Direct Biocatalytic Processes for CO2 Capture as a Green Tool to Produce Value-Added Chemicals

Abstract

Direct biocatalytic processes for CO2 capture and transformation in value-added chemicals may be considered a useful tool for reducing the concentration of this greenhouse gas in the atmosphere. Among the other enzymes, carbonic anhydrase (CA) and formate dehydrogenase (FDH) are two key biocatalysts suitable for this challenge, facilitating the uptake of carbon dioxide from the atmosphere in complementary ways. Carbonic anhydrases accelerate CO2 uptake by promoting its solubility in water in the form of hydrogen carbonate as the first step in converting the gas into a species widely used in carbon capture storage and its utilization processes (CCSU), particularly in carbonation and mineralization methods. On the other hand, formate dehydrogenases represent the biocatalytic machinery evolved by certain organisms to convert CO2 into enriched, reduced, and easily transportable hydrogen species, such as formic acid, via enzymatic cascade systems that obtain energy from chemical species, electrochemical sources, or light. Formic acid is the basis for fixing C1-carbon species to other, more reduced molecules. In this review, the state-of-the-art of both methods of CO2 uptake is assessed, highlighting the biotechnological approaches that have been developed using both enzymes.

Full text

Citation: Villa, R.; Nieto, S.; Donaire,
A.; Lozano, P. Direct Biocatalytic
Processes for CO2Capture as a Green
Tool to Produce Value-Added
Chemicals. Molecules 2023,28, 5520.
https://doi.org/10.3390/
molecules28145520
Academic Editor: Hua Zhao
Received: 31 May 2023
Revised: 14 July 2023
Accepted: 16 July 2023
Published: 19 July 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
molecules
Review
Direct Biocatalytic Processes for CO2Capture as a Green Tool to
Produce Value-Added Chemicals
Rocio Villa 1,2 , Susana Nieto 1, Antonio Donaire 3,* and Pedro Lozano 1 ,*
1Departamento de Bioquímica y Biología Molecular B e Inmunología. Facultad de Química,
Universidad de Murcia, 30100 Murcia, Spain; rocio.villa@um.es (R.V.); susanani@um.es (S.N.)
2Department of Biotechnology, Delft University of Technology, 2629 HZ Delft, The Netherlands
3Departamento de Química Inorgánica. Facultad de Química, Universidad de Murcia, 30100 Murcia, Spain
*Correspondence: adonaire@um.es (A.D.); plozanor@um.es (P.L.)
Abstract:
Direct biocatalytic processes for CO
2
capture and transformation in value-added chem-
icals may be considered a useful tool for reducing the concentration of this greenhouse gas in the
atmosphere. Among the other enzymes, carbonic anhydrase (CA) and formate dehydrogenase (FDH)
are two key biocatalysts suitable for this challenge, facilitating the uptake of carbon dioxide from
the atmosphere in complementary ways. Carbonic anhydrases accelerate CO
2
uptake by promoting
its solubility in water in the form of hydrogen carbonate as the first step in converting the gas into
a species widely used in carbon capture storage and its utilization processes (CCSU), particularly
in carbonation and mineralization methods. On the other hand, formate dehydrogenases represent
the biocatalytic machinery evolved by certain organisms to convert CO
2
into enriched, reduced,
and easily transportable hydrogen species, such as formic acid, via enzymatic cascade systems that
obtain energy from chemical species, electrochemical sources, or light. Formic acid is the basis for
fixing C
1
-carbon species to other, more reduced molecules. In this review, the state-of-the-art of both
methods of CO
2
uptake is assessed, highlighting the biotechnological approaches that have been
developed using both enzymes.
Keywords:
carbonic anhydrase; formate dehydrogenase; carbon capture storage and its utilization;
cofactor regeneration
1. Reducing Carbon Dioxide from the Air: The Challenge
One of the main challenges faced by humanity in the 21st century is climate change.
Over the last two centuries, the temperature of the Earth’s crust has risen progressively
and, since 1980, alarmingly, at a rate of 0.18
◦
C per decade. Indeed, last year’s average
temperature was 1.04
◦
C higher than the median temperature in the period prior to 1880 [
1
].
Temperature elevation drives an increase in extreme weather evidenced by a series of
well-known events (draughts, floodings, torrential downpours, etc.), the melting of large
extensions of frozen water, with the subsequent ascent of the sea, changes in ecosystems
with undefined outcomes and, in this sense, uncertainty on how these changes will affect
our way of living and welfare, with estimations that are clearly detrimental [
2
]. Moreover,
the acidification of seas and oceans is also a problem, with coral reef weakening already
having been detected, as well as the low level of oxygen present in marine life [3,4].
At the end of the nineteenth century, S.A. Arrhenius quantified the contribution of
“carbonic acid” (nowadays, carbon dioxide) to the greenhouse effect and was the first
in indicating that “The production of carbonic acid by the combustion of coal would therefore
suffice to cover the loss of carbonic acid by weathering and by peat formation seven times over.
Those are the two chief factors deciding the consumption of carbonic acid, and we thus recognize
that the percentage of carbonic acid in the air must be increasing at a constant rate as long as the
consumption of coal, petroleum, etc., is maintained at its present figure, and at a still more rapid
rate if this consumption should continue to increase as it does now”. He also concluded that
Molecules 2023,28, 5520. https://doi.org/10.3390/molecules28145520 https://www.mdpi.com/journal/molecules

Molecules 2023,28, 5520 2 of 52
this would lead to an increase in the temperature of Earth’s atmosphere [
5
,
6
]. Since then,
a huge amount of evidence correlating both air CO
2
concentration and global warming
has accumulated [
2
,
7
]. Moreover, there is a direct relationship between human activity,
carbon dioxide concentration, and climate change, that is, the anthropogenic origin of
global warming is well established. Atmospheric CO
2
concentration has increased from
280 ppm (year 1750) to 415 ppm (2021), this value being the highest concentration reached
in the last three million years [
8
]. This carbon dioxide increase is essentially related to
the emissions of this gas to the atmosphere as a consequence of the use of fossil fuels by
humans [
9
]. Other gases, such as methane and nitrous oxide, also contribute to global
warming, albeit to a minor extent (11 and 7%, respectively) [
10
]. The objective reached at
the Paris Climate Agreement in 2015 to maintain an increase in overall temperature below
2.0
◦
C with respect to preindustrial levels has recently been revised in the sense that such
increments should not exceed 1.5 ◦C [11].
In this scenario, any scientific strategy that allows for reducing the concentration of
CO
2
in the atmosphere is an object of interest, although the most relevant solution is to
avoid burning fossil energy sources that release CO
2
, and to substitute them with other
sustainable ones. Although more efficient and responsible use of fossil energy sources by
society is also essential to contributing to decreasing CO
2
emissions, the responsibility of
capturing the excess CO2already emitted is also inescapable.
Among other approaches, biocatalysts are useful tools for reducing the accumulation
of atmospheric CO
2
through either carbon capture and storage (CCS) or carbon capture
and its utilization (CCU, Figure 1A). Both approaches are often applied together, known
as carbon capture storage and utilization (CCSU) [
12
]. These methodologies require the
passing of CO
2
from gas to carbon solid and/or chemically reduced forms, a task that
directly implies chemistry in all its fields.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 56
consumption should continue to increase as it does now”. He also concluded that this would
lead to an increase in the temperature of Earth’s atmosphere [5,6]. Since then, a huge
amount of evidence correlating both air CO2 concentration and global warming has accu-
mulated [2,7]. Moreover, there is a direct relationship between human activity, carbon
dioxide concentration, and climate change, that is, the anthropogenic origin of global
warming is well established. Atmospheric CO2 concentration has increased from 280 ppm
(year 1750) to 415 ppm (2021), this value being the highest concentration reached in the
last three million years [8]. This carbon dioxide increase is essentially related to the emis-
sions of this gas to the atmosphere as a consequence of the use of fossil fuels by humans
[9]. Other gases, such as methane and nitrous oxide, also contribute to global warming,
albeit to a minor extent (11 and 7%, respectively) [10]. The objective reached at the Paris
Climate Agreement in 2015 to maintain an increase in overall temperature below 2.0 °C
with respect to preindustrial levels has recently been revised in the sense that such incre-
ments should not exceed 1.5 °C [11].
In this scenario, any scientific strategy that allows for reducing the concentration of
CO2 in the atmosphere is an object of interest, although the most relevant solution is to
avoid burning fossil energy sources that release CO2, and to substitute them with other
sustainable ones. Although more efficient and responsible use of fossil energy sources by
society is also essential to contributing to decreasing CO2 emissions, the responsibility of
capturing the excess CO2 already emied is also inescapable.
Among other approaches, biocatalysts are useful tools for reducing the accumulation
of atmospheric CO2 through either carbon capture and storage (CCS) or carbon capture
and its utilization (CCU, Figure 1A). Both approaches are often applied together, known
as carbon capture storage and utilization (CCSU) [12]. These methodologies require the
passing of CO2 from gas to carbon solid and/or chemically reduced forms, a task that di-
rectly implies chemistry in all its fields.
Figure 1. Three pathways to incorporating CO2 into soluble carbon forms by using enzymes. (A)
Formation of hydrogen carbonate using carbonic anhydrase; (B) photosynthesis performed using
RuBisCO protein; (C) reduction to achieve C1 in reduced form, formic acid, catalyzed using FDHs.
Approaches A and C are the aims of the present work (see text for details).
Figure 1.
Three pathways to incorporating CO
2
into soluble carbon forms by using enzymes. (
A
) For-
mation of hydrogen carbonate using carbonic anhydrase; (
B
) photosynthesis performed using Ru-
BisCO protein; (
C
) reduction to achieve C
1
in reduced form, formic acid, catalyzed using FDHs.
Approaches A and C are the aims of the present work (see text for details).
Research in this field has exponentially increased in the last decade. Indeed,
Figure 2A
shows the number of articles published, directly or indirectly, that relate either to car-

