Responsive Emulsions for Sequential Multienzyme Cascades

Article abstract of DOI:10.1002/anie.202013737

Multienzyme cascade biocatalysis is an efficient synthetic process, avoiding the isolation/purification of intermediates and shifting the reaction equilibrium to the product side. However, multienzyme systems are often limited by their incompatibility and cross-reactivity. Herein, we report a multi-responsive emulsion to proceed multienzyme reactions sequentially for high reactivity. The emulsion is achieved using a CO₂, pH, and thermo-responsive block copolymer as a stabilizer, allowing the on-demand control of emulsion morphology and phase composition. Applying this system to a three-step cascade reaction enables the individual optimal condition for each enzyme, and a high overall conversion (ca. 97 % of the calculated limit) is thereby obtained. Moreover, the multi-responsiveness of the emulsion allows the facile and separate yielding/recycling of products, polymers and active enzymes. Besides, the system could be scaled up with a good yield.

Full text of DOI:10.1002/anie.202013737

Angewandte
Communications
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How to cite: Angew. Chem. Int. Ed. 2021, 60, 8410–8414
International Edition:
German Edition:
doi.org/10.1002/anie.202013737
doi.org/10.1002/ange.202013737
Biocatalysis Hot Paper
Responsive Emulsions for Sequential Multienzyme Cascades
+
+
Zhiyong Sun , Qingcai Zhao , Rainer Haag,* and Changzhu Wu*
Abstract: Multienzyme cascade biocatalysis is an efficient
synthetic process, avoiding the isolation/purification of inter-
mediates and shifting the reaction equilibrium to the product
side.. However, multienzyme systems are often limited by their
incompatibility and cross-reactivity. Herein, we report a multi-
responsive emulsion to proceed multienzyme reactions sequen-
tially for high reactivity. The emulsion is achieved using a CO2,
pH, and thermo-responsive block copolymer as a stabilizer,
allowing the on-demand control of emulsion morphology and
phase composition. Applying this system to a three-step
cascade reaction enables the individual optimal condition for
each enzyme, and a high overall conversion (ca. 97% of the
calculated limit) is thereby obtained. Moreover, the multi-
responsiveness of the emulsion allows the facile and separate
yielding/recycling of products, polymers and active enzymes.
Besides, the system could be scaled up with a good yield.
However, operating multiple enzymes in abiological
conditions often suffers from some intractable limitations.
The first limitation, namely, incompatibility issue, stems from
the fact that different enzymes often have different optimal
reaction conditions, e.g., pH, temperature and co-factors, and
[
6]
therefore cannot be simply combined in one system. The
second issue is the cross-reactivity, which occurs when one
enzyme accepts not only its substrates but also the inter-
mediates/products in the whole cascade route, eventually
[
7]
leading to undesired side reactions.
An elegant solution to the above issues is to compart-
mentalize multienzyme reactions in spatially different
[8]
domains of one system. Emulsions, the dispersion of one
liquid phase within a second immiscible liquid, seem to be
[
9]
a good choice, because they provide not only a large
interfacial area for fast reactions but also heterogeneous
phases to host different reactions. These benefits, especially
their large interface, have promoted rapidly the field of
“emulsion biocatalysis” to address a wide range of synthetic
E
nzymes, the biologic catalysts from nature, have achieved
enormous success in modern chemical and pharmaceutical
[
1]
[10]
[11]
industries. In particular, they are increasingly explored for
challenges to date. For instance, microemulsions, mini-
[
12]
[13]
single-step reactions including oxidation, reduction, and
emulsions, and Pickering emulsions were employed to
encapsulate enzymes for the single-step enantioselective
reduction, hydrolysis, and esterification. Recently, we further
advanced the field by developing multi-compartmentalized
emulsion biocatalysis for benzoin condensation and carbonyl
[
2]
isomerization.
However, in nature, cells do not just
employ single reactions alone but combine them for essential
[
3]
metabolic pathways, e.g., tricarboxylic acid cycle. This
natural design has initiated great efforts in bioinspired
[
4]
[14]
multienzyme reactions, e.g., one-pot cascades, to avoid the
isolation of intermediates and overcome unfavorable reaction
reduction. In principle, polymeric emulsions are attractive
because polymers are easily controlled for desirable applica-
tion properties in addition to good recyclability. An example
of such controllability is that polymeric emulsions can be
[5]
equilibrium, significantly saving time and production costs.
[
15]
made responsive to diverse external stimuli, e.g., pH,
[
+]
[16]
[17]
[18]
[19]
[
*] Dr. Z. Sun, Prof. Dr. C. Wu
temperature,
CO2,
magnetic field,
and light.
