Biotransformations in Pure Organic Medium: Organic- Solvent-Labile Enzymes in the Batch and Flow Synthesis of Nitriles

Article abstract of DOI:10.1002/ejoc.201901168

In recent years, there has been an increasing tendency to use biocatalysts in industrial chemistry, especially in the pharma and fine chemical sector. Preferably, enzymes or whole cells, applied as catalysts for a specific biotransformation, are utilized in aqueous reaction media since water is the natural medium for enzymes. In numerous examples of biocatalytic systems, however, a major problem is the insolubility of hydrophobic substrates in such aqueous reaction media. Apart from lipases, many enzymes are highly sensitive to organic solvents and are inactivated by an organic medium. Therefore, a change of solvent for biotransformations from water to organic solvents is usually challenging. In this study, we investigated the synthesis of nitriles by an organic solvent-labile aldoxime dehydratase in pure organic solvents, exemplified for the dehydration of n-octanaloxime to n-octanenitrile. We present a method for applications in batch as well as flow mode based on an “immobilized aqueous phase” bearing the whole cells in a superabsorber as solid phase, thus enabling the use of a purely organic solvent as “mobile phase” and reaction medium.

Full text of DOI:10.1002/ejoc.201901168

A Journal of
Accepted Article
Title: Biotransformations in pure organic medium with organic solvent-
labile enzymes in batch and flow exemplified for nitrile synthesis
Authors: Alessa Hinzmann, Niklas Adebar, Tobias Betke, Monja
Jochmann, and Harald Gröger
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201901168
Link to VoR: http://dx.doi.org/10.1002/ejoc.201901168
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Biotransformations in pure organic medium with organic solvent-
labile enzymes in batch and flow exemplified for nitrile synthesis
Alessa Hinzmann, Niklas Adebar, Tobias Betke, Monja Jochmann and Harald Gröger*
Abstract: In recent years, there has been an increasing tendency to
use biocatalysts in industrial chemistry, especially in the pharma- and
fine chemical sector. Preferably, enzymes or whole cells, applied as
catalysts for a specific biotransformation, are utilized in aqueous
reaction media since water is the natural medium for enzymes. In
numerous examples of biocatalytic systems, however, a major
problem is the insolubility of hydrophobic substrates in such aqueous
reaction media. Apart from lipases, many enzymes are highly
sensitive to organic solvents and are inactivated by an organic
medium. Therefore, usually a change of solvent for biotransformations
from water to organic solvents is difficult. In this study, we investigated
the synthesis of nitriles by an organic solvent-labile aldoxime
dehydratase in pure organic solvents, exemplified for the dehydration
of n-octanaloxime to n-octanenitrile. We present a method for
applications in batch as well as flow mode based on an “immobilized
aqueous phase” bearing the whole cells in a superabsorber as solid
phase, thus enabling the use of a purely organic solvent as “mobile
phase” and reaction medium.
in terms of enzyme stability when being in contact with
hydrophobic reaction media. Whereas for lipases their use in
solely organic media is widely known (due to their high affinity “by
definition” to organic solvents), for most other enzymes general
methodologies for their utilization in pure organic medium rarely
exist.[
3][4][9][10][13]
Enzymes are often unstable in organic solvents,
biphasic reaction systems consisting of water and an organic
solvent and in many cases even in aqueous reaction media when
containing a large portion of a water-soluble organic co-
solvent.[
1][5][14]
Furthermore, enzymes in crude extracts and in
particular in purified form are even more unstable compared to
whole cell catalysts, which benefit from the cellular matrix
protecting the enzymes. But also for whole cell catalysts,
examples are rare in which they are used in pure organic medium
or biphasic systems.[15] At the same time various strategies to
overcome these stability problems of biocatalysts in contact with
organic solvents have been studied. One opportunity is
compartmentali-zation of the reaction medium. This concept
enables the combination of non-compatible reactions comprising
biocatalytic steps and, e.g., polydimethylsiloxane (PDMS)
membranes were successfully used for this purpose.[
16][17][18]
Introduction
In recent decades biotransformations have emerged towards a
valuable tool in organic synthesis on both lab and industrial
Another solution to overcome the instability problems of
biocatalysts in organic media is immobilization. Besides
immobilization of enzymes in crude extracts or purified enzymes
themselves,[19][20][21][22] techniques of whole cell immobilization are
known as well. Usually, immobilization of “free” enzymes without
protection of the cells is not sufficient to make these biocatalysts
scale.[
1][2][3][4][5][6]
In spite of tremendous success illustrated, e.g.,
by numerous applications in industry, however, a general
challenge related to the reaction medium remained: Typically,
enzyme catalysis is conducted in aqueous medium whereas in
contrast substrates and products are mostly hydrophobic.
