Substrate characterization of a NAD-dependent secondary alcohol dehydrogenase from Rhodococcus sp. GK1 (CIP 105335 (original) (raw)

2002, Journal of Molecular Catalysis B-enzymatic

A NAD-dependent secondary alcohol dehydrogenase (SAD) has been extracted from cells of the sterol-degrading bacterium, Rhodococcus sp. GK1 (CIP 105335). The dehydrogenase was partially purified by means of ammonium sulfate fractionation (60% saturation) and filtration on a Sepharose CL-6B column. The obtained enzyme sample was active with aliphatic secondary alcohols, such as 2-hexanol, and as reductase with aliphatic monoketones and diketones, such as 2-hexanone and 2,3-hexanedione. A hydrophobic environment was required for catalysis: methyl on one side and either methyl, ethyl, propyl, butyl, pentyl or hexyl on the other side of the function being transformed. The K m value for NAD or NADH with, respectively 2-propanol or acetone was around 1.60×10 −4 M at pH 7.0 and 30 • C. The enzyme affinity (1/K m ) for the examined 2-alcohols and 2-ketones (three to eight C atoms) increased with increasing the chain length. Its activity with 2-octanone was somewhat higher than that with 3-octanone, reflecting a better enzyme affinity for a function positioned at C-2. The K m values for the 2-alcohols (pH 7.0, 30 • C) ranged from 6.0 × 10 −2 M for 2-propanol to 1.8 × 10 −3 M for 2-octanol. Reciprocally, the K m values for the 2-ketones ranged from 6.5 × 10 −2 M for acetone to 2.1 × 10 −3 M for 2-octanone. With 2-hexanol as the substrate, the optimal temperature was around 55 • C and the activation energy of the system was 9.49 kcal/mol. The SAD was specific for the (S)-(+)-stereoisomers of 2-butanol.

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