The steroid monooxygenase from Rhodococcus rhodochrous; A versatile biocatalyst

Article abstract of DOI:10.1016/j.tetasy.2013.11.003

The substrate scope of a steroid monooxygenase (STMO) from Rhodococcus rhodochrous DSM 43269 was investigated for a large range of different ketone substrates. These studies revealed that this enzyme not only oxygenates steroids, but also ketone moieties of a series of other open-chain ketones, such as cyclohexyl methyl ketone, cyclopentyl methyl ketone, and 3-acetylindole. Furthermore, the STMO catalyzed the oxygenation of cyclobutanone derivatives. Comparative biotransformations with recombinant Escherichia coli resting cells harboring the STMO, the cycloalkanone monooxygenase (CAMO) from Cylindrocarpon radicicola or the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus revealed that the STMO is enantiodivergent compared to the CHMO-type. Moreover, the STMO resulted in a higher enantiomeric excess of the product lactones compared to the known BVMOs of the same enantiopreference, such as cyclopentanone monooxygenases.

Full text of DOI:10.1016/j.tetasy.2013.11.003

Tetrahedron: Asymmetry 24 (2013) 1620–1624
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
The steroid monooxygenase from Rhodococcus rhodochrous;
a versatile biocatalyst
Friedemann Leipold a, Florian Rudroff b, Marko D. Mihovilovic b, Uwe T. Bornscheuer a,
⇑
a University of Greifswald, Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Felix-Hausdorff Str. 4, 17487 Greifswald, Germany
b Vienna University of Technology, Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163, 1060 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 October 2013
Accepted 5 November 2013
The substrate scope of a steroid monooxygenase (STMO) from Rhodococcus rhodochrous DSM 43269 was
investigated for a large range of different ketone substrates. These studies revealed that this enzyme not
only oxygenates steroids, but also ketone moieties of a series of other open-chain ketones, such as cyclo-
hexyl methyl ketone, cyclopentyl methyl ketone, and 3-acetylindole. Furthermore, the STMO catalyzed
the oxygenation of cyclobutanone derivatives. Comparative biotransformations with recombinant Esche-
richia coli resting cells harboring the STMO, the cycloalkanone monooxygenase (CAMO) from Cylindrocar-
pon radicicola or the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus revealed
that the STMO is enantiodivergent compared to the CHMO-type. Moreover, the STMO resulted in a higher
enantiomeric excess of the product lactones compared to the known BVMOs of the same enantioprefer-
ence, such as cyclopentanone monooxygenases.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
ever, the substrate scope of this enzyme as well as of other steroid
monooxygenases has only been investigated rudimentally and,
Baeyer–Villiger monooxygenases (BVMOs, EC 1.14.13.x) are fla-
vin-dependent oxidoreductases, which convert ketones into esters
or lactones utilizing molecular oxygen and NADPH. Thus, they are
the enzymatic equivalents to peroxyacids or concentrated hydro-
gen peroxide capable of conducting the chemical Baeyer–Villiger
oxidation first described by Adolf von Baeyer and Victor Villiger
in 1899.1 Due to their potential for carrying out Baeyer–Villiger
except for phenylacetone, virtually exclusively for steroids as sub-
strates. The investigation of the substrate scope of the STMO for
further substrates would allow for a more precise classification
into specificity sub-types compared to other BVMOs. Due to the
STMO’s position in a phylogenetic tree separated from well-inves-
tigated BVMOs such as cyclohexanone monooxygenases, sub-
strates could potentially be converted with different enantio- and
regioselectivities. A more comprehensive substrate profile also en-
ables a better assessment of BVMOs with regards to prototype bio-
catalysts for various substrate classes, as recently outlined.9
Herein we investigated the substrate spectrum of the STMO
from R. rhodochrous against a large range of ketones including
steroids as well as (bi)cyclic and open-chain ketones. A whole-cell
recombinant biocatalyst containing the STMO was established and
the enantioselectivities achieved for the oxygenation of 3-phen-
ylcyclobutanone were compared to known BVMOs.
