Sosamma Oommen

Studies on Bacillus stearothermophilus. Part 1. Transformation of progesterone to a new metabolite 9,10-seco-4-pregnene-3,9,20-trione.

When Bacillus stearothermophilus, a thermophilic bacterium isolated from the Kuwaiti desert, was incubated with exogenous progesterone for 24 h, three monohydroxylated metabolites were produced. 20alpha-Hydroxyprogesterone was the major metabolite produced in 60.8 relative percentage yield. The other two monohydroxylated metabolites were identified as 6beta-hydroxyprogesterone and the rare 6alpha-hydroxyprogesterone in 21.0 and 13.6 relative percentage yields, respectively. A new metabolite 9,10-seco-4-pregnene-3,9,20-trione was isolated in 3.7 relative percentage yield. All metabolites were purified by preparative TLC and HPLC followed by their identification using 1H, 13C NMR and other spectroscopic data.

Studies on Bacillus stearothermophilus. Part II. Transformation of progesterone.

Bacillus stearothermophilus, a thermophilic bacterium isolated from Kuwaiti desert, when incubated with exogenous progesterone for 10 days at 65 degrees C produced two new dihydroxy isomers of progesterone, and two known compounds, 5 alpha-pregnane-3,6,20-trione and 6-dehydroprogesterone, along with the earlier reported monohydroxylated metabolites and a B-Seco compound. The two new dihydroxy compounds were identified as 6 alpha,20 alpha-dihydroxyprogesterone and 6 beta,20 alpha-dihydroxyprogesterone. These metabolites were purified by TLC and HPLC followed by their identification through 1H, 13C NMR and other spectroscopic data.

Studies on Bacillus stearothermophilus. Part IV. Influence of enhancers on biotransformation of testosterone.

The impact of chemical enhancers on the biotransformation of testosterone has been exploited. Application of crude cell concentrates to produce Bacillus stearothermophilus-mediated bioconversion of testosterone at 65 degrees C for 72 h has been examined. After incubation, the xenobiotic substrate was added to the concentrated whole cell suspensions. The enhancer molecules were included in the whole cell suspension. The resultant products, after extraction into an organic solvent, were purified by thin layer chromatography and identification was carried out through spectroscopic data. Five steroid metabolites 9,10-seco-4-androstene-3,9,17-trione, 5alpha-androstan-3,6,17-trione, 17beta-hydroxy-5alpha-androstan-3,6-dione, 3beta,17beta-dihydroxyandrost-4-ene-6-one and 17beta-hydroxyandrost-4,6-diene-3-one were identified as biotransformation products of testosterone. A possible biosynthetic route for these bioconversion products is postulated.

Studies on Geobacillus stearothermophilus – Part V: Transformation of 11α-hydroxyprogesterone

The influence of a hydroxyl group on the biotransformation of 11α-hydroxyprogesterone mediated by the thermophile Geobacillus stearothermophilus, was investigated. Bacterial transformation of 11α-hydroxyprogesterone resulted in the formation of previously reported six hydroxylated progesterone metabolites, identified as 11α-hydroxy-5α-pregnane-3, 6, 20-trione 1, 11α, 20α-dihydroxypregnene-3-one 2, 11α, 6β-dihydroxyprogesterone 3, 11α, 6α-dihydroxyprogesterone 4, 11α, 6β, 20α-trihydroxypregnene-3-one 5, 11α, 6α, 20α-trihydroxypregnene-3-one 6. All transformation products were identified through their spectral data and comparison with reference compounds.

Studies on Geobacillus stearothermophilus-Part V1: Transformation of 17α-hydroxyprogesterone and 21-hydroxyprogesterone

Side chain cleavage by biotransformation of 17α-hydroxyprogesterone 1 and 21-hydroxyprogesterone 2 by the thermophile, Geobacillus stearothermophilus was investigated. The side chain cleavage product, androst-4-ene-3,17-dione 1a & 2a, was a common product formed from both substrates. 17α-hydroxy-5α-pregnane-3,6,20-trione 1b and 17α-20α-dihydroxyprogesterone 1c were formed from 1. A rare 17-carboxylic acid analogue of androstene 2b, a transformation product of 2, was also identified. G. stearothermophilus also facilitated hydroxylation of 17α-hydroxyprogesterone at 6α-and 6β-positions resulting in 6β,17α-dihydroxyprogesterone 1d, 6α,17α-dihydroxyprogesterone 1e, 6β,21-dihydroxyprogesterone 2c and 6α,21-dihydroxyprogesterone 2d. All transformation products were identified through their spectral data and comparison with reference compounds.

