Chemo-promiscuity of alcohol dehydrogenases: reduction of phenylacetaldoxime to the alcohol

Article abstract of DOI:10.1016/j.tet.2010.03.050

The reduction of phenylacetaldoxime was catalysed by alcohol dehydrogenases in the presence of NAD(P)H yielding finally the primary alcohol via the imine and aldehyde intermediates. This suggests that the hydride of the cofactor NAD(P)H is transferred to the N-atom of the oxime moiety and not to the carbon atom, as usual stated. This reaction represents the first example of a catalytic chemo-promiscuity of alcohol dehydrogenases.

Full text of DOI:10.1016/j.tet.2010.03.050

Tetrahedron 66 (2010) 3410–3414
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Tetrahedron
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Chemo-promiscuity of alcohol dehydrogenases: reduction of phenylacetaldoxime
to the alcohol
a
a
,
b
b
a
a,
*
Bianca Ferreira-Silva , Iv a´ n Lavandera , Alexander Kern , Kurt Faber , Wolfgang Kroutil
a
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
Research Centre Applied Biocatalysis, c/o Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The reduction of phenylacetaldoxime was catalysed by alcohol dehydrogenases in the presence of
NAD(P)H yielding finally the primary alcohol via the imine and aldehyde intermediates. This suggests
that the hydride of the cofactor NAD(P)H is transferred to the N-atom of the oxime moiety and not to the
carbon atom, as usual stated. This reaction represents the first example of a catalytic chemo-promiscuity
of alcohol dehydrogenases.
Received 11 January 2010
Received in revised form 3 March 2010
Accepted 15 March 2010
Available online 20 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Chemo-promiscuity
Enzymes
Alcohol dehydrogenases
Reduction
Aldoximes
Deoximation
1. Introduction
could be detected. The control experiment showed that the sub-
strate was stable under the reaction conditions employed.
Alcohol dehydrogenases (ADHs) are enzymes, which are re-
sponsible to transform ketone/aldehydes to the corresponding al-
cohols and vice versa at the expense of a nicotinamide cofactor that
Employing the ADH from Rhodococcus erythropolis (RE-ADH) no
transformation of 1 was detected at all. The other five ADHs
showed E/Z-isomerisation [e.g., ADH from Paracoccus pantotrophus
1
8
acts as hydride donor and acceptor, respectively. Baker’s yeast,
DSM 11072 (PpADH)]. Unexpectedly four ADH preparations
2
which is known to possess many ADHs, has been reported to
(HLADH, ADH-A, RasADH, LK-ADH) led to the formation of alcohol
4, whereby in the case of LK-ADH and RasADH phenylacetaldehyde
3 was detected, too. For instance ADH-A led to the formation of 16%
of alcohol 4 within 24 h and 38% within 48 h.
3
transform oximes to carbonyl compounds or alcohols; however,
contradicting reports indicated that Baker’s yeast catalysed the re-
duction of oximes to afford the corresponding hydroxylamines and
4
5
amines. Since amines are valuable intermediates and products, we
envisioned that ADHs might act as suitable catalysts to reduce the
C]N double bond of oximes yielding hydroxylamines, which could
H
ADH
6
easily be transformed to the corresponding amines. Overall, this
would represent a first example of a promiscuous activity of ADHs.
NAD(P)H
7
N
HN
O
Tris-buffer
OH
H
OH
H
E-1
ADH
50 mM, pH 7.5
2
2
2
. Results and discussion
.1. Testing ADHs
ADH
3
Using phenylacetaldoxime 1 (Z/E¼95:5) as a test substrate the
N
+
HO
Z-1
NAD(P)H
desired reduction to hydroxylamine 2 was investigated employing
variousADHpreparationsandNAD(P)Hrecyclingsystems (Scheme 1,
Table 1). Unfortunately, no formation of hydroxylamine or amine
Tris-buffer
5
0 mM, pH 7.5
OH
4
*
Corresponding author. Tel.: þ43 (0)316 380 53 50; fax: þ43 (0)316 380 98 40;
Scheme 1. Biotransformation of oxime 1 employing alcohol dehydrogenases.
e-mail address: wolfgang.kroutil@uni-graz.at.
