Performance of Recombinant-Whole-Cell-Catalyzed Reductions in Deep-Eutectic-Solvent-Aqueous-Media Mixtures

Article abstract of DOI:10.1002/cctc.201500428

Deep-eutectic solvents (DES) are cost-effective, nonhazardous solvents that may be used in biocatalysis as nonconventional media that enable biotransformations with industrially sound high substrate loadings. Based on promising prognoses, this paper explo

Full text of DOI:10.1002/cctc.201500428

DOI: 10.1002/cctc.201500428
Full Papers
Performance of Recombinant-Whole-Cell-Catalyzed
Reductions in Deep-Eutectic-Solvent–Aqueous-Media
Mixtures
Christoph R. Müller,[a, b] Ivµn Lavandera,*[b] Vicente Gotor-Fernµndez,*[b] and
Pablo Domínguez de María*[a, c]
Deep-eutectic solvents (DES) are cost-effective, nonhazardous
solvents that may be used in biocatalysis as nonconventional
media that enable biotransformations with industrially sound
high substrate loadings. Based on promising prognoses, this
paper explores successfully the use of whole cells overexpress-
ing oxidoreductases as biocatalysts for the stereoselective re-
duction of ketones in different DES–aqueous-media solutions.
Enzymes like Ralstonia sp. alcohol dehydrogenase (ADH) and
horse liver ADH, overexpressed in Escherichia coli, remain
active at different DES proportions (for example, DES:buffer,
80:20 v/v) to enable nonconventional biotransformations. Fur-
thermore, the enantiomeric excesses obtained for a broad
range of aromatic substrates increase significantly if DES is
added, which provides a useful tool for combining high sub-
strate loadings and improved enzymatic selectivities while
working with nonhazardous solvents. Different DES are suc-
cessfully employed, which gives hints of the potential that
these emerging solvents may have.
Introduction
The use of oxidoreductases to afford optically active building
blocks is currently a mature technology, with applications both
at academic and industrial scale. Key aspects of those process-
es, such as the need for high substrate loadings, efficient co-
factor-regeneration strategies, and the establishment of effec-
tive overexpression systems, have been tackled adequately
over the last few years.[1] In this respect, the setup of highly ef-
ficient and creative cofactor-regeneration systems has been
sorted out with outstanding results so far, with systems com-
prising both free, isolated enzymes and whole cells,[2] as well
as natural and non-natural cofactors.[3] Likewise, different con-
cepts related to medium engineering, for example, the use of
organic cosolvents or biphasic systems to enhance substrate
loadings, have been assessed in depth as well. In this area,
apart from more classic aqueous-based systems, several re-
search groups have shown that oxidoreductases (lyophilized
whole cells overexpressing alcohol dehydrogenases) can ac-
tually catalyze stereoselective processes in water-free systems
(that is, solvent free or in organic solvents), which thus enables
biocatalytic oxidoreductions in nonconventional media and
opens new possibilities for these enzymes.[2–4] Last but not
least, the identification of novel and useful catalytic performan-
ces for these biocatalysts (for example, imine reductases[5]) pro-
vides even stronger potential for future sustainable solutions
in enzyme catalysis.
From the perspective of neoteric, nonhazardous, and envi-
ronmentally friendly solvents, deep-eutectic solvents (DES)
have appeared recently as a promising alternative for many
chemical applications.[6] DES are typically formed by the combi-
nation of a quaternary ammonium salt (for example, choline
chloride) and hydrogen-bond donor molecules (HBDs), such as
polyols, carboxylic acids, amines, etc. The formation of a deep-
eutectic mixture is caused by disruption of the crystalline struc-
tures of the individual components, which lowers the lattice
energy and leads to liquids at room temperature.[6,7] Among
the emerging DES applications, the use as solvents for biocata-
lytic reactions has been envisaged,[8] and examples covering
hydrolases,[9] lyases,[10] and oxidoreductases[11] have been suc-
cessfully reported over the last few years. Depending on the
biocatalyst type, different amounts of water are often needed
to maintain the enzymes in their optimal active form. In any
case, useful DES–aqueous-media mixtures, for example, DES
containing 10 vol% water, can be applied, which leads to
rather water-saturated neoteric solvents with a (still) large dis-
solving capacity for industrially sound high substrate loadings.
