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    • 专利摘要:
    In a polypeptide for enzymatic detoxification of zearalenone, the polypeptide is a cyclohexanone monooxygenase converting the ketone group at position 7 of zearalenone to an ester group having an amino acid sequence selected from the group of SEQ ID NOs. 1, 2 or 3; or a functional variant thereof, wherein between the functional variant and at least one of the amino acid sequences is a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity.
    公开号:AT516457A2
    申请号:T538/2015
    申请日:2015-08-17
    公开日:2016-05-15
    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 enzymatic detoxification of zearalenone, an isolated polynucleotide encoding a zearalenone detoxifying polypeptide, a transgenic host cell for producing a zearalenone detoxifying monooxygenase, an enzymatic detoxification additive of zearalenone, and a use of the invention and to a method for the enzymatic detoxification of zearalenone.
    Mycotoxins are secondary metabolites produced by filamentous fungi. An important representative of these is the world-wide zearalenone (ZEN), formerly known as F-2 toxin, which is produced by a variety of fish / 'en fungi. These fungi infect, among other crops, such as various cereals, which usually occurs before the harvest of the fungus, wherein the fungus growth or mycotoxin production before or inappropriate storage can also be done after harvesting. The FAO estimates that 25% of agrarian products worldwide are contaminated with mycotoxins, leading to significant economic losses. In a recent global study, a total of 23,781 samples were analyzed from January 2009 to 2011, with 81% positive for at least one mycotoxin and 45% positive for ZEN. ZEN has been found in all regions of the world as well as in all tested cereal and feed categories, such as corn, soybean meal, wheat, wheat bran, DDGS (dry-mash), and ready-to-feed mixes at a frequency of up to 100%. ZEN is a non-steroids, estrogen, macrocyclic, polyketide-metabolically synthesized lactone having the structural formula:
    and the IUPAC nomenclature name (2E, 11S) -15,17-dihydroxy-11-methyl-12-oxabicyclo [12.4.0] octadeca-1 (18), 2,14,16-tetraene-7,13-dione. There are different nomenclatures for ZEN and ZEN metabolites, with the nomenclature of Metzler (2011, Mycotox. Res., 27: 1-3) being used as far as possible in this document. In addition to ZEN, a variety of ZEN derivatives are also found in nature, which are formed by enzymatic or chemical modifications of ZEN. Furthermore, ZEN metabolites are formed, inter alia, in the human or animal organism. ZEN as well as ZEN derivatives such as α-ZEL or β-ZEL may also be used in processed food or feed such as e.g. Bread, beer or dry mash can be detected.
    Although ZEN has relatively low acute toxicity and an oral LD50 of up to 20,000 mg / kg of body weight, subacute and / or subchronic toxic effects, such as teratogenic, carcinogenic, immunosuppressive and estrogenic effects in animals or animals, may be associated with prolonged intake People occur. ZEN binds to the estrogen receptor and can cause hormonal imbalances, therefore detoxification or detoxification of zearalenone is generally understood as a reduction of the metabolite formed compared to ZEN.
    Ingestion of ZEN-contaminated feed leads to developmental disorders in mammals, with pigs, and particularly young animals, being extremely sensitive to ZEN. ZEN concentrations in the feed above 0.5 ppm lead to developmental disorders, e.g. Concentrations above 1.5 ppm may lead to hyperestrogenicity in pigs and concentrations of 12 ppm ZEN have been attributed to miscarriage in cattle. Since zearalenone is absorbed rapidly via the mucous membranes, in particular via the stomach but also via the oral mucosa, an immediate and above all quantitative detoxification is necessary. Just 30 minutes after oral administration of ZEN, this can be detected in the blood. To achieve as complete a detoxification as possible, the use of isolated enzymes against microorganisms has proved to be advantageous, since they have a higher specific activity or a faster action. Due to the harmful effects of ZEN, there are binding ZEN food limits in the European Union and recommendations for ZEN caps in feed (EC NO: 1881/2006).
    The primary strategy to reduce ZEN contamination of food or feed is to limit fungal growth, for example, by following good agricultural practice. This includes, among other things, that seed is free of pests and fungal infestation, or that agricultural waste products are removed from the field in time. 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. Likewise, contaminated material should be removed before further processing by fungal infestation. Despite this list of 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 maize samples were contaminated with ZEN were.
    Other ways of removing ZEN from feed or food are the adsorption or transformation of mycotoxin. For this, it is necessary that the binding of the mycotoxin to the adsorbent is strong and specific over a wide pH range and remains stable throughout the gastrointestinal digestive process. Although some non-biological adsorbents, such as e.g. Activated carbon, silicates or synthetic polymers, such as cholestryamine, can be used efficiently for aflatoxins, their use is limited to other mycotoxins. The main disadvantage of adsorbents is the non-specific binding of other molecules, which are partially essential for nutrition. Biological adsorbents, such as e.g. Yeast or yeast extracts are also described in the literature, but are similarly limited as non-biological adsorbents.
    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 extrusion and treatment with oxidizing agents, e.g. be reduced by 83.9% for 16 h at 80 ° C with 10% hydrogen peroxide solution. The use of extrusion processes and oxidants such as ozone or hydrogen peroxide in feed and food production is limited due to high cost, quality loss, low efficiency and low specificity.
    EP 0 938 575 B1 discloses ZEN-degrading properties of bacteria of the genus Rhodococcus and Nocardia, in particular R. globerulus, R. erythropolis and N. globerula.
    Vekiru et al. (Appl and Environ Microb., 2010, 76, 7, 2353-2359) describes the biotransformation of ZEN into the less estrogen metabolite ZOM-1 by the microorganisms Trichosporon mycotoxinivorans.
    The terms used in the following are taken from the technical language and are each used, unless otherwise stated, in the conventional meanings. Thus, the term "polynucleotide" refers to any type of genetic material of all lengths and 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, as well as 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 a single, specific base or amino acid in the sequence of the polynucleotide or polypeptide.
    The present invention now aims to provide a polypeptide with which it is possible to convert zearalenone quickly and reliably into a zearalenone derivative, the toxicity and estrogenic effect of which has been reduced so far that it is safe for the respective end users in animal feed or food products may remain.
    To solve this object, the present invention is essentially characterized in that the polypeptide is a cyclohexanone monooxygenase converting the ketone group at position 7 of zearalenone into an ester group having an amino acid sequence selected from the group of SEQ ID NOs. 1, 2 or 3; or a functional variant thereof, wherein between the functional variant and at least one of the amino acid sequences a sequence identity is at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity. Cyclohexanone monooxygenases are enzymes belonging to group I of the EC classification which catalyze the incorporation of an oxygen atom from O 2 into the corresponding substrate, in this case in zearalenone. More specifically, in the case of zearalenone, an oxygen atom is incorporated at position 7 of the zearalenone ring structure, yielding a less toxic zearalenone derivative (iZOM). Surprisingly, it has been found that this detoxification proceeds in a single enzymatic reaction step and that the reaction product iZOM despite the continued presence closed-ring structure and the carbon-oxygen double bond at position 7 by a factor of about 100 lower toxicity and has almost no estrogens Effect shows more. A particularly complete conversion of zearalenone into the less toxic derivative iZOM succeeds when the polypeptide has an amino acid sequence selected from the group of the sequence ID Nos. 1, 2 and 3 or a functional variant thereof, wherein between the functional variant and at least one of the amino acid sequences at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity exists. By the presence of at least one such conserved amino acid sequence, such as the sequences of ID Nos. 1, 2 and 3 or a functional variant thereof, it is possible to provide a polypeptide which, in addition to the rapid and complete conversion of ZEN, also has a particularly high activity value compared to hitherto known ZEN-transforming polypeptides.
    To date, it has been thought that efficient detoxification of ZEN requires cleavage of the lactone ring system. Surprisingly, however, it has been found that an effective detoxification can also be achieved by the conversion of the keto group at position 7 of zearalenone into an ester group, in which rearrangement a cleavage of the ring system does not take place.
    Here, the cyclohexanone monooxygenase may be selected to be a Baeyer-Villiger monooxygenase. The use of a Baeyer-Villiger monooxygenase ensures that the reaction can take place exclusively at the desired position 7, since only these are for a so-called Baeyer-Villiger oxidation, i. a conversion of a ketone into an ester by incorporation of an oxygen atom is accessible.
    Due to the specific structure of the zearalenone molecule and in particular due to the keto group present at position 7 of the zearalenone molecule, targeted incorporation of the desired oxygen atom at position 7 of the zearalenone, according to the invention, by means of a cyclohexanone monooxygenase, which is a subgroup of Baeyer Villiger mono-oxygenase (D. Pazmino and M. Fraaije, Future Directions in Biocatalysis, 2007, 107-128). Surprisingly, the ester group resulting from this incorporation has the effect that the cyclic metabolite (iZOM) formed has a significantly lower toxicity than zeralenone.
