Electrochemical control of the catalytic activity of immobilized enzymes

Article abstract of DOI:10.1039/d0cc06190e

Regulation of the catalytic activity of enzymes immobilized on carbon nanotube electrodes was achieved by changing their local pH environment using electrochemical reactions. Reduction of oxygen increased the interfacial pH while oxidation of ascorbate decreased it, thus allowing changing rates of enzymatic reactions of electrode-immobilized amyloglucosidase and trypsin enzymes over a wide activity range.

Full text of DOI:10.1039/d0cc06190e

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Electrochemical control of the catalytic activity
of immobilized enzymes†
Cite this:DOI: 10.1039/d0cc06190e
Vasantha Krishna Kadambar,‡ Madhura Bellare,‡ Paolo Bollella,
and Artem Melman
Evgeny Katz
*
Received 13th September 2020,
Accepted 9th October 2020
*
DOI: 10.1039/d0cc06190e
rsc.li/chemcomm
Regulation of the catalytic activity of enzymes immobilized on These approaches included incorporation of photoresponsive
carbon nanotube electrodes was achieved by changing their local domains into proteins by covalent8–10 or non-covalent11,12 binding
pH environment using electrochemical reactions. Reduction of of a photoisomerizable chromophore, photothermal effect using
oxygen increased the interfacial pH while oxidation of ascorbate enzyme-bound nanoparticles,13–15 incorporation of artificial allo-
decreased it, thus allowing changing rates of enzymatic reactions of steric domains into proteins,16–19 responsive competitive enzyme
electrode-immobilized amyloglucosidase and trypsin enzymes over inhibitors,20 reversible physical nano-confinement of enzymes
a wide activity range.
into a responsive matrix,21,22 and other methods.23 Further flex-
ibility and sophisticated inputs can be achieved using enzymes
Enzymatic networks utilizing concerted work of multiple immobilized at interfaces or in a confined environment24 which
enzymes are the central part of cellular machinery.1 Selective also allows the action of a magnetic field to switch enzymatic
switching and tuning of the catalytic activity of enzymes in these assemblies on and off.25,26 An even more versatile approach for
networks is the key property of living systems allowing detection the regulation of enzymatic activity can be in their integration
and amplification of external signals, retaining cellular homeo- with electronic circuits. Here we report on the regulation of the
stasis, and controlling every aspect of their functioning. Natural activity of enzymes immobilized at electrochemical interfaces
processes in living cells are carefully orchestrated through using the effect of local pH change.27–29
entangled regulation of a large number of enzymes using
For many enzymes, their activity is pH-dependent and varia-
allosteric regulation, covalent modification of enzymes, tions of pH result in large changes in Km and turnover number of
protein–protein interactions, and controlled gene expression. enzymes. However, changes in solution pH are poorly suited for
Artificial enzymatic assemblies possess considerable potential the regulation of enzyme activities in enzymatic networks
for a range of applications including therapeutic applications,2,3 because they affect activities of all enzymes in the network.
biological detoxification,4 chemical synthesis,5,6 and bio- Yet, it is possible to create local pH changes using reactions
sensing.7 While being inspired by life and being assembled from involving release or consumption of hydrogen ions in a variety of
natural biopolymers, artificial dynamic systems have to be built electrochemical reactions at an interface (Fig. 1).29–31 In these
on different principles. Direct replication of cellular regulation
mechanisms relying on the tuning of the enzyme activity is
frequently impractical because of their staggering complexity.
As a viable alternative, the capabilities of artificial enzymatic
assemblies can be extended through their integration with
external stimuli, which can control the activity of key enzymes
in the network.
Several approaches for the dynamic and reversible regulation
of the activity of enzymes by external stimuli have been proposed.
Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam,
NY 13699, USA. E-mail: amelman@clarkson.edu, ekatz@clarkson.edu
Fig. 1 Schematic representation of local pH change at the electrode
surface through electrochemical reactions and its influence on the
catalytic activity of electrode-immobilized enzymes.
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc06190e
‡ These authors contributed equally.
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cases, the local pH change is confined to a very thin layer near
The immobilized enzyme retained its catalytic activity to
the electrochemical interface and does not affect pH in the hydrolyze maltose to glucose (Fig. 3A), which was measured by
solution phase or other interfaces leaving the functioning of a standard assay35 using glucose oxidase (GOx), horseradish
other enzymes there intact. The local pH change upon electro- peroxidase (HRP) and 2,20-azino-bis(3-ethylbenzothiazoline-6-
chemical reactions has been extensively studied and measured sulfonic acid) (ABTS), taking aliquots from the reaction
by various methods.29
mixture. In the assay, the concentration of glucose in the
To produce the local pH change we exploited the electro- solution was measured as the initial rate of ABTS oxidation
chemical reduction of oxygen that is present in aqueous solu- by HRP. The catalytic activity of electrode-immobilized AMG
tions with concentrations around 250 mM. The reduction con- was strongly pH-dependent (Fig. 3B) with the reaction rates
sumes 4H+ per oxygen molecule resulting in pH increase up to 3 dropping rapidly with the increased pH, so, that the reaction
pH units at the electrode surface,30 which can be sufficient for essentially stops at pH ca. 8. The pH dependence was used for
inducing large changes in activities of enzymes immobilized at controlling the catalytic activity of the electrode-immobilized
the electrode.