0
040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.03.050

B. Ferreira-Silva et al. / Tetrahedron 66 (2010) 3410–3414
3411
Table 1
Aldehyde 3 might be obtained by hydrolysis from aldoxime 1 as
generally assumed in literature (Scheme 2, pathway 1).
a
Transformation of aldoxime 1 (Z/E¼95:5) by various ADH preparations
3b,e,13
Entry ADH
NAD(P)H Z-1k (%) E-1k (%) Aldehyde 3k Alcohol 4k
recycling
(%)
(%)
db
Pathway 1: via hydrolysis
ADH
1
2
3
4
5
6
7
d
95
95
58
84
5
5
42
15
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
1
RE-ADHc
2-PrOH
2-PrOH
Ethanol
2-PrOH
d,e
PpADH
HLADH
ADH-A
RasADH
f
H
H
ADH
H O NH OH
2
2
e,g
l
l
l
49 (28) 35 (34) <0.1
16 (38)
N
O
OH
e,h
j
m
GDH
GDH
47
73
46
24
1
1
6
1
OH
NAD(P)H
NAD(P)+
3
4
i
j
LK-ADH
2
H
ADH
Ph
spont.
H O NH3
a
recycling
Reaction conditions: Tris/HCl buffer (pH 7.5, 50 mM), 1 mM NAD(P)H, 6 g/L
H+
ꢀ
H
2
O
+
NH
substrate, ADH preparation and cofactor recycling systems, 30 C, 24 h.
b
NAD(P)H NAD(P)
Control in the absence of enzyme.
RE-ADH: ADH from Rhodococcus erythropolis, NADH.
PpADH: ADH from Paracoccus pantotrophus DSM 11072, NADH, see Ref. 8.
Overexpressed in E. coli, used as lyophilised E. coli powder.
HLADH: horse liver alcohol dehydrogenase, NADH.
ADH-A: ADH from Rhodococcus ruber DSM 44541, NADH, see Ref. 9.
RasADH: ADH from Ralstonia sp. DSM 6428, NADPH, see Ref. 10.
LK-ADH: ADH from Lactobacillus kefir, NADPH.
imine
2
c
d
recycling
e
Pathway 2: via oxime reduction
Scheme 2. Pathways for transformation of aldoxime 1 to alcohol 4.
f
g
h
i
j
Glucose dehydrogenase employing glucose.
Measured by GC–MS.
k
Hydrolysis might be an unspecific spontaneous reaction, how-
ever since the blank experiments (Table 1, entry 1) as well as all
other (hydrolytic) enzyme preparations (Table 2) did not lead to
hydrolysis of aldoxime 1 to afford the corresponding aldehyde,
formation of aldehyde 3 can only be attributed to the activity of the
ADHs. In case the ADHs are indeed catalysing this transformation of
the oxime via hydrolysis, it can be expected that no reducing
equivalents are required: in a test experiment aldoxime 1 was in-
cubated in the presence of various ADHs (ADH-A, PpADH and
RasADH) in the absence of NAD(P)H recycling. To our surprise no
hydrolysis occurred, thus no aldehyde was formed (neither alcohol,
l
48 h reaction time.
m
In the presence of 5% v/v DMSO; without DMSO: 3% alcohol 4.
