Enzyme-Loaded Nanoreactors Enable the Continuous Regeneration of Nicotinamide Adenine Dinucleotide in Artificial Metabolisms

Article abstract of DOI:10.1002/anie.202012023

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme for numerous biocatalytic pathways. While in nature, NAD⁺ is continuously regenerated from NADH by enzymes, all synthetic NAD⁺ regeneration strategies require a continuous supply of expensive reagents and generate byproducts, making these strategies unattractive. In contrast, we present an artificial enzyme combination that produces NAD⁺ from oxygen and water continuously; no additional organic substrates are required once a minimal amount pyruvate is supplied. Three enzymes, i.e., LDH, LOX, and CAT, are covalently encapsulated into a substrate-permeable silica nanoreactor by a mild fluoride-catalyzed sol–gel process. The enzymes retain their activity inside of the nanoreactors and are protected against proteolysis and heat. We successfully used NAD⁺ from the nanoreactors for the continuous production of NAD⁺ i) to sense glucose in artificial glucose metabolism, and ii) to reduce the non-oxygen binding methemoglobin to oxygen-binding hemoglobin. This latter conversion might be used for the treatment of Methemoglobinemia. We believe that this versatile tool will allow the design of artificial NAD⁺-dependent metabolisms or NAD⁺-mediated redox-reactions.

Full text of DOI:10.1002/anie.202012023

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 7728–7734
International Edition:
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Artificial Metabolism
Hot Paper
Enzyme-Loaded Nanoreactors Enable the Continuous Regeneration of
Nicotinamide Adenine Dinucleotide in Artificial Metabolisms
Seong-Min Jo, Frederik R. Wurm,* and Katharina Landfester*
Abstract: Nicotinamide adenine dinucleotide (NAD) is an
essential coenzyme for numerous biocatalytic pathways. While
in nature, NAD+ is continuously regenerated from NADH by
enzymes, all synthetic NAD+ regeneration strategies require
a continuous supply of expensive reagents and generate
byproducts, making these strategies unattractive. In contrast,
we present an artificial enzyme combination that produces
NAD+ from oxygen and water continuously; no additional
organic substrates are required once a minimal amount
pyruvate is supplied. Three enzymes, i.e., LDH, LOX, and
CAT, are covalently encapsulated into a substrate-permeable
silica nanoreactor by a mild fluoride-catalyzed sol–gel process.
The enzymes retain their activity inside of the nanoreactors and
are protected against proteolysis and heat. We successfully used
NAD+ from the nanoreactors for the continuous production of
NAD+ i) to sense glucose in artificial glucose metabolism, and
ii) to reduce the non-oxygen binding methemoglobin to oxy-
gen-binding hemoglobin. This latter conversion might be used
for the treatment of Methemoglobinemia. We believe that this
versatile tool will allow the design of artificial NAD+-
dependent metabolisms or NAD+-mediated redox-reactions.
We demonstrate the continuous production of NAD+ by an
artificial enzyme-combination inside a substrate-permeable
and robust silica nanoreactor by water and oxygen as the
necessary stoichiometric reagents with a minimal amount of
pyruvate, without any additional substrates and without
generating byproducts.
