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 comprising the polypeptide sequence set forth in SEQ ID NO: 22, wherein said recombinant cytochrome P450-BM3 variant oxidizes at least three organic substrates.
2. The recombinant cytochrome P450-BM3 variant of claim 1 , wherein said organic substrate is selected from 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 of claim 3 , wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 21.
5. An expression vector comprising the polynucleotide of claim 3 .
6. The vector of claim 5 , wherein said polynucleotide is operably linked with regulatory sequences suitable for expression of said polynucleotide sequence in a suitable host cell.
7. The vector of claim 6 , wherein said host cell is a prokaryotic cell.
8. The vector of claim 7 , wherein said host cell is E. coli.
9. A host cell comprising the vector of claim 5 .
10. A method for producing a recombinant cytochrome P450-BM3 variant comprising culturing the host cell of claim 9 under conditions such that a recombinant cytochrome P450-BM3 variant is produced.
11. The method of claim 10 , further comprising the step of recovering said recombinant cytochrome P450-BM3 variant.