oxidations in
a more enantioselective and environmentally
friendly manner, BVMOs have been of particular interest for bioca-
talysis and today more than 60 BVMOs have been expressed
recombinantly.2 Detailed investigations of the substrate scopes of
these enzymes revealed that BVMOs convert a wide range of cyclic
(mono-, bi-, and polycyclic) as well as aromatic3 and open-chain4
ketones. Furthermore, some steroid-oxygenating BVMOs have
been described in bacteria5 as well as in fungi.6 A steroid monoox-
ygenase (STMO, EC 1.14.13.54) from Rhodococcus rhodochrous IFO
3338 (identical to R. rhodochrous DSM 43269) was expressed
recombinantly in Escherichia coli7 and the structure of this enzyme
was recently determined. The active site was shown to closely
resemble that of phenylacetone monooxygenase and the enzyme
was also shown to convert phenylacetone.8 This observation indi-
cates that the substrate scope of the STMO from R. rhodochrous
might possibly be more diverse than previously described. How-
2. Results and discussion
In order to fully assess the potential of the STMO for biocatalytic
Baeyer–Villiger oxidation reactions, the enzyme was produced
recombinantly in E. coli BL21 (DE3). This was implemented using
a pET-28b(+)-based construct allowing IPTG induction with the
STMO gene fused to an N-terminal His6-Tag for simplifying the
subsequent purification procedure. The gene was expressed in high
concentration and a high amount of soluble protein was formed.
⇑
Corresponding author. Tel.: +49 3834 86 4367; fax: +49 3834 86 794367.
E-mail address: uwe.bornscheuer@uni-greifswald.de (U.T. Bornscheuer).
0957-4166/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2013.11.003

F. Leipold et al. / Tetrahedron: Asymmetry 24 (2013) 1620–1624
1621
The crude extract obtained from the recombinant E. coli cells,
which were disrupted using a FastPrep24 homogeniser, was lyoph-
ilized without any additives; the activity loss caused by lyophilisa-
tion was determined as relatively low (10%). The pH activity profile
of the STMO measured for the conversion of progesterone was bell-
shaped with an optimal pH value of 10 and a faster activity
decrease at more alkaline pH values. Residual activities were above
60% compared to the optimal pH value at a pH between 8.0 and
10.9 (data not shown).
interest. Cyclobutanone and several of its substituted derivatives
were found to be substrates of this enzyme in contrast to other
cycloaliphatic ketones such as cyclopentanone and -hexanone,
which were not converted. In contrast, cyclopentanone and
-hexanone monooxygenases, which have typically been reported
to catalyze the oxygenation of cyclobutanone10 also convert other
cycloaliphatic ketones.
Whole cell biotransformations established for selected sub-
strates with glucose added for cofactor regeneration using resting
E. coli BL 21 (DE3) producing the STMO allowed us to prove the
conversion of the substrates shown to be active in previous activity
studies. Substrate and product concentrations were monitored
using GC–MS and it was found that the open-chain ketone cyclo-
hexyl methyl ketone was fully converted after 17 h as well as the
steroids progesterone and 11-ketoprogesterone. Additionally, fur-
ther non-steroid open-chain ketones such as cyclopentyl methyl
ketone as well as 3-acetylindole and 2-decanone were converted
in agreement with the spectrophotometric activity data given in
Table 1. Interestingly, also conversion of the cyclobutanone
derivative 3-phenylcyclobutanone was observed and confirmed
by GC–MS analysis.
The kinetic constants of the STMO were determined against a
small panel of steroids, as well as the two non-steroids cyclobuta-
none and cyclohexyl methyl ketone, for which the highest activi-
ties were found in initial activity studies (Table 2). The results
confirmed the previous findings that progesterone is the main sub-
strate of this enzyme,5 being converted with the highest catalytic
efficiency (kcat/Km). However, turnover numbers are in a compara-
ble range of values for all of the substrates tested and mainly the
Michaelis constants varied, resulting in different catalytic efficien-
cies. These data show that steroids are indeed the preferred sub-
strates of this enzyme. Still, the activities against non-steroids
are sufficient enough for biocatalytic applications.
