Abstract
An NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum was purified 15-fold with a yield of 46%. It was homogeneous by gel electrophoresis after three chromatographic steps. The apparent molecular mass was estimated by column chromatography to be 240 kDa. SDS-gel electrophoresis revealed the presence of 33 kDa subunits. Substrates of the enzyme were ethyl and methyl 3-oxobutyrate, 3-oxobutyryl-N-acetylcysteamine thioester, and 3-oxobutyryl coenzyme A. The specific activities were 340 and 10 U (mg protein)-1 for the reduction of 3-oxobutyryl coenzyme A and ethyl 3-oxobutyrate, respectively; the Michaelis constants were 300 μM and 300 mM, respectively. The identity of 12 N-terminal amino acid residues was determined. The ezmyme was used in a preparative reduction of substrate, yielding ethyl (S)-3-hydroxybutyrate (>99% enantiomeric excess).
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Bayer, M., Günther, H. & Simon, H. Purification and characterization of the NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum . Arch. Microbiol. 163, 310–312 (1995). https://doi.org/10.1007/BF00393386
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DOI: https://doi.org/10.1007/BF00393386