We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.

No abstract

No abstract

Cytochrome P450 monooxygenase BM‐3 (EC 1.14.14.1) hydroxylates fatty acids with chain lengths between C12 and C18. It is also known to oxidize the corresponding alcohols and amides. However, it is not known to oxidize alkanes. Here we report that P450 BM‐3 oxidizes octane, which is four carbons shorter and lacks the carboxylate functionality of the shortest fatty acid P450 BM‐3 is known to accept, to 4‐octanol, 3‐octanol, 2‐octanol, 4‐octanone, and 3‐octanone. The rate is much lower than for oxidation of the preferred fatty acid substrates. In an effort to explore the plasticity and mechanisms of substrate recognition in this powerful biocatalyst, we are using directed evolution − random mutagenesis, recombination, and screening − to improve its activity towards saturated hydrocarbons. A spectrophotometric assay has been validated for high throughput screening, and two generations of laboratory evolution have yielded variants displaying up to five times the specific activity of wild‐type P450 BM‐3.

Cytochrome P450 BM-3 variant 139-3 is highly active in the hydroxylation of alkanes and fatty acids (A Glieder, ET Farinas, and FH Arnold, Nature Biotech 2002;20:1135-1139; it also epoxidizes various alkenes, including styrene. Here the authors describe a colorimetric, high-throughput assay suitable for optimizing this latter activity by directed evolution. The product of styrene oxidation by 139-3, styrene oxide, reacts with the nucleophile γ-(4-nitrobenzyl)pyridine (NBP) to form a purplecolored precursor dye, which can be monitored spectrophotometrically in cell lysates. The sensitivity limit of this assay is 50-100 µM of product, and the detection limit for P450 BM-3 139-3 is~0.2 µM of enzyme. To validate the assay, activities in a small library of random mutants were compared to those determined using an NADPH depletion assay for initial turnover