2
.2. Isomerisation
These unexpected biocatalytic activitiesdnamely oxime isomer-
isation as well as reduction of the oxime to the alcoholdprompted us
to investigate further this system. The chemical mechanism for (acid-
and base-catalysed) oxime isomerisation has been elucidated in
literature.11 In general, oximes are stable at neutral pH towards iso-
merisation and hydrolysis as confirmed in our blank experiment. In
order to elucidate whether the observed E/Z-isomerisation of 1 was
a genuine catalytic activity of the ADHs (except RE-ADH) or mediated
in a non-specific fashion, a set of well-known protein preparations
were tested (Table 2). The results showed that isomerisation of oxime
þ
only isomerisation was observed) even if NAD(P) was present. As
12
a consequence it was concluded that the ADH in combination with
the reduced cofactor might be responsible for the observed trans-
formation. Additional tests showed that NAD(P)H itself is not able
to reduce aldoxime 1 spontaneously in the absence of ADH,
therefore both ADH and NAD(P)H were required to transform the
aldoxime 1 to the aldehyde 3 by reduction. The only feasible re-
duced intermediate one can think of is an imine intermediate,
which then spontaneously hydrolyses to yield the aldehyde
1
was not specific for alcohol dehydrogenases, since every prepara-
tion tested led to isomerisation. Therefore we did not investigate this
unspecific reaction in more detail.
(Scheme 2, pathway 2).
Table 2
Attempts to trap the imine intermediate by reduction to the
amine employing NaBH CN, Raney nickel or H /Pd/C were not
3 2
a
E/Z-isomerisation of aldoxime 1 (Z/E¼95:5) catalysed by protein preparations
Z-1c (%)
E-1c (%)
successful; however this was not surprising since additional
chemical reduction experiments employing phenylacetaldehyde in
the presence of huge excess of ammonia showed that the possible
imine intermediate is much too unstable to be trapped at all in
water; thus not a trace of amine could be detected when testing the
chemical reduction of a possible imine in a mixture of phenyl-
acetaldehyde and ammonia in aqueous solution. This is supported
by literature, since no preparative metal-catalysed reductive ami-
nation of comparable aldehydes/ketones with ammonia in water at
reasonable conditions can be found. Anyway, since aldehyde 3 is an
intermediate and since the oxime is reduced (and not hydrolysed),
the imine is the only possible unstable intermediate to be expected.
Related reductive transformations of oximes to imines have pre-
Entry
Protein preparation
db
1
2
3
4
5
6
95
90
90
76
51
55
5
10
10
24
49
45
Lipase Aspergillus niger
Esterase BS3 Bacillus stearothermophilus
Bovine serum albumine
CAL-B Candida antarctica lipase B
E. coli, lyophilised cells
a
Reaction conditions: Tris/HCl buffer (pH 7.5, 50 mM), 1 mM NAD(P)H, 6 g/L
ꢀ
substrate, crude enzyme preparations, 30 C, 24 h.
b
Control: sample without enzymes.
Measured by GC–MS.
c
2
.3. Transformation of oxime to alcohol
14
viously been achieved by chemical methods and have been ob-
served by a reconstituted liver microsomal system of a pig liver
Therefore we turned our attention to the ADH-catalysed trans-
formation of the oxime to the alcohol. Alcohol 4 can only be
obtained here by reduction of the corresponding aldehyde 3
15
CYP2D enzyme (NADH-benzamidoxime reductase). Therefore, we
propose a reduction pathway via the imine for the ADH-catalysed
transformation of oxime 1. Related observations were reported
recently for flavo-proteins for the reduction of aliphatic nitro-
compounds leading finally to aldehydes and not to amines or
(
Scheme 2), which is also indicated by the detected aldehyde 3 in
selected experiments (Table 1, entries 6 and 7). In separate experi-
ments it was proven that the suspected ADH preparations are in-
deed able to reduce aldehyde 3 to the alcohol 4 in the presence of
an NAD(P)H recycling system. As a consequence aldehyde 3 is the
obvious precursor for the formation of the alcohol 4 as depicted in
Scheme 2. The next question concerns the formation of aldehyde 3.
16
hydroxylamines.
