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/polya...
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| Veröffentlicht in: | Applied microbiology and biotechnology 1997-05, Vol.47 (5), p.515-520 |
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| creator | BRUNELLA, A GRAF, M KITTELMANN, M LAUMEN, K GHISALBA, O |
| description | 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] |
| doi_str_mv | 10.1007/s002530050965 |
| format | Article |
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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. 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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. 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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]</abstract><cop>Berlin</cop><pub>Springer</pub><doi>10.1007/s002530050965</doi><tpages>6</tpages></addata></record> |
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| ispartof | Applied microbiology and biotechnology, 1997-05, Vol.47 (5), p.515-520 |
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| source | SpringerLink Journals - AutoHoldings |
| subjects | Ammonium Ammonium sulfate Bacteria Biological and medical sciences Biology of microorganisms of confirmed or potential industrial interest Biotechnology Cations Chelating agents Enzymes Fundamental and applied biological sciences. Psychology Miscellaneous Mission oriented research Rhodococcus equi Sulfates |
| title | Production, purification, and characterization of a highly enantioselective (S)-N-acetyl-1-phenylethylamine amidohydrolase from Rhodococcus equi Ac6 |
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