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

Article abstract of DOI:10.1016/j.steroids.2004.12.003

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°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, 5α-androstan-3,6,17-trione, 17β-hydroxy-5α-androstan-3,6-dione, 3β,17β- dihydroxyandrost-4-ene-6-one and 17β-hydroxyandrost-4,6-diene-3-one were identified as biotransformation products of testosterone. A possible biosynthetic route for these bioconversion products is postulated.

Full text of DOI:10.1016/j.steroids.2004.12.003

Steroids 70 (2005) 327–333
Studies on Bacillus stearothermophilus
Part IV. Influence of enhancers on biotransformation of testosterone
∗
S. Al-Awadi, M. Afzal , S. Oommen
Department of Biological Sciences, Biochemistry Program, Faculty of Science, Kuwait University, P.O. Box 5969, Safat-13060, Kuwait
Received 19 May 2004; received in revised form 1 December 2004; accepted 7 December 2004
Abstract
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 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, 5␣-androstan-3,6,17-trione, 17␤-hydroxy-5␣-
androstan-3,6-dione, 3␤,17␤-dihydroxyandrost-4-ene-6-one and 17␤-hydroxyandrost-4,6-diene-3-one were identified as biotransformation
products of testosterone. A possible biosynthetic route for these bioconversion products is postulated.
©
2005 Elsevier Inc. All rights reserved.
Keywords: Enhancers; Biotransformation; Bacillus stearothermophilus; Cytochrome P-450
1
. Introduction
Hydroxysteroids are important due to their physio-
logical role in many situations. For instance, 6␤-, 14␣-
hydroxyandrost-4-ene-3,17-dione and 14␣-hydroxyandrost-
4-ene-3,6,17-trione both possess androgenic activity and
are useful inhibitors of breast cancer [5]. Similarly, allo-
pregnanalone and allotetrahydrodeoxy-corticosterone are re-
ported to be neuroactive steroids [6].
An important factor in the bioconversion capability of mi-
croorganismsistheinductionofsteroidhydroxylases. Steroid
hydroxylases and cytochrome P-450 are known to be in-
ducible [7,8]. In particular, animal liver microsomes, cy-
tochrome P-450 IA and IIIA forms are highly inducible [9].
Thus, P-450 genes have been induced in laboratory animals
and human liver by a number of chemically unrelated com-
pounds [10]. In this context, the influence of cycloheximide
on the induction of progesterone 11␣-hydroxylating system
has been reported [7]. Rifampicin is a potent CYP3A in-
ducer in rabbit and human hepatocytes [11,12]. Salicylic acid
can also act as a stimulator/enhancer for inducing hydroxy-
lation in many biotransformation processes [13]. Enhanced
biotransformation in enzymatic or microbial media supple-
Metabolism of steroids in healthy and unhealthy human
organs, particularly in the liver, is the subject of extensive
clinical and biomedical investigations. For this kind of work,
it is essential that the authentic reference samples of preg-
nane and androstane based steroids, stereoselectively hy-
droxylated at the ring and bridge positions, become avail-
able. This is not feasible through synthesis. The potential
use of microorganisms as a biomimetic model of xenobi-
otic metabolism in mammalian organisms has been used
as an inexpensive and convenient method for the study of
drug metabolism [1]. Microbial assisted hydroxylation en-
tails an introduction of a hydroxyl group involving activation
of chemically unreactive centers in steroid molecules. Hol-
land has highlighted the industrial importance of microbial
oxidation of steroids [2], and stereoselective hydroxylation
of steroid skeletal positions has been reported [3,4].
∗
Corresponding author. Tel.: +965 4848437; fax: +965 482794.
E-mail address: Afzalkw@yahoo.com (M. Afzal).
0
039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2004.12.003

3
28
S. Al-Awadi et al. / Steroids 70 (2005) 327–333
mented with cyclodextrin has also been investigated [14].
Microorganisms, besides hydroxylation, are also capable of
carrying out a wide variety of other steroid transformation
reactions [15].
