Use of an ionic liquid in a two-phase system to improve an alcohol dehydrogenase catalysed reduction

Article abstract of DOI:10.1039/b401065e

Due to favourable partition coefficients the highly enantioselective reduction of 2-octanone, catalysed by an alcohol dehydrogenase from Lactobacillus brevis, is faster in a biphasic system containing buffer and the ionic liquid [BMIM][(CF-₃SO₂)₂N] compared to the reduction in a biphasic system containing buffer and methyl tert-butyl ether.

Full text of DOI:10.1039/b401065e

Use of an ionic liquid in a two-phase system to improve an alcohol
dehydrogenase catalysed reduction†
a
b
c
a
Marrit Eckstein, Murillo Villela Filho, Andreas Liese and Udo Kragl*
a
Rostock University, Department of Chemistry, 18051 Rostock, Germany.
E-mail: udo.kragl@chemie.uni-rostock.de; Fax: + 49 381 4986452; Tel: + 49 381 4986450
Research Center Jülich, Institute of Biotechnology, 5425 Jülich, Germany; Fax: + 49 2461 613870; Tel: +
b
c
49 2461 613118
University of Münster, Institute of Biochemistry, 49149 Münster, Germany.
E-mail: a.liese@uni-münster.de; Fax: + 49 251 83 32122; Tel: + 251 83 33042
Received (in Cambridge, UK) 22nd January 2004, Accepted 5th March 2004
First published as an Advance Article on the web 30th March 2004
Due to favourable partition coefficients the highly enantiose-
lective reduction of 2-octanone, catalysed by an alcohol
dehydrogenase from Lactobacillus brevis, is faster in a biphasic
system containing buffer and the ionic liquid [BMIM][(CF-
drawn our attention to its potential in an ADH-catalysed reduc-
tion.
In this study we have used the alcohol dehydrogenase from
Lactobacillus brevis (LB-ADH), which catalyses the enantiose-
lective reduction of prochiral ketones to (R)-alcohols. Thereby the
enzyme requires the cofactor NADPH. Since the cofactor NADPH
3
2 2
SO ) N] compared to the reduction in a biphasic system
containing buffer and methyl tert-butyl ether.
is expensive, it is beneficial to regenerate the cofactor during the
reaction.4
,17,18
In the investigated reduction of 2-octanone the
Recently, it was reported that alcohol dehydrogenases (ADH) can
be used in biphasic systems containing an aqueous and an organic
phase to catalyse reductions of prochiral ketones to enantiopure
alcohols.1 While the enzyme is responsible for the high enantiose-
lectivity, the use of an organic phase suppresses limitations caused
by poor substrate-solubility in the aqueous phase leading to higher
volumetric productivities. Among further advantages of this
biphasic approach are an easy work-up by phase separation and the
cofactor regeneration was realised by a substrate-coupled approach,
whereby 2-propanol is oxidised to acetone yielding the reduced
cofactor NADPH (Scheme 1). In a two-phase system the aqueous
phase contains the LB-ADH and the cofactor NADP(H), while the
organic phase can be seen as a reservoir for the poorly water-
soluble substrate 2-octanone ( ~ 1 g/l = 7 mM). During the reaction
the substrate (2-octanone), the co-substrate (2-propanol), the
product (2-octanol) and the co-product (acetone) will partition
between the two phases. The partitioning behaviour of substrates
and products is of great importance for the thermodynamics of
reactions in two-phase systems.7
Since the partition coefficients of 2-propanol and acetone are
approximately equal (P ~ 1) in a two-phase system consisting of
buffer and MTBE (Table 1), their partition behaviour will not lead
to any shift in the thermodynamics of the cofactor regeneration. The
partition coefficients of the same substances conspicuously change
,2
“
free immobilisation” of the enzyme and charged cofactor
NAD(P)H in the aqueous phase allowing a re-use of enzyme and
cofactor.3 However, the drawback of this system is an unfavoura-
ble shift in the thermodynamics of the substrate-coupled cofactor
–6
,19,20
7
regeneration, which will be discussed in detail later.
In this paper we report the use of an ionic liquid as a second phase
in an ADH-catalysed reduction to make use of the advantages of
biphasic reaction conditions, but simultaneously to avoid thermo-
dynamic limitations.
Ionic liquids are defined as salts, which are liquid at temperatures
below 100 °C. Several groups have already shown the use of ionic
3 2 2
when MTBE is replaced by the ionic liquid [BMIM][(CF SO ) N].
8
While 2-propanol preferably remains in the aqueous phase (m =
0.4), acetone is rather removed by the ionic liquid (m = 2.0). In this
case the partition behaviour of the co-substrate and the co-product
will lead to a positive shift in the thermodynamics of cofactor
regeneration. The partition coefficients for 2-octanone and 2-octa-
nol were measured in a range > 100 for both two-phase systems.
Figure 1 demonstrates the advantages of the two-phase system
combining buffer and ionic liquid for the investigated reaction
system. The LB-ADH-catalysed reduction of 2-octanone was
liquids as solvents in biocatalytic applications, which have recently
been reviewed.9 In some cases good activities, enantioselectiv-
ities and stabilities of isolated enzymes and whole cells have been
reported. In biocatalysis ionic liquids have been used as pure
,10
solvents, as additives and even in combination with supercritical
carbon dioxide.1
1,12
However, the use of an ionic liquid in a two-
phase system for a biocatalytic application has only been described
once so far.13
The partition coefficients of 2-propanol and acetone in a two-
phase system containing buffer and ionic liquid [BMIM][(CF-
3 2 2
monitored in a biphasic system of [BMIM][(CF SO ) N] and
buffer and compared to the same reaction in the biphasic system
consisting of MTBE and buffer. The figure shows the conversion of
2-octanone as function of reaction time. Within the first 180 min the
reduction is much faster in the biphasic system containing the ionic
liquid, leading to a conversion of 88%, while the reduction reaches
a conversion of only 61% in the presence of MTBE.
14
3
2 2
SO ) N] significantly differ from their partitioning behaviour in
buffer/organic solvent systems (Table 1). This remarkable fact has
Table 1 Partition coefficients for 2-propanol and acetonea
Partition coefficients m between the two phases
3 2 2
BMIM][(CF SO ) /
N]14
[
Substance
-propanol
MTBE/buffer
buffer
2
1.0
1.1
0.4
2.0
acetone
a
Conditions: 50 mM phosphate buffer (pH 7.0); the ionic liquid was
15,16
prepared referring to Bonh oˆ te et al. and Eckstein et al.;
at 25 °C; analysis by GC-FID.
measurements
Scheme 1 Reduction of 2-octanone catalysed by alcohol dehydrogenase
from Lactobacillus brevis (LB-ADH). Substrate-coupled NADPH-re-
generation with 2-propanol.
†
Electronic supplementary information (ESI) available: experimental
section. See http://www.rsc.org/suppdata/cc/b4/b401065e/
1
084
C h e m . C o m m u n . , 2 0 0 4 , 1 0 8 4 – 1 0 8 5
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