The sequential reduction of the aldehyde to the alcohol was
much faster than the aldoxime reduction, since only traces of the
aldehyde could be detected in selected cases (LK-ADH and

3412
B. Ferreira-Silva et al. / Tetrahedron 66 (2010) 3410–3414
Table 3
Testing the promiscuous oxime reducing activity with purified strep-RasADH
Entry
Catalyst
Reagents
Z-1c (%)
E-1c (%)
Aldehyde 3c (%)
Alcohol 4c (%)
1
2
3
4
d
NADPH
Glucose
GDH
95
95
93
58
5
5
7
<0.1
<0.1
<0.1
1
<0.1
<0.1
<0.1
6
d
d
a,b
strep-RasADH
NADPH, glucose, GDH
33
a
ꢀ
Reaction conditions: Tris/HCl buffer (pH 7.5, 50 mM), 1 mM NADPH, 6 g/L substrate, GDH, glucose, 30 C, 24 h.
ADH from Ralstonia sp. DSM 6428 with strep-tag, purified by affinity chromatography.
Measured by GC–MS.
b
c
RasADH). Since carbonyl reduction is the ‘natural’ activity of ADHs,
4. Experimental section
4.1. General
it is understandable that it proceeds considerably faster than the
promiscuous aldoxime reduction. This is in agreement with pre-
vious enzyme promiscuity studies comparing natural and pro-
17
miscuous activity showing that the latter is much slower.
.4. Tests with purified ADH
To ensure that the observed activity is not catalysed by other
Phenylacetaldehyde, and hydroxylamine hydrochloride and all
other reagents and solvents were purchased with the highest pu-
rity and used as received either from Aldrich–Sigma–Fluka (Vienna,
Austria) or Lancaster (Frankfurt am Main, Germany). Alcohol de-
hydrogenase from R. erythropolis (RE-ADH, Nr. 04.11, 33.8 U/g),
glucose dehydrogenase (GDH) from Bacillus megaterium, and es-
terase BS3 from Bacillus stearothermophilus (Nr. 16 U/g) were pur-
chased from J u¨ lich Chiral Solutions GmbH, a Codexis company
2
interfering enzymes possibly present in the crude (commercial)
ADH preparations, a strep-tagged ADH from Ralstonia sp. DSM 6428
(
strep-RasADH) was prepared and purified by affinity chromatog-
raphy. This pure strep-RasADH was used for the transformation of
aldoxime employing NADPH as cofactor and glucose
(
(
Germany). Alcohol dehydrogenase from Lactobacillus kefir
LK-ADH, Nr. 06543, 0.4 U/mg), lipase from Aspergillus niger (194 U/
1
g), alcohol dehydrogenase from equine liver (HLADH, Nr. A8435,
.33 U/mg), and bovine albumin (BSA, A-2153) were obtained from
dehydrogenase (GDH) with glucose for cofactor recycling (Table 3).
Only in the presence of strep-RasADH and NADPH recycling the
formation of alcohol 4 and a trace of aldehyde 3 could be detected
1
Aldrich–Sigma–Fluka (Vienna, Austria). Lipase from Candida ant-
arctica B (CAL-B) was purchased from Novozymes. Alcohol de-
hydrogenase from Rhodococcus ruber DSM 44541 (ADH-‘A’) was
commercially available from Codexis Inc or can be prepared as
(
entry 4), proving that indeed only the promiscuous activity of the
alcohol dehydrogenase is responsible for reducing aldoxime 1 to
finally yield the primary alcohol 4.
9
Escherichia coli/ADH-A preparation as previously reported. Over-
Since the reduction step of the aldoxime did not afford the
stable hydroxylamine 2, but the unstable imine (which hydrolysed
spontaneously), it can be concluded that the hydride of NAD(P)H is
transferred to the nitrogen atom of the oxime moiety and not to the
carbon atom, probably due to the poorer electrophilicity of the
carbon atom of the oxime moiety and the changed polarisation of
the nitrogen atom by the proximity of the negatively polarised
hydroxyl group. As observed for the reduction of carbonyls, ADHs
transfer the hydride to the atom next to the negative polarised
oxygen, which is in the case of carbonyls a carbon but for oximes it
is the nitrogen atom; another reason for the hydride transfer to the
nitrogen might result from the spatial arrangement between sub-
strate, NAD(P)H and enzyme.
