Organic Process Research & Development 2009, 13, 1202–1205
A Continuously Operated Bimembrane Reactor Process for the Biocatalytic
Production of (2R,5R)-Hexanediol
Kirsten Schroer† and Stephan Lu¨tz*,†
Institute of Biotechnology 2, Research Centre Ju¨lich, 52425 Ju¨lich, Germany
Abstract:
described. One of them is the substrate-coupled approach, where
the producing ADH also catalyzes the cofactor-regenerating
reaction. Usually 2-propanol is applied as cosubstrate for that
purpose.
The major disadvantage of substrate-coupled cofactor re-
generation is the thermodynamic limitation that occurs since
there is an equilibrium between all involved educts and products.
The thermodynamic equilibrium can be shifted towards higher
product yields either by increasing the concentration of substrate
and/or cosubstrate or by removing the product or the coproduct
acetone from the reaction process.8
Alcohol dehydrogenase-catalyzed reductions of prochiral ketones
to chiral alcohols require the regeneration of consumed cofactors
such as NADH or NADPH. In the substrate-coupled cofactor
regeneration approach, where 2-propanol is oxidized to acetone,
complete conversion is inhibited by a thermodynamic limitation.
This can be overcome by applying methods of in situ product
removal techniques such as pervaporation. Here we present a new
reactor concept which enables a continuous biocatalytic ketone
reduction process with concurrent in situ removal of the byproduct
acetone. In such a bimembrane reactor system recombinant
Escherichia coli cells expressing alcohol dehydrogenase from
Lactobacillus breWis were applied for the continuous reduction of
2,5-hexanedione. The product (2R,5R)-hexanediol could be syn-
thesized with exceedingly high space-time yield of >170 g/(L ·d)
and catalyst usage (17.9 gP/gwetcellweight).
Results and Discussion
The production of (2R,5R)-hexanediol starting from the
diketone 2,5-hexanedione in combination with substrate-coupled
cofactor regeneration is shown in Figure 1. For every molecule
of 2,5-hexanedione that should be converted to (2R,5R)-
hexanediol two molecules of NAD(P)H are needed, and thus
at least a 2-fold excess of the cosubstrate 2-propanol related to
the substrate 2,5-hexanedione is also needed. However, as
pointed out in Figure 2, a much higher excess of 2-propanol is
necessary to achieve sufficient yield since remarkable amounts
of the intermediate 5-hydroxyhexane-2-one are produced which
are not converted to the product 2,5-hexanediol. From the results
presented in Figure 2 an equilibrium constant of keq ≈ 0.1 can
be calculated. Thus, this reaction system is a particularly good
example to demonstrate the impact of acetone removal due to
its thermodynamic properties.
There are already a couple of methods described in the
literature dealing with in situ removal of products or byproducts
during biocatalytic processes. In situ acetone removal has
already been applied for enzyme-catalyzed processes9 and also
for whole-cell processes.10,11 In all cases significantly higher
yields could be achieved compared to yields from processes
without removal of the byproduct acetone from the biotrans-
formation process, but this strategy has only been applied to
biotransformation processes operated in batch mode.
Introduction
Reduction of prochiral ketones to optically active compounds
by biocatalytic methods is of particular interest due to the high
regio-, stereo-, and enantioselectivity of biocatalysts.1 This type
of reaction is usually catalyzed by alcohol dehydrogenases
(ADH) which are dependent on nicotineamide dinucleotide
cofactors such as NADH or NADPH.2 In contrast to enzyme-
catalyzed processes, where NAD(P)H must be added, whole-
cell catalysts already deliver a certain amount of intracellular
cofactors. The reduction of 2,5-hexanedione to optically pure
(2R,5R)-hexanediol is of particular interest since (2R,5R)-
hexanediol is a versatile building block for the synthesis of
various chiral phosphine ligands, which are used in chiral
Wilkinson catalysts.3-5
To run a NAD(P)H-dependent process economically ef-
ficiently there is a need for cofactor-regenerating reactions which
reduce NAD(P)+ to NAD(P)H by oxidizing a cosubstrate.6,7 In
the literature different methods for cofactor regeneration are
* Corresponding author. Telephone: (+49)(0)2461-61-4388. Fax: (+49)(0)2461-
61-3870. E-mail: s.luetz@fz-juelich.de.
Whole cells are particularly suitable catalysts to be applied
in continuously operated processes.12 Due to the natural
compartmentation achieved by the cell membrane, there is a
high retention of cofactors within the cytosol, and thus such a
† Present address: Novartis Institutes for Biomedical Research, 4056 Basel,
Switzerland.
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Vol. 13, No. 6, 2009 / Organic Process Research & Development
10.1021/op9001643 CCC: $40.75 2009 American Chemical Society
Published on Web 10/16/2009