Tryptophan Oxidative Metabolism Catalyzed by Geobacillus Stearothermophilus: A Thermophile Isolated from Kuwait Soil Contaminated with Petroleum Hydrocarbons

Tryptophan metabolism has been extensively studied in humans as well as in soil. Its metabolism takes place mainly through kynurenine pathway yielding hydroxylated, deaminated and many other products of physiological significance. However, tryptophan metabolism has not been studied in an isolated thermophilic bacterium. Geobacillus stearothermophilus is a local thermophile isolated from Kuwait desert soil contaminated with petroleum hydrocarbons. The bacterium grows well at 65 °C in 0.05 M phosphate buffer (pH 7), when supplied with organic compounds as a carbon source and has a good potential for transformation of steroids and related molecules. In the present study, we used tryptophan ethyl ester as a carbon source for the bacterium to study the catabolism of the amino acid at pH 5 and pH 7. In this endeavor, we have resolved twenty one transformation products of tryptophan by GC/LC and have identified them through their mass spectral fragmentation.

Modification of progesterone and testosterone by a food-borne thermophile Geobacillus kaustophilus.

Abstract The present work was carried out to study structural modification of steroids by Geobacillus kaustophilus, a bacterial thermophile present in milk and the environment. Incubation of progesterone and testosterone with G. kaustophilus at 65°C resulted in oxygenated steroid nuclei. The oxygenation of the steroid molecule was stereo specific. Seven metabolites of progesterone─6β/6α-hydroxytestosterone, 20-hydroxyprogesterone, 6β-/6α-20-dihydroxyprogesterone, 5α-pregnane-3,6,20-trione, and 3β-hydroxy-5α-pregnane-6,20-dione─were identified. Four compounds─namely, 66-/6--hydroxytestosterone and 6β/6α-hydroxyandrostenedione─and androst-4-en-3,17-dione were identified as testosterone metabolites. This shows that G. kaustophilus is capable of modifying steroid nuclei at elevated temperatures. G. kaustophilus is a stable thermophile first isolated from milk. Our results show that endogenous steroids present in milk can be modified by G. kaustophilus, causing detrimental effect on human health.

Transformation of chenodeoxycholic acid by thermophilic Geobacillus stearothermophilus

We performed a series of experiments with Geobacillus stearothermophilus, a thermophile isolated from oil‐contaminated soil in the Kuwaiti desert. The organism has a good potential for the transformation of a broad spectrum of organic molecules such as steroids, amino acids, and aromatic hydrocarbons. In the present study, we tested its potential for the transformation of a bile component, chenodeoxycholic acid (CDCA). Five transformed products, namely, cholic acid, methylcholate, methylchenodeoxycholate, 3α‐hydroxy‐7‐oxo‐5β‐cholanic acid, and 7α‐hydroxy‐3‐oxo‐5β‐cholanic acid, were the major transformation products catalyzed by G. stearothermophilus. Under aerobic conditions, no evidence of side chain degradation, ring cleavage, or dehydrogenation was found among the metabolites of CDCA. CDCA transformation by a thermophile is reported for the first time.

Antioxidant activity of biotransformed sex hormones facilitated by Bacillus stearothermophilus.

Bacillus stearothermophilus, a thermophilic bacterium isolated from Kuwaiti desert, when incubated with exogenous progesterone for 10 days at 65 degrees C produced two monohydroxylated, two dihydroxy isomers of progesterone and a B-Seco compound. These metabolites were purified by TLC and HPLC followed by their identification through (1)H, (13)C NMR and other spectroscopic data. Microbial hydroxylation of 17beta-estradiol resulted in the production of estrone. The effect of some inducers resulted in the production of two metabolites from 17beta-estradiol, one of which was identified as 3,6beta,17beta-trihydroxyestra-1,3,5,14(10)-tetrene and the other metabolite remains unidentified. The transformation products were identified through their spectral data and comparison with reference compounds. Antioxidant activities of progesterone transformed mixture and purified metabolites of 17beta-estradiol were studied by linoleic acid/beta-carotene assay. An enhanced antioxidant activity for progesterone transformation products was observed when compared to progesterone. A comparison of antioxidant activity of progesterone and 17beta-estradiol transformation products is reported.