8
expressed P. pantotrophus ADH (PpADH) and Ralstonia sp. ADH
10
(
RasADH) were obtained as previously described and used as
lyophilised powder.
TLC plates were run on silica gel Merk 60 F254 and visualised by
4
UV or by spraying with KMnO solution. NMR spectra were mea-
sured on a Bruker AMX spectrometer at 360 MHz.
4.2. GC–MS analysis
GC–MS analyses were performed on an HP 6890 Series GC
system equipped with a 5973 mass selective detector and a 7683
Series injector. The following column was used: Agilent 19091S-
4
33 (30 m, 0.25 mm, 0.25
m
m film) and the method used was:
ꢀ
ꢀ
ꢀ
1.3 mL/min, 100 C, then 10 C/min until 300 C (held 0.5 min).
3
. Conclusion
In summary, we have described the first example of a catalytic
4.3. Synthesis of E- and Z-phenylacetaldoxime 1
chemo-promiscuous activity of alcohol dehydrogenases, namely
the reduction of aldoxime 1 to the unstable imine intermediate,
which hydrolyses to give the aldehyde intermediate. ADH-cata-
lysed reduction of the aldehyde yielded finally the corresponding
primary alcohol resembling overall reductive bio-deoximation.18
As a consequence the results suggest that the hydride of
NAD(P)H is transferred to the nitrogen atom of the oxime moiety
and not to the carbon atom; only transfer of the hydride to the
nitrogen can lead to the proposed imine, which can then hydrolyse
to the aldehyde; transfer of the hydride to the carbon would result
in the formation of stable hydroxylamine, which was not ob-
served. The results explain why various attempts of microbial
oxime reductions aiming at the preparation of amines were un-
successful,3 since the promiscuous ADH activity interfered. Fur-
thermore, it could be deduced that the transformation of oximes
to aldehydes/ketones by fermenting yeast possessing a lot of ADHs
did not proceed via hydrolysis but through reduction catalysed by
the ADHs.
Phenylacetaldehyde (200
anol (5 mL). Hydroxylamine hydrochloride (249 mg, 3.58 mmol)
was dissolved in pyridine (435 L, 5.37 mmol) and added to the
phenylacetaldehyde solution. The reaction mixture was stirred for
24 h at room temperature and monitored by thin-layer chroma-
tography (ethyl acetate/petroleum ether 1:3, v/v). The reaction
mixture was evaporated, water (20 mL) was added and the mixture
was extracted twice with ethyl acetate. The combined organic
mL, 1.79 mmol) was dissolved in eth-
m
2 4
layers were washed with water and brine and dried (Na SO ). After
filtering, the organic solvent was concentrated by evaporation un-
der reduced pressure and the product was purified via flash chro-
matography (ethyl acetate/petroleum ether 1:5, v/v) leading to
a mixture of E- and Z-1 isomers. Upon recrystallisation with chlo-
roform and hexane, the Z-isomer crystallised in about 95% purity.
The yield was 47% [R
f
¼0.24 (ethyl acetate/petroleum ether 1:3, v/v).
þ
ꢁ
þ
1
GC–MS (EI): 135 (M ), 117 (M ꢂOH)]. H NMR (CDCl
3
) showed
3.78
a mixture of E- and Z-isomers. Compound Z-1 (88%):
d

B. Ferreira-Silva et al. / Tetrahedron 66 (2010) 3410–3414
3413
(
d, J¼5.3 Hz, 2H), 6.94 (t, J¼5.3 Hz, 1H), 7.33 (m, 4H), 8.88 (br s, 1H)
BioTAGnology). Then, the supernatant was microfiltered and
passed through the column. The column was washed with buffer W
(25 mL) to remove the remaining E. coli proteins.
and Compound E-1 (12%):
1
d
3.58 (d, J¼6.3 Hz, 2H), 7.57 (t, J¼6.4 Hz,
H), 7.33 (m, 4H), 8.35 (br s, 1H).