    By the term "sequence identity" is meant a percentage sequence identity. For amino acid sequences and nucleotide sequences, the sequence identity with each other can be determined visually, but preferably be calculated with a computer program. As a reference sequence, the amino acid sequences having the sequence ID Nos. 1, 2 and 3 as well as the nucleotide sequences with the sequence ID Nos. 3, 4 and 5 defined. The sequence comparison is also carried out within sequence sections, whereby the section is understood as a continuous sequence of the reference sequence. The length of the sequence sections for nucleotide sequences is normally 18 to 600, preferably 45 to 200, more preferably 100 to 150 nucleotides. The length of the sequence sections for peptide sequences is usually 3 to 200, more preferably 15 to 65, most preferably 30 to 50 amino acids. There are a large number of commercially available or freely available bioinformatic programs that can be used for homology determination and are continuously being further developed. Examples include GCG Wisconsin Bestfit package (Devereux et al., 1984), BLAST (Altschul et al., 1990), or BLAST 2 (Tatusova and Madden, 1999). Due to the different setting options of these algorithms, it is possible that these result in the same input sequences to different results, which is why the search algorithm and the associated setting must be defined. In the present case, the sequence identity was determined using the program NCBI BLAST (Basic Local Alignment Search Tool), in particular with BLASTP for polypeptides and BLASTN for polynucleotides, in the version of October 20, 2014, which is available on the homepage of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). Thus, it is possible to compare two or more sequences with each other according to the algorithm of Altschul et al., 1997 (Nucleic Acids Res., 25: 3389-3402). Here, the programs were used in the version of 15 May 2013. The basic settings were used as program settings, but in particular for the amino acid sequence comparison: "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"; as well as for the nucleotide sequence comparison Word Size: 11; Expect value: 10; Gap costs; Existence = 5, extension = 2; Filter = low complexity activated; Match / Mismatch Scores: 2, -3; Filter String: L; m.
    The terms "functional polypeptide variant" or "functional variant" refer, on the one hand, to "allelic variants" of the polypeptide and to "functional fragments" of the polypeptide, and, on the other hand, to a "modified polypeptide", wherein the enzymatic function is substantially unchanged compared to Polypeptide with the sequence ID no. 1 is. The term "allelic variant" refers to a polypeptide produced by naturally occurring mutation (s) of the nucleotide sequence which causes a change in the amino acid sequence, the enzymatic function of which is unaffected. The term "functional fragment" refers to a portion or a partial sequence of a polypeptide or a portion or a partial sequence of a functional variant thereof, wherein the enzymatic function is substantially retained. "Modified polypeptides" may be C- or N-terminal fusion proteins or deliberately mutated polypeptides, mutations being by substitution, insertion or deletion of at least one amino acid, in particular by site-directed mutagenesis or random mutagenesis, recombination and / or any other protein engineering "Process can be obtained, wherein the enzymatic function is substantially maintained. The terms substitution, insertion and deletion are used in the usual way in genetic engineering and the skilled person meaning. An enzymatic function is then substantially retained if the enzymatic reaction mechanism remains unchanged, i. the keto group at position 7 of the mycotoxin ZEN is converted to a corresponding ester group as shown in the above reaction mechanism and the specific residual activity is at least 5%, preferably at least 10%, especially at least 50%, based on the original polypeptide.
    In the polypeptides having the amino acid sequences with the sequence ID Nos. 1 and 3 are functional allelic variants, the sequences each originating from different microorganisms. This is clearly evident from close affinity, as measured by percent sequence identity, as well as the fact that the polypeptides act on ZEN through the same mechanism. Polypeptides here are each cyclohexanone monooxygenases and the terms "polypeptide", "monooxygenase" and "cyclohexanone monooxygenase" are used synonymously and interchangeably. The polypeptide having the sequence ID-No. 1 is completely contained in the sequence with SEQ ID NO: 2, but the polypeptide having the sequence ID no. 2, an N-terminus extended by 28 amino acids.
    By the term "detoxification" or "detoxify" is meant the reduction of the estrogenic activity of ZEN. In this case, the measurement of the estrogenic activity preferably takes place according to the method described in the exemplary embodiments. Other methods can also be used, the crucial factor being always the reduction of the estrogenic activity of ZEN by its enzymatic conversion to iZOM. Measured with the estrogenicity assay described in the working examples, iZOM has an estrogenic activity about 100 times lower than that of ZEN.
    The present invention further aims to provide an isolated polynucleotide capable of producing a polypeptide for the rapid and reliable detoxification of ZEN.
    To achieve this object, the invention is essentially characterized in that the isolated polynucleotide has a nucleotide sequence coding for a polypeptide which is a monooxygenase converting the keto group at position 7 of zearalenone to an ester group, and / or a degree of sequence identity to at least one nucleotide sequence selected from the group of SEQ ID Nos. 4, 5 and 6 of at least 60%. Here, the nucleotide sequence under moderate stringency conditions with at least one of the group of the sequence ID Nos. 4 to 6 nucleotide sequence and / or hybridize with a partial sequence thereof of at least 200 nucleotides, in particular of at least 100 nucleotides and / or with a complementary strand of the nucleotide sequence or partial sequence thereof. Expression of such polynucleotide can ensure that the resulting polypeptide transforms ZEN into iZOM.
    Nucleotide sequences to be expressed, in particular their triplets (codons) are usually changed depending on the host cell, so that the codon bias is optimized depending on the host cell. This results in that also polynucleotides with a degree of sequence identity of well below 80%, but also below 70% or below 60% can encode the same polypeptide. The sequence comparison for determining the degree of sequence 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 is usually 15 to 600 for nucleotide sequences.
    Using the present isolated nucleotide sequences or sequence sections, it is possible to generate nucleic acid probes which have a length of usually at least 15, 30 or 40 nucleotides. By means of such designated as probes nucleotide sequences, which are usually additionally labeled, e.g. by means of 3H, 32P, 35S, biotin or avidin, nucleotide sequences encoding polypeptides having ZEN-degrading activity can be identified using standard methods. As starting material for the identification of such sequences, for example, DNA, RNA or cDNA of individual microorganisms, genomic DNA libraries or cDNA libraries can be used. For nucleotide sequences of at least 100 nucleotides in length, mean stringency conditions are defined as prehybridization and hybridization at 42 ° C in 5X NaCl-provided Na-EDTA buffer (SSPE, 0.9M NaCl, 60mM NaHaPCU, 6mM EDTA) containing 0 , 3% sodium dodecyl sulfate (SDS), 200 pg / ml sheared and denatured salmon sperm DNA and 35% formamide, followed by standard Southern blotting conditions, with the substrate ending up with 15x 15x 2x sodium chloride citrate buffer (SSC , 300 mM NaCl and 30 mM trisodium citrate, 0.2% SDS) at 55 ° C. For nucleotide sequences of 15 nucleotides to 100 nucleotides in length, mean stringency conditions are defined as prehybridize and hybridize in the buffer consisting of 0.9 M NaCl, 0.09 M Tris-HCl pH = 7.6, 6 mM EDTA, 0.5%. NP-40, 1-fold Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium dihydrogen phosphate, 0.1 mM ATP and 0.2 mg / ml yeast RNA, at a temperature which is 5 ° C to 10 ° C below the calculated Tm), prehybridized and hybridized, Tm being determined by calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48: 1390). The experiment is then continued under standard Southern blot conditions (J. Sambrook, E.F. Fritsch and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York). The support material is finally washed once for 15 minutes with 6X SCC buffer containing 0.1% SDS and twice for 15 minutes with 6X SSC buffer at 5 ° C to 10 ° C below the calculated Tm.
    Another object of the invention is to provide a transgenic host cell for the production of a zearalenone detoxifying monooxygenase.
    To achieve this object, the invention is substantially characterized in that the host cell is a polynucleotide having a nucleotide sequence coding for at least one polypeptide which is a cyclohexanone monooxygenase converting the ketone group of zearalenone at position 7, and / or having a degree of sequence identity to at least one nucleotide sequence selected from the group of SEQ ID Nos. 4, 5 and 6 of at least 60%, wherein the polynucleotide is chromosomally integrated or extrachromosomal, and the host cell is a prokaryotic or eukaryotic cell, in particular a Yeast or plant cell, and that the host cell optionally additionally overexpressing an enzyme which recycles a cofactor required for the oxygenase, in particular converts NADP + or NAD + into NADPH or NADH. NADP + or NAD + in NADPH or NADH converting enzymes and cofactors required for this purpose, the state of the art, for example in Torres Pazmino et al. 2008 (Angew Chem. 47 (12); 2275-8). Preferably, the NADP + or NAD + in NADPH or NADH converting enzymes are chosen so that they can be used easily in the food and / or feed industry, in particular that they are compatible with the relevant food and / or feed regulations. For example, as NADP + in NADPH converting enzyme, an NADPH-dependent xylose reductase, in particular from Pichia stipitis can be used, which uses as a substrate approved as a feed additive xylitol. Alternatively, a NAD (P) H converting mannitol dehydrogenase from Lactobacillus sp. can be used, which can be used as a substrate approved as a feed additive mannitol.