It is recognized that thermostable enzymes of thermo-
philes are resistant to chemical denaturation [16]. However,
little work has been reported about microbial transformation
of steroids using thermophiles [17], and to our knowledge,
0.05 M sodium phosphate buffer pH 7. The cells were mixed
with 10 ml of the same buffer and transferred to 500 ml of
the phosphate buffer containing 10% Castenholtz mineral
salt solution [21] in a 2-L flask. An ethanolic solution of
testosterone and salicylic acid, made to final concentrations
of 50 and 12.5 ␮g/ml, respectively, was added and the cul-
◦
tures were re-incubated at 65 C for 72 h. Similarly, a final
concentration of 50 ␮M rifampicin, 100 ␮g/ml of cyclohex-
imide, 50 ␮g/ml chloramphenicol, 2 moles per one mole of
substrate cyclodextrin, 2.5 ␮M dexamethasone, 50 ␮M be-
tanaphthoflavone, 0.75 mM phenobarbitone, 1 mg/50 ml ri-
boflavin and 50 ␮M rifampicin were also tested as enhancers
for the biotransformation of testosterone. The transforma-
tion experiments of testosterone with enhancers were car-
ried out in 0.05 M phosphate buffer pH 7, containing 10%
Castenholtz mineral salt solution. Cycloheximide enhancer
was used for biotransformation in 0.05 M phosphate buffer
pH 7, containing 1% glucose. The inoculated flasks were
1
7␤,5␣-androstane derivatives, 6-dehydro derivatives and
B-seco-androstene compounds have not been explored using
thermophiles. Thermophile-mediated transformation of
progesterone [18,19] and testosterone, without enhancers,
has been previously reported from our laboratory [20]. These
results encouraged us to further explore the potential of B.
stearothermophilus to metabolize testosterone 1 in the pres-
ence of a variety of hydroxylase enhancers. In this endeavor,
metabolism of 1 induced by salicylic acid, cycloheximide,
rifampicin, chloramphenicol, cyclodextrin, dexamethasone,
betanaphthoflavone, phenobarbital, riboflavin and the
biotransformation products of 2 are reported in this study.
◦
incubated on a rotary shaker at 65 C and 100 rev/min for
72 h.
2.4. Extraction, isolation and purification of metabolites
2
. Experimental
The fermentation mixture was harvested and exhaustively
2
.1. Materials
extracted three times with an equal volume of chloroform.
The pooled extract was dried over anhydrous Na2SO4 and
evaporated to dryness using a rotary evaporator to get a
semi-solid residue. TLC separation of the residue on silica
plates (Merck 60 F254) with benzene: methanol: aceticacid
20:2:1 by volume) followed by second development in
diethylether: toluene: ethylacetate (15:25:10 by volume)
revealed the formation of a variety of metabolites. Since
Culture media were purchased from Difco Becton Dick-
inson and company (Sparks, USA) and Scharlau (Barcelona,
Spain). All chemicals used were of analytical grade and
were obtained from Fluka Riedel-deha e¨ n, Merck (Darm-
stadt, Germany) and Scharlau. Kieselgel-60 F254 fluorescent
thin layer chromatographic plates (TLC) were obtained from
Merck (Darmstadt, Germany). Steroid testosterone 1 and
enhancers, cycloheximide, cyclodextrin, dexamethasone, ␤-
naphthoflavone, chloramphenicol, riboflavin and rifampicin
were obtained from Sigma-Aldrich Co. UK. Androstene-
(
5
␣-androstanes were undetected under UV light at 254 nm,
the TLC chromatograms were stained with anisaldehyde
reagent to get a spectrum of colors before separation and
purification. The induced transformation of testosterone
resulted in the formation of five compounds that were
identified from their spectral data. These metabolites
were identified as 9,10-seco-4-androstene 3,9,17-trione 3,
␣-androstan-3,6,17-trione 4, 17␤-hydroxy-5␣-androstan-
,6-dione 5, 3␤,17␤-hydroxyandrost-4-ene-6-one 6 and
7␤-hydroxyandrost-4,6-diene-3-one 7.
3
,17-dione 2 was purchased from Steraloids Inc. (Newport,
RI, USA). Salicylic acid was from BDH Laboratories (Poole,
England) and phenobarbitone was from Courtin and Warner
Ltd. (Lewes, East Sussex, England).
5
3
1
2
.2. Organism
The details of B. stearothermophilus such as its mainte-
nance and growth conditions have been described previously
20].
