Lipoxygenase

Abstract

The inventors have found a novel fungal lipoxygenase from Manaporthe salvinii and determined its sequence. They have sequenced the gene and cloned it into E. coli and deposited the clone. Oligonucleotides probes based on the sequence information are useful for screening a eukaryotic library to obtain a lipoxygenase. The lipoxygenase is useful in baking and in a detergent.

1 . An isolated lipoxygenase which is: 
 a) a polypeptide encoded by a DNA sequence cloned into plasmid pUC19 present in  Escherichia coli  deposited as DSM 14139.    b) a polypeptide having an amino acid sequence as the mature peptide shown in SEQ ID NO: 1, or which can be obtained therefrom by substitution, deletion, and/or insertion of one or more amino acids,    c) an analogue of the polypeptide defined in (a) or (b) which: 
 i) has at least 50% homology with said polypeptide,  
 ii) is immunologically reactive with an antibody raised against said polypeptide in purified form,  
 iii) is an allelic variant of said polypeptide, or  
   d) a polypeptide encoded by DNA that hybridizes under low stringency conditions with a complementary strand of 
 i) the DNA sequence cloned into plasmid pUC19 present in  Escherichia coli  deposited as DSM 14139 or  
 ii) the DNA sequence of SEQ ID NO: 1 encoding the mature polypeptide or a subsequence thereof having at least 100 nucleotides.  
   
     
     
         2 . The lipoxygenase of  claim 1  which is derived from a filamentous fungus, e.g. an Ascomycota, such as a strain of  Magnaporthe,  particularly  M. salvinii,  more particularly strain IFO 6642.  
     
     
         3 . Isolated DNA comprising a nucleic acid sequence which encodes the lipoxygenase of  claim 1 .  
     
     
         4 . An isolated polynucleotide which comprises: 
 a) the partial DNA sequence encoding a mature lipoxygenase cloned into a plasmid present in  Escherichia coli  DSM 14139.    b) the partial DNA sequence encoding a mature lipoxygenase shown in SEQ ID NO: 1,    c) an analogue of the sequence defined in a) or b) which encodes a lipoxygenase and 
 i) has at least 60% homology with said DNA sequence, or  
 ii) hybridizes at high stringency with a complementary strand of said DNA sequence or a subsequence thereof having at least 100 nucleotides,  
 iii) is an allelic variant thereof, or  
   d) a complementary strand of a), b) or c).    
     
     
         5 . A nucleic acid construct comprising the nucleic acid sequence of  claim 3  operable linked to one or more control sequences capable of directing the expression of the lipoxygenase in a suitable expression host.  
     
     
         6 . A recombinant expression vector comprising the nucleic acid construct of  claim 5 , a promoter, and transcriptional and translational stop signals.  
     
     
         7 . A recombinant host cell comprising the nucleic acid construct of  claim 5 .  
     
     
         8 . A method for producing a lipoxygenase comprising cultivating the host cell of  claim 7  under conditions conductive to production of the lipoxygenase, and recovering the lipoxygenase.  
     
     
         9 . An oligonucleotide probe which consists of at least 20 nucleotides and which encodes a partial polypeptide sequence of SEQ ID NO: 2.  
     
     
         10 . A method for obtaining a polypeptide with lipoxygenase activity, comprising: 
 a) preparing a eukaryotic DNA library,    b) screening the library to select DNA molecules which hybridize to the probe of  claim 8 ,    c) transforming host cells with the selected DNA molecules,    d) cultivating the transformed host cells to express polypeptides encoded by the DNA molecules, and    e) assaying the expressed polypeptides to select polypeptides having lipoxygenase activity.    
     
     
         11 . A method for preparing a dough or a baked product made from dough, comprising adding the lipoxygenase of  claim 1  to the dough.  
     
     
         12 . A dough composition comprising the lipoxygenase of  claim 1 .  
     
     
         13 . A detergent composition comprising a surfactant and the lipoxygenase of  claim 1 .  
     
     
         14 . The detergent composition of the preceding claim wherein the surfactant is anionic.  
     
     
         15 . A process for oxidizing a polyunsaturated fatty acid comprising contacting the acid with the lipoxygenase of  claim 1  in the presence of air.  
     
     
         16 . (canceled)