title: Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds.
abstract: 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.
authors.affiliation: International University Bremen (IUB), Campus Ring 8, 28759 Bremen, Germany.
authors.forename: T S
authors.initials: TS
authors.lastname: Wong
authors.forename: N
authors.initials: N
authors.lastname: Wu
authors.forename: D
authors.initials: D
authors.lastname: Roccatano
authors.forename: M
authors.initials: M
authors.lastname: Zacharias
authors.forename: U
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: 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
chemical_list: 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