For the determination of the substrates converted and the
kinetic parameters, the enzyme was purified to homogeneity by
Ni2+-based metal ion affinity chromatography, yielding a pure pro-
tein of 61 kDa on a SDS gel. Since only a small series of steroid com-
pounds have been identified as substrates of this enzyme so far,
and no extensive investigation of the enzymatic activity toward
substrates other than steroids has been reported in the literature,
a wide range of potential substrates was investigated (Table 1, Sec-
tion 4). Since a steroid monooxygenase from Cylindrocarpon radici-
cola converts open-chain ketones, such as progesterone, preferably
at higher pH values than cycloaliphatic steroids, such as 4-andro-
stene-3,17-dione,5 this was assumed to be also applicable to the
STMO from R. rhodochrous, in case it would convert any cycloali-
phatic ketones. Therefore, in order not to impede the discovery
of cycloaliphatic ketone substrates, all activity measurements were
performed in a sodium phosphate buffer (50 mM, pH 8.0). Results
show that the enzyme exhibits its highest activities against a small
panel of steroids as previously described, particularly progesterone
and 11-a
-hydroxyprogesterone.5 Furthermore, 11-ketoprogester-
one and, with a rather low activity, corticosterone were also
converted, which had not been shown so far for this enzyme. More
interestingly, the enzyme also converted other open-chain ketones
such as 2-decanone, 3-acetylindole as well as cyclohexyl- and
cyclopentyl methyl ketone. These substrates are all open-chain
subterminal ketones such as progesterone and particularly the
two latter substrates might be regarded as a type of minimal sub-
Table 2
Kinetic constants determined for STMO substrates using purified enzyme
strates mimicking the steroidal D-ring. This is also supported by the
fact that cyclohexyl- and cyclopentyl methyl ketones are the non-
steroids, which were converted with the highest activity. Further-
more, the conversion of 3-acetylindole might open up a possibility
for screening mutant libraries of the STMO for their activity toward
open-chain ketones. Besides the converted non-steroid open-chain
ketones, an oxygenation of cycloaliphatic ketones was of particular
Substrate
Vmax
(U/mg)
Km
(mM)
kcat
kcat/Km
(sꢀ1
)
(sꢀ1 mMꢀ1
)
Progesterone
11-a-Hydroxyprogesterone
11-Ketoprogesterone
Cyclohexyl methyl ketone
Cyclobutanone
0.676
0.665
0.485
0.457
0.540
0.085
0.299
0.323
1.461
18.79
0.702
0.690
0.504
0.474
0.561
8.25
2.31
1.56
0.33
0.03
Since the STMO exhibits an interesting activity in the formation
of butyrolactones, and considering that the substrate scope of this
enzyme differs significantly from BVMOs typically reported to con-
vert cycloaliphatic ketones, it was anticipated that the STMO might
possibly exhibit different enantiopreferences and -selectivities.
Since a cycloalkanone monooxygenase (CAMO) from C. radicicola
ATCC 11011 has been expressed and characterized recently exhib-
iting an interestingly high catalytic efficiency against cyclobuta-
none11 the selectivity of the STMO was compared to this enzyme
as well as to the cyclohexanone monooxygenase (CHMO) from Aci-
netobacter calcoaceticus. In order to establish these properties, the
enantioselectivity of these three enzymes was investigated
through biocatalytic reactions using the substrate 3-phenylcyclob-
utanone and compared to the values measured for the CHMO from
A. calcoaceticus (see Scheme 1).
Table 1
Substrate scope of recombinant R. rhodochrous STMO
Substratea
Activity
(mU/mg)
Activityb
(%)
Steroids
Progesterone
11-a-Hydroxyprogesterone
11-Ketoprogesterone
460
2
100
54.9
38.5
1.7
253 23
177
8
2
0
Corticosterone
Cyclic and (bi)cyclic ketones
Cyclobutanone
2-Hydroxy cyclobutanone
Bicyclo[3.2.0]-hept-2-ene-6-one
38
53
8
6
4
1
8.3
11.5
1.7
Open-chain ketones
Cyclohexyl methyl ketone
Cyclopentyl methyl ketone
2-Acetyl cyclopentanone
3-Acetylindole
2-Decanone
3-Decanone
4-Decanone
189
23
43
35
41
11
11
5
1
5
2
1
2
1
41.1
5.1
9.4
7.5
8.9
2.3
2.3
O
O
O
O
O
recombinant E. coli
producing BVMO
or
a
All measurements were made at 1 mM concentration, except for the steroids
(200 M) with purified enzyme and in triplicate.
