Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds

T. S. Wong, N. Wu, D. Roccatano, M. Zacharias, Ulrich Schwaneberg

Research output: Contribution to journalArticlepeer-review

46 Scopus citations


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 hydroxy 1 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 μM 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.

Original languageEnglish
Pages (from-to)246-252
Number of pages7
JournalJournal of Biomolecular Screening
Issue number3
StatePublished - Apr 2005
Externally publishedYes


  • Directed evolution
  • High-throughput screening
  • Hydroxylation
  • Monooxygenase
  • P450


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