Biotechnology andApplied Biochemistry

Transformation of chenodeoxycholicacid by thermophilic Geobacillusstearothermophilus

Mohammad Afzal,∗ Sosamma Oommen, and Samira Al-Awadi

Department of Biological Sciences, Faculty of Science, Kuwait University, Kuwait City, Kuwait

Abstract.We performed a series of experiments with Geobacillusstearothermophilus, a thermophile isolated fromoil-contaminated soil in the Kuwaiti desert. The organism has agood potential for the transformation of a broad spectrum oforganic molecules such as steroids, amino acids, and aromatichydrocarbons. In the present study, we tested its potential forthe 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 byG. stearothermophilus. Under aerobic conditions, no evidenceof side chain degradation, ring cleavage, or dehydrogenationwas found among the metabolites of CDCA. CDCAtransformation by a thermophile is reported for the first time.

C© 2011 International Union of Biochemistry and Molecular Biology, Inc.Volume 58, Number 4, July/August 2011, Pages 250–255 •E-mail: afzalq8@gmail.com

Keywords: chenodeoxycholic acid, transformation,G. stearothermophilus

1. IntroductionBile acids (BAs) are potent digestive surfactants that help lipidabsorption and are used as markers in health and disease [1].Besides their established role in metabolism, they also act assignaling molecules with systemic endocrine functions [2]. Thus,BAs are involved in cell-signaling receptors such as farnesoid Xreceptors and G-protein-coupled receptors, making them impor-tant for the biotechnological production of new steroid drugs [3].Antihypercholesterolemic agents such as lithocholic acid mayalso promote or function as cocarcinogenic agents [4]. It hasbeen estimated that for each million cattle slaughtered, morethan 25 tons of BA conjugates become available as by-products[5]. Thus, there is a need for new bacterial species that can beexploited in biotechnology for the transformation of BAs intophysiologically active new agents.

Intestinal and soil anaerobic bacteria can transform BAsby deconjugation, dehydroxylation, stereospecific hydroxyla-tion, oxidation of hydroxyl groups, epimerization, and by othermiscellaneous reactions [6],[7]. Other transformation productsof BAs may arise from ring cleavage, degradation of side chain,

Abbreviations: BA, bile acid; CA, cholic acid; CDCA, chenodeoxycholic acid; EI, electronimpact; GLC, gas–liquid chromatography; MS, mass spectrometry; NADPH/NADH,nicotinamide adenine dinucleotide phosphate-nicotinamide adenine dinucleotidereduced; NMR, nuclear magnetic resonance; TLC, thin-layer chromatography.∗Address for correspondence: Mohammad Afzal, PhD, Department of Biological Sciences,Faculty of Science, Kuwait University, Kuwait City, Kuwait. Tel.: + 965 249 85712; Fax:+ 965 248 48437; e-mail: afzalq8@gmail.com.Received 26 February 2011; accepted 29 April 2011DOI: 10.1002/bab.34Published online 27 July 2011 in Wiley Online Library(wileyonlinelibrary.com)

and esterification of hydroxyl and carboxyl groups, yieldingnovel metabolites [6–8]. BA transformation products thus ob-tained may be used as lead compounds for the synthesis of bio-logically active steroidal drugs or their precursors. Ursodeoxy-cholic acid, also known as ursodiol, is microbially producedfrom lithocholic acid and is a good example of a therapeu-tic agent used in gallstone dissolution [9],[10]. The forma-tion of ursodeoxycholic acid from lithocholic acid by ediblebasidomycetes, Pleurotus, Flammulina, or Lentinus spp., andthe development of many other catabolites have been patented[11]. In addition, transposon mutagenesis has been employedfor making anti-inflammatory corticosteroids, and for the forma-tion of hydroxy-3-oxo-1,4-pregnadiene-20-carboxylic acid frombile components. This compound is a useful intermediate for theformation of steroid hormones and other therapeutically usefulsteroids [5].

Fungal transformation of BAs using Cunninghamellablakesleeana has resulted in unique metabolites of lithocholicacid [12],[13]. Secosteroids with novel molecular structures havealso been obtained after the ring cleavage [14],[15]. Thus, mi-crobial cleavage of steroid rings A–D has been reported to giveunusual products that could not be synthesized by conventionalchemical syntheses [16]. A sequential ring opening and modi-fication of the steroid nucleus resulting in innovative productshas been proposed by Hayakawa [17].