Molecules 2023,28, 5520 3 of 52
bon storage or carbon utilization per year, while Figure 2B,C display the percentages of
these articles classified by research area. As observed, research on carbon capture is espe-
cially intense in areas such as catalysts, synthesis, electrochemistry, and energy and fuels.
Nowadays, there are two main approaches for CO
2
transformation, namely biological and
chemical transformations [
13
]. In turn, biological CO
2
fixation can be photosynthetic or
not photosynthetic, while the chemical uptake can be divided into hydrogenation, car-
boxylation, mineralization, chemical reduction, and photochemical reduction. Although
hydrogenation is a well-established technology, nowadays, it is still mostly not green, as
it is obtained from the cracking of fossil fuels. The sustainable synthesis and transport of
hydrogen is indeed one of the main challenges related to energy sources. The main target
of hydrogenation is the production of methanol (see Section 7.2), although other reduced
molecules can also be obtained.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 56
Research in this field has exponentially increased in the last decade. Indeed, Figure
2A shows the number of articles published, directly or indirectly, that relate either to car-
bon storage or carbon utilization per year, while Figure 2B,C display the percentages of
these articles classified by research area. As observed, research on carbon capture is espe-
cially intense in areas such as catalysts, synthesis, electrochemistry, and energy and fuels.
Nowadays, there are two main approaches for CO2 transformation, namely biological and
chemical transformations [13]. In turn, biological CO2 fixation can be photosynthetic or
not photosynthetic, while the chemical uptake can be divided into hydrogenation, carbox-
ylation, mineralization, chemical reduction, and photochemical reduction. Although hy-
drogenation is a well-established technology, nowadays, it is still mostly not green, as it
is obtained from the cracking of fossil fuels. The sustainable synthesis and transport of
hydrogen is indeed one of the main challenges related to energy sources. The main target
of hydrogenation is the production of methanol (see Section 7.2), although other reduced
molecules can also be obtained.
Carboxylation is the process of directly converting CO2 into organic value-added
compounds. Organic carbonates and polymers are obtained through this process. An ex-
ample is the production of non-isocyanate polyurethanes (NIPUs) from glycerol car-
bonates derivatives obtained through CO2 cycloaddition to glycidol moieties [14]. In this
case, the authors designed a sustainable chemoenzymatic protocol for the synthesis of
glycerol carbonate acrylate (GCA) and glycerol carbonate methacrylate (GCMA) from
glycidol and CO2 by using ionic liquid (IL) technologies and enzymes, providing conver-
sions of up to 100% under low-pressure values (1–10 bar). These methods are still in their
first stages of development and hence a relatively novel field to work in.
Figure 2. (A) Number of articles published per year related to carbon capture storage (red) and
carbon capture utilization (blue); the insert is a vertical expansion, scale 0–15, by year, published in
the last century (the year 2023 contains the number of articles at halfway through the year). (B,C)
Percentage of articles related to CCS (B) and CCU (C) classified by research area. Source: compiled
by the authors, based on the Web of Science database (hps://clarivate.com), accessed 2 July 2023.
Figure 2.
(
A
) Number of articles published per year related to carbon capture storage (red) and carbon
capture utilization (blue); the insert is a vertical expansion, scale 0–15, by year, published in the last
century (the year 2023 contains the number of articles at halfway through the year). (
B
,
C
) Percentage
of articles related to CCS (
B
) and CCU (
C
) classified by research area. Source: compiled by the
authors, based on the Web of Science database (https://clarivate.com), accessed on 2 July 2023.
Carboxylation is the process of directly converting CO
2
into organic value-added
compounds. Organic carbonates and polymers are obtained through this process. An ex-
ample is the production of non-isocyanate polyurethanes (NIPUs) from glycerol carbonates
derivatives obtained through CO
2
cycloaddition to glycidol moieties [
14
]. In this case,
the authors designed a sustainable chemoenzymatic protocol for the synthesis of glycerol
carbonate acrylate (GCA) and glycerol carbonate methacrylate (GCMA) from glycidol and
CO
2
by using ionic liquid (IL) technologies and enzymes, providing conversions of up to
100% under low-pressure values (1–10 bar). These methods are still in their first stages of
development and hence a relatively novel field to work in.

Molecules 2023,28, 5520 4 of 52
Mineralization is described in Section 6.3 and is used in construction, for instance,
for generating cement. This technique allows for the uptake of large quantities of CO
2
for obtaining sustainable materials, although it is a highly consuming energy at a high
scale. Chemical, electrochemical and photochemical reduction combined with enzymes is
described below (see Sections 7.2 and 7.3).
The genuine physical and chemical properties of CO
2
make it a molecule that cannot
be easily captured or retained. Thus, CO
2
is a nonpolar molecule that resides in a gaseous
state under P and T standard conditions because of the weak van der Waals interactions
established between the molecules themselves. In addition, due to its null polarity, CO
2
has
a low diffusion coefficient in polar solvents (1.26
×
10
−5
cm
2
/s in water under standard
conditions), while its solubility follows Henry’s law (76.5 mM in water at 0
◦
C and partial
pressure of 1 atm) [
15
]. More importantly, its kinetics of capture by water, while increasing
with pH, is extremely low at neutral or acidic pH values, behaving as a Brönsted acid
according to Equations (1) and (2) (Scheme 1). The K
h
value (Equation (2)) indicates that
only a minimal fraction of CO
2
(aq) is present in an aqueous solution of carbonic acid
(ca. 1/600 of the molecules at neutral pH values). Both hydrated carbon dioxide and
carbonic acid behave as weak Brönsted acids because of the low deprotonation constants
(see Equations (3) and (4)) [16].
CO2(g)+H2OCO2(aq)(1)
CO2(aq)+H2OH2CO3→Kh=0.00159 (2)
CO2(aq)+H2OHCO−
3+H+→Ka1 =4.3 ×10−7M (3)
HCO−
3+H2OCO2−
3+H+→Ka2 =4.84 ×10−11 M (4)
CO2+NADH +H+HCOOH +NAD+(5)
Scheme 1.
Acid–base equilibria for CO
2
water-soluble species as a function of the pH
(Equations (1)–(4)
)
and reduction CO2equation to generate formic acid using the cofactor NADH (Equation (5)).
The equilibrium shown in Equation (3) is essential for the role of CO
2
as a buffer
between blood and cells, where the interconversion between CO
2
and HCO
3−
(hydro
carbonate or bicarbonate) forms should be fast in living beings. Under neutral or weak basic
conditions, the formation of bicarbonate anions is slow (the first order kinetic constant, k
1
, is
of the order 10
−2
s
−1
), while it increases in basic media [
17
]. Consequently, living organisms
must have a biocatalyst that allows for fast exchange between acid and basic species. Carbon
dioxide gas can be fixed either in aqueous soluble (hydrogen carbonate) or much more
insoluble (carbonate) forms using only alkalization or using other chemical processes
that incorporate carbon into value-added compounds. In nature, carbonic anhydrase
(CA) catalyzes the CO
2
/hydrogen carbonate reaction (Equation (3)), enabling CO
2
capture
within reasonable times [
17
,
18
]. Thus, CA is used as a tool for incorporating CO
2
as a
hydrogen carbonate anion in soluble species in a multitude of chemical approaches [
19
,
20
].
In contrast, carbonates are usually highly insoluble. It follows that the use of alkaline
reactants (Equation (4)) is the easiest and, consequently, main method for sequestering CO
2
by forming the corresponding salts or their derivatives.
Alternatively, CO
2
can be fixed to hydrogen-enriched energy forms via several natural
mechanisms [
21
,
22
]. Although the most extended and productive mechanism in nature
(green plants and algae) is photosynthesis, where ribulose-1,5-bisphosphate carboxylase-
oxygenase (RuBisCo) converts CO
2
and water into C
3
-carbohydrates by taking energy from
light (Figure 1B) [
22
–
24
], here, the focus is on nonphotosynthetic enzymes. The conversion
of CO
2
into formate anion (C
1
species, Equation (5), Scheme 1) is the simplest way in which
nature captures CO
2
into reduced, highly energetic molecules, and formate dehydrogenases
(FDH) are the main actors in this performance (Figure 1C) [25–29].

