Transformations of Steroids by Beauveria bassiana

Abstract

The course of transformations of testosterone and its derivatives, including compounds with an additional C1,C2 double bond and/or a 17alpha-methyl group, a 17beta-acetyl group or without a 19-methyl group, by a Beauveria bassiana culture was investigated. The fungi promoted hydroxylation of these compounds at position 11alpha, oxidation of the 17beta-hydroxyl group, reduction of the C1,C2 or C4,C5 double bonds and degradation of the progesterone side-chain, leading to testosterone. The structure of 4-ene-3-oxo-steroids had no influence on regio- and stereochemistry of hydroxylation. In a similar manner, dehydroepiandrosterone was hydroxylated by Beauveria bassiana at position 11alpha, however, a small amount of 7alpha-hydroxylation product was also formed.

Full text

Transformations of Steroids by Beauveria bassiana
Ewa Huszcza
*
, Jadwiga Dmochowska-Gładysz, and Agnieszka Bartman
´ska
Department of Chemistry, Agricultural University, Norwida 25, 50-375 Wrocław, Poland.
Fax: 0048-071-3 283576. E-mail: huszcza@ozi.ar.wroc.pl
* Author for correspondence and reprint requests
Z. Naturforsch. 60 c, 103Ð108 (2005); received July 26/September 6, 2004
The course of transformations of testosterone and its derivatives, including compounds
with an additional C1,C2 double bond and/or a 17α-methyl group, a 17β-acetyl group or
without a 19-methyl group, by a Beauveria bassiana culture was investigated. The fungi pro-
moted hydroxylation of these compounds at position 11α, oxidation of the 17β-hydroxyl
group, reduction of the C1,C2 or C4,C5 double bonds and degradation of the progesterone
side-chain, leading to testosterone. The structure of 4-ene-3-oxo-steroids had no influence on
regio- and stereochemistry of hydroxylation. In a similar manner, dehydroepiandrosterone
was hydroxylated by Beauveria bassiana at position 11α, however, a small amount of 7α-
hydroxylation product was also formed.
Key words: Beauveria bassiana, Biotransformation, Steroids
Introduction
Beauveria, belonging to the Moniliaceae family
of fungi imperfecti, is a naturally occurring soil
fungus (Bidochka et al., 1998). It is a recognized
pathogen of more than 100 insect species (Hajek
and St. Leger, 1994), which has found an applica-
tion in agricultural biocontrol programs (Bing and
Lewis, 1991, 1992; Krueger and Roberts, 1997; Mu-
lock and Chandler, 2000).
The fungus Beauveria bassiana ATCC 7159
(also known as B. sulfurescens or Sporotrichum
sulfurescens) is one of the most frequently used
biocatalysts capable of performing reactions of a
different type, e.g. hydroxylation of saturated and
aromatic carbon atoms, keto-alcohol redox reac-
tion, alkene redox reaction, sulfide oxidation,
Baeyer-Villiger oxidation, glucosidation, epoxide
and ester hydrolysis and heteroatom dealkylation.
These results have been summarized in the review
article of Grogan and Holland (2000). The most
significant is the use of B. bassiana for selective
hydroxylation of a wide range of organic com-
pounds.
In contrast to many other fungi currently used
for biocatalysis, Beauveria has not been exten-
sively used for transformations of steroids. Previ-
ous research showed that B. bassiana promotes hy-
droxylation of 4-ene-3-oxo-steroids mainly at
position 11α(Griffiths et al., 1993; Bayunova et
al., 1989; C
ˇapek and Fassatiova, 1977; C
ˇapek et al.,
1966; Schubert et al., 1962) and, rarely, at positions
0939Ð5075/2005/0100Ð0103 $ 06.00 ”2005 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com ·
D
6βand 11α(Griffiths et al., 1993; C
ˇapek and Fas-
satiova, 1977; C
ˇapek et al., 1966). Hydroxylation
at positions 6α,11α,11βand 15αwas observed in
the B-norsteroid 17α,21-dihydroxy-B-nor-pregn-4-
en-3,20-dione (Sanada et al., 1977). Also the abil-
ity of B. bassiana to reduce the 17-ketone to a 17β-
alcohol (Bayunova et al., 1989), and to degrade the
progesterone side-chain leading to testosterone
(Schubert et al., 1962) was reported.