In our earlier studies, we applied thermophilic Geobacil-lus stearothermophilus for the transformation of steroids, aminoacids, and aromatic compounds [15],[18],[19]. In this study, wereport transformation of chenodeoxycholic acid (CDCA) by ther-mophilic G. stearothermophilus.

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2. Materials and methods2.1. MaterialsAll solvents used for extraction of the metabolites were ofanalytical grade. Solvents, thin-layer chromatography (TLC)plates, and phosphomolybdic were purchased from E. Merck(Darmstadt, Germany). Media components and salts were ofhigh purity and were purchased from Becton Dickinson andCompany (Franklin Lakes, NJ, USA) and E. Merck (Darmstadt,Germany). CDCA with minimum 98% purity was purchased fromSigma–Aldrich (St. Louis, MO, USA).

2.2. Methods2.2.1. Cell growthStarter cultures (100 mL) of G. stearothermophilus were incu-bated in sterilized TYE (tryptone + yeast extract + 10% mineralsalts) media adjusted to pH 7.2 in cotton-plugged Erlenmeyerflasks (250 ML) and were allowed to grow for 14 H in a shakerincubator (110 rpm) at 65◦C. Fifty microliters of the culture wasadded to 500 mL of the above-mentioned media in a 2-L Er-lenmeyer flask and was allowed to grow at 65◦C. Cells werecollected at the end of the log phase by centrifuging at 5,000gfor 20 Min at 4◦C, and were washed with 50 mM phosphatebuffer (pH 7.2) followed by their suspension in 10 mL of thesame buffer.

2.2.2. BiotransformationBiotransformation was carried out in cotton-plugged Erlen-meyer flasks (250 mL), each containing 100 mL of 50 mM phos-phate buffer (pH 7.2). CDCA (98% purity, from Sigma–Aldrich)was added in three flasks (20 mg BA/100 mL), and flasks weresterilized by autoclaving at 120◦C for 30 Min. The flasks werecooled to 20◦C; bacterial cells and 10% mineral salt solutionwere added followed by incubation at 65◦C for 120 H, whichwas found to be the best time, for biotransformation, from thetime course studies. Large-scale transformation was carried outfor CDCA in 2-L Erlenmeyer flasks for 3.5 L (7 × 500 mL) ofthe same buffered media under similar conditions as for small-scale experiments. A flask containing cells in buffer and anotherflask containing CDCA in buffer were incubated along with theexperimental flasks. These served as control flasks.

2.2.3. Purification and identificationof BA metabolitesAfter the incubation, the aqueous phase was adjusted to pH2 with 0.5 N HCl and it was exhaustively extracted with ethylacetate. The pooled organic extract was dried over anhydroussodium sulfate, filtered, and the solvent was evaporated un-der reduced pressure on a rotary evaporator. The semisolidresidue, thus obtained, was redissolved in methanol and chro-matographed on 20 × 20 cm Kieselgel-60 TLC plates. TLC plateswere developed in ethylacetate–isooctane–acetic acid (20:5:1,v/v/v) solvent mixture. The metabolites of CDCA that did not re-solve in the above-mentioned solvent mixture were resolved inanother solvent system benzene–dioxane–methanol (30:20:1,v/v/v). BAs were visualized by staining the TLC chromatogramswith a 10% ethanolic solution of phosphomolybdic acid fol-

lowed by heating at 120◦C for 10 Min. Large-scale separationof the products was achieved on TLC chromatograms stainedwith phosphomolybdic acid at one edge of the plate while theremaining plate was masked with a glass sheet. Well-resolvedbands, on the corresponding unstained plates, were markedand cut from the plates. Transformed BAs were eluted from theadsorbent by washing with a mixture of chloroform–methanol(4:1, v/v). Fractions containing identical products were pooledtogether, and the solvent was carefully evaporated to drynessunder a gentle stream of dry nitrogen gas. Purity and authen-ticity of the individual metabolites were confirmed by TLC andspectral data were obtained for identification.