Molecules 2023,28, 5520 5 of 52
Formic acid, or its basic form formate, is a simple molecule for efficient hydrogen
transport. Moreover, formate synthesis is the first step for obtaining other more complex
and energetically enriched molecules [
22
,
30
]. Carbon dioxide and formic acid have similar
formation energies (Figure 3A), with the most similar energetic states for C
1
-carbon forms.
This converts formic acid as the easiest starting point for obtaining other C1 more reduced
carbon species. Nevertheless, reaction 5, in the forward sense, is highly endergonic under
physiological conditions. Indeed, the redox potential at pH 7 for CO
2
reduction to formate
is −430 mV [31], while that of NADH is only −320 mV (Figure 3B). More importantly, for
the reasons discussed below, this first step is kinetically, energetically, and economically
expensive, and it becomes the bottleneck for obtaining value-added products in CO
2
regen-
eration. Several approaches for circumventing this issue are discussed below. However,
other no less relevant problems arise when this reaction is performed for biotechnological
purposes. FDHs from different organisms can solve these problems and incorporate for-
mate anions into their biosynthetic routes. Thus, nature provides solutions to the previous
difficulties. It is a researcher’s task to adjust these solutions at laboratory and industrial
levels for the benefit of society.
Molecules 2023, 28, x FOR PEER REVIEW 5 of 56
in which nature captures CO2 into reduced, highly energetic molecules, and formate de-
hydrogenases (FDH) are the main actors in this performance (Figure 1C) [25–29].
Formic acid, or its basic form formate, is a simple molecule for efficient hydrogen
transport. Moreover, formate synthesis is the first step for obtaining other more complex
and energetically enriched molecules [22,30]. Carbon dioxide and formic acid have similar
formation energies (Figure 3A), with the most similar energetic states for C1-carbon forms.
This converts formic acid as the easiest starting point for obtaining other C1 more reduced
carbon species. Nevertheless, reaction 5, in the forward sense, is highly endergonic under
physiological conditions. Indeed, the redox potential at pH 7 for CO2 reduction to formate
is −430 mV [31], while that of NADH is only −320 mV (Figure 3B). More importantly, for
the reasons discussed below, this first step is kinetically, energetically, and economically
expensive, and it becomes the boleneck for obtaining value-added products in CO2 re-
generation. Several approaches for circumventing this issue are discussed below. How-
ever, other no less relevant problems arise when this reaction is performed for biotechno-
logical purposes. FDHs from different organisms can solve these problems and incorpo-
rate formate anions into their biosynthetic routes. Thus, nature provides solutions to the
previous difficulties. It is a researcher’s task to adjust these solutions at laboratory and
industrial levels for the benefit of society.
Figure 3. (A) Free formation energies of some C1 compounds under standard conditions. (B) Redox
potentials vs. the normal hydrogen electrode (NHE) at pH 7 of some relevant compounds, photo-
synthesizers, and sacrificial agents (see Section 6.4) commented in the text [32,33]. The redox poten-
tial values are shown between parentheses. The HOMO and LUMO energy orbitals for some dyes
are indicated in green and purple, respectively.
In the following pages, the enzymes that participate in both processes of CO2 capture
using CA (CCUS processes) and FDH (formate synthesis) are described. How these natu-
ral systems circumvent the thermodynamics and kinetics problems derived from reactions
3 and 5 are also discussed, paying special aention to the state-of-the-art concerning the
biotechnological applications of these enzymes for CCS and CCU technologies, as well as
for reducing CO2 to formic acid.
2. Carbonic Anhydrases: Efficient Devices for CO2 Uptake
2.1. Classification and Structure of Carbonic Anhydrases
Carbonic anhydrases (EC 4.2.1.1) are found in all living kingdoms [18,34–36]. They
catalyze the reaction of interconversion between CO2 and HCO3- (Equation (3)) in nature
and are generally monomeric proteins with molecular weights roughly comprised
Figure 3.
(
A
) Free formation energies of some C
1
compounds under standard conditions. (
B
) Redox
potentials vs. the normal hydrogen electrode (NHE) at pH 7 of some relevant compounds, photosyn-
thesizers, and sacrificial agents (see Section 6.4) commented in the text [
32
,
33
]. The redox potential
values are shown between parentheses. The HOMO and LUMO energy orbitals for some dyes are
indicated in green and purple, respectively.
In the following pages, the enzymes that participate in both processes of CO
2
capture
using CA (CCUS processes) and FDH (formate synthesis) are described. How these natural
systems circumvent the thermodynamics and kinetics problems derived from reactions
3 and 5 are also discussed, paying special attention to the state-of-the-art concerning the
biotechnological applications of these enzymes for CCS and CCU technologies, as well as
for reducing CO2to formic acid.
2. Carbonic Anhydrases: Efficient Devices for CO2Uptake
2.1. Classification and Structure of Carbonic Anhydrases
Carbonic anhydrases (EC 4.2.1.1) are found in all living kingdoms [
18
,
34
–
36
]. They
catalyze the reaction of interconversion between CO
2
and HCO
3−
(Equation (3)) in nature
and are generally monomeric proteins with molecular weights roughly comprised between
30–50 kDa, depending on their class. CAs have been classified into:
α
-CAs (found in animal
cells, algae, and eubacteria),
β
-CAs [
37
] (found in higher plants, microalgae, eubacteria,
archaebacteria, and fungi),
γ
-CAs [
37
] (algae),
δ
-CA [
38
–
40
] (found in the marine diatom

Molecules 2023,28, 5520 6 of 52
Thalassiosira weissflogii),
ε
-CAs [
41
] (found, for example, in the carboxysomal shell of
Halothiobacillus neapolitanus),
ζ
-CA [
42
] (found in Thalassiosira weissflogii),
η
-CA [
43
](found
in Plasmodium falciparum),
θ
-CA [
44
], and
ι
-CA [
45
–
47
] (found in Burkholderia territorii and
Phaeodactylum tricornutum, among other bacteria). CAs are zinc(II) enzymes, although some
of them can contain other metal ions active in physiological roles: Cd(II) has been found in
δ-CA, although its role is disputed [48,49]; Fe(II) is present in some γ-CAs growing under
anaerobic conditions [
50
]; Co(II) has also been replaced in many
α
-CAs with excellent
activities [
51
], although its original role has not been proven. Remarkably, a group of
proteins called “COG4337” has been described as
ι
-CAs, and strikingly, those from the
cyanobacterium Anabaena sp. PCC7120 and the chlorarachniophyte alga Bigelowiella natans
display activity without any metal involved [
52
]. Table 1lists the main features of some
representative CAs.
Table 1. Features of representative carbonic anhydrases.
Type Organism MW (kDa) Activity
(WAU/mg) Activity (k−1)Metal
Coordination Ref.
α
Bovine 29.8 2540
Zn(II), 3 His, H
2
O
[53]
Homo sapiens (HCAI) 28.7 920 2.0 ×105[54]
Homo sapiens (HCAII) 29.1 8000 1.4 ×106[54]
Persephonella marina 26.9 1748 [55]
Thermosulfurimonas dismutans 27.9 2032 [53]
Thermovibrio ammonificans 25.3 1016 [53]
Bacillus halodurans 37.0 3425 [56]
Sulphurihydrogenibium yellowstonense 26.0 7254 [57]
β
Bacillus subtilis 37.0 714
Zn(II), His, 2 Cys
[58]
Acetobacterium woodii 22.0 1814 [55]
Methanobacterium thermoautotrophicum 19.9 580 [55]
Aspergillus fumigatus 23.0 20 [59]
γ
Geobacillus kaustophilus 22.0 179
Zn(II) or Fe(II), 3
His, H2O
[60]
Thermus thermophilus HB8 24.3 a0.9 [61]
Methanosarcina thermophila 40.0 4872 [62]
Burkholderia pseudomallei 28.2 5.3 ×105[63]
Vibrio cholerae 26.3 7.39 ×105[63]
Porphyromonasgingivalis 26.2 4.1 ×105[63]
δThalassiosira weissflogii, TWCA1 27.0 1.3 ×105
Zn(II), 3 His, H
2
O
[64]
δEmiliania huxleyi 18.3 1.3 ×106 (b)
Zn(II), 3 His, H
2
O
[65]
ζThalassiosira weissflogii, CDCA1 69 a1.5 ×106Cd(II) or Zn(II),
His, 2 Cys [42,66]
ηPlasmodium falciparum 26.2 1.4 ×105Zn(II), 2 His, Gln,
H2O[43,67]
θ
Thalassiosirapseudonana 26.0 122 Cd(II) or Zn(II),
His, 2 Cys
[38]
Phaeodactylum tricornutum 31.1 30.9 [47]
i
Burkholderia territorii 28.2 3.0 ×105
Cd(II) or Zn(II),
His, 2 Cys
[46]
Anabaena sp. PCC 7120 c19.3 16.7 [52]
Bigelowiella natans d55.3 85.8 [52]
a
A trimer formed by three chains, R1, R2, and R3.
b
CA activity in the cytoplasm.
c
Activity of the GOG4337
recombinant protein all2909 (ref. [52]). dActivity of the GOG4337 recombinant protein Bn86287 (ref. [52]).
Mammalian CAs belong to the
α
-CA class. Depending on their location and primary
sequences, several isoforms of human carbonic anhydrases (HCA) have been described,