On the basis that DES are nonhazardous, biodegradable, and
cost effective, the emerging strategies are promising for future
practical applications in biocatalysis.
[a] C. R. Müller, P. Domínguez de María
Institut für Technische und Makromolekulare Chemie (ITMC)
RWTH Aachen University
Worringerweg 1, 52074 Aachen (Germany)
E-mail: dominguez@itmc.rwth-aachen.de
[b] C. R. Müller, I. Lavandera, V. Gotor-Fernµndez
Departamento de Química Orgµnica e Inorgµnica
Instituto Universitario de Biotecnología de Asturias
Avenida Juliµn Clavería 8, 33006 Oviedo (Spain)
E-mail: lavanderaivan@uniovi.es
vicgotfer@uniovi.es
[c] P. Domínguez de María
Sustainable Momentum SL.
Ap. Correos 3517
35004 Las Palmas de Gran Canaria, Canary Islands (Spain)
E-mail: dominguez@sustainable-momentum.net
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201500428.
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With regard to oxidoreductases, reported examples
in DES have focused on wild-type whole cells (baker’s
yeast, Acetobacter sp.). DES exert a beneficial effect
on whole-cell biocatalysis, with significant improve-
ments in stereoselectivity, for example, if using
baker’s yeast.[11c] With a wild-type cell containing sev-
eral oxidoreductases, most of them overexpressed at
analogous low levels,[1a] the observed improvements
were attributed to the inhibition of some of these en-
zymes by DES, whereas others would remain fully
active in such DES–aqueous-media mixtures.
For application-driven biocatalysis, the use of re-
combinant overexpressing whole cells appears as
a powerful option, because the desired enzyme is
then present at high loadings within the cell. By
means of these highly optimized biocatalysts, high
productivities have been reported in asymmetric syn-
Figure 1. Redox performance in DES–aqueous-media mixtures of TeSADH (with 2-octa-
none), HLADH (with benzaldehyde), and RasADH (with propiophenone). Reaction condi-
tions: enzyme (3 U), alcohol (30 mL, 0.39 mmol of propan-2-ol, 0.51 mmol of ethanol in
the case of HLADH), carbonyl compound (3 mL, 0.02–0.03 mmol), 50 mm phosphate
buffer (pH 7.5, 60 mL) with 10 mm cofactor, ChCl/glycerol DES (1:2)– 50 mm phosphate
buffer at pH 7.5 (540 mL), t=24 h, T=308C, total volume: 630 mL.
thesis, even at the industrial scale, by using microaqueous sol-
utions, solvent-free processes, and biphasic systems to en-
hance the substrate loadings.[4,12]
cant observed difference of activities in DES–buffer media de-
pending on the overexpressed enzyme provides support for
the whole cells maintaining their integrity in the DES.[16,17] Ex-
periments performed with free enzymes led actually to very
low conversions (for example, 12% vs. 90% in the case of
HLADH), presumably as a result of deactivation of the biocata-
lyst by the DES outside the whole cell. Therefore, cell integrity
secures the stability of the enzyme to conduct practical biocat-
alysis. Actually, preliminary experiments focusing on whole-cell
recycling led to positive results, with a remaining activity of
70% after three reaction cycles (see Table S5 in the Supporting
Information).
With consideration of that point, and the potential impor-
tance that DES may have for future (bio)catalytic processes,
the use of recombinant whole cells overexpressing oxidoreduc-
tases in DES–aqueous-media mixtures is assessed in this work
for the first time.