2.5. Transformation of androstene-3,17-dione 2 by B.
stearothermophilus
[
2
.3. Influence of enhancers on the biotransformation of
The starter culture and cells were grown and collected as
described above, and the suspension was added to 500 ml
sodium phosphate buffer (0.05 M and pH 7) containing 10%
Castenholz mineral salt solution. An ethanolic solution of
androstene-3,17-dione was added to achieve a final con-
centration of 50 ␮g/ml, and the culture was re-incubated
for 72 h. Collection, separation, purification and identifica-
tion of metabolites were carried out as described above for
testosterone.
testosterone
Starter cultures (50 ml) of B. stearothermophilus were
grown overnight on tryptone yeast extract (TYE) medium
at 65 C in a shaker incubator. The culture was transferred to
5
same conditions until the end of log phase growth (4–4.5 h).
The cells were collected by centrifugation and washed with
◦
00 ml of fresh TYE media in a 2-L flask and kept under the

S. Al-Awadi et al. / Steroids 70 (2005) 327–333
329
2
.6. Quantification of the compounds
Relative percentage yield of each metabolite was mea-
sured from its TLC spot density using Shimadzu Dual Wave-
length Flying Spot Scanner model CS-9000, interfaced with
Quantascan 2D analysis program P/N 206-18597.
2
.7. Analytical methods
Details of the analytical methods have been described pre-
viously [20].
3
. Results and discussion
We have previously reported the biotransformation
of testosterone 1 by B. stearothermophilus, and the ma-
jor metabolite androst-4-ene-3,17-dione 2 was found in
90.2% relative yield along with four other monohydroxy
metabolites [20]. However, the addition of hydroxylase
enhancers like salicylic acid, chloramphenicol, cyclodextrin,
cycloheximide, dexamethasone, betanaphthoflavone, pheno-
barbitone, riboflavin and rifampicin in B. stearothermophilus
cultures resulted in the formation of additional metabolites.
Enhanced yields of 9,10-seco-4-androstene-3,9,17-trione 3,
5
3
␣-androstane-3,6,17-trione 4, 17␤-hydroxy-5␣-androstan-
,6-dione 5 and other monohydroxy derivatives [20] were
obtained using these enhancers (Table 1). Addition of
cycloheximide in the presence of glucose resulted in the
formation of 3␤,17␤-hydroxyandrost-4-ene-6-one 6 as a
major metabolite. This compound was produced as a minor
metabolite with other enhancers, such as salicylic acid, chlo-
ramphenicol, cyclodextrin, cycloheximide, dexamethasone
and ␤-naphthoflavone. The metabolite 17␤-hydroxyandrost-
4
,6-diene-3-one 7 was formed only with the addition of
rifampicin. The molecular structures of these metabolites
are shown in Fig. 1, and the influence of different enhancers
on the biotransformation of 1 is shown in Table 1.
An extensive use of 13C NMR and H NMR spectroscopy
assisted identification of these metabolites. The metabo-
lite 9,10-seco-4-androstene-3,9,17-trione 3 was identified by
1
1
comparison of its mass spectral fragmentation and H NMR,
1
3
C NMR and IR spectral data with those reported in the lit-
erature [18,22,23]. The spectral details of this compound are
1
shown in Tables 2–5. In the H NMR spectrum of the B-Seco
compound 3, the fundamental signals for testosterone were
missing, confirming that the metabolite did not have the an-
drostene skeleton. For instance, the 4-H at ␦6.24 ppm was too
high for the 4-H that normally appears at ␦5.74 ppm in 1 and
at ␦5.76 ppm in androstenedione 2. The C19 methyl protons
showed a split triplet centered at ␦1.22 ppm due to coupling
with the methylene protons at C1 and the methyne proton at
C10. This confirmed the bond cleavage between C9 and C10 of
androstene with a concurrent oxygenation at C9 to give the B-
seco compound 3 [18]. However, the C18 CH3 group showed
a characteristic singlet at the expected position ␦0.93 ppm.