Therefore, it was of interest to us performing
comparative studies on various 4-ene-3-oxo-ste-
roids. We wanted to check whether the additional
C17 methyl group, the lack of the C19 methyl
group, and the additional C1,C2 double bond do
not alter the localisation of the hydroxylation pro-
cess.
Because the knowledge of biotransformations of
5-ene-steroids is much less documented compared
to 4-ene-3-oxo-steroids, we have chosen dehy-
droepiandrosterone (DHEA) as an additional
substrate for the tests with Beauveria bassiana.
There are no previous reports on 19-nortestoster-
one and dehydroepiandrosterone transformation
in a Beauveria bassiana culture.
Materials and Methods
Microorganism
Beauveria bassiana AM446 was obtained from
the Institute of Biology and Botany of the Medical
University of Wrocław. It was isolated from the

104 E. Huszcza et al. · Steroid Transformations by Beauveria bassiana
insect Pyrrhocoris apterus (Pyrrhocoridae) (ima-
go).
Conditions of cultivation and transformation
The fungi were incubated in 3% glucose and 1%
peptone, pH 5.9, and shaken at 27 ∞C in 2 l Erlen-
meyer flasks with 300 ml of medium. After 3 d of
growth, 120 mg of a substrate, dissolved in 5 ml
of acetone or ethanol, were added, and the flasks
returned to the shaker. The products were ex-
tracted with chloroform after 3Ð10 d of trans-
formation (until the substrate was metabolized).
Product analysis
The composition of crude biotransformation
mixtures was analysed by TLC and GC. TLC was
carried out using silica-gel 60 plates (Merck) with
hexane/acetone (2:1 or 1:1 v/v) as eluent. Steroids
were detected by spraying the plates with H
2
SO
4
/
EtOH (1:1 v/v) followed by heating. Analytical
GC analysis was performed on a Hewlett Packard
5890A Series II GC instrument, using a HP-5 cap-
illary column (cross-linked 5% Ph-Me-Silicone,
30 m ¥0.53 mm ¥0.88 µm film thickness; temper-
ature program: 240 ∞CÐ1 min, gradient 5 ∞C/min
to 300 ∞CÐ5 min). Biotransformation products
were separated by column chromatography using
silica gel 0.05Ð0.2 mesh (Merck) with a hexane/
acetone mixture (2:1 v/v) as eluent. Structures of
biotransformation products were determined on
the basis of
1
H NMR spectra, which were recorded
on a DRX 300 Bruker 300 MHz spectrometer in
Table I. Biotransformation of steroids by Beauveria bassiana.
Substrate Products Yield
a
(%)
Testosterone (1)11α-hydroxytestosterone (8) 55.4
5α-androstan-11α,17β-diol-3-one (9) 14.8
11α-hydroxyandrost-4-ene-3,17-dione (10) 10.5
5α-androstan-11α-ol-3,17-dione (11) 8.4
17α-Methyltestosterone (2)11α-hydroxy-17α-methyltestosterone (12) 95.3
19-Nortestosterone (3)11α-hydroxy-19-nortestoterone (13) 40.5
1-Dehydrotestosterone (4)11α-hydroxy-1-dehydrotestosterone (14) 56.4
11α-hydroxyandrost-1,4-diene-3,17-dione (15) 13.2
11α-hydroxytestosterone (8) 11.4
11α-hydroxyandrost-4-ene-3,17-dione (10) 9.3
1-Dehydro-17α-methyltestosterone (5)11α-hydroxy-1-dehydro-17α-methyltestosterone (16) 86.7
Progesterone (6)11α-hydroxytestosterone (8) 94.1
Dehydroepiandrosterone (7) 5-androsten-3β,11α,17β-triol (17) 59.6
7α-hydroxydehydroepiandrosterone (18) 13.1
androstenediol (19) 8.3
a
Yield determined by GC.
CDCl
3
, CD measurements, which were done on a
JASCO-715 spectropolarimeter in chloroform,
and optical rotation measurements, which were
performed on an AUTOPOL IV polarimeter in
acetone at 25 ∞C.
Results and Discussion
In order to examine different structural factors
of a steroid on the biotransformation course, in-
cluding the presence of the C1,C2 double bond
and/or the additional 17α-methyl group and the
absence of either C19-methyl or C17-acetyl
groups, the following substrates have been chosen
for transformations by Beauveria bassiana AM446:
testosterone (1), 17α-methyltestosterone (2), 19-
nortestosterone (3), 1-dehydrotestosterone (4), 1-
dehydro-17α-methyltestosterone (5) and proges-
terone (6). As there is much less information
about hydroxylation of 5-androstenes compared to
4-ene-3-oxo-steroids, we also decided to explore
the bioconversion of dehydroepiandrosterone (7).
