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AssigneeCalifornia Institute of Technology
InventorsFrances H. Arnold, Matthew W. Peters, Peter Meinhold
Estimating the term…
Cytochrome P450 BM-3 from Bacillus megaterium was engineered using a combination of directed evolution and site-directed mutagenesis to hydroxylate linear alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. Mutant 9-10A-A328V hydroxylates octane primarily at the 2-positio to form S-2-octanol (40% ee). Another mutant, 1-12G, hydroxylates alkanes larger than hexane primarily at the 2-position, but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active for alkane substrates and support thousands of product turnovers. These regio- and enantio-selectivities are retained in whole-cell biotransformations with E. coli, where the engineered P450s can be expressed at high levels and the expensive cofactor is supplied endogenously.
1-50. (canceled)
A recombinant P450 enzyme comprising an amino acid sequence having at least 90% identity to SEQ ID NO:2, wherein the sequence comprises at least one non-naturally occurring mutation relative to SEQ ID NO:2 selected from the group consisting of V78A, T175I, A184V, H236Q, E252G, R255S, and L353V, and at least one non-naturally occurring mutation relative to SEQ ID NO:2 at a position selected from the group consisting of R47, K94, P142, F205, S226, and A290, and wherein the enzyme hydroxylates an alkane.
Dependencies are inferred from the claim text.