Living cells need a continuous supply of NAD+ but also
have the capability to regenerate the NAD+.[1] If the NAD+/
NADH couple is used in synthetic chemistry for either
oxidation or reduction, stoichiometric use of NAD+/NADH is
necessary.[2] A sustainable regeneration strategy of the
coenzymes would also reduce the cost[3] of natural or artificial
metabolisms.[4] Chemical, electrochemical, photocatalytic,
and enzymatic strategies for both NAD+ and NADH
regeneration have been proposed.[3,5] Chemical regeneration
of NADH by reducing agents such as NaBH4 is effective,[6] but
its high reactivity is problematic. Electrochemical regener-
ation is usually mediator-dependent, side reactions to 1,6-
NADH or the NAD2 dimer have been described, and fouling
of electrodes can occur.[7] Photocatalytic regeneration became
a recent focus that uses light as a green energy source but UV-
light might be harmful to some processes.[8] Most methods for
the NADH regeneration are non-selective, and other redox-
sensitive compounds interfere.[3,5]
Introduction
Nicotinamide adenine dinucleotide (NAD+; the oxidized
form of NAD) is an attractive bio-based oxidizing agent in
synthesis, however, its regeneration from NADH (the
reduced form of NAD) requires stoichiometric amounts of
reagents or the use of organometallic catalysts. To date, no
regeneration strategy that works without additional reagents
in stoichiometric amounts has been reported. It is desirable to
use NAD+ as an oxidizing agent only in small amounts and
that the NAD+ can be regenerated throughout the synthesis.
In contrast, the enzymatic regeneration of NADH is the
only selective method reported to date.[3,5] However, the low
stability of enzymes and their difficult isolation from a homo-
geneous reaction mixture are significant challenges. Encap-
sulation or immobilization of the enzymes is essential to
increase stability and ease purification. However, it is
accompanied by denaturation and reduced enzyme activity,
for example, acidic/basic pH, non-selective chemistry, or
organic solvents.[9]
We present a NAD+-regeneration strategy that relies on
three enzymes co-encapsulated into semipermeable silica
nanoreactors (SiNRs) prepared via a mild fluoride-catalyzed
sol–gel chemistry in the microemulsion. High encapsulation
efficiencies with conserved enzyme activity were achieved.
The artificial enzyme combination of lactate dehydrogenase
(LDH), lactate oxidase (LOX), and catalase (CAT) only need
a minimal amount of pyruvate to continuously produce
NAD+ from NADH as the additional substrates oxygen and
water are available in the mixture (Figure 1). The SiNRs were
obtained as an aqueous dispersion and were combined
exemplarily with the NAD+-dependent i) fluorometric glu-
cose detection and ii) the recovery of hemoglobin from
methemoglobin. In combination with the possibility of
reusing the SiNRs, the herein presented strategy for contin-
uous NAD+ regeneration is an attractive strategy for the use
in various NAD+-dependent reactions.
[*] Dr. S.-M. Jo, Prof. Dr. F. R. Wurm, Prof. Dr. K. Landfester
Max Planck Institute for Polymer Research
Ackermannweg 10, 55128 Mainz (Germany)
E-mail: landfester@mpip-mainz.mpg.de
Prof. Dr. F. R. Wurm
Sustainable Polymer Chemistry Group, MESA+ Institute for Nano-
technology, Universiteit Twente
PO Box 217, 7500 AE Enschede (The Netherlands)
E-mail: frederik.wurm@utwente.nl
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.202012023.
ꢀ 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 License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
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Results and Discussion
which demonstrates the recycling of pyruvate by LOX
(Figure 1c).[10]
Subsequently, the three enzymes were covalently encap-
We realized the continuous production of NAD+ by the
combination of lactate dehydrogenase (LDH), lactate oxidase
(LOX), and catalase (CAT) encapsulated in a substrate-
permeable silica nanoreactor (Figure 1a). From these en-
zymes, LDH produces NAD+ and lactate from NADH and
pyruvate. The lactate is recycled by LOX to pyruvate without
the need for a coenzyme or cofactor, producing hydrogen
peroxide as the only byproduct (Figure 1b), which is removed
by disproportionation with CAT to oxygen and water. The
enzyme cascade was initiated by the addition of NADH and
a minimal amount of pyruvate to continuously produce
NAD+ (if consumed by a reaction) (Figure 1b). Reproduction
of pyruvate by LDH/LOX, “self-fueling”, enables the con-
tinuous NAD+ regeneration without the need to further
supply pyruvate. The remaining substrates water and oxygen
are present in the open aqueous system.
