Applied Microbiology and Biotechnology

, Volume 66, Issue 4, pp 393–400

Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining

  • Marco W. Fraaije
  • Jin Wu
  • Dominic P. H. M. Heuts
  • Erik W. van Hellemond
  • Jeffrey H. Lutje Spelberg
  • Dick B. Janssen
Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-004-1749-5

Cite this article as:
Fraaije, M.W., Wu, J., Heuts, D.P.H.M. et al. Appl Microbiol Biotechnol (2005) 66: 393. doi:10.1007/s00253-004-1749-5

Abstract

Baeyer–Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer–Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (kcat = 1.9 s−1, KM = 59 μM). The enzyme exhibits moderate enantioselectivity with α-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer–Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52°C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Marco W. Fraaije
    • 1
  • Jin Wu
    • 1
  • Dominic P. H. M. Heuts
    • 1
  • Erik W. van Hellemond
    • 1
  • Jeffrey H. Lutje Spelberg
    • 1
  • Dick B. Janssen
    • 1
  1. 1.Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology InstituteUniversity of GroningenGroningenThe Netherlands
  2. 2.Institute of MicrobiologyChinese Academy of SciencesBeijingChina