usable in pure organic solvent. In case of whole cell
immobilization[15][23][24][25] some methods showed stabilizing
[
1][2][3][4][5][6][7][8]
Thus, the option to run biotransformations in purely
organic medium would offer significant advantages. Besides the
benefits from solubilizing hydrophobic substrates in pure organic
medium, emulsions as a result from two-phase aqueous/organic
solvent systems could be avoided. Such emulsions often lead to
effects and biocatalysts were found to be more stable in presence
of organic solvents.[15][23] Encapsulation of whole cells in
polyurethanes, as shown by von Langermann et al., turned out as
a highly suitable technique to prevent contact of the biocatalyst
a tedious work-up due to, e.g., difficulties in phase separation. In
contrast, when using pure organic medium[9][10][11] the work-up
with the organic medium, thus resulting in a significant higher
stability of the catalyst in such media.[23][26] In continuation of our
could consist of a simple filtration or decantation of the organic
solvent from a heterogenized biocatalyst. Such a biocatalytic
process would be particularly interesting for producing bulk
ongoing study on the use of enzymes being immobilized in
superabsorber materials as a technique for immobilization of
“free” enzymes,[
22][27][28]
and our research work on aldoxime
chemicals due to simplification of downstream-processing and
minimizing the unit operation steps therein.[12] A major task for
dehydratase and their application for the synthesis of nitrile bulk
chemicals in the presence of aldoxime dehydratases,[29][30][31] we
obtaining high conversions and productivities with hardly water-
miscible substrates in enzyme catalysis is to overcome limitations
became interested if such superabsorber-immobilized whole cell
catalysts can be successfully used in pure organic medium. In the
following we report the development of such a solution for utilizing
whole-cell catalysts being immobilized in superabsorbers in pure
organic media, which also provides the perspective for a broad
generality in biocatalysis.
A. Hinzmann, N. Adebar, Dr. T. Betke, M. Jochmann, Prof. Dr. H. Gröger
Chair of Industrial Organic Chemistry and Biotechnology
Faculty of Chemistry, Bielefeld University
Universitätsstrasse 25, 33615 Bielefeld (Germany)
E-mail: harald.groeger@uni-bielefeld.de
Results and Discussion
Internet: https://www.uni-bielefeld.de/chemie/arbeitsbereiche/oc1/HG/
We chose an aliphatic aldoxime, namely n-octanaloxime (1), as
substrate because firstly, substrate 1 and product 2 are insoluble
in water and secondly, based on a previous study of Asano et
Supporting information for this article is given via a link at the end of the
document.
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European Journal of Organic Chemistry
10.1002/ejoc.201901168
COMMUNICATION
al.[32][33][34][35] recently we developed a process for transforming
aliphatic aldoximes to the corresponding nitriles at high substrate
loading in the presence of an aldoxime dehydratase from Bacillus
sp. OxB-1 (OxdB) as a catalyst.[30][36] Our first approach towards
conducting aldoxime dehydratase-catalyzed reactions in pure
organic medium consisted of using wet E. coli cells with
2
3
4
Methyl-tert-butyl ether (MTBE)
Toluene
1
2
Dichloromethane (DCM)
Potassium phosphate buffer (PPB)
>1
61b
5
overexpressed OxdB as biocatalyst (Scheme 1) since
previously[30] we utilized such whole cells in aqueous reaction
media.
a
33 mg
wcm/mL OxdB in whole cells were suspended in 20 ml organic solvent
or PPB including 500 mM n-octanaloxime (1). The reactions were performed
at 30 °C for 24 h and conversions to n-octanenitrile (2) were determined by
GC analysis. Conversion was determined after extraction of the reaction
b
mixture with ethyl acetate (EtOAc). The EtOAc phase was analysed by GC
for determination of conversion.