Activity relative to progesterone (100% value).
l
b
Scheme 1. Formation of 3-phenylbutyrolactone using BVMOs.

1622
F. Leipold et al. / Tetrahedron: Asymmetry 24 (2013) 1620–1624
These experiments revealed that the CAMO exhibits a higher
logical Material (DSMZ). Escherichia coli DH5a [DlacU169(U80-
enantioselectivity compared to the CHMO (Table 3). In contrast,
the STMO turned out to be enantiodivergent compared to the
CHMO, and STMO also shows higher enantioselectivity than previ-
ously known (S)-selective BVMOs. Thus, the BVMOs investigated
herein might possibly enable the synthesis of chiral butyrolactone
derivatives, which represent valuable building blocks for the syn-
thesis of natural products.
lacZ M15) hsdR17 recA1 end A1 gyrA96 thi-1 relA1] was
D
purchased from Clontech (Mountain View, USA). E. coli BL21
(DE3) [Fꢀ ompT hsdSB (rB-mB-) gal dcm rne131 (DE3)] and the plas-
mid pET28b(+) were from Novagen (Darmstadt, Germany).
E. coli strains were routinely cultured in a lysogeny broth (LB)
medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1%
(w/v) NaCl) and supplemented with the antibiotic kanamycin
(20 lg/ml) when necessary. R. rhodochrous was grown at 30 °C on
Table 3
GYM Streptomyces medium containing 0.4% (w/v) glucose, 0.4%
(w/v) yeast extract, 1% (w/v) malt extract, 0.2% (w/v) CaCO3, and
1.2% (w/v) agar and adjusted to an pH of 7.2 (DSMZ medium 65).
For cultivation in liquid phase, the same medium, but without
CaCO3 and agar was utilized.
Enantioselectivities and conversions of the BVMOsa
Enzyme
Organism
Selectivity
ee (%)
Conversion (%)
CHMO
CAMO
STMO
A. calcoaceticus
C. radicicola
R. rhodochrous
(R)
(R)
(S)
58.6
90.9
76.3
45.9
70.8
72.4
4.3. Cloning and recombinant expression of the STMO in E. coli
a
After 20 h of biotransformation with whole cells.
The gene sequence of the STMO from R. rhodochrous was ampli-
fied from the genomic DNA of the wild type strain, which was iso-
lated using a TRI reagent (Sigma) according to the manufacturer’s
instructions.13 Amplification was carried out using PfuPlus DNA
Polymerase and primers incorporating restriction sites for cloning
the BVMO gene into the expression vector pET28b(+) containing a
N-terminal (pHisSTMO), C-terminal (pSTMOHis), and no His6-Tag
(pSTMO), respectively. The fragment for cloning pHisSTMO was
amplified using the primers SMO_ClonN_FW (50-CATATGGA
CGGCCAGCATCCCCGATC-30) and SMO_ClonC_RV (50-GAATT
CAGCCCTCGAGGATCGCAAACCCC-30), and pSTMOHis was con-
structed using SMO_ClonN_FW (50-CCATGGACGGCCA GCATCCCC
GATC-30) and SMO_ClonCH_RV (50-AAGCTT GCCCTCGAGGATCG
CAAACCCC-30). The primers SMO_ClonN_FW and SMO_ClonC_RV
were used for the construction of the STMO-bearing construct
without a N-terminal His6-Tag. The amplified products were sub-
cloned into the pCRÒII-TOPOÒ vector, digested with NdeI and EcoRI
(pHisSTMO), NcoI and HindIII (pSTMOHis) as well as NcoI and Eco-
RI (pSTMO), respectively (37 °C, 4 h) and ligated into an appropri-
ately linearized pET28b(+) vector. The level of STMO expression of
the different constructs was evaluated and the results showed that
the highest production of soluble protein was achieved as a fusion
protein with a N-terminal His6-Tag (data not shown). In order to
establish expression of the STMO with a His6-Tag fused to different
termini of the protein, NcoI had to be used for cloning of two of the
variants for which purpose a mutation N2D was introduced
through the N-terminal cloning primers as reported previously.7
In order to generate the wild type sequence of the construct con-
taining an N-terminal His6-Tag, which showed the highest levels
of soluble protein expression, the mutation originating from the
cloning primers was mutated back by QuikChange-PCR using the
primers SMO_D2N_sense (5’-GCGCGGCAGCCATATGAACGGCCAGC
AT-3’) and SMO_D2N_anti (5’-ATGCTGGCCGTTCATATGGCTGCCG
CGC-3’) thereby generating the plasmid pHisSTMO-WT. Expression
level and activity of the wild type enzyme showed no significant
differences compared to the N2D mutant (data not shown) and
the wild type enzyme fused to an N-terminal His6-Tag was used
for all subsequent experiments. Thus, the final vector encoding
the BVMO used for expression was pHisSTMO-WT and carried a
N-terminal His6-Tag coding sequence followed by a recognition
site for thrombin protease resulting in a fusion protein of 569 ami-
no acids, which differs in 20 amino acids at the N-terminus from
native STMO from R. rhodochrous.
