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Edgardo T Farinas

Publications and source records attributed to Edgardo T Farinas.

4 recordsLinked to original sources
PubMed2006

Fluorescence activated cell sorting for enzymatic activity.

Directed evolution is a reliable method for protein engineering and as a tool for investigating structure/function relationships. A key for a successful directed evolution experiment is oftentimes the screen. Fluorescence activated cell sorting (FACS) is powerful high-throughput screening approach to isolate and identify mutants from large protein libraries. FACS has been successful in isolating proteins with improved or altered binding affinity. However, FACS screening for mutants with enhanced catalytic activity has been met with limited success. This review focuses on the FACS screening of protein libraries for enzymatic activity.

Catalysis↗
PubMed2004

Colorimetric high-throughput assay for alkene epoxidation catalyzed by cytochrome P450 BM-3 variant 139-3.

Cytochrome P450 BM-3 variant 139-3 is highly active in the hydroxylation of alkanes and fatty acids (AGlieder, 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 gamma-(4-nitrobenzyl)pyridine (NBP) to form a purple-colored precursor dye, which can be monitored spectrophotometrically in cell lysates. The sensitivity limit of this assay is 50-100 microM of product, and the detection limit for P450 BM-3 139-3 is ~0.2 microM 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 rates.

Alkenes↗
PubMed2002

Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase.

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.

Alcohols↗