Cytochrome P450 ΒΜ-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. BM-3 variant 9-10A-A328V hydroxylates octane at the 2-position to form S-2-octanol (40% ee). Another variant, 1-12G, also hydroxylates alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee). These biocatalysts are highly active (rates up to 400 min -1 ) and support thousands of product turnovers. The regio-and enantioselectivities are retained in whole-cell biotransformations with Escherichia coli, where the engineered P450s can be expressed at high levels and the cofactor is supplied endogenously.

Picking on someone smaller. Cytochromes P450 catalyze the hydroxylation of thousands of substrates, including alkanes. No naturally occurring P450, however, is known to oxidize the smallest alkanes, ethane and methane. Here we report the direct and selective oxidation of ethane to ethanol using dioxygen, catalyzed by a cytochrome P450 BM‐3 variant engineered for high activity towards small alkanes (see scheme). Achieving P450‐catalyzed oxidation of ethane is a key step in the pathway to P450‐catalyzed methane oxidation and opens new opportunities for the bioconversion of natural gas to fuels and chemicals.

Cytochrome P450 BM-3 from Bacillus megaterium was engineered for enantioselective epoxidation of simple terminal alkenes. Screening saturation mutagenesis libraries, in which mutations were introduced in the active site of an engineered P450, followed by recombination of beneficial mutations generated two P450 BM-3 variants that convert a range of terminal alkenes to either (R)- or (S)-epoxide (up to 83 % ee) with high catalytic turnovers (up to 1370) and high epoxidation selectivities (up to 95 %). A biocatalytic system using E. coli lysates containing P450 variants as the epoxidation catalysts and in vitro NADPH regeneration by the alcohol dehydrogenase from Thermoanaerobium brockii generates each of the epoxide enantiomers, without additional cofactor.

A ligand system containing three preorganized carbamoylmethylphosphine oxide (CMPO) moieties anchored onto a rigid C(3)-symmetric triphenoxymethane platform has been developed for facile metal complexation and subsequent extraction from aqueous acidic nuclear waste streams. Intended to mimic the 3:1 CMPO-actinide stoichiometry of the extracted species in the TRUEX nuclear waste treatment process, the CMPO arms on this ligand are oriented such that all three CMPO moieties can cooperatively bind a metal ion. Extractions of simulated nuclear waste streams (10(-4) M metal in 1 M nitric acid) with solutions of this ligand in methylene chloride (10(-3) M) reveal a high affinity for the actinide thorium and a very low, but constant, affinity for the lanthanides across the series. Thorium and five lanthanide (lanthanum, cerium, neodymium, europium, and ytterbium) nitrate complexes of this ligand have been synthesized and fully characterized by X-ray crystallography, (1)H and (31)P NMR spectra, and FT-ICR-MS to elucidate the mechanism of this unique actinide selectivity. All six oxygen donors from the three CMPO arms of the ligand and one or two nitrate counterions coordinate these metals to afford 2+ cationic complexes in every case. Because of the large size of the ligand, both the thorium and lanthanide complexes present similarly charged and sized surfaces to the extraction solvents, but the thorium complex is extracted quantitatively over the lanthanide complexes. A possible rationale for this extraction behavior difference is presented and further illustrated by the extraction properties of this ligand system for the alkali metals (lithium, sodium, potassium, rubidium, and cesium) as picrate salts and by the solid- and solution-state structures of its lithium picrate complex.

Enzymes that catalyze the terminal hydroxylation of alkanes could be used to produce more valuable chemicals from hydrocarbons. Cytochrome P450 BM3 from Bacillus megaterium hydroxylates medium-chain fatty acids at subterminal positions at high rates. To engineer BM3 for terminal alkane hydroxylation, we performed saturation mutagenesis at selected active-site residues of a BM3 variant that hydroxylates alkanes. Recombination of beneficial mutations generated a library of BM3 mutants that hydroxylate linear alkanes with a wide range of regioselectivities. Mutant 77-9H exhibits 52% selectivity for the terminal position of octane. This regioselectivity is octane-specific and does not transfer to other substrates, including shorter and longer hydrocarbons or fatty acids. These results show that BM3 can be readily molded for regioselective oxidation.