Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants

Jelle Reinen, Daniel van Hemert, Nico P E Vermeulen, Jan N M Commandeur

Research output: Contribution to JournalArticleAcademicpeer-review

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.

Original languageEnglish
Pages (from-to)1246-55
Number of pages10
JournalJournal of Biomolecular Screening
Volume20
Issue number10
DOIs
Publication statusPublished - Dec 2015

Fingerprint

Bioassay
Mixed Function Oxygenases
Biological Assay
Organic solvents
Cytochrome P-450 Enzyme System
Enzyme activity
Libraries
Enzymes
Dealkylation
Flow Injection Analysis
2-Propanol
Methanol
Assays
Screening
Fluorescence

Keywords

  • Amitriptyline
  • Bacterial Proteins
  • Cytochrome P-450 Enzyme System
  • Enzyme Assays
  • Flow Injection Analysis
  • Fluorescence
  • Mutation
  • NADPH-Ferrihemoprotein Reductase
  • Solvents
  • Journal Article

Cite this

@article{635b0191c0a140c88fd9bbdb40580c4e,
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.",
keywords = "Amitriptyline, Bacterial Proteins, Cytochrome P-450 Enzyme System, Enzyme Assays, Flow Injection Analysis, Fluorescence, Mutation, NADPH-Ferrihemoprotein Reductase, Solvents, Journal Article",
author = "Jelle Reinen and {van Hemert}, Daniel and Vermeulen, {Nico P E} and Commandeur, {Jan N M}",
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journal = "Journal of Biomolecular Screening",
issn = "1087-0571",
publisher = "SAGE Publications Inc.",
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}

Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants. / Reinen, Jelle; van Hemert, Daniel; Vermeulen, Nico P E; Commandeur, Jan N M.

In: Journal of Biomolecular Screening, Vol. 20, No. 10, 12.2015, p. 1246-55.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants

AU - Reinen, Jelle

AU - van Hemert, Daniel

AU - Vermeulen, Nico P E

AU - Commandeur, Jan N M

N1 - © 2015 Society for Laboratory Automation and Screening.

PY - 2015/12

Y1 - 2015/12

N2 - 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.

AB - 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.

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KW - NADPH-Ferrihemoprotein Reductase

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