Applied Microbiology and Biotechnology

, Volume 47, Issue 5, pp 515–520

Production, purification, and characterization of a highly enantioselective (S )-N-acetyl-1-phenylethylamine amidohydrolase from Rhodococcus equi Ac6

  • A. Brunella
  • M. Graf
  • M. Kittelmann
  • K. Laumen
  • O. Ghisalba
ORIGINAL PAPER

DOI: 10.1007/s002530050965

Cite this article as:
Brunella, A., Graf, M., Kittelmann, M. et al. Appl Microbiol Biotechnol (1997) 47: 515. doi:10.1007/s002530050965

Abstract

Rhodococcus equi Ac6 was found to express an inducible (S )-specific N-acetyl-1-phenylethylamine amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed around pH 6.5. Purification of the enzyme to a single band in a Coomassie-blue-stained sodium dodecyl sulfate/polyacrylamide gel (SDS-PAGE) was achieved by ammonium sulfate precipitation of R. equi Ac6 crude extract and column chromatographies on Fractogel TSK Butyl-650(S) and Superose 12HR. At pH 7.0 and 30 °C the amidohydrolase had a half-life of around 350 days; at 44 °C it was only 10 min. Except for Ni2+ and, to some extent, Zn2+ and Co2+, the enzyme was neither strongly influenced by metal cations nor by chelating agents, but was inhibited by 95% at 0.1 mM phenylmethylsulfonyl fluoride. The molecular mass of the native enzyme was estimated to be 94 kDa by gel filtration and 50 kDa by SDS-PAGE, suggesting a dimeric structure. Specificity experiments revealed a spectrum of related N-acetylated compounds being hydrolyzed with variable enantiomeric selectivities.

Copyright information

© Springer-Verlag Berlin Heidelberg 1997

Authors and Affiliations

  • A. Brunella
    • 1
  • M. Graf
    • 1
  • M. Kittelmann
    • 1
  • K. Laumen
    • 1
  • O. Ghisalba
    • 1
  1. 1.Bioreactions, Core Technology Area, Pharmaceuticals Division, Novartis International Inc., CH-4002 Basel, SwitzerlandCH
  2. 2.R-1060.1.08, Novartis International Inc., CH-4002 Basel, Switzerland. Fax: +41 61 6977379 e-mail: matthias.kittelmann@pharma.novartis.comCH