala Gly Asn ser Ser Gly Gly Leu Leu15 10 15
Ala Ala Trp Leu Ser20 &lt; 210 &gt; 58
&Lt; 211 &gt; & lt 21; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 58
Val Lys Arg Pro Val Val Ala Gly Asn ser Ser Gly Gly Val Leu15 10 15
Ala Ala Trp Leu ser20 &lt; 210 &gt; 59
&Lt; 211 &gt; & lt 21; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 59
Val Arg Arg Pro val Val val Ala Gly Asn Ser Ser Gly Gly val Leu15 10 15
Ala Ala Trp Leu ser20
&Lt; 210 &gt; & lt 60; 211 &gt; & lt 21; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 60
Val Lys Arg Pro val Val Val Ala Gly Asn ser Ser Gly Gly val Leu15 10 15
Ala Ala Trp Leu Ser20
&Lt; 210 &gt; & lt 61; 211 &gt; & lt 21; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 61 Leu Le Thr His His Ala Arg Thr l Asp Pro Glu Thr Gly Glu15 10 15
Leu Leu Gly Ala Leu20 &lt; 210 &gt; & lt 62; 211 &gt; 21
&Lt; 212 &gt; PRT &lt; 21B &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; ¢ 1). (21) 400 &gt; 62
Val Leu Leu Thr His His Met Asp Asp Asp Asp Asp Gly Gly His15 10 15
Leu val Gly Ala Leu 20 &lt; 210 &gt; 63
&Lt; 211 &gt; & lt 21; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 63
Val Leu Leu Thr His His Met Arg Gly Thr Asp Pro Glu Thr Gly Asn15 10 15
Leu Leu Gly Ala Leu20 &lt; 210 &gt; 64
&Lt; 211 &gt; & lt 21; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 64
Val Leu Leu Thr His His Asp Asp Ala Leu His Met Met His Leu Phe15 10 15
Leu Leu Gly Ala Leu20 &lt; 210 &gt; 65
&Lt; 211 &gt; & lt 21; 212 &gt; PRT &lt; 21B &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 65
Val Asp Tyr Gin ser His Pro Asp Ala Leu His Met Met His Leu Phe15 10 15
Asp Pro Ala Arg Tyr20
&Lt; 210 &gt; & lt 66; 211 &gt; & lt 21; 212 &gt; PRT &lt; 21B &gt; Artificial sequence &lt; 220 &gt; &Lt; 223 &gt; amino acid motif &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 66 val Asp Tyr Ala ser val Pro Asp Ala Leu His Met Met His Gin Phe15 10 15
Asp Pro Pro Arg Tyr20 &lt; 210 &gt; 67
&Lt; 211 &gt; & lt 21; 212 &gt; PRT &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &Lt; 221 &gt; PEPTIDE &lt; 222 &gt; (1). (21) &lt; 400 &gt; 67 val Asp Tyr Glu Ser val Pro Asp Ala s His Met Met His Gin Ser15 10 15
Ala Pro Ala Arg Tyr20