The fungus Beauveria bassiana was incubated
with the substrates until they were metabolised
(3Ð10 d). The results of the biotransformations
are presented by Fig. 1. The yield of products was
determined by GC analysis of the chloroform ex-
tract (Table I).
The structures of biotransformation products
were assigned mainly based on
1
H NMR spectra.
Both location and configuration of the newly in-
troduced hydroxyl group were determined by ana-
lysing differences between NMR spectra of the

E. Huszcza et al. · Steroid Transformations by Beauveria bassiana 105
O
OH
O
OH
O
OH
O
OH
O
OH
O
O
O
OH
OH
OH
OH
O
OH
OH
O
OH
OH
O
O
OH
O
O
OH
O
OH
OH
O
OH
OH
O
OH
OH
O
O
OH
O
OH
OH
O
O
OH
O
OH
OH
O
OH
OH
OH
OH OH
O
OH
+
+++
1
2
3
4
5
6
7
8910 11
12
13
14 15
16
17
+
+
810
8
++
18 19
Fig. 1. Metabolism of testosterone (1), 17α-methyltestosterone (2), 19-nortestosterone (3), 1-dehydrotestosterone
(4), 1-dehydro-17α-methyltestosterone (5), progesterone (6) and dehydroepiandrosterone (7)byBeauveria bassiana.
starting material and products (Table II, III), sup-
ported by literature data (Jones, 1973; Kirk et al.,
1990).
All the products obtained from 4-ene-3-oxo-ste-
roids transformations contained a 11α-hydroxyl
group, which was proved by a large downfield shift
of the 19-H
3
signal (but not 18-H
3
) and by the
broad multiplet profile for the 11β-H signal in the
region of δ3.86 ppm to 4.11 ppm as was reported
by Jones (1973) and Kirk et al. (1990). The spectral

106 E. Huszcza et al. · Steroid Transformations by Beauveria bassiana
Table II.
1
H NMR data for B. bassiana 4-ene-3-oxo-steroids transformation products and some substrates
a
.
Compound 4-H 17α-H 17α-CH
3
18-H 19-H 11β-H Other significant signals
85.72 3.69 t, J= 8.3 Hz Ð0.82 1.32 4.03 m Ð
9Ð3.69 t, J= 8.5 Hz Ð0.76 1.13 3.98 m Ð
10 5.73 ÐÐ0.93 1.32 4.05 m Ð
11 ÐÐ Ð0.89 1.15 4.02 m Ð
25.71 Ð1.20 0.90 1.19 ÐÐ
12 5.72 Ð1.23 0.92 1.32 4.05 m Ð
13 5.80 3.64 t, J= 8.5 Hz Ð0.78 ÐÐ Ð
13 5.81 3.68 t, J= 8.3 Hz Ð0.80 Ð3.86 m Ð
46.04 3.61 t, J= 8.4 Hz Ð0.79 1.21 Ð7.03 d, J= 10.2 Hz (1-H)
6.19 d, J= 10.1 Hz (2H)
14 6.06 3.63 t, J= 8.4 Hz Ð0.81 1.30 4.03 m 7.79 d, J= 10.3 Hz (1-H)
6.13 dd, J= 10.2 Hz, 2 Hz (2-H)
15 6.10 ÐÐ0.93 1.32 4.11 m 7.74 d, J= 10.3 Hz (1-H)
6.14 dd, J= 10.5 Hz, 2 Hz (2-H)
56.05 Ð1.18 0.92 1.24 Ð7.04 d, J= 10.2 Hz (1-H)
6.21 dd, J= 10.2 Hz, 2 Hz (2-H)
16 6.08 Ð1.20 0.93 1.32 4.09 m 7.79 d, J= 10.3 Hz (1-H)
6.13 dd, J= 10.2 Hz, 2 Hz (2-H)
a
Chemical shifts in ppm relative to Me
4
Si; solvent, CDCl
3
;J, coupling constant.
Table III.
1
H NMR data for dehydroepiandrosterone and its transformation products
a
.
