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 cytochrome P450-BM3 activity and comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth in SEQ ID NO:18, 24, 26, 28, 30, 32, 46, or 62.
1 . ecombinant 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 nifedipine, propranolol, verapamil, and diclofenac.
4 . An isolated polynucleotide sequence encoding a recombinant cytochrome P450-BM3 variant of claim 1 .
5 . The isolated polynucleotide sequence of claim 4 , wherein said sequence comprises a polynucleotide sequence selected from of SEQ ID NO:17, 23, 25, 27, 29, 31, 45 or 61.
6 . An expression vector comprising the polynucleotide sequence of claim 4 .
7 . The vector of claim 6 , wherein said polynucleotide sequence is operably linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell.
8 . The vector of claim 6 , wherein said host cell is a prokaryotic or eukaryotic cell.
9 . The vector of claim 8 , wherein said host cell is a prokaryotic cell.
10 . The vector of claim 8 , wherein said host cell is E. coli.
11 . A host cell comprising the vector of claim 6 .
12 . A method for producing at least one recombinant cytochrome P450-BM3 variant comprising culturing the host cell of claim 11 under conditions such that a recombinant cytochrome P450-BM3 variant is produced.
13 . The method of claim 12 , further comprising the step of recovering said at least one recombinant cytochrome P450 variant.