权利要求:
Claims (15)
[1]
1. Polypeptide for the hydrolytic cleavage of zearalenone and / or of zearalenone derivatives, characterized in that it has an amino acid sequence which has a degree of sequence identity to amino acid sequences +24 to +50, and / or +52 to +77, and / or +79 to +87, and / or +89 to +145, and / or +150 to +171, and / or +177 to +193, and / or +223 to +228, and / or +230 to +237, and / or +239 to +247, and / or +249 to +255, and / or +257 to +261, and / or +263 to +270, and / or +272 to +279, and / or +297 to +301, and / or +303 to +313, and / or +24 to 328, and / or +1 to +328 of a sequence SEQ ID NO: 1 of at least 70%, preferably at least 84%, more preferably at least 92%, and more preferably at least 98%, or a variant thereof, or contains a functional fragment thereof or a variant of the functional fragment.
[2]
2. Polypeptide according to claim 1, characterized in that the variant has a amino acid modification selected from the group substitution, deletion and insertion of one or more amino acids.
[3]
3. Polypeptide according to one of claims 1 or 2, characterized in that the enzyme has a specific activity of at least 0.01 U / mg, preferably at least 0.1 U / mg, in particular at least 1 U / mg, and / or a KM Value of the zearalenone hydrolytic cleavage of at most 50 μΜ, preferably at most 3.5 μΜ, especially at most 0.5 μΜ, and / or a kcar value of the hydrolytic cleavage of zearalenone of at least 0.05 s'1, preferably at least 0.6 s &. 1, in particular at least 5 s'1, and / or a vmax value of the hydrolytic cleavage of zearalenone of at least 0.00001μΜ'1 s'1, preferably at least 0.0001 μΜ'1 s'1, in particular at least 0.001 μΜ ' 1 s'1 owns.
[4]
A polypeptide according to any one of claims 1, 2 or 3, characterized in that the polypeptide is an α / β-hydrolase suitable for oxygen-independent and cofactor-free hydrolytic cleavage of the ester grouping of zearalenone and / or zearalenone derivatives in that it is an amino acid catalyzing the hydrolytic cleavage. Triad consisting of serine, an acidic amino acid selected from glutamic acid and aspartic acid, in particular aspartic acid and histidine, and that the catalytic triad is S128, D264 and H303, wherein the positioning relative to the sequence is represented by SEQ ID NO: 1.
[5]
5. Polypeptide according to claim 4, characterized in that in one of 3 amino acids before and 3 amino acids after the serine of the catalytic triad existing Sequenz¬ section, at least one polar amino acid selected from Y, Q, N, T, K, R, E, D and at least one non-polar amino acid selected from F, M, L, I, V, A, G, P.
[6]
6. Polypeptide according to one of claims 1 to 5, characterized in that it at least one mutation of the amino acid sequence with respect to SEQ ID NO: 1 at least one of the following positions: 22, 23, 25, 26, 27, 29, 31st , 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, 205 206,207,220,209,210,212,213,214,216,217,220,221,222,229,231,233,238,240,244,245,246,248,249,251,254 , 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.
[7]
7. Polypeptide according to claim 6, characterized in that it comprises at least one mutation of the amino acid sequence with respect to SEQ ID NO: 1 selected from: 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, 1118V, K119R, V123I, L132V, A141S, 1146V, I146L, A148G, A149V, A154P, P163T, A164T, Y165C, Y165H, V169I, L170R, A172G, A176M, A176V, Y180F, D182T, F183Y, 1190V, G191S, K194T, K194E, F196Y, V197C, V197R, E198R, E198S, K201D, K201G, P204S, P204A, A205S, K206P, A207M, M208A, Q209R, L210A, L210S, ΔΡ212, 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.
[8]
8. Polypeptide according to one of claims 1 to 7, characterized in that at least one of the following amino acid motifs selected from the group SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO : 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 67 is included.
[9]
9. Polypeptide according to one of claims 1 to 8, characterized in that at least one amino acid sequence selected from the group SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; or at least one zearalenone and / or zearalenone derivative hydrolyzing variant thereof, or at least one functional fragment thereof.
[10]
10. Polypeptide according to claim 9, characterized in that the polypeptide contains at least one conservative amino acid substitution in at least one position, and the conservative amino acid substitution is selected from substitutions of 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, Μ, 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 to 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, Η, K, R.
[11]
An isolated polynucleotide, characterized in that the nucleotide sequence encodes a polypeptide having a zearalenone and / or zearalenone derivative hydrolyzing property and encodes at least one polypeptide according to any one of claims 1 to 10 and / or a degree of sequence identity to at least a nucleotide sequence from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 having at least 70% and / or under mean stringency conditions with at least one nucleotide sequence to be selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29 and / or with a Partial sequence of at least 2 00 nucleotides, in particular of at least 100 nucleotides thereof and / or hybridized with a complementary strand of the nucleotide sequence or partial sequence thereof.
[12]
Additive characterized in that at least one polypeptide capable of hydrolytically cleaving zearalenone and / or zearalenone derivatives is contained in any of claims 1 to 10.
[13]
13. Additive according to claim 12, characterized in that in addition at least one inert carrier and optionally further constituents, such as vitamins and / or mineral substances and / or enzymes and / or further components for detoxification of mycotoxins are contained.
[14]
14. An additive according to any one of claims 12 or 13, characterized in that the polypeptide according to one of claims 1 to 10 in a concentration of at most 10,000 U / g, preferably at most 1,000 U / g, particularly preferably at most 100 U / g and Be More preferably at most 10 U / g is contained therein.
[15]
An additive according to any one of claims 12, 13 or 14, characterized in that the polypeptide according to any one of claims 1 to 10 is in encapsulated or coated form.

