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

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 pH6.5. 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 pH7.0 and 30°C the amidohydrolase had a half-life of around 350 days; at 44°C it was only 10 min. Except for Ni^sup 2+^ and, to some extent, Zn^sup 2+^ and Co^sup 2+^, the enzyme was neither strongly influenced by metal cations nor by chelating agents, but was inhibited by 95% at 0.1mM phenylmethylsulfonyl fluoride. The molecular mass of the native enzyme was estimated to be 94kDa by gel filtration and 50kDa by SDS-PAGE, suggesting a dimeric structure. Specificity experiments revealed a spectrum of related N-acetylated compounds being hydrolyzed with variable enantiomeric selectivities.[PUBLICATION ABSTRACT]