Molecules 2023,28, 5520 7 of 52
with HCAII being the most studied and best-characterized CA. The HCAII tertiary structure
(Figure 4A) consists of a unique domain containing ten
β
-strands that twist to form a
β
-sheet (eight of them organized in an antiparallel arrangement and the other two in
parallel) [
68
,
69
]. Surrounding these
β
-sheets, up to eight other
α
-helixes are located on the
surface of the protein.
Molecules 2023, 28, x FOR PEER REVIEW 7 of 56
Mammalian CAs belong to the α-CA class. Depending on their location and primary
sequences, several isoforms of human carbonic anhydrases (HCA) have been described,
with HCAII being the most studied and best-characterized CA. The HCAII tertiary struc-
ture (Figure 4A) consists of a unique domain containing ten β-strands that twist to form a
β-sheet (eight of them organized in an antiparallel arrangement and the other two in par-
allel) [68,69]. Surrounding these β-sheets, up to eight other α-helixes are located on the
surface of the protein.
The structure of HCA It’s aI is the most studied of all HCA isoforms, and it’s aactive
site can be defined as a cone-shaped cleft, 15 Å deep, formed by a hydrophilic region (Tyr7,
Asn62, His64, Asn67, Thr199, and Thr200) and a hydrophobic region (Val121, Val143,
Leu198, Val207, and Trp209). At the boom of the cavity lies the Zn(II) ion, tetrahedrally
coordinated by three conserved histidines (His94, His96, and His119) and a solvent mole-
cule. Although the core of the active site in α-CAs is highly conserved, there is variability
in the polarity and hydropathicity of its periphery [70].
Figure 4. (A) Three-dimensional structure of human CA (5ZXW pdb code [68]). (B) Active center of
the enzyme showing the most relevant amino acids (see text for details). (C) CA mechanism of ac-
tion.
The HCA family includes a subclass of three noncatalytic isoforms (HCAs VIII, ×,
and XI) called CA-related proteins (CA-RPs), whose classification is based on their se-
quence. The noncatalytic behavior is due to the absence of one or more histidines that
coordinate the Zn(II) ion of a catalytic HCA isoform. For instance, in HCA-RP VIII, the
Zn-coordinating His94 (HCA II numbering) is replaced by an arginine (Arg116, according
to HCA-RP VIII numbering). This residue avoids CO2 hydration in the first step of CA
catalysis. [71]. Although the biological functions of CA-RPs have not been defined, these
isoforms are of high interest in different scientific research fields. Recently, the X-ray crys-
tal structure of only one HCA-RP (HCA-RP VIII) was determined [72]. HCA-RP VIII is
expressed in the cerebellum [73] and has been identified as a binding partner for the ino-
sitol 1,4,5 triphosphate (IP3) receptor type [74]. It should be mentioned that the stability
and structures of other HCAs such as HCA III or extracellular HCAs (i.e., IV, VI, IX and
XIV) have not been as extensively studied as HCAs I and II. For instance, the bovine CA
III showed a similar unfolding profile to that of HCA II, providing a molten globule inter-
mediate and an unfolded state at a Cm of 2.6 M GuHCl concentration [75]. On the contrary,
the isoforms VIII, ×, and XI showed two distinct transitions, and their sensitivity to guan-
idinium chloride chemical denaturalization was higher than that of HCA II (Cm 0.4 M for
HCA-RP VIII and 0.9 M for HCA II) [71].
The catalytic Zn(II) ion is located in a deep-centered slot that is accessible to the sol-
vent. The Zn(II) ion is coordinated to three histidine residues (Figure 4B). While His94 and
96 are coordinated through their Nε2 imidazole nitrogen to the metal ion, His119 is bound
through its Nδ1 imidazole nitrogen (HCAII numeration) [48]. A water molecule completes
Figure 4.
(
A
) Three-dimensional structure of human CA (5ZXW pdb code [
68
]). (
B
) Active center of
the enzyme showing the most relevant amino acids (see text for details). (
C
) CA mechanism of action.
The structure of HCA It’s aI is the most studied of all HCA isoforms, and it’s aactive
site can be defined as a cone-shaped cleft, 15 Å deep, formed by a hydrophilic region
(Tyr7, Asn62, His64, Asn67, Thr199, and Thr200) and a hydrophobic region (Val121, Val143,
Leu198, Val207, and Trp209). At the bottom of the cavity lies the Zn(II) ion, tetrahedrally co-
ordinated by three conserved histidines (His94, His96, and His119) and a solvent molecule.
Although the core of the active site in
α
-CAs is highly conserved, there is variability in the
polarity and hydropathicity of its periphery [70].
The HCA family includes a subclass of three noncatalytic isoforms (HCAs VIII,
×
, and
XI) called CA-related proteins (CA-RPs), whose classification is based on their sequence.
The noncatalytic behavior is due to the absence of one or more histidines that coordinate the
Zn(II) ion of a catalytic HCA isoform. For instance, in HCA-RP VIII, the Zn-coordinating
His94 (HCA II numbering) is replaced by an arginine (Arg116, according to HCA-RP VIII
numbering). This residue avoids CO
2
hydration in the first step of CA catalysis. [
71
].
Although the biological functions of CA-RPs have not been defined, these isoforms are
of high interest in different scientific research fields. Recently, the X-ray crystal structure
of only one HCA-RP (HCA-RP VIII) was determined [
72
]. HCA-RP VIII is expressed
in the cerebellum [
73
] and has been identified as a binding partner for the inositol 1,4,5
triphosphate (IP3) receptor type [
74
]. It should be mentioned that the stability and structures
of other HCAs such as HCA III or extracellular HCAs (i.e., IV, VI, IX and XIV) have not
been as extensively studied as HCAs I and II. For instance, the bovine CA III showed a
similar unfolding profile to that of HCA II, providing a molten globule intermediate and an
unfolded state at a C
m
of 2.6 M GuHCl concentration [
75
]. On the contrary, the isoforms VIII,
×
, and XI showed two distinct transitions, and their sensitivity to guanidinium chloride
chemical denaturalization was higher than that of HCA II (C
m
0.4 M for HCA-RP VIII and
0.9 M for HCA II) [71].
The catalytic Zn(II) ion is located in a deep-centered slot that is accessible to the solvent.
The Zn(II) ion is coordinated to three histidine residues (Figure 4B). While His94 and 96
are coordinated through their N
ε
2 imidazole nitrogen to the metal ion, His119 is bound
through its N
δ
1 imidazole nitrogen (HCAII numeration) [
48
]. A water molecule completes
the tetrahedral coordination of Zn(II). A second coordination sphere is formed by amino
acids that are not directly coordinated with the metal ion but are essential in the catalytic
process. Through the formation of a network of hydrogen bonds, residues Tyr7, Asn62,

Molecules 2023,28, 5520 8 of 52
His64, Asn67, Glu106, Thr199, and Thr200 stabilize the mediator species in such a way that
the reaction can occur.
2.2. CA Mechanism of Action
Carbonic anhydrase accelerates reaction 3 by more than six orders of magnitude
(Table 1) with respect to its rate without the biocatalyst [
18
,
76
] (from ca.
3.6 ×10−2M−1s−1
to 1.0
×
10
6
M
−1
s
−1
in the absence and presence of CA) [
17
], allowing for the reaction
to take place under physiological conditions. Otherwise, CO
2
cannot be assimilated as
bicarbonate by living organisms.
The kinetic parameters of CA can be determined by measuring hydratase activity
(Equation (3), Scheme 1). The method basically consists of saturating a buffered water
solution (typically Tris.HCl 0.020 M, pH 8.3) kept in an ice bath with CO
2
at a fixed high pH
value, then adding CA, and measuring the time that the solution takes to reach a low pH
value (generally ca. 6.3) because of the conversion of carbon dioxide to hydrogen carbonate
anion that takes the capture of protons (Equation (3)) and hence lowers pH. The Wilbur–
Anderson hydratase activity unit [
77
] is calculated as the ratio ((t
0−
t)/t
0
)/(mg enzyme),
where t
0
and tare the measured times that an indicator present in the solution shifts its
color for the control (without CA) and the sample (in the presence of CA), respectively.
This method provides a qualitative measurement of CA activity, but it is not strictly
transferable from one set of experiments to another [
78
]. This method depends on the
degree of saturation of CO
2
(which in turn changes with temperature and the time at
which CO
2
is bubbled), the nature of the buffer, its concentration, and its initial pH value.
Together, these units are roughly—not strictly—comparable. Most classes of CAs also
exhibit esterase activity (
δ
-CA lacks esterase activity), whose measurement is more direct
and contrastable [
79
]. This is generally performed by the hydrolysis of p-nitrophenyl
acetate, which releases free p-nitrophenol, with maximum absorption at 400 nm, which
is easily measurable [
80
]. Accurate experimental requirements for the latter experiments
(concentration of the reactants, enzyme, and pH) are easily reproducible and should depend
only on contrastable and exchangeable conditions.
The mechanism of action of CA has been deciphered [
81
] and basically consists of two
stages (Figure 4C). In the first phase, a CO
2
molecule, partially stabilized by interactions
with groups of the enzyme active site, reaches the active center, and subsequently, the
hydroxide group bound to the Zn(II) ion attacks the CO
2
carbon atom (nucleophilic attack).
Then, hydrogen carbonate is formed, and a water molecule replaces it through Zn(II)
coordination. In the second step, a proton is transferred from the Zn(II)-coordinated water
molecule to the solvent. This is the rate-limiting step. His64 is the amino acid responsible
for accepting this proton, which is finally transferred to the bulk solvent. Due to this
mechanism, at neutral/soft acid pH levels, the rate of CO
2
/HCO
3−
conversion is enhanced
by more than six orders of magnitude, making life possible. At pH values higher than 9.5, a
direct hydroxide attack can form carbonate anions at rates comparable to those performed
by CA at neutral pH values.
3. Formate Dehydrogenases: Natural Machines for Reducing CO2
The specific reduction in carbon dioxide converts it into formate/formic acid
(
Equation (5)
, Scheme 1). This C
1
metabolism reaction occurs in hydrogenotrophic
methanogens (Euryarcheota) and autotrophic acetogens (bacteria) and is carried out by
the enzyme formate dehydrogenase (EC 1.17.1.9) [
24
,
82
–
85
]. Energy and high reduction
power (i.e., a cofactor in the NAD(P)H form) are required to perform this process. In
contrast, formate dehydrogenases also catalyze the backward reaction (5), oxidizing for-
mate anions and obtaining energy from them. FDHs are divided into two main groups:
nonmetal- or NAD-dependent FDHs, and metal-dependent FDHs. The structure, nature,
and activity of these two FDH sets are completely different. More importantly, the ability
of these two groups to reduce CO2is manifestly diverse, as discussed below.

