Hydroxylation of steroids with 11 alpha-hydroxylase of Rhizopus nigricans

Three groups of 3-keto-4-ene steroids with different side chains were used as substrates for the induced 11 alpha-hydroxylase of Rhizopus nigricans. The highest total bioconversion as well as the highest yield of 11 alpha-hydroxylated product is found using progesterone as substrate. By changing the polarity of the side chain, much higher yields of 6 beta- and 7 beta-hydroxylated products relative to 11 alpha-hydroxylated product are obtained. Our results thus provide evidence for the importance of the side chain in steroid-enzyme interactions.     

Effect of shear on membrane-associated enzymes: Studies of the release of intracellular protein and of the progesterone 11alpha-hydroxylase complex from rhizopus nigricans

Rhizopus nigricans ATCC 6227b grown in either shake flasks or a fermentor was sheared in a concentric cylinder viscometer. The cells grown in shake flasks were found to be more susceptible to disruption by shear than those grown in the fermentor. Cells resuspended in a medium containing reduced glutathione and EDTA were found to be more easily disrupted than cells resuspended in 0.5% NaCl. The optimum condition for disruption of shake flask cells grown in the former medium with retention of progesterone 11alpha-hydroxylase activity was a laminar shear rate of 4300 s(-1), for a period of 3 min at 4 degrees C. During the first 30 s the apparent viscosity was found to decrease significantly with applied shear.     

Hydroxylation of steroids with nonpolar side chains with 11 alpha-hydroxylase of Rhizopus nigricans

Steroids with nonpolar side chains with 2, 4 and 8 C atoms were used as substrates for the 11 alpha-hydroxylase of Rhizopus nigricans. Their bioconversion was compared to that of progesterone, which was found to be far the best substrate giving the highest total bioconversion. 3-keto-4-ene steroids with nonpolar side chains were converted to their hydroxylated products in a small yield or not at all. The absence of an oxygen function in the side chain did not affect the regio-specificity of the hydroxylation, but resulted in a much lower total bioconversion. The strong effect of the oxygen function and of the length of the side chain on hydroxylation with the 11 alpha-hydroxylase of Rhizopus nigricans was demonstrated.     

Inhibition of prostaglandin biosynthesis by c-5,c-8,c-11-eicosatrienoic acid.

The enzyme prostaglandin H leads to E-isomerase (EC 5.3.99.3), which is present in sheep vesicular gland and needs glutathione as cofactor, is inhibited by c-5,c-8,c-11-eicosatrienoic acid, the fatty acid accumulating during essential fatty acid deficiency. The EFA-deficiency syndrome can partly be explained from a prostaglandin deficiency caused by lack of precursors. The present finding indicates that 5,8,11-eicosatrienoic acid could well be an additional factor in modifying the symptoms of EFA-deficiency.     

Optimization of the production of progesterone 11α-hydroxylase by Rhizopus nigricans

The progesterone 11α-hydroxylase of Rhizopus nigricans ATCC 6227b is an inducible enzyme system that is primarily induced by its substrate progesterone. Maximum induction was found at a progesterone concentration of 0.5 g/liter or above. Oxygen is the other substrate for the hydroxylation and this was found to have a major effect on the amounts of hydroxylase synthesized. Optimum induction of the hydroxylase in a fermentation with a 3.1 m/sec impeller tip speed was found to occur at a dissolved oxygen tension (DOT) of 10% of air saturation. The agitation rate also effects the amount of hydroxylase synthesized with an apparent maximum at 3.1 m/sec impeller tip speed. The DOT for a maximum hydroxylation rate was much higher than for enzyme synthesis so that it was preferable to increase the DOT after induction was completed.     

Microbial transformations of steroids--I. Rare transformations of progesterone by Apiocrea chrysosperma