3
30
S. Al-Awadi et al. / Steroids 70 (2005) 327–333
Table 3
1
3C NMR of testosterone and its metabolites in CDCl3
Carbon atom
Chemical shifts (ppm)
(3) (4) (5)
38.6
(
1)
(2)
36.0
34.3
(6)
34.5
30.9
(7)
34.5
34.5
1
2
3
4
5
6
7
8
9
36.1
34.1
36.07 38.0
34.48 37.5
37.9
198.0 199.8
124.2 124.5
170.4 170.8
199.6 211.3 210.2
67.4 200.2
126.2 128.6
161.30 164.3
202.5 124.3
46.9 140.9
126.5
163.9
41.7
58.1
41.8
58.1
32.8
32.2
36.1
54.6
39.0
21.2
37.1
43.2
51.1
23.8
30.7
32.9
31.1
35.5
54.1
39.0
20.7
36.1
47.9
51.2
22.1
31.6
40.0 208.7 208.6
30.3
46.0
208.2
51.5
20.8
31.5
47.5
52.8
22.2
36.0
45.9
37.9
54.1
38.6
21.2
31.6
48.6
52.1
22.2
36.2
46.6
36.8
54.1
38.7
21.9
37.5
44.0
51.8
23.8
30.9
82.0
11.7
13.2
34.6
51.6
36.5
21.1
36.1
39.7
51.8
23.7
30.9
81.9
11.6
18.1
38.2
48.8
36.9
20.9
30.9
44.4
51.4
23.5
30.2
81.9
11.6
16.9
10
11
12
13
14
15
16
17
18
19
81.3 220.9
11.3
17.3
218.5 220.1
14.2
18.2
14.1
17.7
14.2
13.2
of compound 3. Except for the signal at 30.3 ppm for C7, all
other signals for carbon positions 4-, 5-, 6-, 8-, 9- and 10- had
shifted upfield. The additional 13C signals, including the C17
carbonyl carbon at 220.1 ppm instead of 181.3 ppm in testos-
terone 1, were all comparable to those of androstenedione 2
and the values are given in Table 3. Mass spectral fragmen-
tation of the B-seco compound also supported the proposed
molecular structure with a parent ion at m/z 302 and a base
peak at m/z 149 (Table 4).
Fig. 1. Structures of testosterone metabolites. Testosterone (1) Androst-
-en-3,17-dione (2) 9,10-Seco-4-Androstene-3,9,17-trione (3) 5␣-
Androstane-3,6,17-trione (4) 17␤-Hydroxy 5␣-androstane-3,6-dione
5) 3␤,17␤-hydroxyandrost-4-ene-6-one (6) 17␤-Hydroxyandrost
,6-diene-3-one (7).
4
The metabolite 4 was identified as 5␣-androstane-3,6,17-
trione. All spectroscopic details of the compound corre-
(
4
1
sponded to its molecular structure. Its H NMR spectral
data showed that the fundamental signals of the androstene
ring A had an upfield shift confirming the loss of the an-
drostene ring structure resulting in 5␣-androstane as shown
in Table 2. The compound was undetectable at 254 nm on
TLC chromatograms but showed a bright yellow color with
anisaldehyde stain that was used for its detection on TLC
The three carbonyl groups at C3, C9 and C17 were also ap-
−
1
parent at 1680.09, 1698.43 and 1738.11 cm respectively
in the FTIR spectrum. Other IR absorption frequencies in-
−
1
−1
cluded 2924.01 cm for C H stretching and 2851.41 cm
for CH CH3 stretching for bridge methyl groups (Table 5).