Compound 6-H 3α-H CHOR 17α-H 18-H 19-H
75.34 d, J= 5.1 Hz 3.51 m, Wh = 23 Hz ÐÐ0.86 1.02
17 5.41 d, J= 5.8 Hz 3.53 m, Wh = 22 Hz 4.08 m (11β-H) 3.69 t, J= 8.5 Hz 0.78 1.16
18 5.62 d, J= 5.3 Hz 3.56 m, Wh = 22 Hz 3.95 m, Wh = 11 Hz (7β-H) Ð0.87 1.00
19 5.33 d, J= 5.2 Hz 3.50 m, Wh = 22 Hz Ð3.63 t, J= 8.5 Hz 0.75 1.01
a
Chemical shifts in ppm relative to Me
4
Si; solvent, CDCl
3
;J, coupling constant.
data of these compounds (Table II) correspond
very closely to those described in the literature for
11α-hydroxytestosterone (8) (Smith et al., 1990;
Kirk et al., 1990), 11α-hydroxyandrost-4-ene-3,17-
dione (10) (Kirk et al., 1990), 11α-hydroxy-17α-
methyltestosterone (12) (Huszcza and Dmochow-
ska-Gładysz, 2003), 11α-hydroxy-1-dehydrotes-
tosterone (14) and 11α-hydroxyandrost-1,4-diene-
3,17-dione (15) (Ahmed et al., 1996).
Apart from the hydroxylation, two other redox
reactions took place in the transformations pro-
moted by B. bassiana. The metabolites with satu-
rated C4,C5 or C1,C4 bonds were identified in tes-
tosterone (1) and 1-dehydrotestosterone (4)
transformations, respectively. The configuration of
5βfor compound 9was determined from the nega-
tive Cotton effect observed in the CD spectrum
([Θ]
304
=Ð1450). The presence of C3 and C17
carbonyl groups in the metabolite 11 resulted a
total positive Cotton effect, therefore the 5βcon-
figuration was confirmed by optical rotation mea-
surement ([α]
25
589
= + 72.9∞), which was in a good
agreement with the literature data (Allard, 1965).
Similar reduction of the C4,C5 double bound to the
5βconfiguration was observed for Beauveria globu-
lifera (Protiva et al., 1968).
Conversion of the alcohol at C17 into the ketones
1and 4also occurred. The presence of C17αmethyl
group inhibited reduction of both C4,C5 double bond
in 17α-methyltestosterone (2) and C1,C2 double
bond in 1-dehydro-17α-methyltestosterone (5).
It is noteworthy that the products of C4,C5
double bond reduction and/or C17 oxidation were
not found by transformation of 19-nortestosterone
(3). This substrate was relatively poorly metabo-
lized, which is in agreement with the results ob-
tained by Shibahara et al. (1970). They observed
that in spite of the induction of Aspergillus ochra-
ceus hydrolase only a low level of 11α-hydroxylation
of 19-nortestosterone could be achieved.
We have found that the main profile of biotrans-
formation of progesterone (6)byB. bassiana is the

E. Huszcza et al. · Steroid Transformations by Beauveria bassiana 107
side chain cleavage. Thus, the metabolite of proges-
terone was found to be the derivative of testoster-
one 8. Interestingly, 11α-hydroxytestosterone (8),
which was formed as the single product in high
yield, was not observed for other B. bassiana strains.
The main result of our study is the fact that struc-
tural differences in the 4-ene-3-oxo-steroids sub-
strates not effect the regio- and stereoselectivity of
the hydroxylation process. The steroid skeleton was
always attacked only at α-face of C11. Although the
6βand 11αpositions are expected to be equivalent
in enzyme-substrate complexes, 6β-hydroxy and
6β,11α-dihydroxy products were not found in any
our experiments.
The correlation between the structure of the sub-
stituent at C17 and the site specifity of hydroxyla-
tion of different steroid compounds by known 11α-
hydroxylators e.g. Rhizopus nigricans (Z
ˇakelj-Nar-
vic
ˇand Belic
ˇ, 1987), Aspergillus ochraceus (Tan and
Smith, 1968) and Cephalosporium aphidicola
(Boynton et al., 1997) was previously investigated.
Unlike in our study, it was shown that the side chain
at C17 had a strong influence on the position and
yield of hydroxylation by these fungi.