    Preferably, it is possible to prepare or express the polypeptide for detoxification of ZEN and an enzyme capable of recycling the cofactor as a fusion protein. As a result, the two enzymatic activities can be produced in a common process, in particular a biotechnological up- and downstream process. Furthermore, application of such a fusion protein ensures that the cofactor circulating between the two oxidation states remains in close proximity to the polypeptide for detoxification of ZEN.
    Finally, the present invention aims to provide an additive with which rapid and reliable detoxification, defined as reduction of the estrogenic activity, of ZEN in a defined or complex matrix, for example in feed or foodstuffs, is achieved.
    To solve this problem, a zearalenone detoxifying additive is provided, wherein the additive contains at least one polypeptide which is a cyclohexanone monooxygenase converting the ketone group at position 7 of zearalenone to an ester group, which cyclohexanone monooxygenase has an amino acid sequence selected from Sequence ID- Nos. 1, 2 and 3 or a functional variant thereof, wherein a sequence identity between the functional variant and at least one of the amino acid sequences is at least 60% and optionally contains at least one adjuvant. With such an additive, in a food or feed, the one-step biochemical conversion of ZEN to a zearalenone derivative, iZOM, whose toxicity is a factor of about 100 lower than that of zearalenone, succeeds directly.
    According to a preferred embodiment of the invention, the additive is designed so that the at least one excipient from the group of inert carriers, vitamins, minerals, enzymes, other components for detoxification of mycotoxins and cofactors, in particular NADPH and / or NADH is selected. The use of such an additive ensures that the vast majority of the amounts of ZEN contained, for example, in food or feed are certainly oxidized to iZOM, thereby ensuring that it has a deleterious effect on the organism of a subject receiving that feed or food absent.
    In this case, the additive in addition to a polypeptide according to the invention may also contain, for example, an enzyme preparation as adjuvant, in which further at least one enzyme is involved, which is involved for example in the degradation of proteins, such. Proteases, or involved in the metabolism of starch or fiber or fat or glycogen, such as e.g. Amylases, cellulases or glucanases, and for example hydrolases, lipolytic enzymes, mannosidases, oxidases, oxidoreductases, phytases, xylanases and / or combinations thereof.
    Further possible auxiliaries which can be used according to 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 other than zearalenone, such as a mycotoxin detoxifying enzyme, such as e.g. Aflatoxin oxidase, ergotamine hydrolases, ergotamine amidases, ochratoxin amidases, fumonisin carboxylesterases, fumonisin amino-transferases, aminopolyol amine oxidases, deoxynivalenol epoxide hydrolases; and / or at least one mycotoxin-detoxifying microorganism and / or at least one mycotoxin-binding component, for example microbial cell walls or inorganic materials, such as bentonites.
    According to the invention, the polypeptide is contained in the additive in a concentration which enables ZEN to be rapidly and in particular already before its absorption by the body, of a contaminated feed or food consuming subject, in particular of a mammal, into or significantly less toxic metabolite to convert iZOM.
    Here, the polypeptide may be present in the composition in encapsulated or coated form, with standard encapsulation or coating methods, such as e.g. in WO 92/12645 described, can be used. By encapsulating or coating it is possible to transport the polypeptide without change, in particular without degradation and damage to its site, so that only after dissolution of the protective cover, for example in the digestive tract of animals, the polypeptide begins to act, creating an even more targeted, faster and more complete transformation of ZEN can also be achieved in the acidic, protease-rich and anaerobic environment. Furthermore, the encapsulation or coating also increases the temperature stability of the polypeptide in the additive.
    The encapsulated or coated polypeptide can be further processed into premixes with adjuvants customary in the feed industry.
    The present invention further aims at a use of the additive for detoxification of zearalenone in feed, in particular for pigs, poultry and
    aquaculture; in food; or in dry sludge. By using the additive according to the invention, it is possible to oxidize and thus detoxify the ZEN contained in the food or feed or in dry pasture by converting the keto group at position 7 into an ester group, such detoxification already taking place at low polypeptide concentrations in contaminated feed - or food succeeds.
    In this case, the polypeptide is in a range of about 1 g to about 500 g per ton of feed or food or in dry pasture, the encapsulated or coated polypeptide in a range of about 20 g to about 3000 g, the premix in a range of about 200 g to about 10 kg or Zusatzsoff in a range of about 5 mg to about 10 kg before. This will allow the ZEN present in feed or food, which can be present therein up to a concentration of 10,000 pg / kg, to be converted to iZOM for the most part.
    If the additive is used in liquefaction processes of starch, in saccharification processes, in biogas processes or in fermentation processes, for example in the mashing or fermentation process of bioethanol production, it can be ensured that ZEN can be used in the products used for or from the processes, for example Dry fume or starch is converted to iZOM, leaving no deleterious amounts of ZEN intact.
    The present invention further aims to provide a method enabling rapid and reliable conversion of ZEN to a non-toxic derivative, iZOM.
    To solve this object, the method is carried out so that zearalenone with at least one keto group at position 7 of the Zearalenions converting into an ester group, an amino acid sequence selected from the group of the sequence ID Nos. 1, 2 or 3 or a functional variant thereof and having a sequence identity between the functional variant and at least one of the amino acid sequences of at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% cyclohexanoin monooxygenase optionally at least one further enzyme, which is reduced by the enzymatic transformation of the oxidized cofactor, in particular NADP + or NAD +, in NADPH or NADH is brought into contact with water and optionally additionally with at least one excipient. The one-step enzymatic reaction not only ensures rapid incorporation of the oxygen atom, which forms the ester group together with the keto group in position 7, but above all ensures that the reaction is complete and zearalenone is completely converted into the less toxic iZOM by about a factor of 100 becomes. NADP + or NAD + can be converted or recycled into NADPH or NADH by the enzymatic systems already described. When using a polypeptide having an amino acid sequence selected from the sequence ID Nos. 1, 2 and 3, a particularly complete transformation, in particular a particularly complete detoxification of zearalenone is observed.
    The method according to the invention, as corresponds to a further development of the invention, can be used both in an additive per se and in a transgenic host cell, a transgenic plant or in seed and leads to consistent good results and in particular complete transformation of zearalenone, regardless of the site of use in iZOM.
    The method may be performed by mixing the polypeptide or additive with a feed or food contaminated with zearalenone, contacting the resulting mixture with moisture, and subjecting the polypeptide or additive to contamination in the contaminated feed or food Food containing zearalenone detoxi-fied. In wet foods or foods such as mash or porridge, the conversion of zearalenone to iZOM will take place prior to ingestion in the feed or food, thereby ensuring that the harmful effects of zearalenone on humans and animals are largely eliminated. In this case, moisture is understood to mean the presence of water or water-containing liquids, including also e.g. Saliva or other fluids present in the digestive tract fall.
    The inventive method may also be conducted so that the feed or food is pelleted prior to oral ingestion.
    According to a development of the invention, the method is carried out so that at least 70%, preferably at least 80%, in particular at least 90% of the zearalenone are detoxified. Transformation and detoxification are synonymous herein. This may prevent subacute and / or subchronic toxic effects, such as teratogens, carcinogens, estrogens and immunosuppressive effects in animals or humans.
    The invention will be explained in more detail with reference to embodiments and drawings. In these shows:
    Fig. 1 shows the partial conversion of ZEN in the negative control by S. cerevisiae.
    2 shows the conversion of ZEN in the experimental batch by S. cerevisiae which the polypeptide with the sequence ID no. 1 expressed.
    3 shows the chemical structure of iZOM.
    Figure 4 shows the estrogenic activity of ZEN (Figure 4A) as well as iZOM (Figure 4B).
    Unless otherwise specified, all molecular biological or microbiological work has been carried out using standard methods (Methods in Enzymology, Volume 194, Pages 3-933 (1991); Guide to Yeast Genetics and Molecular Biology. Edited by Christine Guthrie, Gerald R Fink, ISBN: 978-0-12-182095-4, J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring Harbor).
    Example 1: Formation of iZOM from ZEN in a one-step reaction
    The yeast strain YZGA515 (Saccharomyces cerevisiae modified by inactivation of the ABC transporter gene PDR5 (Poppenberger et al., 2003, J. Biol. Chem., 278 (48) 47905-14) for improved uptake of zearalenone (ZEN)) was analyzed by standard methods with the Plasmid pCS57 transformed. pCS57 is a plasmid derived from the yeast expression plasmid pADH-FW (Mitterbauer et al 2002, Appl Environ Microbiol, 68 (3), 1336-1346) with LEU2 as the selection marker and ADH1 promoter.