This
Department of Physics, Chemistry and Pharmacy
University of Southern Denmark
Campusvej 55, 5230 Odense (Denmark)
E-mail: wu@sdu.dk
stimuli responsiveness allowed the controllable emulsification
and demulsification, which were successfully explored for oil
recovery and chemocatalysis. However, until now, stim-
[20]
[21]
uli-responsive emulsions for biocatalysis have been relatively
[
+]
Dr. Q. Zhao, Prof. Dr. R. Haag
Institute of Chemistry and Biochemistry
Freie Universitꢀt Berlin
[13c,22]
underdeveloped,
and to the best of our knowledge, they
have never been explored for multienzyme cascades despite
their potential to improve reaction compatibility and reduce
cross-reactivity.
Takustr. 3, 14195 Berlin (Germany)
E-mail: haag@chemie.fu-berlin.de
Herein, we present a scalable multi-responsive emulsion
as controllable liquid scaffolds to proceed efficient multi-
Prof. Dr. C. Wu
Danish Institute for Advanced Study
University of Southern Denmark
Campusvej 55, 5230 Odense (Denmark)
enzyme cascades. A pH-, CO -, and thermo-responsive
2
polymeric emulsion is obtained by utilizing a diblock copo-
lymer, poly(N-[2-(dibutylamino)ethyl]acrylamide)-b-poly(N-
isopropylacrylamide) (PDBAEAM-b-PNIPAm), as the sta-
bilizer. This multi-responsiveness allows not only switching
the biocatalysis on/off by on-demand emulsification/demulsi-
fication, but also the control of phase composition in the
demulsified status (Scheme 1). As a result, the exchange of
enzymes and a facile yet high conversion of biocatalysis can
be achieved by applying different stimuli. Furthermore,
+
[
] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202013737.
ꢁ
2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution Non-Commercial
License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used
for commercial purposes.
[
23]
utilizing this system in a sequential approach,
each
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Figure 1. a) An organic phase containing PDBAEAM -b-PNIPAm was
3
9
22
added to an aqueous phase (upper), and the emulsion was obtained
by handshaking (lower). b) Confocal laser scanning microscopy
(CLSM) image of the emulsion, aqueous phase was in green and
organic phase was in red. c, d) Emulsion’s stability at different pH and
temperatures. e) Responsiveness of emulsion to temperature change
and CO /N .
2
2
Scheme 1. Multi-responsive emulsion is constructed by a responsive
polymer, PDBAEAM-b-PNIPAm, for controllable multienzyme cas-
cades: Increasing temperature switches polymers from amphiphilic to
hydrophobic, leading to the demulsification with polymers localized in
the organic phase, which allows the exchange of enzyme solution for
next biocatalysis. On the other hand, demulsification by lower pH or
than 6. In lower pH conditions, the whole diblock copolymer
became hydrophilic and therefore cannot work as an emulsi-
fier. The thermal stability of the emulsion was investigated at
the temperature from 20 to 408C (Figure 1d). When the
temperature was lower than 258C, the emulsion was stable for
more than 12 h (Figure S2). At 308C, slight phase separation
was observed over 12 h. At 358C, the emulsion was stable for
about 3 hours, while at 408C the emulsification was unsuc-
cessful, which was due to the change of PNIPAm block from
hydrophilic to hydrophobic over LCST.
using CO results in a hydrophilic polymer and an organic phase
2
without the polymer, which facilitates the product purification.
biocatalytic step undergoes under its optimal condition, and
therefore, the performance of the multienzyme reactions can
be improved without incompatibility and cross-reactivity
issues.
To design the multi-responsive block copolymer, we opted
to use PNIPAm and PDBAEAM as the temperature- and pH/
To investigate the responsiveness of the emulsion, tem-
perature change and CO /N treatment were applied, respec-
2
2
tively (Figure 1e). Demulsification could be achieved by two
CO -responsive segments because of their biocompatibility to
methods via, for example, keeping the emulsion at 408C or
2
[24]
enzymes and orthogonal responsiveness in different stimuli
bubbling CO to the emulsion for 10 minutes. Although in
2
[
25]
state.
Moreover, due to their characteristic molecular
both cases demulsification was achieved, the location of the
polymer was different, which was confirmed by the yellowish
color of the toluene or water phase, respectively. After the
demulsification, the system could not be emulsified anymore
by shaking, thus suggesting the change of the block copolymer
amphiphilicity. However, after cooling down the system to
structures, the hydrophilic PNIPAm and hydrophobic
PDBAEAM can be respectively switched to the hydrophobic
and hydrophilic segment in response to environmental
[26]
changes, thus allowing the precise control of the hydro-
phobicity of the block copolymer for multi-responsive
emulsions. The block copolymer was synthesized by rever-
sible addition-fragmentation chain-transfer polymerization
room temperature or bubbling N , the emulsion could be
2
obtained again by handshaking. The reversibility of emulsi-
fication/demulsification was demonstrated by repeating the
heating/cooling and CO /N cycles. Besides CO and temper-
(
see ESI), and the ratio of PDBAEAM/PNIPAm was
[
27]
controlled as 3:1 to stabilize a water-in-oil (W/O) emulsion.