2.2.4. Mutation, biotransformation, purification,and identification of metabolitesStarter culture (50 mL) of G. stearothermophilus, grown for 14 Hin a shaker incubator at 65◦C, was added to 450 mL TYE mediain 2-L baffled flasks and was allowed to grow until the mid-logphase under the same conditions. The cells were collected aftercentrifugation for 20 Min at 5,000g. Nine milliliters of the dilutedcell suspension was added to three empty Petri dishes and ex-posed to ultraviolet (UV) light (λ = 254 nm) for 3 Min. After theexposure, the cell suspension was transferred to a sterilized vialcontaining 1 mL of saturated solution of the chemical mutagen(1-methyl nitrosoguanidine). The vials with chemical mutagenwere allowed to undergo mutation for 15 Min. Cell suspensionwas subsequently streaked on agar plates. Diluted cell suspen-sion, before exposure to UV radiation, was streaked as control.All the plates were labeled and allowed to grow in an incubatorat 60◦C for 24 H. Microbial colonies were examined under a mi-croscope to observe changes in the structural cell morphologyof the organism. Colonies with morphological differences werepicked up, cultured, and used for biotransformation studies.

Large-scale biotransformation, extraction, and purifica-tion of the transformed products were carried out under iden-tical conditions as for the parent strain. Purified metaboliteswere analyzed from their mass spectral fragmentation, 1H and13C nuclear magnetic resonance (NMR), and infrared (IR) data(Tables 1–4).

2.2.5. Spectral analysesInfrared spectra were run on a Jasco (Tokyo, Japan) FT/IR-6300.1H and 13C NMR spectra were obtained as D4-methanolic solu-tions on Bruker 400 and 600 MHz AC 400 and Bruker AvanceII 600 (Bruker, Switzerland) spectrometers. Mass spectra wereobtained in electron impact (EI) mode on GC/MS DFS-ThermoFinnegan and V.G mass spectrometer-2025, model 305, fromAnalytical (Manchester, UK) at an ionization potential of 70 eV.The mass spectrometers were interfaced with a National Insti-tute of Standards and Technology (NIST) library database.

2.2.6. Gas chromatography separationof CDCA metabolitesAn Agilent GC model 6890 series interfaced with an AgilentMSD model 5973 network mass selective detector and an Agi-lent autosampler model 7683 were used in a 1/50 split ratio with

Transformation of CDCA by G. stearothermophilus 251

Table 11H NMR data for CDCA and its metabolites as D4 methanolic solution

Compound COOH 18-CH3 19-CH3 21-CH3 CHOH Others

CDCA 8.3 0.709 0.942 0.976 3.81 (>CH-OH) C73.38 (>CH-OH) C3

1. – 0.706 0.944 0.974 3.81 (>CH-OH) C7 3.66-OCH3

3.52 (>CH-OH) C32. 8.5 0.747 0.933 1.05 3.55 (>CH-OH) C33. 8.5 0.635 0.938 0.882 3.75 (.CH-OH) C74. 8.5 0.727 0.930 1.024 3.93 (.CH-OH) C12

3.79 (>CH-OH) C73.53 (>CH-OH) C3

5. – 0.724 0.931 1.024 3.96 (>CH-OH) C12 3.67-OCH3

3.81 (>CH-OH) C73.4 (>CH-OH) C3

Values are given in ppm.

Table 213C NMR of CDCA and its metabolites in methanol

Carbon # CDCA standard 1 2 3 4 5

1 34.83 35.37 34.54 38.1 35.15 35.642 29.97 29.9 29.30 34.61 29.79 29.93 67.66 68.2 70.07 216.6 67.69 68.24 39.38 39.89 40.76 46.77 39.05 39.615 41.78 42.3 43.72 45.02 41.8 42.356 34.51 35.01 40.99 37.87 34.53 35.057 71.46 72.01 215.8 69.06 71.50 72.18 39.05 39.6 46.64 41.11 39.63 40.169 32.65 33.18 36.48 33.48 26.47 27.0310 35.19 35.19 37.22 36.59 34.48 35.011 21.1 20.92 24.61 22.29 28.2 28.7312 39.66 40.17 38.00 40.88 72.64 73.1613 42.29 42.81 44.97 43.85 46.12 46.6314 50.12 50.67 51.49 51.63 41.58 42.1515 23.25 23.7 23.86 24.73 22.89 23.3716 27.85 28.39 29.30 29.42 27.30 27.8117 55.93 56.38 57.60 57.65 46.65 47.1318 10.85 11.29 12.24 12.37 12.71 12.1119 22.07 22.5 22.16 22.5 21.88 22.320 35.37 35.9 35.11 35.25 35.38 35.9121 17.48 17.9 18.99 19.08 16.33 16.7322 30.97 30.9 30.79 30.77 30.64 31.023 30.66 30.4 30.73 30.72 30.94 31.3924 176.83 179.51 180.02 179.19 176.81 175.7

