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Preparative scale Baeyer–Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process

Nature Protocols volume 3, pages 546–554 (2008)Cite this article

Abstract

An efficient biocatalytic process based on the use of adsorbent resin (in situ substrate feeding and product removal) makes experiments at high substrate concentration possible by overcoming limitations due to substrate and product inhibition. This process was successfully applied to the preparative scale Baeyer–Villiger biooxidation of (−)-(1S,5R)-bicyclo[3.2.0]hept-2-en-6-one (25 g). Whole cells of recombinant E. coli (1 liter) overexpressing cyclohexanone monooxygenase were used as a biocatalyst and the substrate was preloaded onto the adsorbent resin. The corresponding lactone was obtained in 75–80% yield. Time for cell growth and biotransformation is about 24 h each and oxygen supply can be improved by using a tailor-made bubble column.

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Figure 8: Kinetic profile of the BV oxidation of 25 g of (−)-(1S,5R)-1 in the 1-liter bubble column over two cycles: exchange of biocatalyst after 10 h.
Figure 9: GC analysis (Chirasil-Dex column, 110 °C, 1.2 bar He) of (−)-(1R,5S)-2 after bulb-to-bulb distillation.

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Acknowledgements

This work was supported by European Community Fifth Framework Program (QLK3-CT2001-00403). The Swiss Federal Office for Education and Science (BBW) and the Foundation Ramón Areces are acknowledged for postdoctoral fellowships.

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Authors and Affiliations

  1. Biosciences CNRS 3005, Aix-Marseille Université, Avenue Escadre Normandie-Niemen, Marseille, 13397, France

    Iris Hilker, Maria C Gutiérrez, Roland Furstoss & Véronique Alphand

  2. Department of Biochemistry and Molecular Biology, University College of London, Gower Street, London, WC1E 6BT, UK

    John Ward

  3. Research Specialties, Sigma-Aldrich, Industriestrasse 25, Buchs, CH-9470, Switzerland

    Roland Wohlgemuth

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  1. Iris Hilker
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Correspondence to Véronique Alphand.

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Competing interests

One of the authors (RW) is employed by Sigma Aldrich Ldt. Another one (IH) was employed at that time. We collaborated via a European “demonstration” program (EC Framework 5). This company also provided us with most of chemicals and resins.

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Hilker, I., Gutiérrez, M., Furstoss, R. et al. Preparative scale Baeyer–Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process. Nat Protoc 3, 546–554 (2008). https://doi.org/10.1038/nprot.2007.532

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