Molecules 2023,28, 5520 9 of 52
3.1. Metal-Independent/NAD-Dependent FDHs
Metal-independent FDHs belong to the family of D-specific 2-oxoacyd dehydrogenases.
They are present in bacteria, yeast, plants, and mammals, are globular proteins with ca.
350–400 amino acids
, depending on the species, and usually form homodimers [
25
,
26
,
86
].
Each FDH monomer contains two domains, one destined to allocate the substrate (the
catalytic domain) and another pocket that allows for the binding of the NADH cofactor.
The function of metal-independent FDHs is generally associated with mechanisms for ob-
taining energy from the oxidation of formate anions in methanogenic pathways (backward
reaction 5), that is, these FDHs are machines efficient in catalyzing backward reaction,
although much less effective in performing CO
2
reduction [
25
,
26
,
84
,
87
]. The cofactor of
all known metal-independent FDHs is NAD(P)
+
/NAD(P)H, and, consequently, they are
also called NAD(P)-dependent FDHs. FDH from the methylotropic yeast Candida boidinii
(Cb) is the most studied and best characterized nondependent FDH, since it was the first
FDH expressed in Escherichia coli, and it is commercially available and relatively inexpen-
sive
[88,89]
. As shown in Figure 5A, the enzyme (364 amino acids) consists of 15
α
-helices
and 13
β
-strands [
90
]. There is a deep groove between both domains that allows both the
substrate and the cofactor to be bound in this cavity with short contacts between them.
Figure 5B displays the active site of the enzyme, including both the NAD
+
binding site and
the most significant amino acids concerning catalytic properties. NAD
+
cofactor strongly
binds the protein through its adenine, ribose, and phosphate moieties in such a way that
it can only be removed by extensive washing with, for instance, 2 M sodium chloride.
Formate binds closely to the nicotinamide group through the positively charged residue
Arg258. The mechanism of formate oxidation (Figure 5C) consists of hydride transfer to
the nicotinamide oxidized group and the release of the CO
2
formed. The intermediated
anion is stabilized by an arginine (Arg258 CbFDH numbering). The groove where the
nicotinamide group and the formate anion are located is hydrophobic; hence, the hydride
anion cannot interact with the solvent, so the reaction can take place. Both high formate
and NAD
+
strong binding with positive residues of the enzyme (Gln287, His311) reduce
the ability to exchange the products of the reaction. In turn, this is one of the main factors
that decrease the efficient recycling of the cofactor, making these FDHs, in general, not
excellent biocatalysts for CO2reduction.
Molecules 2023, 28, x FOR PEER REVIEW 10 of 56
Figure 5. (A) Three-dimensional structure of CbFDH with NAD+ and azide anion (6D4C pdb code
[90]). The two dimers are colored in red and green, while the NAD+ cofactor is colored in orange.
(B) Active center of CbFDH showing the arrangement of the cofactor (pink) as well as the most rel-
evant amino acids closest the NAD+, as discussed in the text. (C) Mechanism of action of NAD+-
dependent FDHs for formate oxidation (blue arrows) or formate reduction (inverse sense, red ar-
rows); the NAD nicotinamide group in its oxidized and reduced state is displayed. (D) Active center
of CtFDH (6T8Z pdb code [91]) showing the four residues mutated in both works cited in the text
[92,93], the numbering corresponding to CtFDH.
With wild-type CbFDH being not ideal for forward reaction 5 [94,95], specific mutants
increase its capability to capture CO2. For instance, the double mutant V120S-N187D was
shown to substantially increase CO2 reduction [96]. Other FDHs have kcat and kM values
that are higher than wild-type CbFDHs and its mutants (Table 2). Choe et al. studied Thi-
obacillus sp. KNK65MA FDH (TsFDH) and concluded that it presented 84-fold higher cat-
alytic efficiency for CO2 reduction than CbFDH [67]. NAD-FDH mutants with higher effi-
ciency have been successfully designed [89,96]. Binay and coworkers obtained and puri-
fied mutants of Candida methylica (CmFDH) and another four mutants from Chaetamium
thermophilum (CtFDH) in amino acid positions close to the cofactor binding [92,93]. The
highest activity was found for the Asn120Cys mutant in CtFDH, for which the kcat value
increased 6.5-fold, the same increment observed for the kM value, which indicated that the
efficiency of CO2 reduction was due to the lower affinity for the substrate, that is, for the
ability to release formate. As observed in Figure 5D, Asn120 is located close to the nicotin-
amide NAD group; specifically, the amide nitrogen of Asn120 is as close as 3.8 Å from the
nicotinamide ring. Its mutation by a smaller cysteine residue introduces more space in the
active site and, consequently, higher conformational flexibility, which facilitates the re-
lease of formate anion [92]. His96 interacts with the hydrogen carbonate anion stabilizing
the hydride in the transition state, as confirmed by molecular dynamics performed on the
double mutant. Based on kinetic and molecular dynamic studies, the authors concluded
that subtle structural changes around the Asn120 position allowed for the location of two
molecules of hydrogen carbonate instead of one of formate, favoring the CO2 forward re-
action taking place. On the other hand, replacing key residues G93H/I94Y in CmFDH,
Figure 5.
(
A
) Three-dimensional structure of CbFDH with NAD
+
and azide anion (6D4C pdb
code [90]). The two dimers are colored in red and green, while the NAD+cofactor is colored in

Molecules 2023,28, 5520 10 of 52
orange. (
B
) Active center of CbFDH showing the arrangement of the cofactor (pink) as well as the
most relevant amino acids closest the NAD
+
, as discussed in the text. (
C
) Mechanism of action of
NAD
+
-dependent FDHs for formate oxidation (blue arrows) or formate reduction (inverse sense,
red arrows); the NAD nicotinamide group in its oxidized and reduced state is displayed. (
D
) Active
center of CtFDH (6T8Z pdb code [
91
]) showing the four residues mutated in both works cited in the
text [92,93], the numbering corresponding to CtFDH.
With wild-type CbFDH being not ideal for forward reaction 5 [
94
,
95
], specific mutants
increase its capability to capture CO
2
. For instance, the double mutant V120S-N187D was
shown to substantially increase CO
2
reduction [
96
]. Other FDHs have k
cat
and k
M
values
that are higher than wild-type CbFDHs and its mutants (Table 2). Choe et al. studied
Thiobacillus sp. KNK65MA FDH (TsFDH) and concluded that it presented 84-fold higher
catalytic efficiency for CO
2
reduction than CbFDH [
67
]. NAD-FDH mutants with higher
efficiency have been successfully designed [
89
,
96
]. Binay and coworkers obtained and
purified mutants of Candida methylica (CmFDH) and another four mutants from Chaetamium
thermophilum (CtFDH) in amino acid positions close to the cofactor binding [
92
,
93
]. The
highest activity was found for the Asn120Cys mutant in CtFDH, for which the k
cat
value
increased 6.5-fold, the same increment observed for the k
M
value, which indicated that
the efficiency of CO
2
reduction was due to the lower affinity for the substrate, that is,
for the ability to release formate. As observed in Figure 5D, Asn120 is located close to
the nicotinamide NAD group; specifically, the amide nitrogen of Asn120 is as close as
3.8 Å from the nicotinamide ring. Its mutation by a smaller cysteine residue introduces
more space in the active site and, consequently, higher conformational flexibility, which
facilitates the release of formate anion [
92
]. His96 interacts with the hydrogen carbonate
anion stabilizing the hydride in the transition state, as confirmed by molecular dynamics
performed on the double mutant. Based on kinetic and molecular dynamic studies, the
authors concluded that subtle structural changes around the Asn120 position allowed for
the location of two molecules of hydrogen carbonate instead of one of formate, favoring the
CO
2
forward reaction taking place. On the other hand, replacing key residues G93H/I94Y
in CmFDH, located in the catalytic pocket of FDH, increased the catalytic efficiency (k
cat
/k
M
)
of the wild-type protein 5.4-fold for the reduction of HCO
3−
. Here, K
M
values do not vary
significantly, while k
cat
/k
M
does. The authors suggested that there is a reorganization
in the active site that enlarges the space and allows for the reactant (carbonate anion) to
adopt a better orientation for catalysis, and so it becomes easier for the HCO
3−
to reach the
nicotinamide ring and the reaction is produced in a faster way [93].

