A NAD-dependent secondary alcohol dehydrogenase has been isolated from the sterol-degrading bacterium Rhodococcus sp. CIP 105335 (strain GK1) [1, 2]. Some of the enzyme properties were determined. The aim of the present study is to describe further enzyme characterization. This dehydrogenase was partially purified by means of ammonium sulfate fractionation and Sepharose CL-6B gel filtration. It was found to be active against a broad range of substrates, particularly mid-chain secondary aliphatic alcohols, such as 2-hexanol, and 2-octanol. It is also active against diol substrates. It reduced monoketones or diketones, such as 3-octanone and 2,3-hexanedione. However, the enzyme is inactive against primary or aromatic alcohols. The apparent Km value for NAD⁺ with 2-propanol as the substrate was 1.69 x 10^-4 M at 30°C and pH 7.0. In similar conditions, the Km value determined for NADH with acetone was 1.60 x 10^-4 M. NADP(H) were not coenzymes for this dehydrogenase. Solvents, examined in order to dissolve enzyme substrates, such as acetonitrile [CH₃CN] or dimethylsulfoxyde [(CH₃-)2S–>O], used at 15% (v/v) inhibited the oxidative activity by 60-80%. Thermal denaturation study showed that the dehydrogenase is stable at 50°C for 1 hour at least. However, 50 % of the initial activity was lost in 10 min at 60°C. The partially purified enzyme sample could be stored at - 20°C for 2 months at least without noticeable loss in activity. Enzyme specific production* from cells grown in a chemically defined medium on sitosterol as the sole carbon and energy source was 4 folds higher than that from cells grown on acetate under similar conditions. The microorganism growth on cholate also resulted in enzyme specific production 2 folds higher than that from acetate-grown cells. The present dehydrogenase seems to resemble to that isolated from the alkane-degrading bacterium Rhodococcus erythropolis ATCC 4277 [3]. Its physiological function remains to be determined. Hypothetically, it may be involved in the catabolism of aliphatic hydrocarbon derivatives.

* Estimated for crude extracts and expressed in unit/mg protein.

[1] Kreit J., Lefebvre G. and Germain P. (1994) Membrane-bound cholesterol oxidase from Rhodococcus sp. cells. Production and extraction. J. Biotechnol. 33, 271-282.

[2] Krier F., Kreit J. and Millière J.-B. (1998) Characterization of partially purified alcohol dehydrogenase from Rhodococcus sp. strain GK1. Lett. Appl. Microbiol. 26, 283-287.

[3] Ludwig B., Akundi A. and Kendall K. (1995) A long-chain secondary alcohol dehydrogenase from Rhodococcus erythropolis ATCC 4277. Appl. Environ. Microbiol. 61, 3729-3733.