Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures

M.P. Machielsen, N.G.H. Leferink, A. Hendriks, S.J.J. Brouns, H. Hennemann, T. Daussmann, J. van der Oost

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33 Citations (Scopus)

Abstract

There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30°C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30°C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of ~1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30°C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183.
Original languageEnglish
Pages (from-to)587-594
JournalExtremophiles
Volume12
Issue number4
DOIs
Publication statusPublished - 2008

Keywords

  • directed evolution
  • escherichia-coli
  • protein
  • (2r,5r)-hexanediol
  • biocatalysis
  • catalysis
  • nadp(+)
  • complex
  • design
  • acid

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