The use of any type of organic solvent as reaction medium directly
led a strong drop of activity, indicating the high degree of
deactivation of the enzyme in these cases. Thus, we found very
low conversions in the range of <1% to 2% of n-octanaloxime (1)
to n-octanenitrile (2) when using OxdB in pure organic medium
(Table 1, entries 1-4). In contrast, in the control reaction using the
aqueous buffer as reaction medium a conversion of 61% was
observed (entry 4). These experiments revealed that wet cells are
not applicable for a use in pure organic solvent. Thus, as a next
step we studied the use of lyophilized cells of OxdB as a catalyst
in pure organic medium (Scheme 2).
Scheme 1. OxdB in wet whole cells catalyzed dehydration of n-octanaloxime (1)
to n-octanenitrile (2) in pure organic medium.
In our experiments, we utilized n-octanaloxime (1) at
a
concentration of 0.5 M in combination with various types of
organic solvents (cyclohexane, methyl-tert-butyl ether (MTBE),
toluene and dichloromethane (DCM)) as reaction medium and 33
mg of wet cell mass (wcm) of E. coli cells including OxdB as
biocatalyst per ml of reaction medium. A control reaction was
performed using potassium phosphate buffer (PPB) as reaction
medium. The reactions were performed at 30 °C reaction
temperature for 24 h in round bottom flasks and 20 mL scale.
Scheme 2. OxdB in lyophilized cells catalyzed dehydration of
n-octanaloxime (1) to n-octanenitrile (2) in pure organic medium.
Again, we used n-octanaloxime (1) in a concentration of 0.5 M in
different solvents (cyclohexane, MTBE, toluene and DCM) and at
3
0 °C reaction temperature for 24 h. In this case, 7.5 mg dry cell
mass (dcm) per ml of reaction medium was used, corresponding
to 33 mg bio wet mass. We again performed a control reaction
using resuspended dry cells in aqueous buffer. As shown in
Figure 2, in all solvents the lyophilized whole cells were
homogeneously suspended. After 24 h reaction time, the
conversion of n-octanaloxime (1) to the corresponding nitrile was
analyzed by GC (Table 2). In case of the control reaction in
aqueous buffer the reaction was extracted with EtOAc beforehand.
Figure 1. Pictures of reaction mixtures with OxdB in wet whole cells in different
organic solvents.
During the chosen reaction time, the whole cell catalyst behaved
very different in each solvent, as visualized in the photos shown
in Figure 1. In case of cyclohexane and toluene, the cells were
suspended in the solvent, but in case of DCM and especially
MTBE, the cells precipitated and were not suspended in the
organic medium (Figure 1). After a reaction time of 24 h, the
conversion of n-octanaloxime (1) to the corresponding nitrile 2
was analyzed by GC (Table 1). In case of the control reaction in
phosphate buffer, the reaction was extracted with ethyl acetate
(EtOAc) beforehand.
Figure 2. Pictures of reaction mixtures with OxdB in lyophilized cells in different
organic solvents.
Table 1. OxdB in wet whole cells catalyzed dehydration of
n-octanaloxime (1) in pure organic medium.
#
1
Reaction medium/solvent
Cyclohexane
Conversion to 2 /%a
However, in spite of a homogeneous biocatalyst suspension
also in these cases a srong deactivation of the Oxd-enzyme
was observed as soon as the reaction is conducted in an
2
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European Journal of Organic Chemistry
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COMMUNICATION
organic solvent, thus underlining that these enzymes are labile
towards the presence of an organic medium (Table 2, entries
active in the cells (see comparison of experiments shown in entry
1 of the Tables 1-3). However, still such a biphasic system has a
negative impact on the activity and/or stability of the enzyme.
1
-4). In detail we found very poor conversions below 1% of
n-octanaloxime (1) to the corresponding nitrile 2 independent
of the type of organic solvent as reaction medium. In contrast,
the control reaction gave 23% conversion showing that the
enzyme in the lyophilized cells is still active after lyophilization.
Table 3. OxdB in wet whole cells catalyzed dehydration of
n-octanaloxime (1) in biphasic systems.
#
1
2
3
4
Organic phase
Conversion to 2 /%a
Table 2. OxdB in dry whole cells catalyzed dehydration of n-octanaloxime in
pure organic medium.