Molecules 2023,28, 5520 11 of 52
Table 2.
Kinetic and thermodynamic parameters of most relevant FDHs, classified into their two groups. Molecular weights and pH conditions in which these
conditions were determined are also indicated. For metal-dependent FDHs, the types and number of their subunits as well as the cofactors of their active centers are
also indicated.
A. NAD-Dependent FDHs.
Formate Oxidation CO2reduction
Organism MW (kDa) apH kcat (s−1)
(U mg−1)bKM(mM) kcat/KM
(mM−1s−1)pH kcat (s−1)
(U mg−1)KM(mM) kcat/KM
(mM−1s−1)ref
Myceliophthora thermophila 42 10.5 0.32 7.2 0.04 7.0 0.10 0.43 0.23 [97]
Ancylobacter aquaticus 45 6.0 (21.6) 6.0 (23) 4.5 [98]
Candida boidinii 41 7.0 1.081 (6.1) 8.55 0.13 5.5 0.015 2.6 0.006 [98]
Thiobacillus sp. KNK65MA 45 6.5 1.769 (10.9) 16.24 0.11 5.5 0.32 0.95 0.34 [98]
Candida methylica 42 8.0 1.31 (13.2) 7.01 0.19 8.0 0.008 0.078 0.01 [97]
Chaetomium thermophilum 45 5.0 2.04(3.1) 3.30 0.62 5.0 0.023 3.29 0.069 [93,99]
Ceriporiopsis subvermispora 40 6.5 (1.3) 6.0 (0.8) [98]
Moraxella sp. C-1 45 5.5 (14.3) 5.5 (2.8) [98]
Paracoccus sp. 12-A 45 5.5 (12.2) 5.5 (6.5) [98]
B. Metal-Dependent FDHs
Formate Oxidation CO2reduction
Organism MW a(kDa) Subunits bCofactors pH kcat(s−1)
(U mg−1)aKM(mM) kcat/KM
(mM−1s−1)pH kcat(s−1)
(U mg−1)aKM(mM) kcat/KM
(mM−1s−1)ref
Syntrophobacter
fumaroxidans FDH-1 175 (αβγ)2 W, SeCys
4 [Fe2S2]
[Fe4S4] 7.0 (700) 0.04 - 282 (900) - - [100]
Syntrophobacter
fumaroxidans FDH-2 125 (αβ)2 W, SeCys
2 [Fe2S2] 7.0 (2700) 0.01 - 282 (89) - - [100]
Desulfovibrio
desulfuricans 135 (αβγ)3
Mo, SeCys
2 MGDs
4c-heme
2 [Fe4S4]
8.0 543 0.0571 9526 7.0 46.6 0.0157 2968 [101]
Escherichia coli
FDH-H 79 αβ
Mo, SeCys
2 MGDs
1 [Fe4S4] 7.5 2800 26 107.7 7.5 1.0 8.3 0.12 [102,103]
Desulfovibrio
vulgaris
Hildenborough 97.4 (αβχ)3 Mo, SeCys
4 [Fe4S4]
4c-heme 7.6 1310 (77) 0.017 77.06 315(1.0) 0.42 750 [104,105]
Acetobacterium
woodii 169 (αβ)3 Mo SeCys
[4Fe-4S]) 7.0 (600) 1.0 - 7.0 372 (132) 3.8 97.9 [106]

Molecules 2023,28, 5520 12 of 52
Table 2. Cont.
B. Metal-Dependent FDHs
Formate Oxidation CO2reduction
Organism MW a(kDa) Subunits bCofactors pH kcat(s−1)
(U mg−1)aKM(mM) kcat/KM
(mM−1s−1)pH kcat(s−1)
(U mg−1)aKM(mM) kcat/KM
(mM−1s−1)ref
Cupriavidus necator 178 (αβγ)3
Mo,
4 [Fe4S4]
FMN
3 [Fe2S2]
7.0 140 0.082 1707 7.0 11 2.7 4.07 [107]
Rhodobacter
capsulatus 180 (αβγ)2 Mo, 4 Fe4S4,
1Fe2S2,
2 MGD, FMN 5.0 36.5 281 0.13 7.7 1.48 - - [108,109]
Pseudomonas
oxalaticus 315 - Mo,
2 FMN - 0.135 - 6.2 3.0 40 0.075 [110,111]
Clostridium
Ijungdahlii 80 - W Cys
2 MGD
[Fe4S4] 9.0 14.77 1.40 10.55 7.0 0.73 7.27 0.17 [112,113]
Clostridium
autoethanogenum 74 W
2 MGD
[Fe4S4] 9.0 1.04 4.51 0.231 7.0 4.00 23.15 0.17 [113]
Clostridium coskatii 62 W
2 MGD
[Fe4S4] 9.0 0.62 5.57 0.111 7.0 5.62 59.65 0.094 [113]
Clostridium ragsdalei 74 W 9.0 11.88 44.83 0.265 7.0 3.28 31.20 0.11 [113]
Desulfovibrio gigas 121 (αβ)2 W, SCys
4 [Fe4S4] 8.0 (34.1) - - - - - - [114]
Moorella
thermoacetica (αβ)2 W, SeCys
4 [Fe4S4] 7.5 (1100) - - - - - - [115]
Sub-Table (A):
a
Molecular weight of the monomer (most FDHs are associated with dimers).
b
Data between parenthesis refer to the activity in units (
µ
mol min
−1
) of formate/CO
2
oxidized/reduced per mg of FDH; Sub-Table (B):
a
Molecular weight of the monomer.
b
The number refers to the number of subunits, that is, the composition of dimers or trimers of
each FDH. MGD: molybdopterin guanidine dinucleotide. FMN: flavin mononucleotide.

Molecules 2023,28, 5520 13 of 52
3.2. Metal-Dependent FDHs
FDHs containing metals constitute the other large set of FDHs [
28
,
84
,
116
,
117
]. All
metal-dependent FDHs catalyze the interconversion between formate and CO
2
. The sense
of the reaction (Equation (5), Scheme 1) depends on the external conditions. In general, in
biological conditions, formate oxidation (backward reaction 5) is favored, and thus, some
organisms obtain their energy from this exergonic reaction. However, metal-dependent
FDHs can also catalyze CO
2
reduction, and most of them do so, although to a different
extent. There are a small number of microorganisms (hydrogenotrophic methanogens,
Euryarcheota, and autotrophic acetogens, bacteria) that use FDH not for generating energy
(i.e., not for oxidizing formate), but for using C
1
carbon species as a primary source of their
carbon metabolism [116,117]. All these microorganisms have metal-dependent FDHs.
This type of FDH is much more complex than NAD-FDHs. Indeed, they have more
than 700 amino acids arranged in different domains that, in turn, contain several cofactors
and/or metal centers such as ferredoxins, heme groups, flavin mononucleotides, etc.,
depending on the species [
118
]. For instance, FDH N from E. coli comprises three domains
(Figure 6A): the
α
-domain, which contains the Mo cofactor (see below) and one [4Fe-4S]
cluster; the
β
-domain with four [4Fe-4S] centers; and the
γ
-domain, with two b-hemes
(Figure 6B) [
119
]. These enzymes receive the electrons from these metal clusters and not
necessarily from NADH (although, in some cases, NADH can also be the cofactor), and
thus, these FDHs are called nondependent NADHs. Excellent reviews describing the
three-dimensional structures of these FDHs, their metal centers, their functions, and the
biotechnological achievements of these enzymes have been published [28,84,116,117].
Molecules 2023, 28, x FOR PEER REVIEW 13 of 56
Figure 6. (A) Up: three-dimensional structure of FDH N from E. coli (1KQF pdb code [119]). The
three polypeptide chains are shown in blue (chain A), pink (chain B), and green (chain C); the mo-
lybdopterin (in chain A), the five F4S4 clusters (in chains A and B), and the two heme (chain C) metal
cofactors are displayed in pink, green, and red, respectively. (A) Down: a detailed picture of the
cofactors at the same scale. (B) Up: graphic of the molybdenum cofactor in FDH N from E. coli. (B)
Down: chemical formula of the Mo cofactor. (C) FDH mechanism of action for CO2 reduction (red
arrows) or formate oxidation (inverse sense, blue arrows) [27]; the direction of the reaction depends
on the experimental conditions (see text for details).
Importantly, despite their heterogeneity, these FDHs share common features con-
cerning the active center. They all contain a molybdenum or a tungsten metal ion bound
to two dithiolene atoms provided by two pyranopterin guanidine dinucleotides, a sulfur
or selenium donor atom, and a disulfide anion (Figure 6B) [104]. CysSe residue is present
in both Mo- and W-FDHs; hence, it is not specific to a determined metal. On the other
hand, according to kinetics parameters (kcat and kM) the presence of CysSe instead of the
native amino acid cysteine, is not crucial for FDH activity.
Metal-dependent FDHs catalyze Equation (5) (Scheme 1) in both directions; however,
unlike wild-type NAD-dependent FDHs, they are much more efficient in catalyzing for-
ward Equation (5) than the laer, clearly evident in Table 2. This table presents the kinetic
parameters, as well as the composition of the active site and cofactors, specifically for
metal-dependent FDHs. Hence, from biotechnological applications, that is, for reducing
CO2, metal-dependent FDHs are much more active and, hence, more aractive than non-
metal FDHs.
What are the key factors that enable metal-dependent FDHs to reduce CO2 effi-
ciently? These issues have been extensively studied in the literature [26–29,84]. Here, some
notes on the crucial aspects are commented upon. First, the existence of different redox
centers acts as a corridor for efficient electron transfer toward the CO2 molecule. Second,
and importantly, there is presence of a sulfido group that accepts a hydride anion (see
Figure 2C). It is well known that, in Mo/W enzymes, a sulfido group accepts a hydride. In
FDHs, metal oxidation states change from IV to VI; when they are in a reduced state, lig-
ands tend to be protonated, while tending to deprotonate in the Mo/W(VI) oxidation state.
Figure 6.
(
A
)
Up
: three-dimensional structure of FDH N from E. coli (1KQF pdb code [
119
]). The
three polypeptide chains are shown in blue (chain A), pink (chain B), and green (chain C); the
molybdopterin (in chain A), the five F
4
S
4
clusters (in chains A and B), and the two heme (chain C)
metal cofactors are displayed in pink, green, and red, respectively. (
A
)
Down
: a detailed picture of
the cofactors at the same scale. (
B
)
Up
: graphic of the molybdenum cofactor in FDH N from E. coli.
(
B
)
Down
: chemical formula of the Mo cofactor. (
C
) FDH mechanism of action for CO
2
reduction (red
arrows) or formate oxidation (inverse sense, blue arrows) [
27
]; the direction of the reaction depends
on the experimental conditions (see text for details).

