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.
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Received: 20 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996
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Brunella, A., Graf, M., Kittelmann, M. et al. Production, purification, and characterization of a highly enantioselective (S )-N-acetyl-1-phenylethylamine amidohydrolase from Rhodococcus equi Ac6. Appl Microbiol Biotechnol 47, 515–520 (1997). https://doi.org/10.1007/s002530050965
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DOI: https://doi.org/10.1007/s002530050965