sulated into the substrate-permeable silica nanoreactor by
a mild sol–gel process (Figure 2a). Unlike the conventional
sol–gel process that relies on alkaline or acidic catalysts, we
used fluoride (FÀ) as a catalyst to retain the enzyme activity
(Figure 1b).[11] This artificial enzyme combination, confined
in the same nanoreactor, enabled an efficient cascade
reaction, modular handling, and easy separation from reac-
tants with the possibility for reuse.[12]
Encapsulation of LDH, LOX, and CAT into the semi-
permeable silica nanoreactor was achieved by a fluoride-
catalyzed sol–gel process (Figure 2). As a control, a silica
nanoreactor, loaded with LDH alone, was prepared. We co-
condensated tetraethyl orthosilicate (TEOS) as the matrix
To investigate the compatibility of the artificial enzyme
set, the NAD+ production was conducted in a solution
containing LDH or a mixture of LDH, LOX, and CAT. When
the ratio of NADH:pyruvate was set to 2:1, 50% conversion
of NADH to NAD+ by LDH alone was achieved, as pyruvate
was converted to lactate. In contrast, the mixture of LDH,
LOX, and CAT achieved the full conversion of NADH,
Figure 1. Continuous NAD+-production and regeneration by an artifi-
cial enzyme set. a) The combination of LDH (lactate dehydrogenase),
LOX (lactate oxidase), and CAT (catalase) encapsulated in semiperme-
able silica nanoreactors catalyze an NAD+-dependent reaction A!B.
b) Enzyme reactions inside the silica nanoreactor: LDH converts
NADH to NAD+ and pyruvate to lactate. LOX recycles the pyruvate
from lactate using water and oxygen and produces H2O2. CAT partially
recycles H2O2 to water and oxygen. c) The reaction of lactate to
pyruvate: if the pyruvate is added in nonstoichiometric amounts
(NADH:pyruvate=2:1) the single enzyme (LDH) can oxidize 50% of
the NADH to NAD+ (blue curve), while the enzyme combination
(LDH, LOX, CAT) achieves the full conversion (red curve).
Figure 2. Preparation of silica nanoreactors (SiNRs) with covalently
encapsulated enzymes. a) Fluoride-catalyzed sol–gel chemistry and
one-pot procedure to enzyme-loaded silica nanoreactors. b) A trans-
mission electron microscope image of enzyme-loaded silica nano-
reactors. c) Solid-state 29Si NMR of LDH/LOX/CAT@SiNRs.
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material with 3-aminopropyl-trimethoxysilane (APTMS) as
a covalent anchor for the enzymes in a water-in-oil micro-
emulsion with potassium fluoride (KF) as the catalyst,
preserving the pH-value at 7.4. During the process, the
enzymes were covalently attached to the silica network by N-
(3-dimethyl aminopropyl)-N’-ethyl carbodiimide hydrochlo-
ride (EDC) and N-hydroxysuccinimide (NHS) chemistry.
Circular dichroism (CD) spectra of enzymes in the fluoride
solution show no significant change in their secondary
structure (LDH and LOX for Figure S1), (CAT for the
previous report[13]). Conventional sol–gel chemistry relies on
acidic or alkaline catalysis, which may result in the denatura-
tion of proteins and loss of enzymatic activity in some cases.[11]
For example, when ammonia was used as a catalyst for the
condensation, no LDH enzyme activity was detected in the
resulting nanoreactor (Figure S2). Besides, with the fluoride-
catalyzed sol–gel chemistry, a higher enzyme loading effi-
ciency than conventional surface-immobilization of enzymes
was achieved (see below and ref. [14]).
As the LOX reaction produces hydrogen peroxide, a high
amount of CAT is essential to disproportionate hydrogen
peroxide into water and oxygen in the same nanoreactor and
there is a need to protect LDH from the risk of oxidative
denaturation. In the following, we adjusted the molar
equivalents of the enzymes to LDH:LOX:CAT= 3:1:15.