When Apiocrea chrysosperma is incubated with progesterone for 7 days in a peptone, yeast-extract medium, eight major metabolites are produced. Each compound has been purified and its structure determined by high-field 1D and 2D 1H nuclear magnetic resonance (NMR) spectroscopy. A clear synthetic pattern is recognisable. The products have been formed by multiple transformation reactions, usually double hydroxylations. Seven compounds are tertiary alcohols in which the hydroxyl group is located on the underside of the progesterone skeleton at either the axial 9 alpha- or the axial 14 alpha-site. One compound has hydroxyl groups at both these sites. Five metabolites are also secondary progesterone alcohols, the hydroxyl groups being at the 6 beta-, 15 alpha- or 15 beta-sites. Two compounds are monohydroxy metabolites; one is dehydrogenated in ring B and the other has lost the pregnane side-chain. The structures of the eight metabolites are 6 beta, 9 alpha-dihydroxyprogesterone; 6 beta, 14 alpha-dihydroxyprogesterone; 9 alpha, 14 alpha-dihydroxyprogesterone; 9 alpha, 15 beta-dihydroxyprogesterone, 14 alpha, 15 alpha-dihydroxyprogesterone; 14 alpha, 15 beta-dihydroxyprogesterone; 14 alpha-hydroxypregna-4,6-diene-3,20-dione and 15 alpha-hydroxyandrostene-3,17-dione. All compounds, except the last one, are biologically rare because they are not products of mammalian progesterone or androstenedione metabolism. They would be difficult to synthesise chemically. We believe that the compounds, 9 alpha, 15 beta-dihydroxyprogesterone; 14 alpha, 15 alpha-dihydroxyprogesterone and 14 alpha-hydroxypregn-4,6-diene-3,20-dione, have not been reported previously as microbial transformation products of progesterone.     

Hydroxylation of progesterone by Cunninghamella blakesleeana NCIM 687

Progesterone was completely hydroxylated, principally to 11,14 dihydroxyprogesterone by Cunninghamella blakesleeana NCIM 687 in 48h.S.B. Chincholkar is with the Microbiology Division, Department of Life Sciences, North Maharashtra University, Jalgaon 425 001, India. R. Seeta Laxman is with the Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, India. R.D. Wakharkar is with Organic Chemistry. Technology Division, National Chemical Laboratory, Pune 411 008. India     

Microbiological Aspects in the Hydroxylation of Estrogens by Fusarium moniliforme

A strain of Fusarium moniliforme (IH4), isolated from soil, showed outstanding enzymatic abilities to hydroxylate a number of estrogens. Estrone and estradiol were transformed into the 15alpha-hydroxy derivatives, and estradiol 3-methyl ether was transformed into the corresponding 6beta-hydroxy derivative. Delta(6)-Estrone was not hydroxylated. The accumulation of 15alpha-hydroxyestrone was influenced by the nutritional conditions of the fungus. Maximal yield was obtained when the organism grew in Czapek solution supplemented with yeast extract, although good conversion was also found in a peptone-corn molasses medium. Substitution of NO(3)-N in Czapek medium with NH(4)-N, lactalbumin hydrolysate, Casitone, or Casamino Acids resulted in limited hydroxylation of estrone. A remarkable strain specificity was demonstrated in this conversion. Of 13 strains of F. moniliforme and Gibberella fujikuroi under investigation, only 2 strains (IH4 and ATCC 9851) accumulated substantial amounts of the 15alpha-hydroxylated product. However, marked quantitative variations were observed which are attributable to a different ability of the organisms to degrade the steroid nucleus. Biochemical instabilities were also found through the appearance of spontaneous variants lacking steroid-hydroxylating activity. Replacement culture studies revealed that 15alpha-hydroxylation of estrone was dependent on the supply of external phosphate; exogenous nitrogen or energy sources were not required. Most of the enzymatic activity was confined to the mycelia. Microconidia showed a very limited hydroxylating activity, even in the presence of supplements or energy sources.     

Microbial transformation of steroids--VII. Hydroxylation of progesterone by extracts of Phycomyces blakesleeanus

Post mitochondrial supernatants (S-12 extracts) were prepared from Phycomyces blakesleeanus by grinding washed and frozen mycelial cakes in fine sand and extracting the paste produced with buffer containing Tris-HCl pH 7.8 (0.1 M), EDTA (0.01 M), dithiothreitol (5 mM) and glycerol (10% v/v). The S-12 extracts, obtained in this way, reproducibly hydroxylated progesterone, producing 7 alpha- and 15 beta-hydroxyprogesterone the major products of whole-cell transformation. Cell-free progesterone hydroxylation was found to be approximately linearly dependent on extract concentration, to require reduced NADP (partly replaceable by NADH), and to be dependent on progesterone (apparent Km calculated to be 4 mM). K+ and Mg2+ were found not to be required. Maximum progesterone hydroxylation occurred after 2 h at pH 7.8 and at 24 degrees C. Using optimum conditions S-12 extracts were capable of hydroxylating between 5 and 15% of added progesterone (0.2 mM). Hydroxylation was found to be partially inhibited by carbon monoxide (ca 40%) and almost completely inhibited by azoles, ketoconazole and diconazole. The NADPH and molecular oxygen requirements were replaceable by NaIO4. These findings strongly suggest that hydroxylation was being catalyzed by cytochrome P-450. This was confirmed by preparing progesterone-hydroxylating microsomes and Triton N-101-solubilized microsome extracts, and by obtaining a dithionite-reduced carbon monoxide-difference absorption spectrum peak at 455 nm in the solubilized microsome extracts.     