Cyclohexane
9
Methyl-tert-butyl ether (MTBE)
Toluene
4
#
1
2
3
4
5
Reaction medium/solvent
Cyclohexane
Conversion to 2 /%a
3
<1
<1
<1
<1
23b
Dichloromethane (DCM)
<1
Methyl-tert-butyl ether (MTBE)
Toluene
a
33 mgwcm/mL OxdB in whole cells were suspended in 10 ml PPB and an
organic solvent including 500 mM n-octanaloxime (1) was added. The
reactions were performed at 30 °C for 24 and conversions to
n-octanenitrile (2) were determined by GC analysis.
Dichloromethane (DCM)
Potassium phosphate buffer (PPB)
As a concept to overcome these limitations, we envisioned that
encapsulating the whole cells in a solid and immobilized aqueous
phase could make such biotransformations possible, although
being conducted in a pure organic solvent as mobile phase. As
such an “immobilized aqueous phase” we used superabsorber,
which is basically polyacrylic acid having a very high affinity to
bind water. It was already shown, that this technique is useful for
a
7
.5 mgdcm/mL OxdB in whole cells were suspended in 20 ml organic solvent
or PPB including 500 mM n-octanaloxime (1). The reactions were performed
at 30 °C for 24 and conversions to n-octanenitrile (2) were determined by
GC analysis. Conversion was determined after extraction of the reaction
with ethyl acetate (EtOAc). The EtOAc phase was analysed by GC for
determination of conversion.
b
the immobilization of alcohol dehydrogenases as a crude extract
Since we found that the use of any type of whole cells in a pure
organic solvent does not lead to significant conversions, as a next
step we studied this type of biotransformation running in a
biphasic aqueous-organic media as this media has been utilized
often in enzyme catalysis.[4][5] In these experiments we used wet
whole cells containing OxdB as a biocatalyst at a concentration of
[22][28][37]
for ketone reduction.
Although such enzymes are known
to be tolerant to organic solvents, we became interested to
evaluate this methodology also for the entrapment of whole cells
bearing enzymes which are labile against organic solvents such
as the Oxd-enzyme used in this study. By means of such an
immobilized aqueous phase we expected that the contact
between the organic solvent and the Oxd-containing whole cells
is minimized, thus leading to a stabilizing effect on the whole cells
and enzymes therein, and to the opportunity to conduct
biotransformations with organic solvent-labile enzymes in a pure
organic reaction medium as liquid phase. Furthermore,
separation of the reaction mixture from the biocatalyst would be
simplified. The concept of this approach is visualized in Scheme
33 mgwcm/ml of total reaction medium (1:1(v/v) aqueous:organic,
total 20 mL). As aqueous phase, a phosphate buffer containing
the whole cells was used. The organic phase consisted of the
organic solvents cyclohexane, MTBE, toluene or DCM, containing
1
M of the substrate n-octanaloxime (1) as substrate (thus,
leading to an overall concentration of 500 mM). The reactions
were performed in a batch mode utilizing a round bottom flask with
magnetic stirrer at 30 °C for 24 h (Scheme 3).
4
.
Scheme 3. OxdB in whole cells catalyzed dehydration of n-octanaloxime (1) to
n-octanenitrile (2) in a biphasic system.
Scheme 4. OxdB in whole cells immobilized in superabsorber catalyzed
dehydration of n-octanaloxime (1) to n-octanenitrile (2) using a pure organic
solvent as reaction medium.
After a reaction time of 24h, the organic phases were analysed by
GC for the determination of the conversion of n-octanaloxime (1)
to n-octanenitrile (2) (Table 3). While MTBE, toluene and DCM
again gave very low conversions not exceeding 4% (Table 3,
entries 2-4), the use of cyclohexane led to 9% conversion of
n-octanaloxime (1) to the corresponding nitrile 2 (entry 1), which
at that time was the best result for this biotransformation in the
presence of an organic solvent. Furthermore, this experiment
illustrates that an aqueous phase is necessary to keep OxdB
In the resulting experiment, 33 mgwcm whole cells containing OxdB
per ml of total reaction medium (1:1(v/v) aqueous:organic, total
2
0 mL) were dissolved in 10 mL of aqueous buffer, and the
resulting aqueous phase was immobilized by addition of the
superabsorber under stirring in a round bottom flask. Then, 10 mL
organic phase containing 1 M n-octanaloxime (1) (0.5 M overall
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European Journal of Organic Chemistry
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COMMUNICATION
concentration) was added, and the reaction was performed at
3
0 °C for 24 h. We were pleased to find that after 24 h reaction
time with cyclohexane as organic solvent an excellent conversion
of >99% of n-octanaloxime (1) to n-octanenitrile (2) was achieved
(Table 4, entry 1). When utilizing toluene a conversion of 11% was
detected and in case of MTBE (5%) and DCM (<1%) lower
conversions were obtained (entries 2-4), thus indiciating a
significant impact on the type of organic solvent and the high
suitability of cyclohexane as organic reaction medium.