AMG. Applying ꢀ0.5 V potential to the AMG-modified electrode
For immobilization of enzymes and conducting all experiments, in a solution of 3 mM HEPES and 0.1 M Na2SO4 in the presence
we used an electrode made of compressed carbon nanotubes of O2 with the bulk pH 6.0 (Fig. 3C, arrow ‘‘a’’) we induced a
(buckypaper), which is frequently used in bioelectrochemical local pH increase resulting in essentially complete suppression
systems.32 It should be noted that the buckypaper electrode was of the AMG-catalyzed hydrolysis of maltose as no traces of
selected for experimental convenience and other electrode materi- glucose in the cell were detected by the HRP–ABTS assay of the
als can be used applying different methods for the enzyme solution. Note that the bulk pH value was not affected by the
immobilization.33 The buckypaper electrode was treated with electrochemical process despite the low buffer concentration.
N-hydroxysuccinimidyl ester of pyrene-1-butyric acid (PBSE), which Disconnection of the potential (Fig. 3C, arrow ‘‘b’’) resulted in a
is known to bind to aromatic surfaces due to strong p–p stacking.34 rapid increase of glucose concentration, which was stopped by
Incubation of the resultant electrode with amyloglucosidase reapplying ꢀ0.5 V potential to the electrode. As can be seen
(AMG; E.C. 3.2.1.3) enzyme resulted in the immobilization of the from Fig. 3C, the process is reversible and can be repeated
enzyme at the electrode surface through a reaction of lysine multiple times actuating the AMG activity by applying and
residues of AMG with the N-hydroxysuccinimidyl ester group of disconnecting the electrical potential on the enzyme-modified
PBSE. Experiments were performed in 3 mM, pH 6.0, HEPES buffer electrode.
containing 0.1 M sodium sulfate. At that bulk pH the electrode with
The mechanism of these reversible changes in the AMG
the immobilized enzyme showed a reduction of oxygen starting activity involving changes in the local pH at the electrode
from a potential of ca. ꢀ0.35 V (vs. Ag|AgCl|KCl, 3 M; all other
potentials are reported vs. this reference) (Fig. 2A). This reduction
results in the consumption of hydrogen ions near the electron
surface increasing the local pH (Fig. 2B). It should be noted that
using bioelectrocatalytic reduction of oxygen with co-immobilized
bilirubin oxidase, the overpotential for the O2 reduction process
can be substantially lowered, if needed.31
Fig. 3 (A) AMG-catalyzed hydrolysis of maltose. (B) Dependence of the
relative catalytic activity of the electrode-immobilized AMG on the bulk pH
value. (C) Suppression of the catalytic activity of the electrode-
immobilized AMG upon applying ꢀ0.5 V potential (a), and resumption of
the AMG catalytic activity in the absence of the potential (b) (OCP is an
open circuit potential produced on the electrode when the potential is not
externally applied). The experiment was performed in the presence of
2 mM maltose and O2 (under equilibrium with air). (D) Analogous experi-
ment in the absence of oxygen (under Ar). The background electrolyte was
3 mM, pH 6.0, HEPES buffer and 0.1 M Na2SO4.
Fig. 2 (A) Cyclic voltammograms of the AMG-modified buckypaper
electrode recorded under anaerobic (under Ar) and aerobic (under air)
conditions. The background electrolyte was 3 mM HEPES buffer with 0.1 M
Na2SO4. Potential scan rate, 10 mV sꢀ1. (B) The scheme of producing a local
pH increase at the electrode surface upon O2 electrochemical reduction.
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surface due to the reduction of O2 was confirmed by conducting AMG-modified electrode results in a sharp increase of the
analogous experiments in the absence of oxygen (Fig. 3D). AMG enzymatic activity (Fig. 4C, arrow ‘‘b’’), which is stopped
Indeed, the local pH change was not possible under anaerobic by the removal of the applied potential (Fig. 4C, next arrow
conditions, thus preserving the AMG activity regardless of the ‘‘a’’). The activation–inactivation cycles can be repeated
potential applied.
multiple times by changing the electrical potential on the
The opposite process of activation of the electrode immobi- enzyme electrode.