The hydrodynamic diameters of the silica nanoreactors were
determined by dynamic light scattering (DLS) to be between
200 and 270 nm (LDH@SiNRs: average diameter: 270 nm,
PDI = 0.404; LDH/LOX/CAT@SiNRs average diameter:
200 nm, PDI = 0.420, Figures S3 and S4). From thermogravi-
metric analysis (TGA), a high enzyme loading was estimated
by a weight loss of ca. 20% organic contents in the self-fueled
nanoreactor during the thermal degradation compared to the
empty nanoreactor (Figure S5). 29Si solid-state NMR spectra
showed the successful formation of the silica network by
condensation of TEOS (Q2, Q3, and Q4) and APTMS (T2 and
T3) with a molar ratio of 2:1 by the sol–gel process (Fig-
ure 2c). Besides, FT-IR confirms the formation of silicon
oxide by detecting two strong bands for Si-OH (780 cmÀ1) and
Si-O-Si (1040 cmÀ1) (Figure S6). Additionally, distinct amide
vibrations at 1550 cmÀ1 were detected for the loading of
enzymes. Measurements of the surface area of the nano-
reactors by Brunauer–Emmett–Teller (BET) gas adsorption
showed a BET surface area of 31.5 m2 gÀ1 and pore volume of
0.08 cm3 gÀ1, which was sufficient for the diffusion of small
molecular substrates and products to the enzymes through the
silica matrix.[15]
Figure 3. Enzyme reactions of silica nanoreactors. a,b) Michaelis–
Menten kinetics of LDH/LOX/CAT@SiNRs (a), LDH@SiNRs (b) (LDH
activity was measured). c) Thermal stability of encapsulated LDH and
native LDH. d) Proteolytic stability of encapsulated, native, and sur-
face-immobilized LDH after incubation with Proteinase K.
product.[16] Our results show a slightly decreased velocity for
the forward reaction of native LDH (10% for 1:20 of
pyruvate:lactate), which is probably caused by the increasing
amount of lactate in the mixture. Interestingly, much stronger
effect of the product inhibition was observed for the
LDH@SiNRs (25% for a 2:1 of pyruvate:lactate ratio,
Figure S7), presumably due to the local accumulation of the
produced lactate inside the nanoreactors. In contrast, lactate
was quickly eliminated by the reaction of LOX in the LDH/
LOX/CAT@SiNRs, which we believe the main contribution
to the increased kcat value.
The activity of LOX did not differ remarkably for both
single LOX-loaded nanoreactors and LDH/LOX/CAT@-
SiNRs in terms of Km and kcat (Figures S8 and S9), because
no substrate recycling (by LDH) occurred during the
reaction.
We determined the changes of the enzymatic activity after
exposure to 60 and 708C (below melting temperature (Tm) of
the native enzyme) and 808C (above Tm) for 15 min (Fig-
ure 3c). The enzymatic assay was performed at room temper-
ature. The native LDH significantly lost its activity after
heating to 808C (above Tm) for 30 min to less than 10% of the
initial activity. In contrast, the encapsulated LDH showed
much higher stability when heated to 808C for 2 h with ca.