    In the experiment, the yeast strain YZGA515 was transformed with the pCS57 vector, which additionally contained the polynucleotide with the sequence ID No. 4 as BamHI-XhoI fragment after the strong constitutive ADH1 promoter (alcohol dehydrogenase 1). This allowed the formation of the polypeptide of Sequence ID No. 1.
    As a negative control, the yeast strain YZGA515 was transformed with the empty vector (pADH-FW without the polynucleotide with the sequence ID No. 4).
    The transgenic yeasts (experimental and negative control) were grown in SC-LEU medium (Sherman 1991, Methods Enzymol., 194, 3-21), centrifuged off and resuspended in fresh SC-LEU medium at a concentration of 2 mg / L ZEN, in both cases a cell density of OD600 = 4 was set. After various incubation times, samples were taken, which were mixed with 1 volume of methanol, thereby stopping the intracellular processes. By centrifugation, the cell suspension was clarified and the resulting supernatants used for measurement by HPLC-MS. The starting substance zearalenone (ZEN) was measured as described above, as well as the transformation product ZOM-1 formed by Trichosporon mycotoxinivorans (Vekuri et al., 2010, Appl. Environ. Microbiol., 76 (7) 2353-9).
    In the negative control transformed with the empty vector no ZOM-1 and in particular no iZOM was formed. The corresponding HPLC analysis data are shown in Figure 1, where the y-axis indicates nanograms per milliliter (ng / ml) and the x-axis indicates the time in hours (h).
    In contrast, zearalenone was significantly faster transformed in the experimental approach and it was a new metabolite, with the expected mass of Vekiru et al. (2010) postulated Intermdiates, identified below called iZOM. In addition, ZOM-1 was also detectable, which is a clear indication that hydrolysis of iZOM to ZOM occurs to a slight extent. The corresponding HPLC analysis data are shown in FIG. Compared to the negative control, a much smaller amount of ZEN was measured, with the y-axis indicating nanograms per milliliter (ng / ml) and the x-axis indicating the time in hours (h).
    In a further experimental approach, the metabolite iZOM was additionally measured quantitatively. Table 1 shows the molar metabolic rate of ZEN. It can clearly be seen that the majority of the zearaleon (ZEN) used was converted into iZOM after only 6 hours. The small deficit in the balance sheet can be explained by the formation of further metabolites as well as the methodically induced analytical blurring. These results clearly demonstrate that the polypeptide of SEQ ID NO: 1 is capable of converting ZEN into the metabolite iZOM.
    Table 1: Molar Assessment of the ZEN Metabolism in the Experimental Approach ~ Time ZEN ZOM-1 iZOM " [h] [nM] [nM] [nM] 0 3364 0 0 6 172.1 0 2163.1 24 4.8 214.8 2068.2
    Example 2: Identification of the Chemical Structure of iZOM
    The chemical structure of the metabolite iZOM (Figure 3) was determined by NMR measurements of the isolated and purified metabolite. For the measurement of the NMR spectra, a sufficient amount of iZOM was prepared from several liters of the culture filtrate of a yeast culture (SC-LEU as described in Example 1). By solid phase extraction followed by preparative HPLC iZOM was purified.
    On the one hand, the conversion of ZEN to iZOM can be followed with the aid of the isolated reference substance for iZOM (see Example 1) and, on the other hand, the chemical structure can be elucidated by means of NMR.
    The NMR spectra for identification of iZOM were obtained in a CD30D solution with a Bruker Avance DRX-400 FT-NMR spectrometer at room temperature (20 ° C) with a 5 mm inverse broad z-gradient probe head. The chemical shifts were established on the basis of residual solvent resonance (3.31 ppm for 1H NMR, 49.15 ppm for 13C NMR). All pulse programs were taken from the Bruker Software Library. The NMR data were evaluated using TopSpin 1.3 (Bruker BioSpin GmbH). Complete structure determination and assignment was performed based on 1H, 13C-APT, 1H1H correlation spectroscopy (COZY), 1H13C heteronuclear single-quantum correlation (HSQC), and 1H13C heteronuclear multi-band correlation spectra (HMBC).
    The NMR spectroscopic data from iZOM show two significant differences compared to ZEN. These confirmed the transformation of the keto group into an ester group. The differences are essentially the shift of the carbonyl carbon atom from 212 ppm in ZEN (characteristic of a ketone) to 175 ppm (characteristic of a carboxylic acid derivative) and the displacement of the adjacent CH2 group of 36 ppm and 2.90 / 2.30 ppm (position 8 in ZEN) to 65 ppm and 4.20 / 4.05 (position 9 in iZOM) for 13C and 1H, respectively, caused by the adjacent oxygen atom. In contrast to the open ring of ZOM1, the macrocyclic ring in iZOM is still intact, as evidenced by a long-distance correlation between C7 and H9. The NMR data are given in Table 2. The chemical structure of iZOM is shown in FIG.
    Table 2: 1H and 13C NMR data to identify iZOM.
    Position 1H 13C δ (ppm) Multiplicity, J (Hz) δ (ppm) 1: 172.9 3 5.14 m 74.2 3-CH3 1.36 d, 6.2 20.2 4 1.65-1.80 m 36.5 5 1.90,1.76 m 23.1 6 2.30-2.45 m 35.8 7 - 175.4 9 4.18,4.05 m 64.6 10 1.75-1.90 m 28.9 11 2.30-2.50 m 30.8 12 5.93 ddd, 15.4, 8.3, 6.1 132.4 13 7.00 d, 15.4 133.2 14 6.40 d, 2.2 109.6 15 - 165.0 16 6.19 d, 2.2 103.3 17 - 165.6 18 - 104.9 19 - 143.9
    Example 3: Significantly reduced estrogenic activity of iZOM compared to ZEN
    The purified metabolite iZOM, as described in Example 2, was used to determine its toxicity expressed in estrogenic activity and to compare it to zearalenone.
    To measure the estrogenic activity, a reporter yeast strain prepared specifically for this purpose was used, YZHB817 (Bachmann, H .: Phenotypic detection of zearalenone in Saccharomyces cerevisiae, Master thesis BOKU Vienna, 2003). This strain is derived from Yeast-two hybrid strain PJ69-4a (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4- (deleted) gal80 (deleted) LYS2 :: GAL1-HIS3 GAL2-ADE2 met2 :: GAL7- lacZ) (James et al., 1996, Genetics; 144 (4), 1425-1436). PJ69-4a was further developed by the disruption of the ABC transporters PDR5 and SNQ2. Strains with this double mutation pdr5 and snq2 show particularly high ZEN uptake (Mitterbauer et al., 2003, Appl., Environ., Microbiol., 69 (2), 805-811). This strain was then transformed with an expression vector (pTK103) which elicits the production of a fusion protein containing the hormone-dependent activation domain of the human estrogen receptor alpha and the DNA binding domain of the yeast Gal4 protein around strain YZHB817.
    In the presence of estrogenic substances, the yeast strain YZHB817 induces the expression of the GAL7 lacZ reporter gene whose product, the enzyme beta-galactosidase, can be readily measured by hydrolysis of the chromogenic substrate ONPG (ortho-nitrophenyl-beta-galactoside) at 420 nm. (Current Protocols in Molecular Biology, Chapter 13, Yeast (Eds. Lundblad V, Struhl, K.)).
    100 μl of the estrogenic test solution (ZEN or iZOM) dissolved in 50% ethanol was mixed with 1.9 ml of the yeast culture of strain YZGA817 in SC-TRP medium with a cell density of OD6oo = 0.1 and mixed at 30 ° and 180 rpm Incubated for 18 hours. Thereafter, the ODeO of the culture was determined and the cell pellet (10 min, 13krpm) of 1 ml in 500 ml of Z buffer resuspended and permeabilized with 25 μΙ chloroform. The enzyme reaction was started by addition of 100 .mu.l ONPG solution (4 mg / ml) and stopped after yellowing by adding 250 .mu.l of a 1 M Na2CÖ3 solution. The supernatant was measured at 420 nm and the "relative units" (based on the amount of cells used) was calculated as: RU = (OD420 X 1000) / (Οϋβοο X incubation time in min)
    As shown in Figure 4, in the low ppb region, ZEN induces expression of the GAL7-lacZ reporter gene, so about 45 ppb of ZEN are necessary to express 7.5 relative units of beta-galactosidase (Figure 4 A). iZOM, on the other hand, has a much lower estrogenicity. In comparison, about 500 ppb were needed to achieve about the same effect (Figure 4 B). As a result, iZOM is approximately 100 times less estrogen than ZEN compared to ZEN.