13
13
C NMR of compound 3 was very distinctive when com-
pared to the parent compound 1. Thus, signals at 126.5 (C4),
chromatograms. The C NMR spectral data (Table 3) when
compared with the reported values [24–26], confirmed the
structure 4 for this metabolite. Mass spectral fragmentation,
as reported earlier [24], was also in full agreement with the
proposed structure. The FTIR absorption spectrum also sup-
1
63.9 (C ), 40.0 (C ), 30.3 (C7), 46.0 (C8), 208.2 (C9) and
5 6
5
1.5 (C10) ppm were all different compared to 1. This cluster
of signals was the main evidence for the molecular structure
Table 2
1
H NMR data for testosterone metabolites produced by the addition of enhancers
Compounds
4-H
17␣-OH
18-CH3
19-CH3
CHOH
Other significant signals
1
2
3
4
5
6
7
5.74
5.76
6.24
3.67
–
–
0.81
0.93
0.92
0.88
0.80
0.82
0.86
1.21
1.22
1.22
1.01
0.99
1.20
1.14
–
–
–
–
–
–
2.11 (4␣-H), 2.38 (4␤-H)
2.11 (4␣-H), 2.39 (4␤-H)
6.20
5.69
–
1.59 (5␣-H)
1.63 (5␣-H)
3.64
3.66
3.69
3.64 (17␣-H)
4.07 (3.66)
3.69 (17␣-H)
6.12 (6-H), 6.12 (7-H)

S. Al-Awadi et al. / Steroids 70 (2005) 327–333
331
Table 5
Infrared spectra of testosterone and its metabolites
Compounds
Group frequencies
CO
OH
CH
CH CH3
1
2
1656.62
1736.08
3383.06
2943.92
2939.77
2846.75
2856.26
–
1670.95
3
1738.11
–
2924.01
2851.41
1
1
698.43
680.09
4
5
6
7
1735.38
713.11
1733.33
713.00
–
2926.27
2917.51
2930.31
2941.95
2855.68
2849.19
2854.67
2841.73
1
3418.03
1
1684.34
3648.44
596.88
3466.92
3
1729.55
ported 5␣-pregnane structure through absorption frequencies
−
1
at 1713.00 and 1735.38 cm for the overlapped carbonyl
functions at C3, C and C17, respectively (Table 5).
6
The metabolite 17␤-hydroxy-5␣-androstan-3,6-dione 5
was identified by comparing its spectral data with those of
compounds reported in the literature [27,28]. The presence
13
of the 17␤-hydroxyl group was apparent from its C NMR
1
and H NMR and mass spectrum of 5. The other signals for
5
␣-androstane and the 3,6-keto groups were obvious from
1
13
the H NMR, C NMR and FTIR spectra when compared
to the metabolite 4 (Tables 2–5).
The spectral data of metabolite, 3␤,17␤-hydroxyandrost-
4
-ene-6-one 6 was compared with the reported data of sim-
1
ilar compounds [28,29]. In the H NMR spectra, the 3␤-
hydroxylation and the carbonyl function at C resulted in
6
a downfield shift for the 4-H signal to ␦6.20 ppm. The 3␤-
and 17␤-hydroxyl groups were evident from the signals
at ␦4.07 ppm and ␦3.66 ppm, respectively (Table 2). In its
13
C NMR spectrum, values for the C carbonyl function
6
(
202.5 ppm) and the 3␤- and 17␤-hydroxyl signals (67.4
and 81.9 ppm, respectively) confirmed the proposed molec-
ular structure. A shift for C (161.3 ppm) and C7 signals
5
(
46.9 ppm) also supported the carbonyl group at C (Table 3).
6
The mass spectral analysis for 6 showed a parent ion at m/z
+
3
02 that confirmed addition of an oxygen atom to the origi-
nal molecule after its transformation. The additional oxygen
−1
appeared as a carbonyl group at 1684.34 cm in the FTIR
spectrum.
From the spectroscopic details, metabolite 7 was iden-
tified as 17␤-hydroxyandrost-4,6-diene-3-one (Tables 2–5).
1
The H NMR features of the 4,6-dienone systems were in
full agreement with the 6-dehydrotestosterone. The reported
1
3
1
C NMR, H NMR spectral data [30–32] and the infrared ab-
−1
sorption frequencies at 1604.17 and 1646.75 cm were all in
agreement with the conjugated dienone system. The presence
of a 17␤-hydroxyl group was supported from the position of
1
the 17␣-H in its H NMR spectrum (Tables 2 and 3). The
mass spectral data of compound 7 showed the parent ion at

3
32
S. Al-Awadi et al. / Steroids 70 (2005) 327–333
+
m/z 286 with loss of a water molecule from the CH(OH)
[5] Yoshioka H, Asada S, Fujita S. Process for production of 6␤,14␣-
hydroxyandrost-4-ene-3,17-dione, European Patent 5: 99, 658.1994.
[6] Barbaccia ML, Serra M, Purdy RH, Biggio G. Stress and neuroactive
steroids. Int Rev Neurobiol 2001;46:243–72.
+
group at C17with a fragment ion at m/z 268 . The carbonyl
group at C3 was confirmed from its FTIR and 13C NMR
spectrum.
[
7] Breskvar K, Hudnik-Plevnik T. Inducibility of progesterone hy-
droxylating enzymes in Rhizopus nigricans. J Steroid Biochem
1978;9:131–4.