Introduction of the C5,C6 double bond to the ste-
roid skeleton slightly alters the transformation
course by the B. bassiana culture. Apart from the
major metabolite, 5-androstene-3β,11α,17β-triol
(17), incubation of dehydroepiandrosterone (7) with
B. bassiana gave a small quantity of 7α-hydroxyde-
hydroepiandrosterone (18). 7α-Hydroxylation was
confirmed by a down-field shift of the 6-H signal
(0.28 ppm as compared to substrate) and the exis-
tence of a narrow signal of 7β-H at 3.95 ppm. The
presence of the other minor product, androstenediol
(19), suggests that the 11α-hydroxylation was fol-
lowed by the reduction of the 17-ketone to the 17β-
alcohol. The hydroxylation of dehydroepiandroster-
one (7) at position 11αwithout further oxidation at
C3 is a typical feature of 11α-hydroxylating fungi
such as Rhizopus nigricans (Raspe and Richler,
1960), Rhizopus arrhizus (Holland and Diakow,
1979) and Aspergillus niger (Bell et al., 1972).
7α-Hydroxydehydroepiandrosterone (18) and an-
drostenediol (19) are metabolites of dehydroepian-
drosterone (7), which were detected in several mice
tissues. Compound 18 was described as a more po-
tent activator of immune processes in mice than 7
(Morfin and Courchay, 1994).
To sum up, we have found Beauveria bassiana to
be an efficient 11α-hydroxylator of dehydroepian-
drosterone (7) and 4-ene-3-ones, especially of 17α-
methyltestosterone (2) and 1-dehydro-17α-methyl-
testosterone (5). Compared to many other microor-
ganisms, Beauveria bassiana showed particularly
high regioselectivity and very low substrate specifity
in steroid hydroxylation. This phenomenon was ob-
served also for B. bassiana catalysed hydroxylations
of a variety of substrates e.g. amides, lactams, carba-
mates, azides and sulfonamides (Grogan and Hol-
land, 2000). Additionally, we have identified a new
dehydroepiandrosterone (7) transformation pro-
duct: 5-androstene-3β,11α,17β-triol (17).

108 E. Huszcza et al. · Steroid Transformations by Beauveria bassiana
Ahmed F., Williams A. D., and Smith K. E. (1996), Mi- Huszcza E. and Dmochowska-Gładysz J. (2003), Trans-
crobial transformations of steroids ÐX. Cytochromes formations of testosterone and related steroids in
P-450 11α-hydroxylase and C17-C20 lyase and A 1-ene Absidia glauca culture. J. Basic. Microbiol. 43, 113Ð
dehydrogenase transform steroids in Nectria haemato- 120.
cocca. J. Steroid Biochem. Mol. Biol. 58, 337Ð349. Jones E. R. H. (1973), The microbiological hydroxyla-
Allard S. (ed.) (1965), Tables of Constants and Numeri- tion of steroids and related compounds. Pure Appl.
cal Data. 14. Selected Constants, Optical Rotary Chem. 32,39Ð52.
Power, Ia. Steroids. Pergamon Press, Oxford, UK. Kirk D. N., Toms H. C., Douglas C., White K. A., Smith
Bayunova V. I., Gabinskaya K. N., Kolyanova T. S., Ko- K. E., Latif S., and Hubbard R. W. (1990), A survey
robova Y. N., and Grinenko G. S. (1989), Beauveria of the high-field
1
H NMR spectra of the steroids
sp. II. Transformation of androst-4-ene-3,17-dione and hormones, their hydroxylated derivatives, and re-
androst-1,4-diene-3,17-dione. Khim. Farm. Zh. 23, lated compounds. J. Chem. Soc. Perkin Trans. 2,
471Ð473. 1567Ð1594.
Bell A. M., Browne J. W., Denny W. A., Jones E. R. H., Krueger S. R. and Roberts D. W. (1997), Soil treatment
Kasal A., and Meakins G. D. (1972), Microbial hy- with entomopathogenic fungi for corn rootworm
droxylation of steroids. Part VI. Hydroxylation of sim- (Diabrotica spp.) larval control. Biol. Control 9,67Ð
ple mono- and di-oxygenated 5α-androstanes and of 74.
