The V,, and KM of various forms of lipase from Pseudomonas cepacia ( powder, adsorbed onto Celite or covalently linked to polyethylene glycol ) were determined in organic solvents preequilibrated to water activities ( a, ) from ~0.1 to 0.84. The model reaction was the transesterification between n-octanol and vinyl butyrate. It was found that Ki,., for the nucleophile increased with increasing a, for all three lipase forms. V,, increased with increasing a, for polyethylene glycol-lipase, whereas there was an optimum at intermediate a, values ( 0.11 -0.38 ) for lipase powder and Celite-immobilized lipase.

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NAD(P)-dependent hydroxysteroid dehydrogenases were used as catalysts for the oxidoreduction of the hydroxyl-keto groups of cholic acid (3cu,7a,l2a-trihydroxy-5~-cholan-24-oic acid) and dehydrocholic acid (3,7,12-trioxo-5~-cholan-24-oic acid). Cholic acid was regiospecifically oxidized, on a preparative scale, at each of the three possible positions, and dehydrocholic acid regio-and stereospecifically reduced at each of the three positions. The compounds were quantitatively transformed and the producta were 97-99% pure. The assignment of product structure was made by NMR. The nicotinamide cofactors were enzymatically regenerated, in situ, with the a-ketoglutarate/glutamate dehydrogenase, formate/formate dehydrogenase or glucose/glucose dehydrogenase systems. The enzymes were employed in the free form or immobilized on Sepharose CL-4B.

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Cholic acid (3a.7a12a-trihydroxy-5~cholanoic acid) was completely and selectively transformed into 1Zketomsodeoxycholic acid (3a,7@lihydroxy-12-oxo-Sf&cholanoic acid) by means of hvo consecutive enzymatic steps catalyzed, the fifit. by 7u-and 12a-hydroxysteroid dehydrogenase and, the second, by 76-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with a-ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.