    Example 4: Determination of sequence identity
    The determination of the percent sequence identity of the polypeptides having the amino acid sequences with the sequence ID Nos. 1, 2 and 3 relative to each other was compared by comparing two sequences to each other using the program BLAST (Basic Local Alignment Search Tool), in particular with BLASTP, which is available on the homepage of the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov/) can be used. Thus, it is possible to compare two or more sequences with each other according to the algorithm of Altschul et al., 1997 (Nucleic Acids Res. (1997) 25: 3389-3402). 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 sequence identity between the sequences of SEQ ID NO: 1 and SEQ ID NO: 3 is 73%. SEQUENCE LISTING < 110 > Erber Aktiengesellschaft < 120 polypeptide for detoxification of zearalenone, isolated polynucleotide thereof and an additive containing polypeptide, use thereof < 130 P05359 < 160 6 < 170 patent in version 3.5
    ≪ 210 > 1 < 211 > 630 < 212 > PRT < 213 > Trichosporon mycotoxinivorans < 400 > 1
    Met Thr Leu Glu Ala Ile Pro Val Pro Glu Glu Thr Pro Ala Gin Thr 15 10 15
    Val Pro Thr Ala Pro Leu Ser Glu Glu Glu Arg Glu Ala Leu Lys His 20 25 30
    Arg Tyr Arg Ala Glu Arg Asp Lys Arg Ile Arg Glu Asp Gly Ile Lys 35 40 45
    Gin Tyr Arg Ser Leu Glu Gly Leu Leu Asp Val Asp Asp Thr Lys Asp 50 55 60
    Pro Tyr Thr Pro Val Lys Pro Arg Glu Pro Leu Asn Asp His Val Asp 65 70 75 80
    Phe Leu Phe Leu Gly Gly Gly Phe Ala Gly Leu Ile Val Cys Ser His 85 90 95
    Leu Lys Lys Thr Gly Phe Asn Asp Phe Arg Ile Ile Glu Ser Gly Gly 100 105 110
    Asp Phe Gly Gly Val Trp Tyr Trp Asn Arg Phe Pro Gly Ala Met Cys 115 120 125
    Asp Thr Ala Ala Met Val Tyr Leu Pro Leu Leu Glu Glu Thr Gly Thr 130 135 140
    Val Pro Ser Ala Lys Tyr Val Arg Gly Pro Glu Ile Leu Ala His Ala 145 150 155 160
    Gin Arg Ile Ala Arg Thr Phe Asp Leu Tyr Pro His Ala Leu Phe His 165 170 175
    Thr His Leu Glu Ser Leu Thr Trp Asp Glu Glu Gin Gly Val Trp Lys 180 185 190
    Val Lys Thr Arg Gin Gly Asp Ser Phe Thr Ala Thr His Val Gly Met 195 200 205
    Gly Thr Gly Pro Leu Asn Lys Pro His Leu Pro Gly Ile Pro Gly Ile 210 215 220
    Glu Lys Phe Lys Gly Lys Ala Met His Thr Ala Arg Trp Asp Phe Ala 225 230 235 240
    Thr Thr Gly Gly Gly Trp Asn Gly Glu Val Met Glu Gly Leu Lys Asp 245 250 255
    Lys Arg Val Gly Val Ile Gly Thr Gly Ala Thr Gly Ile Gin Ala Ile 260 265 270
    Pro Glu Leu Gly Arg Asp Ser Gly Glu Leu Phe Val Phe Gin Arg Thr 275 280 285
    Pro Ser Ala Val Ala Val Arg Gly Asn His Pro Ile Asp Pro Glu Trp 290 295 300
    Phe Ala Ser Leu Asp Lys Gly Trp Gin Thr Lys Trp Asn Arg Asn Phe 305 310 315 320
    Ile Gin Leu Met Ser Ser Gly Met Ala Glu Asn Asp Tyr Val Arg Asp 325 330 335
    Gly Trp Thr Asp Gly Val Lys Arg Ile Thr Ala Arg Met Phe Ala Glu 340 345 350
    Ala Ala Lys Ala Gly Arg Asp Pro Ser Thr Leu Ser Phe Asp Asp Phe 355 360 365
    Leu Ala Ala Tyr His Leu Ser Asp Asp Glu Tyr Met Thr Ala Val Arg 370 375 380
    Asn Arg Thr Asn Glu Val Val Asn Asp Pro Lys Thr Ala Asp Gly Leu 385 390 395 400
    Lys Ala Trp Tyr Arg Gin Phe Cys Lys Arg Pro Cys Phe His Asp Glu 405 410 415
    Phe Leu Ala Thr Phe Asn Arg Pro Thr Val His Leu Val Asp Thr Asn 420 425 430
    Gly Gin Gly Val Thr Glu Val Asp Glu Thr Gly Val Trp Ala Asn Gly 435 440 445
    Gin His Tyr Asp Leu Asp Ile Ile Val Tyr Ala Thr Gly Phe Glu Phe 450 455 460
    Asn Ser Glu Trp Thr Tyr Lys Ser Gly Met Glu Val Tyr Gly Arg Asp 465 470 475 480
    Gly Leu Thr Leu Thr Gin Ala Trp Lys Asp Gly Met Lys Thr Tyr His 485 490 495
    Gly Met His Ile Asn Gly Phe Pro Asn Leu Phe Met Leu Gin Phe Gin 500 505 510
    Gin Gly Ser Ser Leu Ala Ser Asn Ile Thr Ser Asn Tyr Val Asp Ser 515 520 525
    Gly Tyr Thr Ile Ala Ala Ile Leu Asn Lys Lys Lys Glu Leu Gly Ala 530 535 540
    Lys Thr Val Glu Val Pro Ala Asp Val Gin Ser Lys Trp Ile Glu His 545 550 555 560
    Leu Leu Thr Ala Asn Lys Gly Leu Ile Gly Gly Pro Glu Cys Thr Pro 565 570 575
    Gly Tyr Tyr Asn Asn Glu Gly Gin Glu Glu Gly Leu Lys Glu Lys Leu 580 585 590
    Asn Gly Ala Arg Tyr Pro Ala Gly Ser Leu Ala Phe Phe Asp Tyr Ile 595 600 605
    Ala Glu Trp Arg Thr Asn Gly Lys Phe Glu Gly Leu Ala Phe Asp Gly 610 615 620
    Lys Gin Ile Ala Val Gin 625 630
    ≪ 210 > 2 < 211 > 709 < 212 > PRT < 213 > Trichosporon mycotoxinivorans < 400 > 2
    Met Ser Ala Ser Arg Arg Arg Gly Ser Pro His Tyr Leu Gly Asp Arg 15 10 15
    Ser Thr Lys Ser Gly Leu Arg Arg Pro Phe Lys Arg Ser Lys Lys Asn 20 25 30
    Arg Ala Thr Asn Ala Val Phe Ser Gin Arg His Arg Lys Pro Thr Glu 35 40 45
    Gly Ala Gly Val Arg Gly Lys His Ser Cys Ser Val Ile Pro Asp Lys 50 55 60
    His Thr Ala Pro Trp Gly Pro Ala Ala Ala Glu Pro Gly Trp His Met 65 70 75 80
    Thr Leu Glu Ala Ile Pro Val Pro Glu Glu Thr Pro Ala Gin Thr Val 85 90 95
    Pro Thr Ala Pro Leu Ser Glu Glu Glu Arg Glu Ala Leu Lys His Arg 100 105 110
    Tyr Arg Ala Glu Arg Asp Lys Arg Ile Arg Glu Asp Gly Ile Lys Gin 115 120 125
    Tyr Arg Ser Leu Glu Gly Leu Leu Asp Val Asp Asp Asp Asp Lys Asp Pro 130 135 140
    Tyr Thr Pro Val Lys Pro Arg Glu Pro Leu Asn Asp His Val Asp. Phe 145 150 155 160
    Leu Phe Leu Gly Gly Gly Phe Ala Gly Leu Ile Val Cys Ser His Leu 165 170 175
    Lys Lys Thr Gly Phe Asn Asp Phe Arg Ile Ile Glu Ser Gly Gly Asp 180 185 190
    Phe Gly Gly Val Trp Tyr Trp Asn Arg Phe Pro Gly Ala Met Cys Asp 195 200 205
    Thr Ala Ala Met Val Tyr Leu Pro Leu Leu Glu Glu Thr Gly Thr Val 210 215 220
    Pro Ser Ala Lys Tyr Val Arg Gly Pro Glu Ile Leu Ala His Ala Gin 225 230 235 240
    Arg Ile Ala Arg Thr Phe Asp Leu Tyr Pro His Ala Leu Phe His Thr 245 250 255
    His Leu Glu Ser Leu Thr Trp Asp Glu Glu Gin Gly Val Trp Lys Val 260 265 270
    Lys Thr Arg Gin Gly Asp Ser Phe Thr Ala Thr His Val Gly Met Gly 275 280 285
    Thr Gly Pro Leu Asn Lys Pro His Leu Pro Gly Ile Pro Gly Ile Glu 290 295 300
    Lys Phe Lys Gly Lys Ala Met His Thr Ala Arg Trp Asp Phe Ala Thr 305 310 315 320
    Thr Gly Gly Gly Trp Asn Gly Glu Val Met Glu Gly Leu Lys Asp Lys 325 330 335
    Arg Val Gly Val Ile Gly Thr Gly Ala Thr Gly Ile Gin Ala Ile Pro 340 345 350
    Glu Leu Gly Arg Asp Ser Gly Glu Leu Phe Val Phe Gin Arg Thr Pro 355 360 365
