NOVEL P450-BM3 VARIANTS WITH IMPROVED ACTIVITY
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.
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.