title: Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants.
abstract: A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3-mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small library of novel CYP BM3 mutants in flow-injection analysis mode in the presence of the organic modifiers methanol, acetonitrile, and isopropanol. Mutants with enhanced tolerability toward all three solvents were identified, and the EAD setup was adapted to facilitate CYP BM3 activity screening against a gradient of an organic modifier to study the behavior of the small library of CYP BM3 mutants in more detail. The simple methodology used in this study was shown to be a very powerful tool to screen for novel CYP BM3 mutants with increased tolerability toward organic solvents.
authors.affiliation: Division of Molecular Toxicology, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Jelle
authors.initials: J
authors.lastname: Reinen
authors.affiliation: Division of Molecular Toxicology, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Daniel
authors.initials: D
authors.lastname: van Hemert
authors.affiliation: Division of Molecular Toxicology, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.affiliation: Division of Molecular Toxicology, Amsterdam Institute for Molecules, Medicines and Systems, Faculty of Sciences, VU University Amsterdam, Amsterdam, The Netherlands j.n.m.commandeur@vu.nl.
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
mesh_terms: D000639:Amitriptyline / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D017022:Flow Injection Analysis / Q000379:methods*; D005453:Fluorescence; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D012997:Solvents; D000639:Amitriptyline; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases
keywords: CYP BM3; continuous-flow enzyme activity detection; cytochrome P450 monooxygenase BM3; flow-injection analysis; organic solvent tolerability