3-oxoestranes with the fungus Aspergillus ochraceus. Morfin R. and Courchay G. (1994), Pregnenolone and
J. Chem. Soc. Perkin Trans. I 23, 2930Ð2936. dehydroepiandrosterone as precursors of native 7-hy-
Bidochka M. J., Kasperski J. E., and Wild G. A. M. droxylated metabolites which increase the immune re-
(1998), Occurrence of the entomopathogenic fungi sponse in mice. J. Steroid Biochem. Mol. Biol. 50,
Metarhizium anisopliae and Beauveria bassiana in 91Ð100.
soils from temperate and near-northern habitats. Can. Mulock B. and Chandler L. (2000), Field-cage studies of
J. Bot. 76, 1198Ð1204. Beauveria bassiana (Hyphomycetes: Moniliaceae) for
Bing L. A. and Lewis L. C. (1991), Suppression of Os- the suppression of adult western corn rootworm, Dia-
tinia nubilalis (Huebner) (Lepidoptera: Pyralidae) by brotica vigifera vigifera (Cleoptera: Chrysomelidae).
endophytic Beauveria bassiana (Balsamo) Vuillemin. Biocontrol Sci. Technol. 10,51Ð60.
Environ. Entomol. 20, 1207Ð1211. Protiva J., Schwarz V., and Martinkova J. (1968), Forma-
Bing L. A. and Lewis L. C. (1992), Endophytic Beau- tion of 5β-saturated steroid derivatives by the 11α-
veria bassiana (Balsamo) Vuillemin in corn: the influ- hydroxylating fungus Beauveria globulifera. Folia
ence of the plant growth stage and Ostinia nubilalis Microbiol. 13, 139Ð145.
(Huebner). Biocontrol. Sci. Technol. 2,39Ð47. Raspe G. and Richler H. (1960), Ger. Patent
Boynton J., Hanson J. R., and Hunter A. C. (1997), The No. 1,080,553.
hydroxylation of some 13α-methylsteroids by Cepha- Sanada V., Fajkos J., and Protiva J. (1977), Microbial ox-
losporium aphidicola. Phytochemistry 45, 951Ð956. ygenation of norsteroids with Beauveria bassiana.
C
ˇapek A. and Fassatiova O. (1977), Some chemical char- Collect. Czech. Chem. Commun. 42, 3646Ð3653.
acteristic of species of the genus Beauveria. Folia Schubert A., Heller K., Onken D., Schwarz S., Siebert
Microbiol. (Prague) 22, 308Ð310. R., and Zaumseil J. (1962), Conversion of steroids
C
ˇapek A., Hanc O., Tadra M., and Tuma J. (1966), Mi- with microorganisms. Z. Chem. 2, 289Ð297.
crobial transformation of steroids XXV. Effect of sub- Shibahara M., Moody J. A., and Smith L. L. (1970),
stituents on the direction of the transformation of ste- Microbial hydroxylations V. 11α-Hydroxylation of
roids by Beauveria bassiana. Folia Microbiol. (Prague) progesterone by cell-free preparations of Aspergillus
11, 159Ð162. ochraceus. Biochim. Biophys. Acta 202, 172Ð179.
Griffiths D. A., Brown D. E., and Jezequel S. G. (1993), Smith K. E., Latif S., and Kirk D. N. (1990), Microbial
Metabolism of xenobiotics by Beauveria bassiana. transformations of steroids ÐVI. Transformation of
Xenobiotica 23, 1085Ð1100. testosterone and androstenedione by Botryosphaerica
Grogan G. J. and Holland H. L. (2000), The biocatalytic obtusa. J. Steroid Biochem. 35, 115Ð120.
reactions of Beauveria spp. J. Mol. Cat 9,1Ð32. Tan L., and Smith L. (1968), Microbial hydroxylations
Hajek A. E. and St. Leger R. J. (1994), Interactions be- II. Oxidative cleavage of the 17β-acetyl side chain of
tween fungal pathogens and insect hosts. Annu. Rev. progesterone by Aspergillus ochraceus. Biochim. Bio-
Entomol. 39, 293Ð322. phys. Acta 152, 758Ð769.
Holland H. L. and Diakow P. R. P. (1979), Microbial hy- Z
ˇakelj-Narvic
ˇM. and Belic
ˇI. (1987), Hydroxylation of
droxylation of steroids. 5. Metabolism of androst-5- steroids with 11α-hydroxylase of Rhizopus nigricans.
ene-3,17-dione and related compounds by Rhizopus J. Steroid Biochem. 28, 197Ð201.
arrhizus ATCC 11145. Can. J. Chem. 57, 436Ð440.