Hydrolysis of bile acid conjugates by selected fungi

Summary Fungi which have been previously shown to hydrolyse glycocholic acid, with liberation of the free bile acid, have now been shown to be similarly capable of hydrolysing glycodeoxycholic acid. Sodium taurocholate, however, is much less susceptible and its hydrolysis has been demonstrated with only one of the selected fungi, Penicillium chrysogenum, growing in a medium containing the conjugate as the sole sulphur source. It is concluded that the nature of the amino acid moiety is important in determining the ease of hydrolysis of bile acid conjugates by whole cells of the fungi under test.     

Transformation of cholic acid by Clostridium bifermentans

Suspensions of washed, resting-stage cells of Clostridium bifermentans were incubated with cholic acid under a variety of conditions in an attempt to manipulate the competing 7α-dehydroxylation and 7α-dehydrogenation reactions. Important variables thus identified were then tested in batch fermentation. Sodium thioglycollate was observed to inhibit dehydrogenation and to delay dehydroxylation. Zinc ion inhibited both dehydrogenation and dehydroxylation. In the presence of EDTA, near quantitative 7α-dehydrogenation was achieved with no associated dehydroxylation. Finally, the presence of air in the fermenter headspace enhanced both transformations, possibly through an effect on the redox potential of the medium.     

Biohydrogenation of linoleic acid by Clostridium sporogenes, Clostridium bifermentans, Clostridium sordellii and Bacteroides sp.

Abstract Several strains of Clostridium bifermentans, Clostridium sporogenes and Clostridium sordellit and one strain of Bacteroides sp. hydrogenate linoleic acid into transvaccenic acid in vitro following the same pathway. Linoleic acid (18:2; 9- cis , 12- cis ) was first isomerised into 9- cis , 11- trans -octadecadienoic acid, after which the 9- cis double bond was reduced. These species also hydrogenated linoleic acid into an octadecenoic acid in vivo when mono-associated with gnotobiotic rats. Several other species of Clostridium and Bacteroides did not hydrogenate linoleic acid.     

Oxidation of primary bile acids by a 7 alpha-hydroxysteroid dehydrogenase elaborating Clostridium bifermentans soil isolate

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)     

Appendages of Clostridium bifermentans Spores

Four distinct spore appendage types were detected in an electron microscope survey of 12 strains of Clostridium bifermentans. A smooth tubular appendage and a feather-like appendage are described in detail. In addition, hirsute tubular appendages and small pin-like appendages are depicted. Spores of four strains apparently lack appendages.     

Differentiation Between Clostridium sordellii and Clostridium bifermentans by Gas Chromatography

Clostridium bifermentans can be differentiated from Clostridium sordellii by gas chromatography on the basis of amines detected after growth for 6 hr in cookedmeat medium. Products detected after exposure of resting cells to amino acids gave evidence for the probable origin of the amines.     

Producing hydrogen from wastewater sludge by Clostridium bifermentans

Excess wastewater sludge collected from the recycling stream of an activated sludge process is biomass that contains large quantities of polysaccharides and proteins. However, relevant literature indicates that the bio-conversion of wastewater sludge to hydrogen is limited and therefore not economically feasible. This work examined the anaerobic digestion of wastewater sludge using a clostridium strain isolated from the sludge as inoculum. A much higher hydrogen yield than presented in the literature was obtained. Also, the effects of five pre-treatments-ultrasonication, acidification, sterilization, freezing/thawing and adding methanogenic inhibitor-on the production of hydrogen were examined. Freezing and thawing and sterilization increased the specific hydrogen yield by 1.5-2.5 times to that of untreated sludge, while adding an inhibitor and ultrasonication reduced the hydrogen yield.     

Lyophilized Clostridium perfringens 3 alpha- and Clostridium bifermentans 7 alpha-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis

Preparations of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form. Purification of the enzyme was achieved using triazine dye affinity chromatography. C. perfringens 3 alpha-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield). Quantitative measurement of bile acids with the purified enzymes, 3 alpha-hydroxysteroid dehydrogenase and 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically. Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20-100 microM. Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3 alpha-hydroxysteroid dehydrogenase and showed also that 7 alpha-hydroxyl groups were independently quantified by the 7 alpha-hydroxysteroid dehydrogenase. Bile acids in Folch extracts of human bile samples were measured using purified preparations of Pseudomonas testosteroni 3 alpha-hydroxysteroid dehydrogenase, C. perfringens 3 alpha-hydroxysteroid dehydrogenase, Escherichia coli 7 alpha-hydroxysteroid dehydrogenase and C. bifermentans (strain F-6) 7 alpha-hydroxysteroid dehydrogenase. Statistical comparison validated the use of C. perfringens 3 alpha- and C. bifermentans 7 alpha-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.     

The hydrolysis of bile acid conjugates

Studies were made of a) the relationship of bile acid structure and analytical recoveries (measured by 3-hydroxysteroid oxidoreductase) following vigorous alkaline hydrolysis of bile acid conjugates and b) the relationship of structure and hydrolysis time of taurine- and glycine bile acid conjugates in a reaction catalyzed by glycocholic acid hydrolase. Alkaline hydrolysis resulted in good recoveries of hydroxy and 7 and 12- oxo-bile acids but poor recoveries of 3-oxo-bile acids. Borohydride reduction of the 3-oxo-acids prevented these losses. Complete enzymatic hydrolysis of glycine conjugated bile acids was about five times more rapid than that of taurine conjugates. Hydrolysis of conjugates containing oxo groups was slow. Borohydride reduction of oxoacids corrected this and did not inhibit enzymatic hydrolysis. It was concluded that both vigorous alkaline and enzymatic hydrolysis are satisfactory in bile acid assays if borohydride reduction is instituted before the hydrolytic step. However, due to the presence of possible enzyme inhibitors and solubility difficulties, strong alkaline hydrolysis is preferable to enzymatic hydrolysis in fecal bile acid determinations at this time.     

Appendage Development in Clostridium bifermentans

The appendages of Clostridium bifermentans UK-A 1003 spores were shown to originate from a substance located just exterior to the outer forespore membrane. The dense spore coat develops along the periphery of this material, and, as the appendages develop in the cytoplasm, the coalescing spore coat intervenes between the appendages and their origin. Freeze etching revealed that the appendages are in the form of distinct fibers in proximity to the mature spore body. These fibers form a network around the spore, seemingly encasing it and insuring that the appendages remain attached to the mature, free spore. The inner wall of each appendage tubule is lined with fibers whereas the outer surface is smooth. The developing exosporium contained several layers consisting of small (3 nm) globular subunits; the outer exosporial surface is composed of relatively unstructured material.     

Biochemical properties of Clostridium bifermentans spores.

As previously found for spores of Bacillus species, dormant spores of Clostridium bifermentans contained essentially no adenosine triphosphate, a high level of adenosine monophosphate, a high level of 3-phosphoglyceric acid, and much transfer ribonucleic acid lacking a 3'-terminal adenosine monophosphate residue. As in spores of Bacillus species, germination of C. bifermentans spores was accompanied by utilization of the 3-phosphoglyceric acid, a large increase in the adenosine triphosphate level, and the disappearance of defective transfer ribonucleic acid. In contrast to spores of Bacillus species, dormant spores of C. bifermentans contained little free amino acid.