1 3
Topics in Current Chemistry (2018) 376:43
combinations of recombinant thermostable enzymes and employed an electrochemically mediated diaphorase system to regenerate NAD
+
and extract electrons from the
sugar mixtures. The first cascade sequence converts complex (poly)saccharides to glucose-6-phosphate via sucrose phosphorylase (SP), xylose isomerase (XI), polyphosphate glucokinase (PPGK), and phosphoglucomutase (PGM) [17, 18]. Both variants
of this sequence require inorganic polyphosphate. The second cascade sequence consists of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) to sequentially oxidize glucose-6-phosphate and 6-phosphogluconate while cleaving CO 2 from the latter, ultimately producing ribulose-5-phosphate
[16]. This sequence requires NAD
+
and enables the electrochemical interface required
for energy conversion. The third cascade sequence employs a series of kinases and
isomerases to convert six equivalents of ribulose-5-phosphate into five equivalents of
glucose-6-phosphate. This sequence consists of ribose-5-phosphate isomerase (RPI),
ribulose-5-phosphate-3-epimerase (RuPE), transketolase (TK), transaldolase (TA),
and phosphoglucose isomerase (PGI) to convert two equivalents of ribulose-5-phosphate into one equivalent of glyceraldehyde-3-phosphate and glucose-6-phosphate
that feeds back into the second (electrochemical) cascade sequence. Finally, three
additional enzymes were included to convert glyceraldhyde-3-phosphate to glucose6-phosphate, thereby preventing it from building up and causing product inhibition.
Those three enzymes were triose phosphate isomerase (TPI), aldolase (ALD), and
fructose-1,6-biphosphatase (FBP) [18] (Fig. 3).
Despite exemplifying the robustness of the multi-enzyme electrode approach to
electrocatalytic cascades, this work importantly highlights an inherent limitation to
the use of overly specialized catalysts. The sequential nature of both Zhang’s pseudoglycolysis pathway and Minteer’s immobilized Kreb’s cycle necessitates that the
overall catalytic rate will be limited by the slowest catalytic step. Furthermore, the
buildup of reaction intermediates and a multitude of enzymatic cofactors due to
unbalanced kinetics can result in multiple competitive inhibition pathways, effectively shutting down the entire cascade. Two solutions to this fundamental problem
have emerged: (1) the use of compartmentalization and microscopic spatial orientation to control the movement of intermediates and cofactors (which will be discussed in a later section), and (2) a collective movement of researchers towards
fewer, but more promiscuous electrocatalysts. Some of the early research using promiscuous enzymatic cascade electrodes has been based on reaction pathways that
mimic glycolysis [19, 20].
Glucose oxidase and glucose dehydrogenase catalyze the oxidation of glucose,
and have been widely investigated for bioelectrode designs, because of the importance of electrochemical glucose sensing. However, in the context of energy conversion, these enzymes are only capable of extracting two electrons per molecule of
glucose via oxidation of the C1 carbon. Gorton and coworkers demonstrated that
pyranose dehydrogenase (PDH) from Agaricus meleagris is capable of electrochemically oxidizing both the C2 and C3 positions of glucose [19]. Therefore, by
co-immobilizing PDH with the dehydrogenase domain of cellobiose dehydrogenase
from Corynascus thermophiles (an enzyme that, like glucose dehydrogenase, oxidizes glucose at C1), a bienzyme-modified electrode can electrochemically oxidize
the C1, C2, and C3 positions while collecting six electrons per molecule of glucose
Reprinted from the journal
153
Topics in Current Chemistry (2018) 376:43
combinations of recombinant thermostable enzymes and employed an electrochemically mediated diaphorase system to regenerate NAD
+
and extract electrons from the
sugar mixtures. The first cascade sequence converts complex (poly)saccharides to glucose-6-phosphate via sucrose phosphorylase (SP), xylose isomerase (XI), polyphosphate glucokinase (PPGK), and phosphoglucomutase (PGM) [17, 18]. Both variants
of this sequence require inorganic polyphosphate. The second cascade sequence consists of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) to sequentially oxidize glucose-6-phosphate and 6-phosphogluconate while cleaving CO 2 from the latter, ultimately producing ribulose-5-phosphate
[16]. This sequence requires NAD
+
and enables the electrochemical interface required
for energy conversion. The third cascade sequence employs a series of kinases and
isomerases to convert six equivalents of ribulose-5-phosphate into five equivalents of
glucose-6-phosphate. This sequence consists of ribose-5-phosphate isomerase (RPI),
ribulose-5-phosphate-3-epimerase (RuPE), transketolase (TK), transaldolase (TA),
and phosphoglucose isomerase (PGI) to convert two equivalents of ribulose-5-phosphate into one equivalent of glyceraldehyde-3-phosphate and glucose-6-phosphate
that feeds back into the second (electrochemical) cascade sequence. Finally, three
additional enzymes were included to convert glyceraldhyde-3-phosphate to glucose6-phosphate, thereby preventing it from building up and causing product inhibition.
Those three enzymes were triose phosphate isomerase (TPI), aldolase (ALD), and
fructose-1,6-biphosphatase (FBP) [18] (Fig. 3).
Despite exemplifying the robustness of the multi-enzyme electrode approach to
electrocatalytic cascades, this work importantly highlights an inherent limitation to
the use of overly specialized catalysts. The sequential nature of both Zhang’s pseudoglycolysis pathway and Minteer’s immobilized Kreb’s cycle necessitates that the
overall catalytic rate will be limited by the slowest catalytic step. Furthermore, the
buildup of reaction intermediates and a multitude of enzymatic cofactors due to
unbalanced kinetics can result in multiple competitive inhibition pathways, effectively shutting down the entire cascade. Two solutions to this fundamental problem
have emerged: (1) the use of compartmentalization and microscopic spatial orientation to control the movement of intermediates and cofactors (which will be discussed in a later section), and (2) a collective movement of researchers towards
fewer, but more promiscuous electrocatalysts. Some of the early research using promiscuous enzymatic cascade electrodes has been based on reaction pathways that
mimic glycolysis [19, 20].
Glucose oxidase and glucose dehydrogenase catalyze the oxidation of glucose,
and have been widely investigated for bioelectrode designs, because of the importance of electrochemical glucose sensing. However, in the context of energy conversion, these enzymes are only capable of extracting two electrons per molecule of
glucose via oxidation of the C1 carbon. Gorton and coworkers demonstrated that
pyranose dehydrogenase (PDH) from Agaricus meleagris is capable of electrochemically oxidizing both the C2 and C3 positions of glucose [19]. Therefore, by
co-immobilizing PDH with the dehydrogenase domain of cellobiose dehydrogenase
from Corynascus thermophiles (an enzyme that, like glucose dehydrogenase, oxidizes glucose at C1), a bienzyme-modified electrode can electrochemically oxidize
the C1, C2, and C3 positions while collecting six electrons per molecule of glucose
Reprinted from the journal
153