Nad-dependent 3α- and 12α-hydroxysteroid dehydrogenase activities from eubacterwm lentum atcc no. 25559

Abstract

Eubacterium lentum (ATCC No. 25559) was shown to contain 3α- and 12 α- hydroxysteroid dehydrogenases both of which were NAD-dependent and active against conjugated and unconjugated bile salts. In addition, the 3 α- hydroxysteroid dehydrogenase was active against members of the Androstan series containing a 3 α- hydroxyl group regardless of the stereo-orientation of the 5-H. No measurable activity against 7α-, 7β-, 11β-, or 17 β- hydroxyl groups was demonstrated. The growth of E. lentum and the production of 3α- and 12 α- hydroxysteroid dehydrogenases were greatly enhanced by the addition of l-, d- or dl-arginine to the medium. Yields of hydroxysteroid dehydrogenase were optimal in the range of 0.50–0.75% arginine; however, the growth of the organisms was further enhanced at arginine concentrations > 0.75%. The 12 α- hydroxysteroid dehydrogenase was heat labile and could be selectively inactivated by heating at 50°C for 45 min. Both the heated enzyme preparation (containing only 3 α- hydroxysteroid dehydrogenase) and the unheated enzyme preparation (containing 3α- and 12 α- hydroxysteroid dehydrogenases) were useful in the spectrophotometric quantification of bile salts. The optimal pH values for 3α- and 12 α- hydroxysteroid dehydrogenases were 11.3 and 10.2, respectively. Kinetic studies have K m estimates of 2 · 10 −5 M and 1.0 · 10 −5 M with 3 α,7 α- dihydroxy-5 β- cholanoyl glycine and 7 α,12 α- dihydroxy-5 β- cholanoate for the two respective enzymes.