Abstract
An NADP-dependent 7β-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7β-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant ( K m) value of 0.05 mM for 3α,7β-dihydroxy-5β-cholanic acid whereas higher values were obtained with 3α,7β-dihydroxy-5β-cholanoyl glycine (0.20 mM), and 3α,7β-dihydroxy-5β-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and has a K m value of 0.30 mM. A molecular weight of 64 000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.
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