Factors affecting the induction of 11 alpha-hydroxylase of progesterone in the filamentous fungus Rhizopus nigricans

The 11 alpha-hydroxylase of progesterone was induced in the filamentous fungus Rhizopus nigricans ATCC 6227b with different steroids as inducers and the induction process was optimized in regard to the age of the mycelium, to the concentration of the inducer and to the time of induction. Deoxycorticosterone and testosterone, steroids with higher polarity of the side-chain than progesterone, although poorer substrates for in vivo hydroxylation than progesterone, induced more enzyme compared to progesterone. Other alterations in the steroidal ring system examined diminished the induction capability of the inducing steroid to different extent. The highest 11 alpha-hydroxylating activity, if expressed on the basis of mycelial wet weight, was achieved with 18 h old mycelium which was induced for 2 h with 0.30 mM deoxycorticosterone.     

Microbiological transformations of lipids: acyl-specific hydrolysis of lard by yeasts.

The fatty acid and positional specificities of Saccharomyces cerevisiae (UI-SACCH) and Schizosaccharomyces octosporus (NRRL Y-854) in the hydrolysis of lard were studied by using gas-liquid chromatography. Synthetic triglycerides were used to determine the positional specificities of the lipases of both organisms. Palmitic acid is specifically cleaved from all three triglyceride ester positions by S. cerevisiae, while S. octosporus was able to cleave stearic acid at either position 1 or position 3 of the glycerol moiety. Preparative scale fermentation with 200 g of lard per liter yielded 48.4 g of palmitic acid per liter with S. cerevisiae and 42 g of stearic acid per liter with S. octosporus. The free fatty acids produced by microbial transformation of lard were characterized spectrally (1H and C nuclear magnetic resonance and mass spectrometry) and chromatographically (thin-layer and gas chromatographies).     

Microbial transformations of steroids--III. Transformation of progesterone by Sepedonium ampullosporum

The 16 alpha-steroid hydroxylating fungus Sepedonium ampullosporum (CMI strain 203 033) transformed progesterone into 16 alpha-hydroxyprogesterone and four other major metabolites which have not been reported previously for this organism, 6 beta-hydroxyprogesterone, 17 alpha-hydroxyprogesterone, 16 alpha-hydroxyandrostenedione and 16-oxotestosterone (16-ketotestosterone). Among the minor metabolites we have been able to identify 15 alpha-hydroxyprogesterone. This compound has not been reported for S. ampullosporum. The conditions used for transformation had comparatively little effect on the relative proportions of products formed, 16 alpha-hydroxyprogesterone always being the predominant metabolite, but had a major effect on the total yields of metabolites isolatable. These findings suggest that one or more constitutive enzyme systems were responsible for the transformations.     

Microbiological transformations of nabilone, a synthetic cannabinoid.

A screening program was conducted to find microorganisms that modify the synthetic cannabinoid nabilone. After purification, the products from three cultures were analyzed by spectral methods to determine their chemical structures. An optically active 9S-hydroxy-6aR,10aR-trans cannabinoid was isolated from a culture of an unidentified soil bacterium designated A24007. From Bacillus cereus cultures were isolated a 9S,6'-dihydroxy-6aR,10aR-trans cannabinoid, a 9S-hydroxy-6'-keto-6aR,10aR-trans cannabinoid, a 9-keto-6'-hydroxy-6aS,10aS-trans cannabinoid, and a 6',9-diketo-6aS,10aS-trans cannabinoid. All of these products were optically active, as was a 9S-hydroxy-6aS,10AS-trans cannabinoid also isolated from B. cereus cultures. A series of acidic products were isolated from cultures of Nocardia salmonicolor. All of these products contained a carboxylic acid group at the terminal end of three-position alkyl side chains having varying numbers of carbon atoms. Two of the acidic products contained a 9-keto group, whereas all other carboxylic acid products were 9-hydroxy cannabinoids. The array of products obtained from incubation of nabilone indicates the usefulness of microbial transformations in the preparation of new cannabinoids.