Molecules 2023,28, 5520 14 of 52
Importantly, despite their heterogeneity, these FDHs share common features concern-
ing the active center. They all contain a molybdenum or a tungsten metal ion bound to
two dithiolene atoms provided by two pyranopterin guanidine dinucleotides, a sulfur or
selenium donor atom, and a disulfide anion (Figure 6B) [
104
]. CysSe residue is present in
both Mo- and W-FDHs; hence, it is not specific to a determined metal. On the other hand,
according to kinetics parameters (k
cat
and k
M
) the presence of CysSe instead of the native
amino acid cysteine, is not crucial for FDH activity.
Metal-dependent FDHs catalyze Equation (5) (Scheme 1) in both directions; however,
unlike wild-type NAD-dependent FDHs, they are much more efficient in catalyzing forward
Equation (5) than the latter, clearly evident in Table 2. This table presents the kinetic
parameters, as well as the composition of the active site and cofactors, specifically for
metal-dependent FDHs. Hence, from biotechnological applications, that is, for reducing
CO
2
, metal-dependent FDHs are much more active and, hence, more attractive than non-
metal FDHs.
What are the key factors that enable metal-dependent FDHs to reduce CO
2
efficiently?
These issues have been extensively studied in the literature [
26
–
29
,
84
]. Here, some notes
on the crucial aspects are commented upon. First, the existence of different redox centers
acts as a corridor for efficient electron transfer toward the CO
2
molecule. Second, and
importantly, there is presence of a sulfido group that accepts a hydride anion (see Figure 2C).
It is well known that, in Mo/W enzymes, a sulfido group accepts a hydride. In FDHs, metal
oxidation states change from IV to VI; when they are in a reduced state, ligands tend to be
protonated, while tending to deprotonate in the Mo/W(VI) oxidation state. Thus, metal
sulfido can act as a donor/acceptor hydride. Indeed, spectroscopic studies are consistent
with the transfer of a hydride from a sulfur atom [
27
,
84
,
116
,
118
]. On the other hand, there
is no evidence of the direct coordination either of CO
2
or formate directly to the metal
center. Altogether, this leads to the conclusion that this facilitates the acceptance/donation
of a hydride directly towards the CO
2
carbon, which has an electronic net deficiency, and so
is prone to attack by anions. It is also remarkable that the tungsten or molybdenum metal
ion are indistinguishable concerning the catalytic activities while the presence of selenium
cysteine does not appear to be relevant in CO2reduction.
Several schemes of the reaction have been proposed for the mechanism of action of
these FDHs [
120
,
121
]. However, it is robustly supported that CO
2
, rather than HCO
3−
,
is the substrate of FDHs for forward Equation (5) [
104
,
122
]. This reaction takes place by
abstracting (or adding, reverse reaction) a hydride anion, without the intervention of any
oxygen atom [
123
]. Concordantly, the mechanism of action should consider these two
fundamental facts. Moura et al. proposed a mechanism for the forward reaction in which
the reduced CysSH coordinated to the Mo ion attacks the carbonyl atom and hydride
transfer takes place (Figure 6C) [
27
]. The formate anion is then stabilized by the positive
charge of Arg446 (FDH N from E. coli numeration), and afterward, when the protein is again
reduced by the other cofactors and by the addition of another hydride to the coordinated
Cys, the formate is released. In contrast, the reverse reaction is also produced by the
opposite hydride attack from the formate anion on the same Cys (in this case, oxidized).
4. Improving CA Performance: Enzyme Immobilization
For industrial and biotechnological applications to be profitable, enzymes must be
as stable and reusable as possible. CA and FDH in solution, like all soluble proteins,
behave as solutes with full mobility in the solvent. Although an aqueous medium is,
in general, the most suitable for enzyme action, the stability and activity of enzymes in
this medium usually decrease rapidly. In addition, the use of enzymes in solution is
always constrained by strict pH and temperature conditions. More importantly, enzymes
in aqueous solutions can only be applied in the cycle of a specific reaction; hence, their
applicability at the industrial level is highly limited. In contrast, enzymes immobilized on
solid or gel supports extraordinarily increase their stability, amplifying the range of action
of the biocatalyst conditions and the possibility of using more drastic, usually more efficient,

Molecules 2023,28, 5520 15 of 52
reaction conditions (for instance, increasing temperature) [
124
–
126
]. This immobilization
allows for their easy separation from reactants and products, and, consequently, enzymes
in this form can be reused for posterior cycles [
127
,
128
]. This drastically reduces the
cost of the whole process, regardless of the biotechnological industrial reaction. Both
CA and FDH have been immobilized on different supports. Based on the immobilization
method, the following approaches can be considered: physical adsorption, covalent binding,
entrapment, encapsulation, and crosslinking.
Excellent reviews on CA and FDH immobilization have recently been published
[129–139]
.
Here, some illustrative cases regarding the relevance of immobilization in enzyme stabilization
are highlighted according to the immobilization method (Figure 7). Because all the examples
provided in Section 6are related to CO
2
reduction by immobilized FDH, here, the focus is
on CA immobilization. Table 3summarizes relevant studies on CA immobilization using
these methods.
Molecules 2023, 28, x FOR PEER REVIEW 15 of 56
Figure 7. Different methods of enzyme immobilization.
Figure 7. Different methods of enzyme immobilization.

Molecules 2023,28, 5520 16 of 52
Table 3. Immobilization of Carbonic Anhydrase using different methods.
Method Support Immobilization Conditions Main Results Ref
Physical adsorption
Mesoporous aluminosilicate Phosphate buffer 100 mM, pH 7, 0.2 mL of enzyme (1 mg/mL) in 10 mg of
support for 6 hr at 120 rpm. CA loading up to 3 mg/mL optimal for CA activity. Carbonation
activity was 55% (8 cycles of reuse [140]
Carboxylic acid group-functionalized
mesoporous silica (FMS)
0.6–2.0 mg FMS with functional groups incubated with 70–1000 IL of
2.0 mg/mL BCA II in water (pH 6.5) for 2 h at 21 ◦C under
1200 rpm shaking.
High protein loading density 0.5 mg of protein/mg. High stability
and activity (95.6%) of the immobilized enzyme [141]
Silver nanoparticles amine-functionalized
mesoporous SBA-15
10 mg dispersed separately in 2 mL free HCA in buffer (3 mg/mL HCA in
100 mM sodium phosphate, pH 6.4) followed by incubation at 25 ◦C with
shaking for 4 h.
The activity of the materials was ∼25-fold higher than the activity of
the free HCA for converting CO2to CaCO3after 30 cycles. [142]
Mesoporous silica nanoparticles with
polydopamine (PDA) and
polyethyleneimine (PEI)
5 mg FDH/1.6 mg CA added to 2 mL of phosphate buffer (0.05 M, pH 6).
PDA/PEI-mSiO2(0.05 g) and 5 µL GA aqueous solution (20 wt%) were
added (30 ◦C for 24 h).
Optimal FDH and CA concentration was 2.5 and 0.8 mg/mL
respectively, (specific activity of 0.045 mM/h/mg). 10 reuse cycles
with 86.7% of activity. [143]
Palmityl-substituted sepharose 4B Denatured Bovine CA solution by incubation for 15–120 min (58–65 ◦C),
Tris-sulfate buffer, pH 7.5 (0.0125–0.5 mg/mL).
Denaturation and renaturation processes of the enzyme in the matrix.
After renaturation at 60 ◦C, 85% of immobilization was achieved
(70% of the original activity after 1 h). [144]
Silica or titania particles The clarified lysates expressed BCA-peptide (0.088 mg protein) were
incubated with 30 mg silica or 20 mg titania for 10 min with shaking at
room temperature in 25 mM sodium phosphate buffer (pH 6.5).
After 10 days, 90 ±4% and 95 ±3% of silica and titania particles’
original activity remained. Immobilized BCA-peptide fusion protein
shows ∼95% of its residual activity with up to 5 cycles of reuse. [145]
Porous polypropylene and a non-porous
polydimethoysilane hollow fiber membrane Application of CA in situ to the shell side surface of each fiber. CO2absorption into K2CO3increased approximately three-fold when
CA is adsorbed onto the Porous polypropylene or non-porous
polydimethoxysilane PDMS membrane surface [146]
Entrapment or encapsulation
Magnetic nanoparticles (sol-gel
ferria hydrosol) 200 mL of ferria hydrosol mixed with CAB solution (10 g/L, 0.05 M Tris,
pH 7.4) and dried at 20 ◦C. Large thermal stability of CA immobilized, active up to 95 ◦C [147]
Polyurethane foam (PUF)
1 mL of CA (1 g/L) or whole-cell solution in 20 mM Tris-sulfate buffer
(pH 8.3) was added to a 50 mL containing 1 g of prepolymer. The swelling
of PUF continued for 30 min, and then an additional 10 min was allowed
for curing.
An immobilization efficiency of 3.4%, 16-fold higher than that for free
enzymes. The reusability of the immobilized whole-cell catalyst
shows no apparent decrease in activity after 9 reuses. The rate of CO2
capture was accelerated by 80%.
[148]
Ni-based MOFs (Ni-BTC) nanorods, 100 mL cell lysate of His-HCA II (0.4 mg protein) and 900 mL Milli-Q
water were incubated with Ni-based MOFs.
His-HCA II from cell lysate obtained an activity recovery of 99%.
After storing for 10 days, the immobilized His-HCA II maintained
40% activity (free enzyme lost 91% activity). Immobilized His-HCA
II retained 65% (8 cycles).
[149]
Supported ionic-liquid membranes (SILMs) 0.25 mg CA/g IL. SILMs resent permeability (PCO2= 733.73 barrier) at high
temperatures (up to 373 K) and a good transport selectivity towards
CO2against N2.[150]
Cholinium-based ionic liquids 0.1 mg CA/g IL CA samples promote an enhancement of 63% in the carbon dioxide
transport rate. [151]