3. Conclusion
An extensive study of the substrate scope of the steroid mono-
oxygenase from R. rhodochrous revealed that this enzyme oxygen-
ates a range of non-steroid ketones in addition to its well-
established steroidal substrates. In particular the conversion of
cyclobutanone derivatives and a series of open-chain ketones such
as cyclohexyl methyl ketone by STMO indicates a significant over-
lap with the substrate scope of a variety of other BVMOs. Hence,
the STMO from R. rhodochrous is a far more versatile biocatalyst
than it was previously assumed. The enzyme converts (aryl-)ali-
phatic ketones such as 3-acetylindole, phenylacetone, and 2-deca-
none, which have previously been described as typical substrates
of 4-hydroxyacetophenone monooxygenase (HAPMO), PAMO, and
aliphatic ketone monooxygenase (AKMO), respectively. The
STMO-catalyzed oxygenation of cyclobutanone derivatives, which
are typically converted by CHMOs and CAMO, was of particular
interest since this enzyme only converts open-chain ketones
except for this substrate group. The STMO produces the enantio-
complementary (S)-3-phenyl-4-butyrolactone product by the oxy-
genation of 3-phenylcyclobutanone compared to CHMO and
CAMO. Furthermore, the STMO shows similar or higher enantiose-
lectivity than the previously described (S)-selective BVMOs indi-
cating the potential of this enzyme for the biocatalytic
preparation of chiral butyrolactones. A whole-cell recombinant
biocatalyst based on E. coli producing the STMO, with glucose
added for cofactor regeneration, has been established with good
conversion and enantioselectivity, pointing the way toward future
applications in non-steroid biotransformations using this STMO.
4. Experimental
4.1. Chemicals
All chemicals were of highest purity and purchased from Fluka
(Buchs, Switzerland), Sigma–Aldrich (Munich, Germany), Roth
GmbH (Karlsruhe, Germany), Alfa Aesar (Karlsruhe, Germany),
and Acros Organics (Geel, Belgium) unless otherwise specified.
Restriction enzymes were obtained from New England Biolabs
(Beverly, MA, USA), DNA polymerases were from Roboklon (Berlin,
Germany). HisTrap™ 5 ml FF columns were purchased from GE
Healthcare (Uppsala, Sweden). 3-phenylcyclobutanone was pre-
pared as described previously.10
For protein production, pHisSTMO-WT containing the BVMO
gene was transformed into E. coli BL21 (DE3) and cultivation was
routinely carried out in 500 ml of the TB medium (2.4% (w/v) yeast
extract, 1.2% (w/v) tryptone, 4% (v/v) glycerol, 10% (v/v) potassium
4.2. Organisms, plasmids and culture conditions
Rhodococcus rhodochrous DSM 43269 (equivalent to IFO 3338)
was purchased from the German National Resource Centre for Bio-
phosphate buffer (pH 7.4, 1 M)) containing 20
shaking flasks. The cells were grown in a shaking incubator at 37 °C
lg/ml kanamycin in

F. Leipold et al. / Tetrahedron: Asymmetry 24 (2013) 1620–1624
1623
and 200 rpm to an optical density at 600 nm (OD600nm) of 0.6–0.8,
4.6. Biocatalysis using resting cells
then induced by the addition of IPTG to a final concentration of
0.2 mM and cultivated at 20 °C for further 15 h. Cells were har-
vested by centrifugation (4 °C, 15 min, 4500g) and washed twice
with sodium phosphate buffer (50 mM, pH 8.0). Cell disruption
was carried out using glass beads (0.1–0.11 mm) with FastPrep24
homogenizer (MP Biomedicals, Solon, OH, USA) three times (4 m/
s for 20 s) and crude extract was obtained by centrifugation
(4 °C, 20 min, 10,000g).