2
2
2
As a result, PDBAEAM -b-PNIPAm was synthesized.
ature, the emulsion was also responsive to pH changing
(Figure S3).
3
9
22
The W/O emulsion, stabilized by the amphiphilic polymer,
can be prepared by the facile mix of water and an organic
solvent, such as toluene or dichloromethane (Figure S1).
Three different enzymes, Candida antarctica Lipase B
[
28]
(CalB), thermophilic alcohol dehydrogenase (ADH-ht),
À1
[29]
Typically, a 500 mL organic phase containing 40 mgmL
and benzaldehyde lyase (BAL), were obtained (see ESI)
to study their catalytic performance in a 1 mL emulsion,
where 2.5 mgmL enzymes and 200 mM substrates resided in
PDBAEAM -b-PNIPAm was added to a 500 mL water
3
9
22
À1
phase, which is followed by handshaking to form a water/oil
emulsion with good stability (Figures 1a,b). The emulsifica-
tion was tested under different pH conditions (Figure 1c), and
the results suggested that the emulsion could be successfully
prepared when the pH value of the aqueous phase was higher
aqueous and organic phases, respectively. Three reactions,
hydrolysis of acetic acid benzyl ester (AABE), oxidation of
benzyl alcohol (BeOH), and benzoin condensation, were
performed individually under their optimal pH conditions in
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the emulsion (Figures S5–7), using a biphasic system as
a control. Product concentration was determined by GC
compared. In the case of demulsification by CO (Figure 2c),
2
the activity of both CalB and ADH-ht had an only slight
decrease, while BAL lost 30% and 50% activity after the first
and second round and finally retained 45% after 20 cycles.
[30]
measurements using an external standard method.
Fig-
ure 2a showed that our emulsion system largely improved the
catalytic efficiency of all reactions compared to the biphasic
control due to its large interfacial area. Furthermore, the
responsive emulsion contributed to facilitated purification.
Most traditional emulsifiers end up in final product, bringing
This was because the introduction of CO lowered the pH
2
value of the system, which demoted BAL performance. On
the other hand, when using temperature change to run the
cycles (Figure S8), the activity of CalB and BAL decreased
gradually after each circle, and finally reached 70% and 60%
after 20 cycles, respectively. In comparison, as a thermophilic
enzyme, ADH-ht was almost not influenced in the first
15 cycles and eventually retained more than 80% activity.
These results suggested that the multi-responsiveness of our
emulsion not only allowed for a facile recycling of products
but also provided more opportunities for enzyme reuse under
their favourable conditions.
[
31]
additional difficulty in product purification. Even though
some polymeric stabilizers can be precipitated from emul-
[
14a]
sions,
it is usually at cost of using a large amount of
antisolvents. Here, we showed the demulsification by CO2
treatment, the obtained colorless organic phase (Figure S4)
contained no polymers and was sent directly for the follow-up
workup. Besides, the facile separation contributed to a higher
conversion than the direct precipitation process (Figure 2a)
because precipitation could lead to a small amount of product
embedded in the polymer sediments. The demulsification,
however, offered clear phase separation and the entire
product was in the organic phase and not wasted. Therefore,
the responsive emulsion provided an easy, economical (less
solvent needed) method with the high bioconversion. The
final conversion of the biocatalytic reactions was about 95%
The accomplishment of single enzyme reactions encour-
aged us to further investigate their cascade reaction (Fig-
ure 3a). To this end, initially, we were curious about whether
our responsive emulsion would be useful for avoiding multi-
enzyme cross-reactivity. Interestingly, no cross-reactivity
could be observed in the one-pot cascade reaction (Fig-
ure S15), which may be either due to the substrate specificity
[28a,32]
(
(
hydrolysis by CalB), 80% (oxidation by ADH-ht), and 40%
benzoin condensation by BAL), respectively.
of enzymes
or because of side-products below our
detection limit. However, one big challenge of this cascade
was that the optimal condition for every step differed largely
from each other (Figures S16–18). For example, CalB and
ADH-ht preferred higher temperatures, while BAL was only
stable at the temperature from 20 to 308C (Figure S18).