Table 4. OxdB in wet whole cells catalyzed dehydration of
n-octanaloxime (1) in the “superabsorber system”.
#
Organic phase
Conversion to 2 (isolated
yield) /%
a
1
2
3
4
Cyclohexane
>99 (82)
Methyl-tert-butyl ether (MTBE)
Toluene
5
Figure 3. Picture and time-course measurement of the “superabsorber-system”
with cyclohexane as organic phase.
11
<1
The time-course measurement indicates that the reaction further
proceeds even after 8 h reaction time. This result shows that the
enzymes within the whole cell-catalyst maintain their activity in the
superabsorber when using cyclohexane as organic solvent. In
order to get further insight into the stability of the OxdB biocatalyst
under the chosen reaction conditions, we performed a stability
assay of OxdB whole cells in superabsorber and in biphasic
reaction medium using cyclohexane as organic solvent. After
different incubation times of the OxdB(-immobilisate) in an
organic solvent or biphasic system, n-octanaloxime (1) was
added and the conversion to n-octanenitrile (2) was determined
after 15 min. The results are shown in Figure 4.
Dichloromethane (DCM)
a
3
3 mgwcm/mL OxdB in whole cells were suspended in 10 ml PPB and
immobilized in superabsorber. An organic solvent including 500 mM
n-octanaloxime (1) was added. The reactions were performed at 30 °C for
24 and conversions to n-octanenitrile (2) were determined by GC analysis.
These results showed that this “superabsorber approach” in
combination with the right choice of organic solvent component is
useful for the biocatalytic synthesis of nitriles in organic medium
although using an enzyme being labile against organic solvents.
We found cyclohexane to be the most suitable solvent for our
purpose. In comparison to the simple use of whole cells in pure
organic solvent, immobilization in superabsorber gave a much
more stabilized catalyst, thus leading to an excellent conversion.
This also indicates that the superabsorber stabilizes the whole
cells by protecting them from a direct contact with the solvent. The
positive impact of cyclohexane can be rationalized by its high logP
value, thus ensuring a low concentration in the aqueous super-
absorber phase as well as a negligible “dry out”-effect of removing
water from the superabsorber to the organic solvent. As we were
interested to explore the suitability of this method to operate at
higher substrate loading as a desired criteria for a technical
process, we investigated substrate concentrations of 500 mM,
which corresponds to a substrate loading of approximately 75 g/L
reaction medium. We were pleased to find that the biocatalyst
remains stable in the superabsorber system over several hours
and that quantitative conversions were obtained in the
biotransformation. The time-course of this reaction with the
superabsorber-immobilized whole cells using cyclohexane as
solvent as well as a photo of this reaction mixture is shown in
Figure 3.
Figure 4. Stability assay of OxdB whole cells immobilized in superabsorber in
cyclohexane and OxdB whole cells in a biphasic system consisting of buffer and
cyclohexane. The superabsorber-approach without previous incubation in
cyclohexane was set as 100%.
As shown in Figure 4, the activity of OxdB whole cells immobilized
in superabsorber does not decrease when being incubated for
one hour. After 5 hours of incubation in cyclohexane still
approximately 80% activity was left. In case of the biphasic
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European Journal of Organic Chemistry
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COMMUNICATION
reaction medium, OxdB showed already lower activity without
incubation beforehand. The activity is approximately 60% of the
activity of superabsorber-immobilized cells without incubation.
The activity drops very fast in case of the biphasic approach,
indicating again that the superabsorber-immobilization protects
the biocatalyst against the organic solvent.