40% residual activity (Figure S10). Regarding such higher
stability of the encapsulated LDH, previous studies have
claimed that immobilized proteins by multipoint attachment
could increase the resistance against heat, organic solvents, or
denaturing agents, presumably due to the prevention of
structural denaturation.[17] It is consistent with our previous
results that the encapsulated glucose oxidase and beta-
glucosidase in silica nanoreactors show higher stability than
their native states.[9,14] The encapsulated LOX also showed
higher preserved activity than their native form at high
temperatures. Unlike encapsulated LDH, the native LDH
All enzyme-loaded silica nanoreactors exhibited a high
enzyme activity, which is evident in the substrate-permeabil-
ity of the silica matrix. The enzymatic activity (kcat/Km) (with
k
cat = turnover number, Km = Michaelis–Menten constant) of
LDH in the self-fueled nanoreactors (LDH/LOX/CAT@-
SiNRs) was 2.3-fold higher than that of the LDH@SiNRs for
forward reactions (pyruvate + NADH ! lactate + NAD+)
(Figure 3a,b). In particular, a two-fold higher kcat value was
observed in LDH in the LDH/LOX/CAT@SiNRs. To explore
the reason for the increased kcat value, we investigated the
product inhibition of LDH. According to the literature, the
forward reaction of LDH can be inhibited by lactate as the
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lost its activity at 708C (Figure S11). On the other hand, both
deactivation (Figure S21). In contrast, the self-fueled nano-
native CAT and encapsulated CAT did not significantly lose
their enzymatic activity after exposure to 808C for 15 min
(Figure S12).
reactors (LDH/LOX/CAT@SiNRs) enabled the continuous
regeneration of NAD+ for at least eight cycles (Figure 4a). To
initiate each cycle, only additional NADH was added to the
dispersion, no further substrates or cofactors were necessary
to produce NAD+. After eight cycles, the dispersion was
centrifuged to separate the nanoreactors from the reactant.
After re-dispersion the nanoreactors in a new reaction
cocktail, four additional NADH additions obtained the
successful conversion to NAD+, which seems not to be the
limit. Notably, the reaction velocity decreased from addition
to addition (Figures 4a and S22), which can be attributed to
the changed NAD+ concentration in the reaction mixture that
alters the equilibrium constant (K) after each addition of
NADH and reduces the reaction rates (Figure S23). This was
supported by conducting the NAD+ regeneration at different
ratios of NADH:NAD+, proving a decreased conversion rate
of NADH, when the amount of NAD+ was increased
(Figure S24). After recycling the nanoreactors, the rate of
the NAD+ production was increased to ca. 80% of the initial
velocity of the 1st cycle; probably only 80% recovery was
obtained due to loss of the material during the washing
procedures (Figure S25). We confirmed the protection-effect
of CAT inside of the nanoreactors. The NADH oxidation was
studied with a nanoreactor, loaded only with LDH and LOX
(but without CAT). The reaction rates decreased gradually,
and the NAD+ production ceased after the 6th cycle due to
side-reactions with hydrogen peroxide (Figure S26).
To further explore the structural stability of encapsulated
enzymes, nano differential scanning fluorimetry (NanoDSF)
measurements were performed.[12,18] NanoDSF is based on
changes in the intrinsic fluorescence of aromatic amino acids
(i.e., Trp) in the protein structure during thermal denatura-
tion. The folded and unfolded proteins exhibit a different
emission ratio at 330 nm and 350 nm. We compared the
structural stability of the enzymes in solution, surface-
immobilized, or encapsulated in silica nanoreactors. In the
case of LDH, the initial emission ratios of 350 nm/330 nm
were not significantly different between LDH in solution and
the encapsulated LDH (0.82 and 0.83, respectively) (Fig-
ure S13), which indicated a similar folding state of native
LDH in solution and the LDH after encapsulation. However,
a significant difference between the pure LDH and the
encapsulated LDH was detected after cooling to room
temperature: while the native enzyme did not show any
residual activity, the encapsulated enzyme in the silica matrix
was still active, which might be attributed to partial refolding
during the cooling procedure (Figure S14). According to the
results, the encapsulated LDH recovered almost its initial
fluorescence ratio of 350 nm/330 nm after the cooling, while
native LDH did not.
For LOX, both the initial emission ratios at 350 nm/
330 nm and melting temperature were similar between
dissolved and encapsulated LOX (688C) (Figure S15). The
stability of the encapsulated CATwas found to be more stable
than native CAT (48C increased melting temperature in
encapsulated CAT; Figures S16 and S17). This further under-
lines the mild conditions of the encapsulation procedure for
a variety of enzymes.