lized AMG enzyme can be stimulated by an electrochemical
Rates of accumulation of glucose produced by the electrode-
decrease of the local pH starting from a neutral or weakly basic immobilized AMG enzyme at bulk solution pH 7.5 when a
pH in the solution phase, which suppresses the catalytic activity potential of +0.2 V was applied were equal to the rates of
of AMG. While pH decrease can be achieved upon electro- glucose production at the same electrode in the same buffer
chemical oxidation of water,29 this process requires excessively with bulk pH adjusted to 6.0. Thus, applying the potential
harsh conditions that are generally not compatible with the results in a local pH change of approximately 1.5 units.
stability of electrode-immobilized enzymes. Instead, we
To confirm the generality of the approach to the electro-
achieved the local decrease in pH through electrochemical chemical control of immobilized enzyme activity via local pH
oxidation of ascorbate, which was present in a physiologically change in the course of electrochemical reactions, we studied a
relevant 1 mM concentration in the electrolyte. Electrochemical reaction catalyzed by trypsin (E.C. 3.4.21.4) immobilized at an
oxidation of ascorbate at the AMG-modified buckypaper elec- electrode surface. Trypsin was immobilized on a buckypaper
trode proceeds at potentials more positive than ꢀ0.05 V electrode using the reaction of lysine acylation with pentafluoro-
(Fig. 4A) resulting in the release of hydrogen ions and solution phenyl 1-pyrenebutyrate.
acidification (Fig. 4B), which can be used for controlling
The catalytic activity of the electrode-immobilized trypsin
the activity of the electrode-immobilized AMG enzyme. In the measured spectrophotometrically using standard BAPNA
absence of the applied potential (the OCP condition) the (Na-benzoyl-DL-arginine 4-nitroanilide) substrate (Fig. 5A)36
catalytic activity of the electrode-immobilized AMG enzyme at was pH-dependent, but in contrast to AMG enzyme the cata-
pH 7.5 is very small (Fig. 3B), so, no accumulation of lytic activity of trypsin has a maximum at pH 9 (Fig. 5B). At the
glucose can be detected. Note that the glucose assay with starting weakly acidic bulk pH 5.6 the immobilized trypsin
GOx–HRP–ABTS is not possible in the presence of ascorbate, possessed smaller, but visible catalytic activity (Fig. 5C, ‘‘a’’).
and thus another assay using NAD+/glucose dehydrogenase was Application of ꢀ0.5 V potential to the trypsin-modified elec-
used to follow the glucose concentration in the solution. The trode in the presence of O2 resulted in a large (3.9-fold)
experiment was started in a 3 mM HEPES buffer solution reversible increase in the catalytic activity of trypsin
(pH 7.5) containing 2 mM of maltose, 1 mM of ascorbate and (Fig. 5C, ‘‘b’’), although the dynamic range of the electroche-
0.1 M of sodium sulfate, when the AMG enzyme is not active mical regulation was smaller than for AMG (compared to
(Fig. 4C, arrow ‘‘a’’). Applying a potential of +0.2 V to the Fig. 3 and 4). However, the dynamic range can be significantly
increased if both the oxidation of ascorbate and the reduction
of oxygen were alternatingly utilized for the enzyme activity
regulation. By applying alternating +0.2 V (Fig. 5E, ‘‘a’’) and
ꢀ0.5 V (Fig. 5E, ‘‘b’’) potentials to the electrode, we achieved
more than 13-fold difference in enzyme reaction rates. Like in
the previous cases switching off and on enzymes can be
repeated multiple times.
In summary, we have established that the catalytic activity of
enzymes immobilized at electrode surfaces can be reversibly
regulated by controlling the interfacial pH in solutions with a
lower buffer capacity. The local pH at an electrode surface can
be increased through electrochemical reduction of dissolved
oxygen or decreased through oxidation of physiologically
relevant concentrations of ascorbate. We believe that this
approach is general and can be used for both fine regulation
and switching of enzymatic activity of separated enzymes or
enzymatic networks immobilized at electrode surfaces. Sophis-
Fig. 4 (A) Cyclic voltammograms in the absence (1) and presence (2) of
1 mM ascorbate. Potential scan rate, 10 mV sꢀ1. (B) The scheme of
generation of a local pH decrease at the electrode surface upon ascorbate
electrochemical oxidation. (C) Catalytically inactive state of the electrode-
immobilized AMG in the absence of the potential applied (OCP) (a) and
activation of the enzyme upon applying a potential of +0.2 V (b). The
experiment was performed in the presence of 1 mM ascorbate and 2 mM
of maltose. The background electrolyte was 3 mM, pH 7.5, HEPES buffer
and 0.1 M Na2SO4.
ticated multi-enzyme/multi-step biocatalytic cascades can be
regulated upon electrochemical signals switching/tuning
branched pathways. While the electrochemical O2 reduction
and ascorbate oxidation are convenient electrochemical
processes resulting in the local pH change and, therefore,
enzyme rate regulation, many other electrochemical processes
can be applied for the same purpose.29
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