Results and Discussion
As the prototypical DES for the oxidoreductase-catalyzed pro-
cesses, a mixture of choline chloride and glycerol (1:2 mol/mol)
was chosen,[7a] because such a system has been shown as ben-
eficial for other biocatalytic processes, including (wild-type)
whole-cell biocatalysis.[8–11] In a first set of experiments, differ-
ent ADHs overexpressed in Escherichia coli were screened with
2-octanone, benzaldehyde, or propiophenone as the sub-
strates, in combination with alcohols as ancillary substrates to
regenerate the cofactor (ethanol for benzaldehyde and
propan-2-ol for the ketones). From that preliminary screening,
three ADHs displayed outstanding activities at different DES–
buffer proportions, namely the ADH from Thermoanaerobacter
ethanolicus (TeSADH),[13] horse liver ADH (HLADH),[14] and Ralsto-
nia sp. ADH (RasADH).[15] The results are depicted in Figure 1.
As can be observed, the three whole-cell overexpressing
ADH systems displayed high biocatalytic reduction conversions
at different DES–aqueous-buffer proportions. Remarkably,
RasADH remained virtually fully active at 60–70 vol% of the
DES and almost half of the activity was still observed even at
95 vol% of the DES. At very high proportions, the conversions
dropped drastically, which suggests the deactivation of the
biocatalyst by high concentrations of DES together with low
water quantities. Thus, in addition to other wild-type whole
cells reported in the literature,[11] recombinant cells overex-
pressing ADHs may also be used in these DES–buffer neoteric
mixtures, which can certainly broaden the applicability of
these systems in nonhazardous unconventional media. The
other two ADHs (TeSADH and HLADH) displayed high-to-mod-
erate activities at different DES–buffer proportions. The signifi-
As a result of its outstanding performance, overexpressed
RasADH was chosen as the biocatalyst for further reactions in
DES–buffer media. In a subsequent set of different experi-
ments, the ancillary cosubstrates (ethanol, propan-1-ol, and
propan-2-ol) were assessed in the DES–aqueous-media mix-
tures, with propiophenone as the model substrate for RasADH.
In this case, not only activities but also stereoselectivities were
evaluated at different DES–buffer proportions. The results are
depicted in Figure 2.
With regard to conversions (Figure 2, middle), they varied
significantly at different DES–buffer proportions, but they also
depended on the ancillary cosubstrate. Thus, ethanol led to
the lowest conversions, irrespective of the amount of DES: an
effect that must be clearly related to the selectivity of RasADH
for this substrate and not to the nonconventional media ap-
plied. On the other hand, both propan-1-ol and propan-2-ol
led to similar results, which showed again the stability of
RasADH in different DES–buffer media.
Quite remarkably, a more interesting trend was obtained
from the stereoselectivity point of view (Figure 2, bottom).
Thus, a significant improvement of the enantiomeric excess
was observed at higher DES proportions. In the cases of
propan-1-ol and propan-2-ol as ancillary substrates, excellent
ee values of >90% were achieved, from a starting point of vir-
tually racemic production in the absence of DES for that
enzyme and substrate (Figure 2, bottom). The observed im-
provement of the stereoselectivity is consistent with previous
cases with wild-type whole cells.[11]
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tivity improvement was observed significantly in all
tested substrates (Figure 3). The effect was particular-
ly clear in bulky substrates, because the enantiomeric
excess was improved from low optical purities in
pure buffer to moderate-to-high values (ee values of
60–90%; enantiomeric ratios of 80:20 to 95:5) at
larger DES proportions. Hence, the combination
of inexpensive and nonhazardous nonconventional
media, such as DES, to dissolve bulky substrates, to-
gether with protein engineering to further improve
enzyme performances, may become a powerful tool
for future practical biocatalysis.
Triggered by the outstanding observed results in
terms of stereoselectivity improvement with DES, fur-
ther experiments were conducted by using different
choline chloride based DES with different HBDs,
namely glycerol (at 1.5 and 2 equiv), urea (2 equiv),
and ethylene glycol (2 equiv). The results comprising
conversions and stereoselectivities are summarized in
Figure 4.