The efficiency of enzyme-loaded nanoreactors was dem-
onstrated by conducting the NADH oxidation at an extremely
Proteolytic resistance of encapsulated enzymes (LDH and
LOX) in nanoreactors was investigated by exposure to
Proteinase K (EC 3.4.21.64; 28.9 kDa): unlike the dissolved
and surface-immobilized enzymes, the nanoreactors with
loaded enzymes did not lose their activity in the presence of
Proteinase K (Figure 3d for LDH and Figure S18 for LOX).
This data further underlines the encapsulation of the enzymes
and indicates that Proteinase K cannot penetrate the silica
matrix due to its high molar mass. Additionally, no leakage of
the enzymes from the nanoreactors was detected: the
enzymatic activity remained unchanged after extensive wash-
ing of the dispersion, proving the covalent attachment of the
enzyme inside of the silica matrix (Figure S19 and S20). In
previous studies, only very little enzyme loading was achieved
when the crosslinker (EDC, NHS) was omitted during the
preparation of the silica nanoreactors.[15]
The continuous production of NAD+ was studied at
different ratios of pyruvate: NADH at an initial molar ratio of
pyruvate: NADH = 2:1. The decreasing absorbance moni-
tored the conversion of NADH into NAD+ at 340 nm.
Additional NADH was added after the first equivalent was
consumed. In the case of LDH@SiNRs, only two cycles
produced NAD+; afterward, no oxidation of NADH was
observed due to the lack of pyruvate and possible enzyme
Figure 4. Continuous NAD+ production and regeneration. a) Continu-
ous production of NAD+ by LDH/LOX/CAT@SiNRs upon the sequen-
tial addition of NADH. For each cycle, 3 mM NADH was used. After 8
additions, the SiNRs were centrifuged, washed, and reused. b) NAD+
regeneration with small amounts of pyruvate (2.5 mM), and NADH
(0.25 mM). The normalized value of 1.0 along the y-axis indicates the
raw absorbance of 0.23. c) Comparison of the reaction kinetics of
LDH/LOX@SiNRs, LDH/LOX separately-encapsulated, and
LDH@SiNRs. The starting concentration of NADH was 3 mM.
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low pyruvate concentration, i.e., pyruvate:NADH = 1:100,
which would lead to only 1% NAD+ conversion by the LDH,
which resembles a change of 0.002 in the absorbance at
340 nm. Using the self-fueled nanoreactors, almost quantita-
tive conversion from NADH to NAD+ was achieved (Fig-
ure 4b), proving that only a minimal amount of pyruvate was
sufficient to produce NAD+, only relying on feeding with
water and oxygen as substrates for the enzyme catalysis
cascade (water and oxygen are present throughout the
reaction cycles as the reaction proceeds under ambient air).
The confinement effect of several enzymes in the same
nanoreactor was compared to separately encapsulated en-
zymes in different nanoreactors. We compared the kinetics for
the NAD+ production for the LDH@SiNRs alone with two
single enzyme nanoreactors (LDH and LOX) and the nano-
reactors encapsulated LDH and LOX. Single LDH nano-
reactors exhibited the lowest reaction rate to NAD+ as the
concentration of pyruvate decreases during the process
(Figure 4c, green). The addition of LOX (in a separate
nanoreactor) increased the reaction kinetics by a factor of ca.
1.1, as pyruvate was constantly regenerated by LOX (Fig-
ure 4c, black). However, the confinement of both LDH and
LOX in the same nanoreactors further increased the NAD+
regeneration by a factor of ca. 2.3 as the local pyruvate
concentration remains high throughout the whole enzyme
cascade (Figure 4c, red).
of 10% conversion would be achieved without NAD+-
regeneration. Using the NAD+-producing nanoreactor, high
conversion to resorufin was achieved (Figure 5b). In contrast,
when the LDH@SiNRs were used, only a low conversion
(< 10%) was achieved, which was equivalent to the amount
of NAD+ in the reaction mixture, as no continuous regener-
ation was possible. The results underline that the NAD+-
regeneration module can be combined with natural or
artificial NAD+-dependent enzymatic pathways, which opens
the possibility for catalysis in artificial cells or reactors.[20]
With the redox potential of “NAD+ + H+ + 2eÀ!
