Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds.
Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 microM phenol and standard deviations below 11% in 96-well plate assays. The monooxygenase P450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a saturation mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.
Wong
T S
TS
International University Bremen (IUB), Campus Ring 8, 28759 Bremen, Germany.
Wu
N
N
Roccatano
D
D
Zacharias
M
M
Schwaneberg
U
U
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D006571:Heterocyclic Compounds / Q000737:chemistry*; D006841:Hydrocarbons, Aromatic / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D001426:Bacterial Proteins; D006571:Heterocyclic Compounds; D006841:Hydrocarbons, Aromatic; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases