Novel p450-bm3 variants with improved activity

Abstract

The present invention provides improved P450-BM3 variants with improved activity. In some embodiments, the P450-BM3 variants exhibit improved activity over a wide range of substrates.

What is claimed is: 
     
         1 . A recombinant cytochrome P450-BM3 variant having at least 90% sequence identity to a polypeptide sequence comprising the sequence set forth in SEQ ID NO: 6 or 8. 
     
     
         2 . The recombinant cytochrome P450-BM3 variant of  claim 1 , wherein said variant oxidizes at least three organic substrates. 
     
     
         3 . The recombinant cytochrome P450-BM3 variant of  claim 2 , wherein said organic substrate is selected from loratadine, imatinib, geftinib, and diclofenac. 
     
     
         4 . A recombinant polynucleotide sequence encoding a recombinant cytochrome p450-BM3 variant having at least 90% sequence identity to a polynucleotide sequence comprising the sequence set forth in SEQ ID NO: 5 or 7. 
     
     
         5 . A recombinant polynucleotide sequence encoding a recombinant cytochrome P450-BM3 variant of  claim 1 . 
     
     
         6 . The recombinant polynucleotide sequence of  claim 4 , wherein said sequence comprises SEQ ID NO: 5 or 7. 
     
     
         7 . An expression vector comprising the polynucleotide sequence of  claim 4 . 
     
     
         8 . The vector of  claim 7 , wherein said polynucleotide sequence is operably linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell. 
     
     
         9 . The vector of  claim 8 , wherein said host cell is a prokaryotic or eukaryotic cell. 
     
     
         10 . The vector of  claim 9 , wherein said host cell is a prokaryotic cell. 
     
     
         11 . The vector of  claim 10 , wherein said host cell is E. coli. 
     
     
         12 . A host cell comprising the vector of  claim 7 . 
     
     
         13 . A method for producing at least one recombinant cytochrome P450-BM3 variant comprising culturing a host cell under conditions such that at least one recombinant cytochrome P450-BM3 variant of  claim 1  is produced by said host cell. 
     
     
         14 . The method of  claim 13 , further comprising the step of recovering said at least one recombinant cytochrome P450-BM3 variant.