NADH” (À320 mV) and hemoglobin
A
(À52 to
À71 mV),[21] the NAD+-regeneration module was also capa-
ble of reducing methemoglobin to hemoglobin. Hemoglobin
is an essential protein in most vertebrates for oxygen-trans-
port, and it is a major component of red blood cells. The heme
group is responsible for the binding and release of oxygen.
Methemoglobinemia is a serious disease in which the hemo-
globin is autoxidized to the methemoglobin, and thus it
cannot bind to oxygen.[22] In natural erythrocytes, the
reduction of methemoglobin to hemoglobin is achieved by
the NADH-dependent methemoglobin reductase.[23] The
reduction of methemoglobin would be of high interest not
only for natural red blood cells but also for the design of
synthetic hemoglobin-based oxygen carriers (Figure 6a).[24]
As the oxidation of NADH to NAD+ by LDH is a two-
electron process, two heme groups might be involved to
reduce Fe3+ into Fe2+. LDH/LOX/CAT@SiNRs were used to
produce hemoglobin from methemoglobin (0.125 mM) by the
help of NADH (5 and 50 mM). The UV/visible spectrum
shows the formation of oxyhemoglobin (oxygen-bound form):
The NAD+-producing nanoreactors can be applied in
artificial enzyme cascades, for example, for glucose detec-
tion.[19] Glucose dehydrogenase is an NAD+-dependent
enzyme and catalyzes the conversion of glucose to glucono-
d-lactone. In a consecutive step, we used peroxidase, which
converted Amplex red into resorufin, generating a fluores-
cence signal at 555 nm excitation and 595 nm emission, which
is a simple glucose sensor (Figure 5a). The molar ratio of
NADH:pyruvate:Amplex red was 1:1:10, that is, a maximum
Figure 5. Utilization of LDH/LOX/CAT@SiNRs in NAD+-dependent
enzyme pathways. a) Glucose-sensing: Glucose is a starting substrate
for glucose dehydrogenase, producing glucono-d-lactone. The perox-
idase converts Amplex red into resorufin. Regeneration of NAD+ from
NADH is necessary for the continuous progress of the reaction.
b) Monitoring of the fluorescence of resorufin during artificial glucose
metabolism using LDH@SiNRs or LDH/LOX/CAT@SiNRs. The molar
ratio of NADH, pyruvate, and Amplex red was 1:1:10
Figure 6. Rapid reduction of methemoglobin to hemoglobin with
NAD+-regeneration nanoreactors. a) A reaction scheme for the reduc-
tion of methemoglobin to hemoglobin. b) UV/Vis spectra showing the
reduction of methemoglobin (8 mgmLÀ1; 125 mM) to oxyhemoglobin
at different concentrations of NADH with LDH/LOX/CAT@SiNRs.
c) Reduction of methemoglobin to oxyhemoglobin with 10 mM NADH
by LDH/LOX/CAT@SiNRs, LDH@SiNRs, and only NADH. Absorb-
ance at 580 nm indicates production of oxyhemoglobin. d) UV/Vis
spectra showing the reduction of metmyoglobin to oxymyoglobin with
10 mM NADH by LDH/LOX/CAT@SiNRs.
(5 mM:5 mM:50 mM). The normalized value of 100% at the y-axis
indicates the raw value for the fluorescence of 33000.