The biotransformation of the androst-4-ene-3,17-dione 2
resulted in five compounds, that were identified from their
spectral data by comparison with testosterone biotransforma-
tion products and also with values reported in the literature.
These compounds were identified as 9,10-seco-4-androstene-
[
8] Breskvar K, Hudnik-Plevnik T. Inducibility of cytochrome P-450 and
NADPH-cytochrome c reductase in progesterone treated filamentous
fungi Rhizopus nigricans and Rhizopus arrhizus. J Steroid Biochem
1981;14:395–9.
3
,9,17-trione, 5␣-androstane-3,6,17-trione, testosterone, 6␤-
[9] Okey AB, Roberts EA, Harper PA, Denison MS. Induction of
drug metabolizing enzymes: mechanisms and consequences. Clin
Biochem 1986;19:132–41.
10] Monshouwer M, van’t Klooster GAE, Nijmeijer SM, Witkamp RF,
van Miert ASJPAM. Characterization of cytochrome P-450 isoen-
zymes in primary cultures of pig hepatocytes. Toxicol In Vitro
1998;12:715–23.
[11] Morel F, Beaune PH, Ratanasavanh D, Flinois JP, Guengerich FP,
Guillouzo A. Effects of various inducers on the expression of cy-
tochromes P-450 II C8910 and IIIA in cultured human hepatocytes.
Toxicology In Vitro 1990;4:458–60.
12] Daujat M, Clair P, Astier C, Fabre I, Pineau T, Yerle M, et al.
Induction, regulation and messenger half-life of cytochromes P450
IA1 IA2 and IIIA6 in primary cultures of rabbit hepatocytes. Eur J
Biochem 1991;200:501–10.
hydroxyandrost-4-en-3,17-dione and 6␣-hydroxyandrost-4-
en-3,17-dione.
[
We have previously reported 6-dehydro- and 5␣-pregnane
derivatives of progesterone transformation products with in-
creased incubation period [19]. From the transformation
products, it is clear that the compound testosterone is trans-
formed into androstenedione 2, which is then transformed
into 3, and 4. At the same time, testosterone is transformed
into compounds 5 and 6. All these metabolites were produced
in very small amounts along with the previously identified
monohydroxy metabolites [20]. With increased incubation
periods, the yield of these metabolites increased many folds
after the addition of enhancers indicating the influence of
these enhancers on the hydroxylation of these steroids. 17␤-
Hydroxyandrost-4,6-diene-3-one 7 probably also arises from
testosterone 1, through a process of dehydrogenation. Forma-
tion of these compounds from a thermophilic bacillus has not
been reported before.
With the increasing knowledge of microbial transforma-
tion, particularly with respect to P-450 hydroxylases, and an
ongoing interest in thermostable enzymes, this work may ini-
tiate a natural progression away from the microbial biotrans-
formation by whole cell cultures. With the progress made in
this study, we are confident that our new isolate can lead to
new approaches for the efficient transformation of potential
biologically active steroids.
[
[
13] Coudray C, Favier A. Determination of salicylic hydroxylation prod-
ucts as an in vivo oxidative stress marker. Free Rad Biol Med
2000;29(11):1064–70.
[14] Jadoun J, Bar R. Microbial transformations in cyclodextrin medium.
Part 4. Enzyme versus microbial oxidation of cholesterol. Appl Mi-
crobiol Biotechnol 1993;40:477–82.
15] Sebek OK, Perlman D. Microbial transformation of steroids and
sterols. In: Pepler HJ, Perlman D, editors. Microbial technology, 1.
New York: Academic Press; 1979. p. 483–96.
[16] Berquist PL, Love RD, Croft JE, Streiff MB. Genetics and
potential biotechnological application of thermophilic and ex-
tremely thermophilic microorganisms. Biotechnol Genetic Eng Rev
[
1987;5:194–9.
[
17] Sideso O, Williams RAD, Welch SG. Smith KE. Progesterone 6-
hydroxylation is catalysed by cytochrome P-450 in the moderate
thermophilic Bacillus thermoglucosidasius Strain 12060. J Steroid
Biochem Mol Biol 1998;67:163–9.