    Ser Ala Val Ala Val Arg Gly Asn His Pro Ile Asp Pro Glu Trp Phe 370 375 380
    Ala Ser Leu Asp Lys Gly Trp Gin Thr Lys Trp Asn Arg Asn Phe Ile 385 390 395 400
    Gin Leu Met Ser Ser Gly Met Ala Glu Asn Asp Tyr Val Arg Asp Gly 405 410 415
    Trp Thr Asp Gly Val Lys Arg Ile Thr Ala Arg Met Phe Ala Glu Ala 420 425 430
    Ala Lys Ala Gly Arg Asp Pro Ser Thr Leu Ser Phe Asp Asp Phe Leu 435 440 445
    Ala Ala Tyr His Leu Ser Asp Asp Glu Tyr Met Thr Ala Val Arg Asn 450 455 460
    Arg Thr Asn Glu Val Val Asn Asp Pro Lys Thr Ala Asp Gly Leu Lys 465 470 475 480
    Ala Trp Tyr Arg Gin Phe Cys Lys Arg Pro Cys Phe His Asp Glu Phe 485 490 495
    Leu Ala Thr Phe Asn Arg Pro Thr Val His Leu Val Asp Thr Asn Gly 500 505 510
    Gin Gly Val Thr Glu Val Asp Glu Thr Gly Val Trp Ala Asn Gly Gin 515 520 525
    His Tyr Asp Leu Asp Ile Ile Val Tyr Ala Thr Gly Phe Glu Phe Asn 530 535 540
    Ser Glu Trp Thr Tyr Lys Ser Gly Met Glu Val Tyr Gly Arg Asp Gly 545 550 555 560
    Leu Thr Leu Thr Gin Ala Trp Lys Asp Gly Met Lys Thr Tyr His Gly 565 570 575
    Met His Ile Asn Gly Phe Pro Asn Leu Phe Met Leu Gin Phe Gin Gin 580 585 590
    Gly Ser Ser Leu Ala Ser Asn Ile Thr Ser Asn Tyr Val Asp Ser Gly 595 600 605
    Tyr Thr Ile Ala Ala Ile Leu Asn Lys Lys Lys Glu Leu Gly Ala Lys 610 615 620
    Thr Val Glu Val Pro Ala Asp Val Gin Ser Lys Trp Ile Glu His Leu 625 630 635 640
    Leu Thr Ala Asn Lys Gly Leu Ile Gly Gly Pro Glu Cys Thr Pro Gly 645 650 655
    Tyr Tyr Asn Asn Glu Gly Gin Glu Glu Gly Leu Lys Glu Lys Leu Asn 660 665 670
    Gly Ala Arg Tyr Pro Ala Gly Ser Leu Ala Phe Phe Asp Tyr Ile Ala 675 680 685
    Glu Trp Arg Thr Asn Gly Lys Phe Glu Gly Leu Ala Phe Asp Gly Lys 690 695 700
    Gin Ile Ala Val Gin 705 < 210 > 3 < 211 > 630
    ≪ 212 > PRT < 213 > Trichosporon asahii < 400 3
    Met Thr Ile Asp His Ile Pro Glu Pro Thr Asp Tyr Lys Gin Thr Gin 15 10 15
    Pro Val Ala Pro Leu Ala Asp Glu Glu Arg Gin Ala Leu Gin Ala Lys 20 25 30
    Tyr Arg Glu Glu Arg Asp Lys Arg Leu Arg Ala Asp Gly Ile Asn Gin 35 40 45
    Tyr Lys Pro Leu Gly Gly Ile Leu Lys Leu Asp Glu Asp Lys Asp Pro 50 55 60
    Tyr Thr Asp Val Gin Pro Arg Glu Pro Val His Asp His Asp Asp Phe 65 70 75 80
    Leu Phe Leu Gly Gly Gly Phe Ala Gly Leu Thr Val Cys Ala Lys Leu 85 90 95
    Lys Gin Ala Gly Phe Asp Ser Ile Arg Ile Leu Glu Ser Gly Gly Asp 100 105 110
    Phe Gly Gly Val Trp Tyr Trp Asn Arg Phe Pro Gly Ala Met Cys Asp 115 120 125
    Thr Ala Ala Met Val Tyr Leu Pro Met Met Glu Glu Val Gly Thr Val 130 135 140
    Pro Ser Ala Lys Tyr Val Arg Gly Pro Glu Ile Leu Ala His Ala His 145 150 155 160
    Arg Ile Ala Arg His Phe Asp Leu Tyr Pro His Ala Leu Phe Ser Thr 165 170 175
    His Leu Glu Glu Leu Arg Trp Asp Glu Asp Arg Ser Val Tyr Val Val 180 185 190
    Lys Thr Arg Glu Gly Asp Glu Phe Thr Ala Thr Asn Val Ala Met Gly 195 200 205
    Thr Gly Pro Leu Asn Arg Pro Leu Pro Gly Ile Pro Gly Leu Glu 210 215 220
    Thr Phe Lys Gly Gin Ala Met His Thr Ala Arg Trp Asp Phe Gly Val 225 230 235 240
    Thr Gly Gly Gly Trp Asp Asp Glu Val Leu Glu Gly Leu Lys Asp Lys 245 250 255
    Arg Val Gly Val Ile Gly Thr Gly Ala Thr Gly Val Gin Cys Ile Pro 260 265 270
    Thr Leu Gly Arg Asp Ser Gly Ser Leu His Val Phe Gin Arg Thr Pro 275 280 285
    Ser Ala Val Ala Ile Arg Gly Asn His Ala Ile Asp Lys Glu Trp Phe 290 295 300
    Ser Gin Leu Asp Lys Gly Trp Gin Thr Lys Trp Leu Arg Asn Phe Cys 305 310 315 320
    Gin Leu Met Ser Thr Gly Tyr Ala Pro Val Asp Tyr Val His Asp Gly 325 330 335
    Trp Thr Asp Gly Val Gin Arg Ile Thr Lys Arg Met Leu Glu Met Cys 340 345 350
    Ala Lys Glu Gly Arg Ala Pro Thr Gly Met Ala Asp Tyr Met Lys Ala 355 360 365
    Tyr His Leu Ser Asp Asp Glu Tyr Thr Thr Ala Val Arg Ala Arg Thr 370 375 380
    Asp Glu Val Val Lys Asp Pro Glu Thr Ala Asp Lys Leu Lys Ala Trp 385 390 395 400
    Tyr Arg Gin Phe Cys Lys Arg Pro Thr Phe His Asp Glu Tyr Leu Gin 405 410 415
    Thr Phe Asn Arg Pro Asn Val Thr Leu Val Asp Thr Asp Gly Lys Gly 420 425 430
    Val Glu Arg Ile Asp Glu Thr Gly Val Trp Ala Asn Gly Lys His Tyr 435 440 445
    Asp Leu Asp Val Leu Val Tyr Ala Thr Gly Phe Glu Phe Asn Ser Glu 450 455 460
    Tyr Thr Tyr Lys Ser Gly Leu Glu Val Tyr Gly Arg Gly Gly Arg Thr 465 470 475 480
    Leu Thr Asp Ala Trp Lys Asp Gly Met Gin Ser Phe Gin Gly Met His 485 490 495
    Val His Gly Phe Pro Asn Leu Phe Val Ile Gly Phe Ala Gin Gly Ser 500 505 510
    Ser Leu Ala Ser Asn Ile Thr Ser Asn Tyr Thr Glu His Gly Pro Thr 515 520 525
    Val Leu Ser Ile Leu Gin Lys Lys Lys Glu Leu Gly Ala Lys Thr Val 530 535 540
    Glu Val Pro Gin Lys Thr Gin Asp Asp Trp Ile Glu Leu Ile Leu Gin 545 550 555 560
    Gly Asp Arg Gly Ile Ile Gly Gly Pro Glu Cys Thr Pro Gly Tyr Tyr 565 570 575
    Asn Asn Glu Gly Gin Glu Glu Gly Arg Arg Glu Arg Leu Asn Val Ala 580 585 590
    Arg Tyr Pro Ala Gly Pro Leu Ala Phe Phe Asp Tyr Ile Ala Glu Trp 595 600 605
    Arg Ala Asn Gly Lys Phe Glu Gly Leu Glu Phe Asp Gly Lys Pro Val 610 615 620
    Glu Ile AlaAlaThr Leu 625 630
    ≪ 210 > 4 < 211 > 1890 < 212 > DNA < 213 > Artificial Sequence < 220 > ≪ 223 > Codon Optimized polynucleotide of the polypeptide of SEQ ID NO: 1 for expression in Saccharomyces cerevisiae < 400 > 4 atgactttgg aagctattcc agttccagaa gaaactccag ctcaaactgt tccaactgct 60 ccattgtctg aagaagaaag agaagccttg aaacatagat acagagccga aagagataag 120 agaatcagag aagatggtat caagcaatac agatccttgg aaggtttgtt ggatgttgat 180 gataccaaag atccatacac tccagttaag ccaagagaac cattgaacga tcatgtcgat 240 tttttgtttt tgggtggtgg ttttgctggt ttgatcgttt gttctcattt gaaaaagacc 300 ggtttcaacg acttcagaat cattgaatct ggtggtgatt tcggtggtgt ttggtattgg 360 aatagatttc caggtgctat gtgtgatact gctgctatgg tttatttgcc tttgttggaa 420 gaaactggta cagttccatc tgctaaatat gttagaggtc cagaaatttt ggctcacgct 480 caaagaattg ctagaacttt tgacttgtac ccacatgctt tgttccatac ccatttggaa 540 tctttgactt gggatgaaga acaaggtgtt tggaaagtta agaccagaca aggtgattct 600 ttcactgcta ctcatgttgg tatgggtact ggtccattga acaaaccaca tttgcctggt 660 attccaggta tcgaaaagtt taaaggtaag gctatgcata ccgctagatg ggattttgct 720 actactggtg gtggttggaa tggtgaagtt atggaaggtt taaaggataa gagagttggt 780 gttattggta ctggtgctac tggtattcaa gctataccag aattgggtag