Fungal peroxygenases and methods of application

Abstract

The invention relates to polypeptides having peroxygenase activity and compositions comprising such polypeptides, their encoding polynucleotides, expression vectors and recombinant host cells comprising such polynucleotides or vectors, methods of producing the polypeptides, as well as methods of application and uses thereof, including a process for enzymatic, regioselective oxygenation of N-heterocycles of the general formula (I) to the corresponding N-oxides of the formula (II), by converting N-heterocycles of the formula (I) with a peroxidase polypeptide in the presence of at least one oxidizing agent in a one-stage reaction process.

1 . An isolated polypeptide having peroxygenase activity, selected from the group consisting of:
 (a) a polypeptide comprising an amino acid sequence having at least 90% identity to the polypeptide of SEQ ID NO: 10; and   (b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 80% identity to the mature polypeptide coding sequence of SEQ ID NO:9   
     
     
         2 . The isolated polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 90% identity to the polypeptide of SEQ ID NO: 10. 
     
     
         3 . The isolated polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 95% identity to the polypeptide of SEQ ID NO: 10. 
     
     
         4 . The isolated polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 97% identity to the polypeptide of SEQ ID NO: 10. 
     
     
         5 . The isolated polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid sequence having at least 98% identity to the polypeptide of SEQ ID NO: 10. 
     
     
         6 . The isolated polypeptide of  claim 1 , wherein the polypeptide is encoded by a polynucleotide comprising a nucleotide sequence having at least 90% identity to the mature polypeptide coding sequence of SEQ ID NO:9. 
     
     
         7 . The isolated polypeptide of  claim 1 , wherein the polypeptide consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 10. 
     
     
         8 . The isolated polypeptide of  claim 1 , wherein the polypeptide encoded by a polynucleotide of SEQ ID NO:9. 
     
     
         9 . A nucleic acid construct comprising the polynucleotide of  claim 1  operably linked to one or several control sequences that direct the production of the polypeptide in an expression host. 
     
     
         10 . An isolated recombinant host cell comprising the nucleic acid construct of  claim 9 . 
     
     
         11 . A method of producing the polypeptide of 1, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide of  claim 1 , under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. 
     
     
         12 . A method of producing the polypeptide of  claim 1 , comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide of  claim 1  under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. 
     
     
         13 . A process for enzymatic, regioselective oxygenation of N heterocycles of the formula (I) in  FIG. 1  to corresponding N oxides of the formula (II) in  FIG. 1 , by converting N hetero┐cycles of the formula (I) in  FIG. 1  with a peroxygenase polypeptide as defined in  claim 1  in the presence of at least one oxidizing agent in a one-stage reaction process. 
     
     
         14 . The process of  claim 13 , characterized in that the N heterocycle used is pyridine. 
     
     
         15 . The process of  claim 13 , wherein further H2O2-generating enzymes are added to the reaction mixture to further accelerate the reaction of the compound of the formula (I) with the peroxygenase polypeptide..