Values are given in ppm.

helium carrier gas at a flow rate of 0.8 mL Min−1. An OV1,30-2502 capillary column (30 m × 0.25 mm × 0.25 μm; LifeScience, Peterborough, Canada) was used for separation of thecomponents. The column oven program was programmed as fol-lows: initial temperature of 50◦C and hold for 1 Min, ramp 25◦CMin−1 to 250◦C Min−1 and hold for 10 Min, ramp 10◦C to a finaltemperature of 300◦C and hold for 10 Min. Biotransformed prod-uct mixture as dicholomethane solution was passed through a

syringe membrane filter (0.45 μm) before injection in gas chro-matography (GC).

3. Results and discussionFive transformation products of CDCA were isolated and iden-tified from their spectral data. These products were methyl-chenodeoxycholate (1), 3α-hydroxy-7-oxo-5β-cholanic acid

252 Biotechnology and Applied Biochemistry

Table 3Infrared absorption frequencies of CDCA and itsmetabolites

Group frequencies

Compound ->CO OH >CH >CH CH3

CDCA 1712 3415.69 2934.20 2867.951 1741.19 3434.99 2928.05 2854.722 1704.76 3441.35 2938.98 2868.593 1710.57 3432.49 2927.81 2866.884 1715.32 3325.81 2932.43 2876.945 1739.69 3431.38 2935.24 2867.23

Values are given in cm−1.

(2), 7α-hydroxy-3-oxo-5β-cholanic acid (3), cholic acid (CA) (4),and methylcholate (5). Their molecular structures are shown inFig. 1.

CDCA and CA are two of the main primary BAs in mostmammalian species, including human, and much work has beenpublished for this BA [20]. Our data show that CDCA was de-graded by G. stearothermophilus, yielding five metabolites.

Table 4Mass spectral fragmentation of CDCA and its metabolites

Compound m/z

CDCA 392(9%); 374(32); 356(100); 341(38); 264(25); 228(77);213(72)

1 408(2%); 388(13); 370(63); 355(42); 273(31); 255(48);228(31); 213(47); 55(100)

2 390(27); 372(100); 385(28); 339(42); 271(49); 229(35);107(37); 95(45); 81(53); 55(65)

3 390(5); 109(14); 95(23); 81(27); 69(47); 55(100)4 408(2%); 390(4); 372(87); 354(81); 343(71); 271(100);

253(98);247(56); 226(58)5 422(2%); 388(13); 370(58); 355(42); 273(29);

255(74);228(31); 213(36); 55(100)

When CDCA was incubated in the media alone, without bacte-rial cells, no evidence of degradation was obtained, suggestingthat all metabolites of CDCA were bacterially catalyzed trans-formation products. The cells remained viable throughout thetransformation experiments as shown by turbidimetric analysisat 600 nm. The pure metabolites, components of the crude mix-ture and bacterial products, as resolved on TLC and gas–liquidchromatography (GLC), are shown in Figs. 2 and 3, respectively.

Fig. 1. Molecular structures of chenodeoxycholic acid and its metabolites: (1) Methyldeoxycholate; (2)3α-Hydroxy-7-oxo-5β-cholanic acid; (3) 7α-Hydroxy-3-oxo-5β-cholanic acid; (4) Cholic acid; (5) Methylcholate.

Transformation of CDCA by G. stearothermophilus 253

Fig. 2. Thin-layer chromatography analysis of 5-daytransformation products of chenodeoxycholic acid (CDCA)by G. stearothermophilus and mutant strain. From left toright: lane 1, control (chenodeoxycholic acid); lane 2, cellswithout chenodeoxycholic acid; lane 3, CDCA standard; lane4, cholic acid (CA) standard; lane 5, CA methyl esterstandard; lane 6, compound 1; lane 7, compound 2; lane 8,compound 3; lane 9, compound 4; lane 10, compound 5;lane 11, CDCA transformation by mutant G.stearothermophilus strain; lane 12, CDCA transformation byG. stearothermophilus strain.