18] Al-Awadi S, Afzal M, Oommen S. Studies on Bacillus stearother-
mophilus. Part I. Transformation of progesterone to a new metabo-
lite 910-seco-4-pregnene-3920-trione. J Steroid Biochem Mol Biol
[
Acknowledgements
2002;78(5):493–8.
[
19] Al-Awadi S, Afzal M, Oommen S. Studies on Bacillus stearother-
mophilus. Part II Transformation of progesterone. J Steroid Biochem
Mol Biol 2002;82:251–6.
This work was supported by a KU grant # SL02/02, which
is thankfully acknowledged. We are also thankful to SAF
in the Faculty of Science at Kuwait University (project no.
GS01/01, GS03/01) for making FTIR, MS, and NMR facili-
ties available for this study.
[
20] Al-Awadi S, Afzal M, Oommen S. Studies on Bacillus stearother-
mophilus. Part III. Transformation of testosterone. Appl Microbiol
Biotechnol 2003;62:48–52.
[21] Ramaley RF, Hixson J. Isolation of non-pigmented thermophilic
bacterium similar to Thermus aquaticus. J Bacteriol 1970;103:527–
8.
[
22] Mahato SB, Banerjee S, Podder S. Oxidative side chain and
References
ring fission of pregnanes by Arthrobactor simplex. Biochem J
1988;255:769–74.
[
1] Clark AM, Hufford CD. Use of microorganisms for the study of
drug metabolism: an update. Med Res Rev 1991;11:471–3.
[23] Pollio A, Pinto G, Della Greca M, De Maio A, Fiorentino A, Pre-
vitera L. Progesterone bioconversion by microalgal cultures. Phyto-
chemistry 1994;37(5):1269–72.
[24] Oberman H, Spiteller-Friedmann M, Spiteller G. Localisation of
functional groups in steroids with the aid of mass spectrometry 1
Ketosteroids. Chem Ber 1970;103:1411–97.
[
[
2] Holland HL. Oxidation of steroids. Chem Soc Rev 1982:371–99.
3] Charney W, Herzog HL. Microbial transformation of, steroids. New
York: A handbook Academic Press; 1967. p. 1–80.
[4] Smith LL. Microbiological reactions with steroids. In: Overton KH,
editor. Terpenoids and steroids, 4. London: The Chemical Society;
[25] Szykula J, Hebda C, Orpiszewski. Isolation and identification of a
new metabolite of microbial conversion of upgraded neutral fraction
1974. p. 30–94.

S. Al-Awadi et al. / Steroids 70 (2005) 327–333
333
of polish tall oil by means of four strains of Mycobacterium sp.
Biotechnol Lett 1990;12(7):505–8.
droxylation of testosterone and related seroids by Gnomonia fructi-
cola ATCC 11430. Can J Chem 1989;67:268–74.
[
26] Schaaf O, Dettner K. Transformation of steroids by Bacillus strains
isolated from the foregut of water beetles (Coleoptera: Dytiscidae).
I. Metabolism of Androst-4-en-317-dione (AD). J Steroid Biochem
Mol Biol 1998;67(5–6):451–65.
[30] Hickey JP, Butler IS, Pouskouleli G. Carbon-13 NMR spectra
of some representative hormonal steroids. J Magnet Resonance
1980;38:501–6.
[31] Holland LH, Chenchaiah PC, Thomas EM, Mader B, Dennis MJ. Mi-
6
[27] Mahato SB, Banerjee S, Sahu NP. Metabolism of progesterone and
testosterone by a Bacillus sp. Steroids 1984;43(5):545–58.
[28] Hanson JM, Nasir H. The biotransformation of some steroids by
Cephalosporium aphidicola. Phytochemistry 1993;33(4):831–4.
[29] Holland LH, Brown FM, Chenchaiah PC, Chernishenko MJ, Khan
SH, Appa Rao J. Microbial hydroxylation of steroids. Part 12. Hy-
crobial hydroxylation of steroids.9 Epoxidation of ꢀ -3-ketosteroids
by Rhizopus arrhizus ATCC 11145 and the mechanism of the 6␤
hydroxylase enzyme. J Chem 1984;62:2740–7.
[32] Smith KE, Latif S, Kirk DN. Microbial transformation of steroids-
IV 6,7-dehydrogenation: a new class of fungal steroid transformation
product. J Steroid Biochem 1989;33(2):271–6.