The cofactor regeneration is the rate-limiting step of the
investigated reduction. This means: the more effective the re-
generation step, the faster the main reaction. The regeneration
equilibrium can be shifted using a higher concentration of
reduction in a biphasic system consisting of buffer and the ionic
liquid [BMIM][(CF SO N]. Due to the favourable partition
3
2 2
)
coefficients of 2-propanol and acetone, this reaction shows higher
reaction rates compared to the same reaction in the presence of
methyl tert-butyl ether (MTBE). To the best of our knowledge, this
is the first report of an alcohol dehydrogenase being used in a two-
phase system with an ionic liquid. Therefore, these results might
represent a novel route to biphasic reaction media, not only for
biocatalysis.
2
1
2-propanol and/or by removing acetone. In this biphasic approach
acetone is continuously removed by the ionic liquid due to its
partition coefficient of 2.0, which leads to a very fast regeneration.
Additionally, 2-propanol is permanently available in the aqueous
phase (P = 0.4).
Acetone is known as an inhibitor for the alcohol dehydrogenase.
Using the ionic liquid as a second phase, the extraction of acetone
from the buffer phase leads to a lower concentration of acetone in
the buffer phase. Thus, the inhibiting effect of acetone can be
reduced.
As confirmed by GC-analysis for the product (R)-2-octanol on a
chiral stationary phase, we could not detect any (S)-2-octanol in any
of the two reaction systems (ee > 99%).
We thank Thomas Daußmann from JFC – Jülich Fine Chemicals
for the alcohol dehydrogenase from Lactobacillus brevis
(www.juelich-chemicals.de). We also thank Heike Offermann and
Ursula Mackfeld for their skilful help in the laboratory and
Christine Fischer (IfOK; Rostock) for her help with the GC
analysis.
Villela et al. have reported that the alcohol dehydrogenase from
Lactobacillus brevis shows high stabilities in biphasic systems.
Thereby, the LB-ADH reaches half life times of ~ 10 hours in the
presence of aliphatic hydrocarbons. Using ethers like MTBE in a
two-phase system, the LB-ADH reaches highest stabilities with
half life times > 1000 h (stabilities were measured at 4 °C). Under
the investigated reaction conditions (30 °C) the LB-ADH showed
good stabilities with half life times of about 50 h in the presence of
Notes and references
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Further investigations will be necessary to understand the
development of the partitioning behaviour of the substrates and
products throughout the whole reaction. Another interesting future
aspect is the recycling of the enzyme and cofactor by replacing the
organic phase respectively the ionic liquid phase with fresh
substrate solution. Furthermore, the applicability of the investi-
gated system for other substrates is of interest.
7
8
9
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1
1
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C h e m . C o m m u n . , 2 0 0 4 , 1 0 8 4 – 1 0 8 5
1085