The ADH was eluted with another buffer (buffer E, Tris 100 mM,
NaCl 150 mM, EDTA 1 mM, 2.5 mM desthiobiotin, pH 8) and eight
fractions of 2.5 mL were collected separately. Afterwards, activity
(propiophenone, NADPH) was tested with every fraction to identify
the ADH. SDS-page analysis showed a single band.
4
.4. Example procedure for the enzymatic screening
Lyophilised cells of E. coli/ADH-‘A’ (20 mg), 2-propanol (32 mL)
as hydrogen donor and the substrate (6 g/L) were suspended in
Tris$HCl buffer [500
m
L, pH 7.5, 50 mM, 1 mM NADH] in Eppendorf
ꢀ
tubes (1.5 mL) and shaken at 30 C and 600 rpm for 24 h. The
reaction was stopped by extraction with ethyl acetate (2ꢃ0.5 mL).
The organic layer was separated from the cells by centrifugation
4.6. Procedure of enzymatic conversion using purified
tagged-RasADH
(
2 min, 13,000 rpm) and dried (Na
2
SO
4
). Composition of the
Purified tagged-RasADH (475
mL), substrate 1 (6 g/L), DMSO
product mixture was determined by GC–MS.
(25 L), NADPH (1 mM), GDH (1 mg), and glucose (13 mg) were
m
placed in an Eppendorf tube (1.5 mL). The reaction mixture was
ꢀ
4
.5. Cloning, expression and purification of tagged Ralstonia
shaken at 30 C and 600 rpm for 24 h and stopped by extraction
sp. ADH
with ethyl acetate (2ꢃ0.5 mL). The organic layer was separated and
2 4
dried (Na SO ). Composition of the product mixture was de-
4.5.1. Construction and cloning of a StrepII tagged Ralstonia sp. ADH
termined by GC–MS.
gene. Standard molecular-biology procedures were performed
1
9
0
according to literature. The StrepII tag was introduced at the 3
Acknowledgements
10
end of the RasADH gene by PCR using pEam_RasADH as template.
The primers were ADH-f 5 -ATGTATCGACTATTAAACAAAACAGC-3
and ADH-StrepII-r5 -TTATTTTTCGAACTGCGGGTGGCTCCAAGCGCT-
GACCTGGGTCAATCCACCGTCC-3 (StrepII Tag sequence in bold). The
PCR product was purified and cloned into pEamTA. The resulting
plasmid pEam_tagged-RasADH was used to transform E. coli DH5
the strain was arbitrarily designated as E. coli/tagged-RasADH.
0
0
B.F.-S. acknowledges the FAPSEP (Fundaçao de Amparo a Pes-
quisa do Estado de Sao Paulo) and the CAPES (Fundaçao Coor-
˜
0
˜
˜
0
denaçao de Aperfeiçoamento de Pessoal de Nıvel Superior).
Financial support by FFG, and Province of Styria is gratefully
acknowledged.
˜
´
2
0
a,
References and notes
4.5.2. Preparation of lyophilised cells containing recombinant cata-
lyst E. coli/RasADH. Cultivation medium LB-amp: Luria broth (25 g/L,
Sigma L-3522), ampicillin sodium salt (100 mg/L, Sigma A9518-
1. (a) de Wildeman, S. M. A.; Sonke, T.; Schoemaker, H. E.; May, O. Acc. Chem. Res.