As can be observed, the improvement in the ste-
reoselectivity was general for all DES mixtures as-
sayed, and excellent stereopreferences were reached
at DES proportions of 90 vol%, albeit often at lower
conversions as a result of enzyme deactivation. As
a compromise, DES:buffer 80:20 (v/v) appeared to be
optimal for these processes from a practical perspec-
tive. Another aspect to be considered was whether
the observed improvement was due to the DES as
a structure or as separate components dissociated in
the aqueous media. Recent literature has shown that
the DES mixtures would retain their structure in
aqueous solutions.[19] Herein, experiments with low
proportions of glycerol or choline chloride (10 vol%)
did not lead to significant improvements in the enzy-
matic performance. Aspects such as the intrinsic glyc-
erol viscosity or the low solubility of choline chloride
in buffer must be considered as well. In this respect,
the synergistic combination of both components in
the DES is the proper way to use them in biocataly-
sis, because low viscosities, high substrate solubilities,
and enhanced enzymatic activities may be reached.
To further investigate the effect of DES in the ste-
reoselectivity improvements, we conducted experi-
ments with hexane as the cosolvent and with “cosol-
vent-free” solutions (that is, a more concentrated
buffer system, because the remnant cosolvent
Figure 2. Performance of RasADH for the bioreduction of propiohenone in DES–aque-
ous-media mixtures with different ancillary substrates to regenerate the cofactor. Top: re-
action scheme; middle: enzyme activities; bottom: stereoselectivities. Reaction condi-
tions: E. coli/RasADH (3 U), alcohol (30 mL, 0.39 mmol of propan-1-ol and propan-2-ol,
0.51 mmol of ethanol), propiophenone (3 mL, 0.02 mmol), 50 mm phosphate buffer
(pH 7.5, 60 mL) with 10 mm nicotinamide adenine dinucleotide phosphate (NADP+),
ChCl/glycerol DES (1:2)–50 mm phosphate buffer at pH 7.5 (540 mL), t=24 h, T=308C,
total volume: 630 mL.
Stimulated by these results, we evaluated the substrate
scope of RasADH in DES–buffer mixtures. RasADH is a recently
explored biocatalyst with a promising substrate range, espe-
cially toward hindered ketones, which makes it an attractive
biocatalyst for practical applications.[4b,15,18] In this case, differ-
ent more-or-less sterically hindered aryl ketones were assessed
at several DES–buffer proportions. The results are depicted in
Figure 3.
volume was not fulfilled), and the stereoselectivity explored.
The results are depicted in Figure 5. For comparison, the re-
sults of ChCl:Gly (1:2) are included.
Interestingly, both the use of a lower-volume batch and
hexane as the cosolvent led to improvements in the stereose-
lectivity. In the case with no additive, an increase in the sub-
strate concentration may lead (kinetically) to better stereose-
lectivities. Although the effect of the DES on stereoselectivity
was much pronounced than that with hexane, the observed
results suggested that the improvement in stereoselectivity
The observed enzymatic conversions remained high in the
overall DES–buffer range (not shown). Gratifyingly, stereoselec-
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Experimental Section
Chemicals and biocatalysts
All chemicals were purchased from Sigma–Aldrich and
used without further purification. With regard to biocata-
lysts, E. coli strains to overexpress the RasADH from Ral-
stonia sp. and TeSADH from Thermoaerobacter ethanoli-
cus were kindly provided by Prof. Wolfgang Kroutil (Uni-
versity of Graz). The E. coli strain overexpressing horse
liver ADH was provided by Prof. Martina Pohl (For-
schungszentrum Jülich GmbH). The activity of the over-
expressed biocatalysts is approximately 0.3 UmgÀ1 for
propiophenone (RasADH), 2-octanone (TeSADH), and
benzaldehyde (HLADH).
DES formation
The components were mixed directly together and
stirred at T=608C until a clear solution was obtained
(t=15–60 min). After cooling down to room tempera-
ture, the formed DES could be used directly.