3.1. Cholic acidThis metabolite must have been formed by a stereospecific α-hydroxylation of CDCA at C12. Hydroxylation of CDCA at C12is known to take place by human intestinal and soil bacteria[5],[17],[21]. Its mass spectrometry (MS) showed that an addi-tional oxygen atom was added to CDCA with m/z 408.58 cor-responding to C24H40O5. Its 1H NMR showed an extra >CH-OHproton at 3.96 ppm, suggesting hydroxylation of CDCA at C12.Other spectral data for CA are given in Tables 1–4, whereas itsseparation on TLC–GLC is given in Figs. 2 and 3.

3.2. Methylcholate and methylchenodeoxycholateCholic acid formed from CDCA can be esterified to give methyl-cholate. Similarly, methylchenodeoxycholate arises by micro-bial esterification of its substrate CDCA. Methylcholate showedits molecular ion at m/z 422.30 corresponding to C25H42O5. ItsIR absorption spectrum showed absorption at 1739.69 cm−1,characteristic of an ester function in the molecule. It was furthersupported by a singlet at 3.67 ppm in 1H NMR spectrum of themolecule. Additional spectral information about the moleculeis given in Tables 1–4 and its TLC–GLC separation is shown inFigs. 2 and 3.

Methylchenodeoxycholate showed its molecular ion atm/z 406.30 corresponding to C25H42O4. It showed an absorptionfrequency at 1741.19 cm−1, characteristic of an ester function inthe molecule. Its 1H NMR also showed a three hydrogen sin-glet at 3.66 ppm, characteristic of an -OMe function. Additionalspectral data for the methylchenodeoxycholate are given in Ta-bles 1–4 and its TLC–GLC separation is shown in Figs. 2 and 3.Microbial esterification of CA and other BAs has been earlierreported [5],[22],[23].

Fig. 3. GC–MS separation of chenodeoxycholic acid transformation products. Compound 1, (40.44); compound 2, (40.84);compound 3, (41.76); compound 4, (38.79); and compound 5, (40.35). Retention time is given in minutes in brackets.Separation on OV1 column. Conditions of separation are given in the text.

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3.3. 3α-Hydroxy-7-oxo-5β-cholanic acid and7α-hydroxy-3-oxo-5β-cholanic acidThese two transformation products are isomers and could arisefrom the individual oxidation of hydroxyl groups at C3 and C7of CDCA. Their molecular ion appeared at 390.27 correspondingto C24H38O4. Both metabolites showed characteristic carbonylabsorption frequencies at 1710.57 and 1704.76 cm−1 for oxogroups at C3 and C7, respectively. This was supported by theappearance of 13C NMR signals at 216.6 and 215.8 ppm forthe oxo groups at C3 and C7 and disappearance of the >CH-OH proton signals in their respective 1H NMR spectra. Addi-tional spectral data for these compounds are given in Tables1–4, whereas their separation on TLC–GLC is shown in Figs. 2and 3. Microbial oxidation of hydroxyl groups in a BA nucleushas been reported by many workers [24],[25]. The main oxoderivative was produced from the oxidation of the C7 hydroxylgroup, whereas the isomeric 3-oxo derivative was only a mi-nor product. The intracellularly located nicotinamide adeninedinucleotide phosphate-nicotinamide adenine dinucleotide re-duced (NADPH–NADH)-dependent dehydrogenases are respon-sible for this type of oxidation, as reported by Midtvedt [8].

Our results indicate that G. stearothermophilus carries hy-droxylases in common with other bacteria but the unique featureof this organism is that it carries specific dehydrogenases foroxidation of hydroxyl groups present only at C3 and C7. This issupported by the fact that CA is a cometabolite of CDCA transfor-mation; however, the oxidation of the C12 hydroxyl group wasnot evidenced in the present study. According to Charney andHerzog [26], microbial hydroxylation of a steroid nucleus can beachieved at 17 positions. Although many bacterial genera arecapable of oxidizing hydroxyl groups at C3, C7, and C12, thespecificity of G. stearothermophilus to oxidize hydroxyl groupsonly at C3 and C7 may be of taxonomic value. We speculate thatthe oxidation of hydroxyl groups at C3 and C7 of the BA nucleusmay be a special feature of G. stearothermophilus, which may beexploited for selective transformation of other BAs to producephysiologically active steroids.

AcknowledgementsThis work was supported by research grant number SL04/05from Kuwait University Research Administration. The authorsthankfully acknowledge their support. The investigators also

thankfully acknowledge the assistance of Science AnalyticalFacilities (General Facility Project numbers GS01/01, GS03/01,and GS01/03) for spectral data.

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