Biotransformations were carried out at 25 °C in Tris–HCl buffer
(50 mM, pH 8.0) using resting cells of E. coli BL21 (DE3) expressing
STMO at a final OD600 nm of 15 in 24-well deep well plates covered
with a breathable AeraSeal™ sealing film (Excel Scientific, Victor-
ville, USA). The reaction mixtures of a total volume of 2.5 ml con-
tained 5 mM 3-phenylcyclobutanone, 10 mM b-cyclodextrine,
100 mM glucose, and 2% (v/v) of dimethylformamide. Samples
were taken after 0, 3, and 20 h, extracted with ethyl acetate and
analyzed by GC–MS.
4.4. Protein purification, concentration and purity
Further biotransformations were carried out in order to verify
the conversion of substrates established through a spectrophoto-
metric assay, using a series of substances against which the
enzyme was shown active alongside with other typical BVMO sub-
strates, which were not shown to be converted in spectrophoto-
Overexpressed BVMO was purified using His6-Tag based metal
ion affinity chromatography. The cell pellet from 100 ml cell cul-
ture was disrupted in a sodium phosphate buffer (50 mM, pH
8.0) containing 300 mM NaCl and 30 mM imidazole as described
in Section 4.3. The filtered crude extract was loaded on a HisTrap™
FF crude column (5 ml) integrated into an Äkta purifier (GE Health-
care) at a flow of 1.5 ml/min. The column was washed with 3 col-
umn volumes of the same buffer and one column volume of
sodium phosphate buffer (50 mM, pH 8.0) containing 300 mM NaCl
and 60 mM imidazole thereafter at a flow of 5 ml/min. The elution
of the His6-Tagged protein was performed with 4 column volumes
of sodium phosphate buffer (50 mM, pH 8.0) containing 300 mM
NaCl and 300 mM imidazole. Subsequently, the active fractions
metric studies. The substances were assayed at
a
final
concentration of 2 mM together with 50 mM glucose for cofactor
regeneration and included progesterone, 11-ketoprogesterone, 4-
androstene-3,17-dione, prasterone, corticosterone, cyclopenta-
none, cyclohexanone, 2-phenylcyclohexanone, bicyclo[3.2.0]-
hept-2-en-6-one, 1-indanone, norcamphor, cyclohexyl methyl ke-
tone, cyclopentyl methyl ketone, 3-acetylindole, acetophenone,
and 2-decanone.
were pooled and desalted into
a
sodium phosphate buffer
4.7. GC analysis
(50 mM, pH 8.0) using a centrifugal filter device (cut off 10 kDa,
Amicon).
GC–MS analysis for the conversion of 3-phenylcyclobutanone
was carried out on a GCMS-QP 2010 (Shimadzu Europa GmbH,
Duisburg, Germany) with a HydrodexÒ-b-TBDac column (Mache-
rey-Nagel, Düren, Germany). Injection temperature was set to
220 °C, detection temperature was initially set to 80 °C followed
by a ramp of 2 °C/min to 220 °C. The enantiomeric configuration
of the ester products formed in biotransformation was established
through comparison to the products of a biotransformation using
the CHMO from A. calcoaceticus which have been reported previ-
ously.9 The substrate was eluted after 32.1 min whereas the peaks
for the (S)- and (R)-lactone products were measured after 54.1 and
54.5 min, respectively.
Protein concentration was determined in triplicate using the
bicinchoninic acid (BCA) assay12 with the protein quantification
kit (Uptima, Montluçon Cedex, France) using bovine serum albu-
min as a standard in sodium phosphate buffer (50 mM, pH 8.0).
Protein size and purity were determined by SDS–PAGE carried
out according to the method described by Laemmli.14 The gels
were stained with 1% (w/v) Coomassie brilliant blue R250 in 10%
(v/v) acetic acid, 30% (v/v) ethanol in water.