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two strong absorbance bands at 540 nm and 580 nm, together
reactions or for the development of artificial mitochondria as
with a decreased absorbance at 630 nm, which are typical for
oxyhemoglobin, were detected (Figure 6b).[25] This finding
suggested that our NAD+ regeneration system can be used for
hemoglobin reconstitution, relying on NADH-consumption
and NAD+ production. Additionally, we demonstrated that
the NAD+-regeneration module (LDH/LOX/CAT) was able
to produce hemoglobin from methemoglobin more rapidly at
the early stage of the reaction, even though NADH alone
would be able to produce hemoglobin gradually (Figure 6c
and Figure S27). The previous reports described the oxidation
of NADH to NAD+ without catalysis triggers the reduction of
Fe3+ to Fe2+,[26] whereas much higher and faster methemo-
globin reduction was obtained when the NAD+-regeneration
module was used in this study. We investigated the consump-
tion of NADH for the reduction of methemoglobin: a rapid
consumption of NADH was observed by the reaction of
LDH/LOX/CAT@SiNRs (Figure S28). In contrast, when
NADH was used alone or only LDH@SiNR was added, no
significant change in the NADH concentration was detected.
This implies that the reduction mainly relies on enzymatic
reactions. These results add a new function to LDH/LOX/
CAT@SiNRs and make it a promising replacement for
methemoglobin reductase in artificial hemoglobin regener-
ation. It will open potential applications for treatment of
Methemoglobinemia, combined with other oxygen carriers as
hemoglobin reconstitution modules, and other enzyme-medi-
ated reactions.
artificial organelles in living or synthetic cells. Moreover, the
self-fuelled module can quickly recover the irreversibly
oxidized methemoglobin (metmyoglobin) to the reduced
hemoglobin (myoglobin), implying the potential for a new
treatment to Methemoglobinemia and catalysing other redox
reactions.
Acknowledgements
This work is part of the MaxSynBio consortium, which was
jointly funded by the Federal Ministry of Education and
Research of Germany and the Max Planck Society. We thank
Dr. Robert Graf (MPIP) for 29Si solid-state NMR measure-
ments. We thank Petra Räder (MPIP) for TGA measure-
ments. We thank Dr. Young-Su Jeong (Agency for Defense
Development in South Korea) for helpful discussion about
enzymatic reactions. Open access funding enabled and
organized by Projekt DEAL.
Conflict of interest
The authors declare no conflict of interest.
Keywords: artificial metabolism ·encapsulation ·
enzyme reactions ·nanoreactors ·
In addition, the rapid conversion of metmyoglobin to
oxymyoglobin was also possible using the LDH/LOX/CAT@-
SiNR in the presence of 10 mM NADH (Figure 6d). A faster
reduction of metmyoglobin by the use of the enzyme modules
(1.76-fold) was achieved when compared to hemoglobin.
Myoglobin is a key oxygen carrier in the skeletal muscle tissue
of many vertebrates and in almost all mammals.[27] Unlike
hemoglobin, myoglobin has a single heme group. Due to
higher oxygen affinity than hemoglobin, myoglobin is an
attractive oxygen carrier for blood substitutes.[27b] Moreover,
the prevention of myoglobin autooxidation is an important
challenge in solving the problem of meat discoloration.[28]
Thus, these results could also possibly open a new avenue in
the meat industry.
nicotinamide adenine dinucleotide
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An enzymatic NAD+-regeneration nanoreactor was de-
veloped. The enzymes LDH, LOX, and CAT were encapsu-
lated into the silica nanoreactors by fluoride-catalyzed sol–gel
chemistry retaining their enzymatic activities and stability.
This “all-in-one” module allows their use in any NAD+-
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reaction mixture and the possibility of recycling. The nano-
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external fuelling or additional reagents through self-recycling
of all organic substrates. Only oxygen and water are needed.
The NAD+-regeneration was implemented into NAD+-de-
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Manuscript received: September 3, 2020
Accepted manuscript online: January 11, 2021
Version of record online: February 25, 2021
7734
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