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 the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 20, 36, 38, 40, 44, 50, 52, 56, 58, 60, 64, 66, or 68.
2. The recombinant cytochrome P450-BM3 variant of claim 1 , wherein said variant oxidizes at least three organic substrate selected from the group consisiting of nifedipine, propranolol, verapamil, and diclofenac.
3. An isolated polynucleotide comprising a polynucleotide sequence encoding the recombinant cytochrome P450-BM3 variant of claim 1 .
4. The isolated polynucleotide sequence of claim 3 , wherein said polynucleotide sequence comprises the polynucleotide sequence of SEQ ID NO: 43.
5. An expression vector comprising the polynucleotide sequence of claim 3 .
6. The vector of claim 5 , wherein said polynucleotide is operably linked with regulatory sequences suitable for expression of said polynucleotide in a suitable host cell.
7. The vector of claim 6 , wherein said host cell is a prokaryotic or eukaryotic cell.
8. The vector of claim 7 , wherein said host cell is a prokaryotic cell.
9. The vector of claim 7 , wherein said host cell is Escherichia coli.
10. A host cell comprising the vector of claim 5 .
11. A method for producing a recombinant cytochrome P450-BM3 variant comprising culturing the host cell of claim 10 under conditions such that the recombinant cytochrome P450-BM3 variant is produced.
12. The method of claim 11 , further comprising the step of recovering said recombinant cytochrome P450-BM3 variant.