100 mM in comparison to 500 mM in batch experiments in order
to ensure a completely dissolved substrate which is needed for
running the reaction in such a flow-reactor without blocking effects.
The reaction was performed at room temperature with a residence
time of 30 min. After an equilibration time of two residence times,
a high conversion of >95% for the formation of n-octanenitrile (2)
was obtained for at least 3 h reactor run time for this flow system
(Figure 6). Although within the chosen reaction time some loss in
activity was observed, thus underlining at least to a low extent a
deactivation effect of the organic solvent on the enzyme, this
superabsorber-based whole cells immobilization method in
combination with a pure organic solvent also turned out to be
robust and suitable to be operated in a flow mode.
Having such an efficient biotransformation in batch mode now in
hand and stimulated by the impressive recent achievements and
increasing importance of flow processes,[
38][39][40][41][42][43][44][45]
our
next goal was to develop a flow process for this superabsorber-
based synthesis of n-octanenitrile (2). Recently, biocatalysis in
continuous processes getting more and more popular due to high
reproducablity and productivity of these processes.[43] Thus, we
were interested in the behavior and performance of our Oxd-
superabsorber immobilizates in flow reactions. We chose a
packed bed reactor bearing the superabsorber-immobilized whole
cells, thus enabling an easy separation and re-use of the catalyst.
Toward this end, the reactor was loaded first with a small cotton
layer (5 mm) and then with the superabsorber granulate, followed
by addition of 1 mL suspension of 66 mgwcm/mL of enzyme. After
all liquid was absorbed by the superabsorber, a second cotton
layer (5 mm) was added and the reactor was sealed. In Figure 5
the set-up of this reactor is shown.
Conclusion
In conclusion, a biocatalytic process running in pure organic
medium has been reported in which an enzyme being labile
towards this organic solvent is used. The process concept is
based on the encapsulation of the enzyme as a whole cell catalyst
in a superabsorber as a “solid aqueous phase” in combination
with an organic solvent with a high logP value as “mobile phase”.
This approach has been exemplified for the aldoxime
dehydratase-catalyzed dehydration of n-octanaloxime (1) under
formation of n-octannitrile (2), leading to excellent conversion of
>
99%. In contrast, very low or no conversion were observed when
using other solvents or other methods such as the direct use of
whole cell catalysts in pure organic medium or biphasic solvent
mixtures. Furthermore, an efficient flow-setup with a fixed-bed
reactor of superabsorber-immoblized whole cells was developed,
which turned out to be also very suitable for the biotransformation
leading to high conversion and low catalyst deactivation.
Figure 5. Picture of the packed bed reactor charged with superabsorber gel.
Acknowledgements
We gratefully acknowledge generous support from the
Fachagentur Nachwachsende Rohstoffe (FNR) and the German
Federal Ministry of Food and Agriculture (BMEL), respectively,
within the funding program on the utilization of biorenewables
(
Grant No. 22001716) as well as from the German Federal
Ministry of Education and Research (BMBF) within the project
Biotechnologie 2020+, Nächste Generation biotechnologischer
„
Verfahren“ (grant number 031A184A). We also thank Charlotte
Mann for technical assistance.
Keywords: Aldoximes • enzyme catalysis • flow chemistry •
immobilization • nitriles
Figure 6. OxdB in wet whole cells catalyzed dehydration of n-octanaloxime (1)
using a superabsorber packed bed approach.
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This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.201901168
COMMUNICATION
Entry for the Table of Contents
Key topic: Enzyme Catalysis
COMMUNICATION
Alessa Hinzmann, Niklas Adebar,
Tobias Betke, Monja Jochmann and
Harald Gröger*
Page No. – Page No.
Biotransformations in pure organic
medium with organic solvent-labile
enzymes in batch and flow
Text for Table of Contents
exemplified for nitrile synthesis
Text for Table of Content
A biocatalytic process in pure organic medium is presented in which an enzyme being labile towards this organic solvent is used. The
process concept is based on encapsulation of the enzyme as a whole cell catalyst in a superabsorber as a “solid aqueous phase” in
combination with an organic solvent as “mobile phase”. This approach has been exemplified for the enzymatic dehydration of n-
octanaloxime, leading to n-octannitrile with excellent conversion of >99%.
This article is protected by copyright. All rights reserved.