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法律状态:

优先权:

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ATA667/2013A|AT514775B1|2013-08-28|2013-08-28|Polypeptide for hydrolytic cleavage of zearalenone and / or zearalenone derivatives, isolated polynucleotide thereof and additive containing the polypeptide|ATA667/2013A| AT514775B1|2013-08-28|2013-08-28|Polypeptide for hydrolytic cleavage of zearalenone and / or zearalenone derivatives, isolated polynucleotide thereof and additive containing the polypeptide|

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PCT/AT2014/000164| WO2015027258A2|2013-08-28|2014-08-27|Polypeptide for the hydrolytic cleavage of zearalenone and/or zearalenone derivatives, isolated polynucleotide thereof, and additive containing polypeptide, use of said polypeptide and method|

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CN201910861330.2A| CN110527674A|2013-08-28|2014-08-27|Polypeptide, its polynucleotides of separation, and the additive comprising polypeptide, its purposes and method|

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ES19152160T| ES2837422T3|2013-08-28|2014-08-27|Procedure for the hydrolytic cleavage of zearalenone and / or zearalenone derivatives by means of a polynucleotide, as well as the use|

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PE2016000319A| PE20160243A1|2013-08-28|2014-08-28|POLYPEPTIDE FOR THE HYDROLYTIC CLEAVAGE OF ZEARALENONE AND / OR DERIVATIVES OF ZEARALENONE, POLYNUCLEOTIDE ISOLATED FROM THE SAME AND POLYEPTIDE CONTAINING ADDITIVE, USE AND PROCEDURE OF THE SAME|

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IL244144A| IL244144A|2013-08-28|2016-02-16|Polypeptide for hydrolytic cleavage of zearalenone and/or zearalenone derivativesf as well as a polypeptide containing an additive and use of same|

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DO2016000051A| DOP2016000051A|2013-08-28|2016-02-18|POLYPEPTIDE FOR THE HYDROLYTIC EXCISION OF ZEARALENONE AND / OR DERIVATIVES OF ZEARALENONE, POLYNUCLEOTIDE ISOLATED FROM THE SAME AND POLYEPTIDE CONTAINING ADDITIVE, USE AND PROCEDURE OF THE SAME.|

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NI201600028A| NI201600028A|2013-08-28|2016-02-19|POLYPEPTIDE FOR THE HYDROLYTIC EXCISION OF ZEARALENONE AND / OR DERIVATIVES OF ZEARALENONE, POLYNUCLEOTIDE ISOLATED FROM THE SAME AND POLYEPTIDE CONTAINING ADDITIVE, USE AND PROCEDURE OF THE SAME.|

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US15/054,232| US10076125B2|2013-08-28|2016-02-26|Polypeptide for hydrolytic cleavage of zearalenone and/or zearalenone derivatives, isolated polynucleotide thereof as well as a polypeptide containing an additive, use of same as well as a process|

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ECIEPI201612693A| ECSP16012693A|2013-08-28|2016-03-28|POLYPEPTIDE FOR THE HYDROLYTIC CLEAVAGE OF ZEARALENONE AND / OR DERIVATIVES OF ZEARALENONE, POLYNUCLEOTIDE ISOLATED FROM THE SAME AND POLYPEPTIDE CONTAINING THE ADDITIVE, USE AND PROCEDURE OF THE SAME|

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HK16114603A| HK1226101A1|2013-08-28|2016-12-22|Polypeptide for the hydrolytic cleavage of zearalenone and/or zearalenone derivatives, isolated polynucleotide thereof, and additive containing polypeptide, use of said polypeptide and method|

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US16/161,266| US10779556B2|2013-08-28|2018-10-16|Polypeptide for hydrolytic cleavage of zearalenone and/or zearalenone derivatives, isolated polynucleotide thereof as well as a polypeptide containing an additive, use of same as well as a process|

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JP2018244394A| JP2019088287A|2013-08-28|2018-12-27|Polypeptide for hydrolyzation of zearalenone and/or zearalenone derivative, isolated polynucleotide thereof, polypeptide containing additive, use of polypeptide and method|

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CL2019001365A| CL2019001365A1|2013-08-28|2019-05-20|Polypeptide for hydrolytic cleavage of zearalenone and / or derivatives of zearalenone, polynucleotide isolated therefrom and additive containing the polypeptide, use and process thereof. |

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JP2020071453A| JP2020127407A|2013-08-28|2020-04-13|Polypeptides for hydrolyzing zearalenone and/or zearalenone derivatives, isolated polynucleotides therefor and polypeptide-containing additives, use of the polypeptides and methods|

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AU2020203904A| AU2020203904B2|2013-08-28|2020-06-12|Polypeptide for the hydrolytic cleavage of zearalenone and/or zearalenone derivatives, isolated polynucleotide thereof, and additive containing polypeptide, use of said polypeptide and method|

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