agactccggt 840 gaattatttg ttttccaa ag aacaccatcc gctgttgcag ttagaggtaa tcatccaatt 900 gatccagaat ggtttgcctc tttggataag ggttggcaaa caaagtggaa cagaaacttt 960 attcaattga tgtcctccgg tatggccgaa aatgattacg ttagagatgg ttggactgat 1020 ggtgttaaga gaattactgc tagaatgttt gctgaagctg ctaaagctgg tagagatcca 1080 tctactttgt ctttcgatga tttcttggct gcctaccatt tgtccgatga tgaatatatg 1140 actgccgtca gaaacagaac taacgaagtt gttaacgatc caaagactgc tgatggtttg 1200 aaagcttggt atagacaatt ctgcaaaaga ccatgcttcc acgatgaatt tttggctact 1260 tttaacagac caaccgttca cttggttgat acaaatggtc aaggtgttac tgaagttgac 1320 gaaaccggtg tttgggctaa tggtcaacat tacgatttgg atattatcgt ttacgccact 1380 ggtttcgaat tcaactctga atggacttac aagtctggta tggaagttta tggtagagat 1440 ggtttgacat tgactcaagc ttggaaagac ggtatgaaaa cctatcatgg tatgcacatt 1500 aacggtttcc caaacttgtt catgttgcaa ttccaacaag gttcctcttt ggcttctaac 1560 atcacttcta actacgttga ttccggttac accattgctg ctattttgaa caaaaagaaa 1620 gaattaggtg ccaagaccgt tgaagttcca gctgatgttc aatctaaatg gatcgaacat 1680 ttgttgaccg ctaacaaggg tttga ttggt ggtccagaat gtactccagg ttactataac 1740 aatgaaggtc aagaagaagg tttgaaagaa aagttgaacg gtgctagata tccagctggt 1800 tcattggctt ttttcgatta cattgctgaa tggagaacta atggtaaatt cgaaggtttg 1860 gccttcgatg gtaaacaaat tgctgttcaa 1890
    ≪ 210 > 5 < 211 > 2127 < 212 > DNA < 213 > Trichosporon mycotoxinivorans < 400 > 5 atgtctgcta gtagaccgcg cggctcccca cattatctcg gtgaccggtc taccaaaagc 60 ggtctacgca gaccgtttaa acggtcgaaa aagaacagag ccacgaacgc cgtgttttcc 120 cagcgccatc gcaagccaac cgaaggtgcc ggtgttcggg gtaaacacag ttgctcggtg 180 atccccgata agcacaccgc cccatggggc ccggccgccg ccgaacctgg atggcacatg 240 acactcgaag ctattcctgt tcccgaggag acgcccgcgc agaccgtccc caccgctccg 300 ctctcggagg aagagcgcga ggccttgaag caccgatacc gcgccgagag ggacaagcgt 360 atccgcgaag acggcatcaa gcagtaccgc tccctggagg gtcttctgga cgtcgacgac 420 accaaggacc cgtacacgcc cgtgaagcct cgtgagcccc tgaacgacca tgtcgacttc 480 ctgttcctgg gtggtgggtt cgccggcctc attgtctgct cccatctcaa aaagaccggc 540 ttcaacgatt tccgcatcat cgagtcgggc ggagactttg gaggtgtatg gtactggaac 600 cgcttccccg gcgccatgtg tgacaccgcc gcgatggtct acttgcccct cctcgaggag 660 acgggcaccg tgccgtcggc caagtatgtc cgtggcccag agattcttgc gcacgcgcag 720 cgcatcgctc gcacgtttga cctgtacccg cacgctctgt tccacacgca cctcgagtct 780 cttacgtggg acgaggagca gggcgtctgg aaggtcaaga cgcgccaggg cgactcgttc 840 acggcgaccc acgtgggc at gggcaccgga cctctcaaca agccccacct tcctggtatc 900 cccggtatcg aaaagttcaa gggcaaggcg atgcacactg cccgctggga ctttgcgacg 960 acgggcggcg gctggaatgg agaagttatg gagggactca aggacaagcg cgtcggcgtg 1020 atcggtaccg gcgctaccgg catccaggcg attcccgagc tcggccgcga cagcggcgag 1080 cttttcgtgt tccagcgcac gccctccgct gtcgccgtcc gaggcaacca ccccatcgac 1140 cccgagtggt ttgcgagcct cgacaagggg tggcagacaa agtggaaccg taactttatc 1200 cagctcatga gctcgggcat ggccgagaac gactatgtcc gtgacggctg gaccgacggc 1260 gtcaagcgca ttacggccag gatgtttgcc gaggcggcaa aggctggtcg ggacccttcg 1320 actctgtcgt ttgatgactt ccttgcggcg taccatctct cggacgacga gtacatgaca 1380 gcggtacgca accgcactaa cgaagtcgtc aacgacccca agaccgccga cggtctcaag 1440 gcgtggtacc gccagttttg caagcgtccg tgcttccacg acgagttttt ggccaccttc 1500 aaccgcccta ccgtccacct cgtggacacc aacggccagg gcgtaaccga ggtcgacgag 1560 acgggcgtct gggccaacgg acagcactac gacctcgaca ttatcgtcta tgccactgga 1620 ttcgagttca actcggagtg gacatacaag tccggcatgg aggtgtacgg ccgcgacggg 1680 ctcaccctca cgcaggcgtg gaagg acggc atgaagacgt accacggcat gcacatcaac 1740 gggttcccca acctgttcat gcttcagttc cagcagggat cgtcgctcgc atccaacatt 1800 acatccaact atgtcgactc gggctacacc attgcggcga ttctcaacaa gaaaaaggag 1860 ctcggagcca agacggtcga ggtccccgcc gacgtccagt caaagtggat cgagcacctc 1920 ctcaccgcga acaagggcct gatcggagga ccagagtgca cccccggata ctacaacaac 1980 gagggccagg aggaaggcct caaggagaag ctcaacggag cacggtaccc tgcgggatct 2040 ctcgcattct ttgactacat cgccgagtgg cgtacaaacg gcaagtttga gggtcttgcg 2100 tttgatggaa agcagatcgc agtgcag 2127
    ≪ 210 > 6 < 211 > 1893 < 212 > DNA < 213 > Trichosporon asahii < 400 > 6 atgacgatcg accacatccc agagccgacg gactacaagc agacgcagcc cgtcgcgcct 60 ctggcggacg aggagcgcca ggcgctgcag gccaaatacc gcgaggagcg cgacaagcga 120 ctccgcgccg atggtattaa ccagtacaag cccctcggcg gcatcttgaa gctggacgag 180 gacaaggacc cgtacacgga cgtccagccg cgcgagccgg tccacgacca tgtcgacttt 240 ctgttccttg gcggtggctt cgcgggcctg acggtgtgcg cgaagctgaa gcaggcgggc 300 tttgactcga tccgcatcct cgagagcgga ggcgactttg gcggcgtctg gtactggaac 360 cgcttccccg gcgccatgtg cgacacggcc gccatggtct acctccccat gatggaggag 420 gttggcacgg tgccctcggc caaatatgtc cgcggacccg agattctcgc ccacgcccac 480 cgcatcgcgc gccacttcga cctctacccc cacgctctgt tcagcaccca cctcgaggag 540 ctgcgctggg acgaggaccg cagcgtctac gttgtcaaga cgcgcgaggg cgacgagttc 600 acggcgacca acgtcgcgat gggcactgga cccctgaacc gtccccacct gcccggtatc 660 cctggactgg agacattcaa gggccaggcg atgcacaccg ctcgctggga ctttggcgtg 720 accggcggag gctgggacga tgaggtcctc gaggggttga aggacaagcg tgtcggtgtc 780 atcggcacgg gcgccacggg tgtgcagtgt atcccgactc ttggacgcga ctctggctcc 840 ctgcatgtgt tccagcgc ac gccctctgcc gtcgctatcc gcggcaacca tgcgattgac 900 aaggagtggt tcagccagct ggacaagggg tggcagacca agtggctgcg caacttctgc 960 cagctcatgt cgaccgggta cgcccccgtc gattacgtcc acgacgggtg gaccgacggc 1020 gtgcagcgca tcacgaagcg catgctcgag atgtgcgcca aggaggggcg cgcgccgacc 1080 ggaatggcag actacatgaa ggcgtaccac ctcagcgacg acgagtacac caccgccgtg 1140 cgcgcccgca ccgacgaggt cgtcaaggat cccgaaacgg ccgacaagct gaaggcgtgg 1200 tacaggcagt tttgcaaacg ccccaccttt catgacgagt acttgcagac gttcaaccgg 1260 ccgaacgtta cgctcgtcga cacggacggc aagggcgtgg agcgcatcga cgagacgggc 1320 gtctgggcga acggcaagca ctacgacctc gacgtacttg tgtacgcgac aggcttcgag 1380 ttcaactcgg agtacacata caagtctggt ctcgaggtgt acggtcgcgg tgggcgcacg 1440 ctcaccgatg catggaagga cggcatgcag tccttccagg gcatgcacgt gcacgggttc 1500 ccgaacctgt tcgtgatcgg ttttgcgcag gggtcctcac tcgcgtccaa cattacgagc 1560 aactacaccg agcacgggcc cacggtcctc agcatcctgc agaagaagaa ggagctcggc 1620 gctaagacgg tcgaggtgcc ccagaaaacg caggacgact ggatcgaact catcctccag 1680 ggcgaccggg gcattattgg cggtc ccgag tgcacacccg gctactacaa caacgagggc 1740 caggaggagg gccgtcgcga gcgcctcaat gttgcgcgat accccgctgg tccgctcgct 1800 ttcttcgact acatcgccga gtggcgcgcg aacggcaagt tcgagggcct cgagtttgat 1860 ggcaagccgg tcgagattgc cgcgactctg day 1893
