Sutherland J D, Williams C N, Hutchison D M, Holdeman L V
Department of Medicine, Dalhousie University, Halifax, NS, Canada.
Can J Microbiol. 1987 Aug;33(8):663-9. doi: 10.1139/m87-116.
A gram-positive, rod-shaped anaerobe (strain F-6) was isolated from soil. This organism was identified by cellular morphology as well as fermentative and biochemical data as Clostridium bifermentans. Strain F-6 formed 7-ketolithocholic acid from chenodeoxycholic acid and 7-ketodeoxycholic acid from cholic acid in whole cell cultures, but did not transform deoxycholic acid, ursodeoxycholic acid, or ursocholic acid. This reaction is reversible. The structures of 7-ketolithocholic acid and 7-ketodeoxycholic acid were verified by mass spectroscopy and by thin-layer chromatography using Komarowsky's spray reagent. When incubated with the strain F-6 glycine and taurine conjugates of the primary bile acids were partially hydrolyzed and transformed to 7-keto products. Optimal yields of 7-ketolithocholic acid and 7-ketodeoxycholic acid were obtained after 78 h of incubation. Culture pH changed with time and was characterized by an initial drop (1.1 pH units) and a gradual increase back to the starting pH (7.3). Corroborating these observations, an inducible, NADP-dependent, 7 alpha-hydroxysteroid dehydrogenase was demonstrated in cell extracts of strain F-6. A trace of NAD-dependent 7 alpha-hydroxysteroid dehydrogenase was also found. A substantial increase in the specific activity of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was observed when either 7-ketolithocholic acid, chenodeoxycholic acid, or deoxycholic acid was included in the growth medium. Optimal induction of the NADP-dependent 7 alpha-hydroxysteroid dehydrogenase was achieved with 0.3-0.4 mM 7-ketolithocholic acid. Production of the enzyme(s) was optimal at 6-8 h of growth and the 7 alpha-hydroxysteroid dehydrogenases had a pH optimum of approximately 11.(ABSTRACT TRUNCATED AT 250 WORDS)
从土壤中分离出一株革兰氏阳性、杆状厌氧菌（菌株F-6）。通过细胞形态学以及发酵和生化数据鉴定该微生物为双发酵梭菌。在全细胞培养物中，菌株F-6可将鹅去氧胆酸转化为7-酮石胆酸，将胆酸转化为7-酮脱氧胆酸，但不能转化脱氧胆酸、熊去氧胆酸或熊胆酸。该反应是可逆的。通过质谱和使用科马罗夫斯基喷雾试剂的薄层色谱法验证了7-酮石胆酸和7-酮脱氧胆酸的结构。当与菌株F-6一起孵育时，初级胆汁酸的甘氨酸和牛磺酸共轭物会部分水解并转化为7-酮产物。孵育78小时后可获得7-酮石胆酸和7-酮脱氧胆酸的最佳产量。培养物的pH随时间变化，其特征是最初下降（1.1个pH单位），然后逐渐回升至起始pH（7.3）。与这些观察结果一致，在菌株F-6的细胞提取物中证实了一种可诱导的、依赖NADP的7α-羟基类固醇脱氢酶。还发现了微量的依赖NAD的7α-羟基类固醇脱氢酶。当生长培养基中包含7-酮石胆酸、鹅去氧胆酸或脱氧胆酸时，观察到依赖NADP的7α-羟基类固醇脱氢酶的比活性大幅增加。用0.3-0.4 mM 7-酮石胆酸可实现对依赖NADP的7α-羟基类固醇脱氢酶的最佳诱导。酶的产生在生长6-8小时时最佳，且7α-羟基类固醇脱氢酶的最适pH约为11。（摘要截短至250字）