4.5. Enzyme assay and kinetics
The STMO activity was determined spectrophotometrically by
Conversions for all of the other substrates from the biocatalysis
using resting cells were measured using a BPX5 column (5% phe-
monitoring the decrease of NADPH at 340 nm (
at 25 °C. Reaction mixtures (1 ml) contained sodium phos-
phate buffer (50 mM, pH 8.0), 160 M NADPH, 10–150 mg/l of
e )
= 6.22 mMꢀ1 cmꢀ1
Table 4
l
GC conditions for measurement/achiral analyses of BVMO substrates
pure enzyme depending on the activity of the enzyme toward
the respective substrate and 1% (v/v) dimethylformamide. The
reaction was started by adding the enzyme to the mixture. One
unit of BVMO is defined as the amount of protein that oxidizes
Substrate
Temperature
program
Retention
time
Retention
time
substrate
(min)
product
(min)
1 lmol of NADPH per min. Substrate specificity and kinetic param-
Cyclohexanone
Cyclopentanone
2-Phenyl cyclohexanone
Cycloheptanone
Bicyclo[3.2.0]hept-2-en-6-one
1-Indanone
Program A
Program B
Program A
Program A
Program A
Program A
Program C
Program A
Program A
Program D
Program A
Program A
Program E
Program E
Program E
Program E
4.71
6.20
26.66
10.68
6.37
22.65
8.71
12.05
5.71
19.64
14.71
30.35
16.56
20.77
—
31.65
12.33
6.05
7.58
—
eters were determined using a purified enzyme. For the compari-
son of the overall activity, substrate concentrations were 0.2 mM
for steroids and 1 mM for all other substrates unless otherwise sta-
ted. Oxygenation activity was determined photometrically for the
steroids progesterone, pregnenolone, 4-androstene-3,17-dione,
Norcamphor
prasterone, 11-a-hydroxyprogesterone, 11-ketoprogesterone,
Cyclohexyl methyl ketone
Cyclopentyl methyl ketone
2-Decanone
Acetophenone
3-Acetylindole
hydrocortisone, and corticosterone. Furthermore, the (bi)cyclic
ketones cyclobutanone, 2-hydroxy cyclobutanone, cyclopenta-
none, cyclohexanone, 2-phenyl cyclohexanone, cycloheptanone,
cyclooctanone, bicyclo[3.2.0]-hept-2-ene-6-one, and 1-indanone
were tested. Activity was also investigated for the open-chain
ketones cyclohexyl methyl ketone, cyclopentyl methyl ketone,
2-acetyl cyclopentanone, cyclobutyl methyl ketone, cyclopropyl
methyl ketone, dicyclopropyl ketone, 3-acetylindole, acetophe-
none, 2-butanone, 2-hexanone, 2-octanone, 2-decanone, 3-deca-
none, 4-decanone, and 2-dodecanone. Kinetic data were analyzed
by non-linear regression analysis based on Michaelis–Menten
kinetics using the program Kaleidagraph.
7.40
15.54
25.67
16.86
19.95
12.94
10.71
29.30
17.96
17.96
—
Progesterone
11-Ketoprogesterone
4-Androstene-3,17-dione
Prasterone
—
Program A: 15 min 60 °C, 10 °C/min to 180 °C, maintain 5 min.
Program B: 10 min 32 °C, 20 °C/min to 140 °C, maintain 2 min.
Program C: 25 min 60 °C, 10 °C/min to 180 °C, maintain 10 min.
Program D: 5 min 85 °C, 20 °C/min to 180 °C, maintain 5 min.
Program E: 5 min 240 °C, 2 °C/min to 270 °C, maintain 5 min.

1624
F. Leipold et al. / Tetrahedron: Asymmetry 24 (2013) 1620–1624
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Acknowledgements
We gratefully acknowledge the financial support from the
German Federal State Mecklenburg-Vorpommern through a gradu-
ate scholarship to F.L. We also thank the DFG (Grant No. Bo1862/6-
1), the FWF (Grant No. I723-N17 & P24483-B20), and Vienna Uni-
versity of Technology (Grant No. GEV-TOP163) for financial sup-
port. We are grateful to Dr. Rainer Wardenga and Lilly Skalden
for their advice and support.
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