Considering that the polymeric emulsion was not stable at
Next, the influence of demulsification on enzymes and
their reusability was investigated. Circular dichroism (CD)
spectrum (Figure 2b) showed negligible changes in enzyme
secondary structure after demulsification by both CO treat-
2
ment and heating. This positive result encouraged us to
perform 20 cycles of emulsification/demulsification, in which
the enzyme activity in emulsion and demulsified status was
Figure 2. Single enzymatic reactions in the 1 mL multi-responsive
emulsion. a) Detected conversion of hydrolysis of acetic acid benzyl
ester (AABE), oxidation of benzyl alcohol (BeOH) and benzoin
condensation in the biphasic system, emulsion system and after
Figure 3. a) Reaction Scheme of the cascade reaction. b) Time course
of the concentration of AABE, BeOH, benzaldehyde (BA), and benzoin
in a one-pot cascade at pH 8, 308C. c) Time course of the concen-
tration of the four compounds in the sequential cascade. d) Overall
conversion of compounds in long-term pH 6 and pH 8 one-pot
cascade, and sequential cascade. e) 800 mL reaction experiment and
yielded product.
demulsification by CO . b) CD spectrum of native CalB, CalB recycled
2
from the emulsion system by temperature and CO . c) Relative activity
2
of CalB, ADH-ht, and BAL in emulsification/demulsification cycles by
N and CO . In each cycle, the catalytic performance was investigated
2
2
in both emulsion and demulsified state.
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high temperature, 308C was, therefore, set for the one-pot
sion allows for not only the encapsulation of three active
cascade reaction in further studies. In addition to the temper-
ature, pH also had a great influence on all enzymatic
performance (Figure S19). The three-step cascade reaction
was carried out under both pH 6 and 8 for 24 hours. In the
pH 6 experiment (Figure S20), the first step of hydrolysis by
CalB was fast and almost fully completed. However, due to
the low activity of both ADH-ht and BAL under an acidic
condition, the production of benzaldehyde (25 mM) and
benzoin was largely limited (4.6 mM, overall conversion 9%).
In comparison, in the case of one-pot cascade at pH 8
enzymes but also a facile product purification via stimuli-
responsive phase separation. This finding is significant
because it provides a simple solution to the longstanding
separation problem in the field of emulsion catalysis. Fur-
thermore, the responsive emulsion allows us to proceed
a three-step enzymatic cascade with each step performed
sequentially under an optimal condition, eventually leading to
a 29% final conversion. In addition, our emulsion can be
scaled up 800 times for the gram-scale production. This proof-
of-principle example, on one hand, suggests the fascinating
perspectives of responsive emulsions for cascade reactions by
avoiding catalyst incompatibility. On the other hand, its
exploration for different cascades, ranging from multienzyme
to chemoenzymatic reactions, is envisioned.
(
Figure 3b), although the first step was slower, the latter two
steps were carried out under suitable pH and were able to
shift the reaction equilibrium. Eventually, the overall con-
version was higher (5.6 mM, overall conversion 11%).
To improve the overall catalytic efficiency, the above
three-step reaction was then performed in a sequential
fashion and operated separately under the individual optimal Acknowledgements
pH and temperature conditions of the corresponding enzyme,
which was achieved by the stepwise heating to allow on-
demand phase separation for changing enzyme solution from
one to another (Scheme 1). Initially, every single biotransfor-
mation was carried out for 8 hours. Figure 3c showed the
concentration change of substrates, intermediates, and final
products. AABE was hydrolyzed rapidly by CalB, and after
We acknowledge financial support from the German
Research Foundation and the Independent Research Fund
Denmark. We acknowledge the assistance of the Core Facility
BioSupraMol supported by the DFG. We thank Prof.
Marion B. Ansorge-Schumacher for valuable discussions
and support. We thank Prof. Wolfgang Kroutil for providing
the plasmid of ADH-ht. Z.S. thanks the support from China
Scholarship Council (CSC). Q.Z. thanks Dahlem Research
School for financial support. Open access funding enabled
and organized by Projekt DEAL.
8
hours, about 65% was converted to BeOH. CalB was then
replaced by ADH-ht for the following oxidation reaction. In
the next 8 hours, more than half of BeOH was consumed to
produce about 40 mM benzaldehyde. Demulsification and
exchange of the enzyme were then performed again to
introduce BAL for benzoin condensation. In the last 8 hours,
7
.3 mM benzoin was obtained as the final product (single-step Conflict of interest
conversion near 35%). The final conversion in this sequential
cascade approach was slightly higher than that of 24-hour
one-pot cascade experiments, and it was 15%.
The authors declare no conflict of interest.
In order to further improve the reaction conversion, we
provided enough reaction time to each step of the cascade.
Specifically, the first-step reaction was conducted for
Keywords: biocatalysis · block copolymers ·
multienzyme reaction · responsive emulsion
2
2
0 hours, while the second and third steps were operated for
4 hours. This prolonged reaction time eventually contributed
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