2
007, 40, 1260–1266; (b) Goldberg, K.; Schroer, K.; L u¨ tz, S.; Liese, A. Appl. Mi-
crobiol. Biotechnol. 2007, 76, 237–248; (c) Moore, J. C.; Pollard, D. J.; Kosjek, B.;
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5
6
G), KH
0355).
2
PO
4
2 4
(1.4 g/L, Fluka 60220) and K HPO (4.4 g/L, Fluka
ꢀ
2. Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J. Am. Chem. Soc. 2004,
E. coli/RasADH was stored at ꢂ86 C in a glycerol/LB-amp 15:85
126, 12827–12832.
solution. Prior to use it was plated on LB-amp, a single colony was
plated again on LB-amp (16 h, 37 C), before a loop of cells were
3
. (a) Kreutz, O. C.; Segura, R. C. M.; Rodrigues, J. A. R.; Moran, P. J. S. Tetrahedron:
Asymmetry 2000, 11, 2107–2115; (b) Kreutz, O. C.; Moran, P. J. S.; Rodrigues, J. A.
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Meshram, H. M. J. Chem. Soc., Perkin Trans. 1 1991, 2056–2057.
ꢀ
used to inoculate 250 mL of LB-amp medium in 1 L baffled shake
ꢀ
flasks. After incubation for 24 h at 30 C at 130 rpm an OD of w5
was reached and the expression of ADHs was induced by the ad-
dition of IPTG (450 mg/L, 2 mM final concentration, preqlab Bio-
4. (a) Chimni, S. S.; Singh, R. J. World J. Microbiol. Biotechnol. 1998, 14, 247–250; (b)
Gibbs, D. E.; Barnes, D. Tetrahedron Lett. 1990, 31, 5555–5558.
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. (a) Buchholz, S.; Gr o¨ ger, H. In Biocatalysis in the Pharmaceutical and Bio-
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(b) Kim, M.-J.; Ahn, Y.; Park, J. In Biocatalysis in the Pharmaceutical and Bio-
technology Industries; Patel, R. N., Ed.; CRC: Boca Raton, FL, 2007; pp 249–272;
ꢀ
(
100 mg/L) was added. The incubation was performed at 20 C to
avoid the formation of inclusion bodies for 24 h at 130 rpm. The
cells were harvested by centrifugation (8000 rpm, 3000 g, 20 min,
(c) Turner, N. J.; Carr, R. In Biocatalysis in the Pharmaceutical and Biotechnology
ꢀ
4
C), the medium was decanted and the cells were resuspended in
Industries; Patel, R. N., Ed.; CRC: Boca Raton, FL, 2007; pp 743–755; (d) Aboul-
Enein, H. Y.; Wainer, I. W. The Impact of Stereochemistry on Drug Development
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water, shock-frozen (liquid nitrogen) and lyophilised.
The same procedure was used for overexpression of tagged-
RasADH.
6. Ashton, M. J.; Hills, S. J.; Newton, C. G.; Taylor, J. B.; Tondu, S. C. D. Heterocycles
1989, 28, 1015–1035.
7. Enzyme promiscuity is the ability of enzymes to perform chemical reactions
other than the ones they are evolved. For reviews, see: (a) Copley, S. D. Nat.
Chem. Biol. 2009, 5, 559–566; (b) Hult, K.; Berglund, P. Trends Biotechnol.
4
.5.3. Purification of tagged-RasADH. The purification was per-
formed following the instruction of the supplier of the column (IBA,
BioTAGnology, purification of strep-tag fusion proteins with Strep-
Tactin matrices, version PR03-0002).
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ampicillin (100 mg/L) at 30 C for 24 h. Afterwards, additional
ampicillin (100 mg/L) and IPTG (2 mM) were added and cells were
grown at 20 C for 24 h. Then, the media was centrifuged, the cells
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were suspended in 50 mL of the same buffer.
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on, 2 s off, 50% amplitude, then centrifuged and the supernatant
was taken for the strep-tag purification.
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