    权利要求:
    Claims (11)
    [1]
    Claims 1. A polypeptide for the enzymatic detoxification of zearalenone, characterized in that the polypeptide comprises a cyclohexanone monooxygenase converting the keto group at position 7 of zearalenone to an ester group having an amino acid sequence selected from the group of SEQ ID NOs. 1, 2 or 3; or a functional variant thereof, wherein between the functional variant and at least one of the amino acid sequences a sequence identity is at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% sequence identity.
    [2]
    2. An isolated polynucleotide which codes for a zearalenone detoxifying polypeptide, characterized in that the polynucleotide has a nucleotide sequence which codes for at least one polypeptide according to claim 1 and / or a degree of sequence identity to at least one nucleotide sequence selected from the group of sequence ID No. 4, 5 and 6 of at least 60%.
    [3]
    3. A transgenic host cell for producing a zearalenone detoxifying cyclohexanone monooxygenase, characterized in that the host cell expresses a polynucleotide according to claim 2, wherein the polynucleotide is chromosomally integrated or extrachromosomal and the host cell is a prokaryotic or eukaryotic cell, in particular a yeast or plant cell, and in that the host cell optionally additionally overexpresses an enzyme which recycles a cofactor required for the oxygenase, in particular converts NADP + or NAD + into NADPH or NADH.
    [4]
    4. Transgenic plant with a plant cell according to claim 3.
    [5]
    5. seed of a transgenic plant according to claim 4.
    [6]
    6. additive for the enzymatic detoxification of zearalenone, characterized in that the additive contains at least one polypeptide according to claim 1 and at least one excipient.
    [7]
    7. An additive according to claim 6, characterized in that the at least one excipient from the group of inert carrier, vitamins, minerals, enzymes, other components for detoxification of mycotoxins and cofactors, in particular NADPH and / or NADH is selected.
    [8]
    8. Use of the additive according to claim 6 or 7 for the enzymatic detoxification of zearalenone in animal feed, in particular for pigs, poultry and aquaculture, and in dry pasture or in foodstuffs.
    [9]
    9. A process for the enzymatic transformation of zearalenone, characterized in that zearalenone with at least one keto group at position 7 of the zearalenone in an ester group converting, an amino acid sequence selected from the group of the sequence ID Nos. 1, 2 and 3 having cyclohexanone monooxygenase or having at least one functional variant thereof having a sequence identity to at least one of the amino acid sequences of at least 60%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90%, optionally with at least one Another enzyme, which is reduced by the enzymatic transformation of the oxidized cofactor, in particular NADP + or NAD +, in NADPH or NADH is brought into contact with water and optionally additionally with at least one excipient.
    [10]
    10. The method according to claim 9, characterized in that the cyclohexanone monooxygenase is used in an additive according to claim 6 or 7, or in a transgenic host cell according to claim 3, or in a transgenic plant according to claim 4 or in a seed according to claim 5 ,
    [11]
    11. The method according to claim 9 or 10, characterized in that thereby at least 70%, preferably at least 80%, in particular at least 90% of the zearalenone are transformed.
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    AT514775B1|2013-08-28|2017-11-15|Erber Ag|Polypeptide for hydrolytic cleavage of zearalenone and / or zearalenone derivatives, isolated polynucleotide thereof and additive containing the polypeptide|
    AT516457B1|2014-11-07|2017-03-15|Erber Ag|Polypeptide for the enzymatic detoxification of zearalenone, as well as isolated polynucleotide, as well as additive, use and method thereof|AT516457B1|2014-11-07|2017-03-15|Erber Ag|Polypeptide for the enzymatic detoxification of zearalenone, as well as isolated polynucleotide, as well as additive, use and method thereof|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    AT8162014|2014-11-07|
    ATA538/2015A|AT516457B1|2014-11-07|2015-08-17|Polypeptide for the enzymatic detoxification of zearalenone, as well as isolated polynucleotide, as well as additive, use and method thereof|ATA538/2015A| AT516457B1|2014-11-07|2015-08-17|Polypeptide for the enzymatic detoxification of zearalenone, as well as isolated polynucleotide, as well as additive, use and method thereof|
    CA2966815A| CA2966815A1|2014-11-07|2015-11-04|Polypeptide for the enzymatic detoxification of zearalenone, isolated polynucleotide, and associated additive, use and method|
    PCT/AT2015/000138| WO2016070206A2|2014-11-07|2015-11-04|Polypeptide for the enzymatic detoxification of zearalenone, isolated polynucleotide, and associated additive, use and method|
    EP15807781.8A| EP3215609B1|2014-11-07|2015-11-04|Polypeptide for the enzymatic detoxification of zearalenone, isolated polynucleotide, and associated additive, use and method|
    BR112017008543A| BR112017008543A2|2014-11-07|2015-11-04|zearalenone enzymatic detoxification polypeptide as well as isolated polynucleotide as well as its additive, use and process|
    DK15807781.8T| DK3215609T3|2014-11-07|2015-11-04|Polypeptide for enzymatic detoxification of zearalenone, isolated polynucleotide and associated additive, use and method|
    US15/524,643| US20180298352A1|2014-11-07|2015-11-04|Polypeptide for the enzymatic detoxification of zearalenone, isolated polynucleotide, and associated additive, use and method|
    CN201580060557.7A| CN107109376A|2014-11-07|2015-11-04|For making the polypeptide that zearalenone detoxifies, and the polynucleotides separated, and related additive, purposes and method with enzymatic|
    US16/829,204| US11136561B2|2014-11-07|2020-03-25|Polypeptide for the enzymatic detoxification of zearalenone, isolated polynucleotide, and associated additive, use and method|
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