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: 48.
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 the at least three organic substrates are selected from the group consisting of nifedipine, propranolol, verapamil, and diclofenac.
4. An isolated polynucleotide comprising a polynucleotide sequence encoding the recombinant cytochrome P450-BM3 variant of claim 1 .
5. The isolated polynucleotide of claim 4 , wherein said polynucleotide comprises the polynucleotide sequence of SEQ ID NO: 47.
6. An expression vector comprising the polynucleotide 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 7 , 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. An isolated 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 the 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-BM3 variant.