Polypeptide for the enzymatic detoxification of zearalenone, isolated polynucleotide, and associated additive, use and method

Abstract

The invention relates to a polypeptide for the enzymatic detoxification of zearalenone, said polypeptide being a monooxygenase which converts the keto group in position 7 of zearalenone into an ester group, the monooxygenase in particular being an amino acid sequence selected from the group comprising sequence ID No. 1, 2 and 3 or a functional variant thereof. The functional variant and at least one of the amino acid sequences has a sequence identity of at least 60%, preferably at least 70%, more preferably at least 80% and most preferably 90%,

1 . A polypeptide for the enzymatic detoxification of zearalenone, wherein the polypeptide is a monooxygenase converting the keto group in position 7 of zearalenone into an ester group, and that the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID Nos. 1, 2 and 3 or a functional variant thereof, the functional variant and at least one of the amino acid sequences having a sequence identity of at least 60% to 90%. 
     
     
         2 . The polypeptide according to  claim 1 , wherein the monooxygenase is a Baeyer-Villiger monooxygenase. 
     
     
         3 . The polypeptide according to  claim 1 , wherein the monooxygenase is a cyclohexanone monooxygenase. 
     
     
         4 . The polypeptide according to  claim 1 , wherein the polypeptide, in particular monooxygenase, converts zearalenone to iZOM in a one-step enzymatic oxidation reaction so as to detoxify at least 70% of the zearalenone within 24 hours at 30° C. by converting the keto group in position 7 of the zearalenone into an ester group, said polypeptide being formed by a transformed yeast strain YZGA515, which is transformed with a pCS57 vector additionally containing a polynucleotide for expressing the polypeptide, and the transformed yeast strain is used at a cell density OD600 of 4, and zearalenone is used as a substrate at a concentration of 2 mg/l, and SC-LEU medium is used as a reaction medium. 
     
     
         5 . A transgenic host cell for preparing a zearalenone-detoxifying monoxygenase, wherein the host cell expresses a polynucleotide, that the polynucleotide comprises a nucleotide sequence encoding at least one polypeptide according to  claim 1  and having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6, said polynucleotide being chromosomally integrated or extrachromosomally present and the host cell being a plant cell, and that optionally the host cell additionally overexpresses an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively. 
     
     
         6 . Seed comprising a transgenic plant cell according to  claim 5 . 
     
     
         7 . An additive for the enzymatic detoxification of zearalenone, wherein said additive comprises at least one polypeptide according to  claim 1  and at least one adjuvant selected from the group consisting of vitamins, minerals, enzymes, farther components for detoxifying mycotoxins and cofactors, in particular NADPH and/or NADH, enzyme preparations such as proteases, amylases, cellulases or glucanases, hydrolases, lipolytic enzymes, mannosidases, oxidases, oxidoreductases, phytases, xylanases and/or combinations thereof, mycotoxin-detoxifying enzymes such as aflatoxin oxidase, ergotamine hydrolases, ergotamine amidases, ochratoxin amidases, fumonisin carboxylesterases, fumonisin aminotransferases, aminopolyol aminoxidases, deoxymyalenol epoxide hydrolases; mycotoxin-detoxifying microorganisms; mycotoxin-binding components such as microbial cell walls, and inorganic materials such as bentonite. 
     
     
         8 . The use of an isolated polynucleotide encoding at least one polypeptide according to  claim 1  and having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5and 6 for preparing a zearalenone-detoxifying monooxygenase. 
     
     
         9 . The use of an isolated polynucleotide encoding at least one polypeptide according to  claim 1  and having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5and 6 in a method for preparing a zearalenone-detoxifying monooxygenase. 
     
     
         10 . A method for preparing a zearalenone-detoxifying monoxygenase, wherein an isolated polynucleotide comprising a nucleotide sequence encoding at least one polypeptide according to  claim 1  and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 is chromosomally integrated into a transgenic host cell or extrachromosomally provided, that the host cell is a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, and that optionally an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, is additionally overexpressed in the host cell. 
     
     
         11 . The use of an isolated polynucleotide encoding a zearalenone-detoxifying polypeptide for preparing a transgenic host cell, wherein the host cell expresses the isolated polynucleotide, which comprises a nucleotide sequence encoding at least one polypeptide according to  claim 1  and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6, wherein the polynucleotide is chromosomally integrated or extrachromosomally provided and the host cell is a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, and that optionally the host cell additionally overexpresses an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively. 
     
     
         12 . A method for preparing a transgenic host cell for preparing a zearalenone-detoxifying monooxygenase, wherein an isolated polynucleotide comprising a nucleotide sequence encoding at least one polypeptide according to  claim 1  and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 is chromosomally integrated into the host cell or extrachromosomally provided, that the polynucleotide is expressed in the host cell, that a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, is used as said host cell, and that, optionally an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, is additionally overexpressed in the host cell. 
     
     
         13 . The use of a transgenic host cell for preparing a zearalenone-detoxifying monooxygenase, wherein an isolated polynucleotide comprising a nucleotide sequence encoding at least one polypeptide according to  claim 1  and/or having a degree of sequence identity of at least 60% with at least one nucleotide sequence selected from the group consisting of SEQ ID Nos. 4, 5 and 6 is chromosomally integrated into the host cell or extrachromosomally provided, that the polynucleotide is expressed in the host cell, that a prokaryotic or eukaryotic cell, in particular a yeast cell or a plant cell, is used as said host cell, and that optionally an enzyme recycling a cofactor required for the oxygenase, in particular an enzyme converting NADP+ or NAD+ to NADPH or NADH, respectively, is additionally overexpressed in the host cell.