Standard reaction with oxidoreductases and DES
For HLADH: E. coli/HLADH (15 mg) was added into an
Eppendorf vial and dissolved in 50 mm phosphate buffer
(pH 7.5, 420 mL) and a solution containing 50 mm phos-
phate buffer (pH 7.5, 60 mL) and 10 mm nicotinamide ad-
enine dinucleotide. A ChCl/glycerol DES mixture (1:2,
120 mL) was then added to the mixture, along with ben-
zaldehyde (3 mL) and ethanol (30 mL). The reaction mix-
ture was shaken (300 rpm) for 24 h at T=308C. The
aqueous phase was extracted with ethyl acetate (2”500 mL). After
separation and drying over sodium sulfate, the organic layer was
analyzed by GC (HP-1 column).
Figure 3. Stereoselectivity observed in the aryl substrate scope of RasADH at different
DES–buffer proportions. Reaction conditions: E. coli/RasADH (3 U), propan-2-ol (30 mL,
0.39 mmol), ketone (3 mL, 0.02–0.03 mmol), 50 mm phosphate buffer (pH 7.5, 60 mL) with
10 mm NADP+, ChCl/glycerol DES (1:2)–50 mm phosphate buffer at pH 7.5 (540 mL),
t=24 h, T=308C, total volume: 630 mL.
may be related to internal interactions of the cosolvent (either
hexane or DES compositions) within the enzyme active site.
Analogous results have been recently observed and modeled
for thiamine-dependent lyases and cosolvents,[20] as well as
with ionic liquids added as performance additives.[8a] In any
case, the use of nonhazardous DES offers not only better out-
comes (higher stereoselectivities) but also more environmen-
tally friendly conditions for biocatalysis with recombinant
whole cells.
For RasADH: E. coli/RasADH (15 mg) was added into an Eppendorf
vial and dissolved in 50 mm phosphate buffer (pH 7.5, 420 mL) and
a solution containing 50 mm phosphate buffer (pH 7.5, 60 mL) and
NADP+ (10 mm). A ChCl/glycerol DES mixture (1:2, 120 mL) was
then added to the mixture, along with propiophenone (3 mL) and
propan-2-ol (30 mL). The reaction mixture was shaken (300 rpm) for
24 h at T=308C. The aqueous phase was extracted with ethyl ace-
tate (2”500 mL). After separation and drying over sodium sulfate,
the organic layer was transferred to another Eppendorf vial for de-
rivatization. 4-Dimethylaminopyridine (1.0 mg, 8.2 mmol) and acetic
anhydride (30 mL, 0.32 mmol) were added, and the vial was shaken
for 1 h at T=408C. The reaction was quenched by the addition of
water (500 mL). After extraction of the aqueous phase with ethyl
acetate (2”500 mL), the combined organic layers were dried over
sodium sulfate and filtered. The product solution was analyzed by
GC (Agilent CP-ChiraSil-Dex-CB column).
Conclusions
Herein, the use of whole cells overexpressing oxidoreductases
as biocatalysts in deep-eutectic-solvents (DES)–aqueous media
has been reported for the first time. Different alcohol dehydro-
genases have been proven to remain active at high DES con-
centrations (for instance, DES:buffer 80:20 v/v, with a choline
chloride and glycerol DES mixture), and furthermore, signifi-
cant improvements in the enantiomeric excesses of the alcohol
products have been observed for a broad range of aromatic Acknowledgements
substrates. The results demonstrate that it is possible to
employ these enzyme-friendly and nonhazardous neoteric sol-
vents, with the ability to dissolve high substrate loadings, with
recombinant whole cells. The ease of DES production and the
biodegradability of the solvents are further promising features
for their practical implementation in biocatalyzed processes.
Financial support from DFG training group 1166
“BioNoCo” (“Biocatalysis in Non-conventional Media”) is gratefully
acknowledged. Financial support from MINECO (Project
CTQ2013-44153-P) is also gratefully acknowledged. We thank
Prof. Martina Pohl (Forschungszentrum Jülich GmbH) for the gen-
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erous donation of HLADH plasmids and Prof. Wolfgang
Kroutil (University of Graz) for the generous donation
of RasADH and TeSADH plasmids.
Keywords: biocatalysis · deep-eutectic solvents
·
dehydrogenation
engineering
·
oxidoreductases
·
solvent
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Received: April 18, 2015
Revised: June 8, 2015
Published online on July 31, 2015
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