Molecules 2023,28, 5520 17 of 52
Table 3. Cont.
Method Support Immobilization Conditions Main Results Ref
Covalent binding and
cross-linking
Mesoporous SBA-15 surfaces covalently
functionalized with amines
HCA immobilization was achieved by mixing 10 mg of
amine-functionalized SBA-15 with a 0.1% glutaraldehyde (GA) solution
(50 mM sodium phosphate, pH 8.0, 1 h). The product was treated with
free HCA (3 mg/mL HCA, pH 7.0), incubated and shaking (1 h, 25 ◦C).
Immobilized HCA retained activities after long-term storage,
exposure to high temperatures, and reuse (40 cycles). CO2capture
efficiency of immobilized HCA was 36 times higher than that of free
HCA, 75% of the enzymatic activity was retained after 40 cycles.
[152]
Chitosan CA liquid was slowly added into the pH 5 chitosan solution with
continuous stirring. Ratios of 1:0.05–1:2 chitosan:CA (g:mL)
Textile packing with covalently attached enzyme aggregates retained
100% of the initial 66.7% CO2capture efficiency over 71 days and
retained 85% of the initial capture efficiency after 1-year of ambient
dry storage.
[153]
Polyethyleneimine and polydopamine in
MOF 808
PBS buffer (pH 8, 10 mM), PEI/PDA-MOF-808 or PDA-MOF-808 was
disseminated. 200–400 µL of CA solution (1 mg/mL, Milli-Q) and 10 µL
of aqueous solution of GA (0–25 wt%) were added and shaken for a while
at 28 C.
CaCO3produced by CA@PEI/PDA-MOF-808 was 11.0-fold and
2.5-fold higher than free CA and PEI/PDA-MOF-808, respectively.
After 8 consecutive rounds, the total production of CaCO3by
CA@PEI/PDA-MOF-808 was 92-fold higher than free CA.
[154]
Alumino-siloxane hybrid aerogel beads
100 mg of Al/Si-NH2beads were treated with 0.5% GA for 1 h. 4 mL of
free BCA in the buffer (1 mg/mL 100 mM phosphate buffer) was added to
GA-treated Al/Si-NH2 beads and stirred (3 h, 25 ◦C) for
BCA immobilization.
Free BCA retained 70% of its maximum activity (immobilized BCA,
88%). Free BCA and BCA-Al/Si-NH2 remained 80% of the activities,
after ten days. BCA-Al/Si-NH2retained 89% of their enzyme activity
up to 10 cycles
[155]
Amine-functionalized by co-deposition of
polydopamine (PDA) and polyethyleneimine
(PEI)
Co-deposition of PDA and PEI and CA covalently anchored on the
surface via GA. Surface was treated with a mixture of PDA (2 mg/mL)
and PEI, pH 8,5. Amine-functionalized micro-reactor surface was
contacted with a mixture of CA and GA (concentration 2.0% (v/v)).
A steady CO2absorption rate for several hours and good reusability
of the immobilized enzyme which maintains its original absorption
performance after 10 cycles of operation. [156]
Polypropylene hollow fiber membranes using
GA-activated chitosan
Aminated knitted hollow fiber membranes mats (204 cm2fiber surface
area) were incubated in 5% GA in 100 mM phosphate buffer, pH 8.5 for
1 h under constant rocking at room temperature.
Chitosan/CA coated fibers exhibited accelerated CO2removal in
scaled-down gas exchange devices in buffer and blood (115%
enhancement vs. control, 37% enhancement vs. control, respectively). [157]
Magnetic Cross-Linked Enzyme Aggregates
(CLEAs) to bovine carbonic anhydrase (BCA)
and magnetic nanoparticle
- Adsorption: the NPs suspension (5 and 2 mg solids) added to 1 mL of
10 mM PBS (pH 7.4), 10 g/L BCA.
- Precipitation: NP-enzyme suspension added dropwise to 9 mL
containing the precipitating agent (pH 7.4), 1 h or 0.5 h under mixing.
- Cross-linking: 25% vol GA added. The system kept under mixing for 3,
16 and 22 h.
- CLEAs separated by MF.
BCA-CLEAs can increase the CO2absorption rate concerning the one
observed in the same reactor filled with only alkaline solvent.
Biocatalyst reusability analysis showed that BCA-CLEA retained 95%
of its initial activity after five CO2absorption tests at 1000 mg BCA
CLEA/L and as many liquid-solid separation steps by
membrane filtration.
[158]
Geopolymer micro-spheres (GMS) and
covalent attachment by GA
GMS were introduced into a Tris-HCl buffer solution (0.05 M, pH 8.0)
with CAs solution (1 mg/mL) in a 50 mL centrifuge tube. After shaking
and reacting, the GA was added for the cross-linking between the GMS
and CAs.
Kcat/Kmvalues were 61.50 and 12.36 M−1s−1for the immobilized and
free CAs, respectively. At 60 ◦C, free CAs were inactivated
(immobilized CAs kept 34.8% activity). Immobilized CAs maintain
68.73% of activity (8 cycles).
[159]
Microbial transgluta-minase (MTG) acts as a
“cross-linking medium”
Iso-peptide bond between glutamine and the primary amine group of a
lysine in artificial peptide tags. Equimolar amounts F-CA and M-FDH
were added, and a metal constant temperature oscillator was used for the
cross-linking reaction (reaction time 12 h, 25 °C, 400 rpm. The amount of
MTG used was 1 U/mL.
The remaining CA activity was more than 93%, and the remaining
FDH activity was more than 84%. The efficiency of the cross-linked
enzyme is increased by 5.8 times compared with free enzymes. FDH
thermal stability at different temperatures is improved the optimal
found CA/FDH ratio was 1:2.
[160]

Molecules 2023,28, 5520 18 of 52
4.1. Physical Adsorption
Physical adsorption was the first method used to immobilize enzymes [
161
]. It consists
of affixing the protein onto a solid matrix utilizing hydrophobic (van der Waals), electrostatic
(ionic), or hydrogen bonding interactions [
162
]. Physical adsorption can involve partial
conformational changes and/or denaturation of the enzyme; thus, special attention must be
paid to avoid these events and confirm that the whole activity of the enzyme is retained after
immobilization. The types of functional groups on the surface that produce the adhesion of
the enzyme to the matrix is one of the crucial aspects of this immobilization method [
124
].
These groups can contain hydroxyl, carboxyl, amino, sulfhydryl, or imidazole groups,
among others, and produce interactions with the rest of the amino acids of the protein. Weak
interactions are optimal since strong ones could result in enzyme conformational changes
or even denaturation. Indeed, weak interactions are nonspecific and reversible; therefore,
proteins can be easily recovered. For instance, if the interactions are electrostatic, free protein
can be released into the medium by simply increasing the ionic strength of the solution.
Pore structure can affect enzyme accessibility and, consequently, both the quantity and
activity of the immobilized enzyme. Finally, the surface area is also a critical factor: the
higher the surface area, the larger number of adsorption sites, increasing both the quantity of
immobilized enzyme and the global activity of the carrier. Physical adsorption can increase
the stability of the enzyme against changes in pH, temperature, or organic solvents; the
enzymes can be easily separated from the reaction mixture, making reuse easy. Moreover, in
some cases, adsorption can increase the activity of the enzyme due to stabilization of the
active conformation of the enzyme. On the other hand, physical adsorption can sometimes
result in loss of enzyme activity if the microenvironment is not adequate or in a decrease in
activity due to diffusion limitations of the reactants towards the enzyme active center that
can reduce the rate of substrate conversion. Finally, the cost of the enzyme immobilization
process can be high when applied, in particular, on an industrial scale.
Mesoporous silica and aluminosilicates are excellent candidates for enzyme immo-
bilization using adsorption methods [
163
]. Here, the size and structure of the pores are
crucial. Mesoporous silica with larger pores can allow for higher enzyme accessibility to
the adsorption sites, which typically increases the quantity of the immobilized enzyme and,
consequently, its activity. Wanjari et al. immobilized CA in an ordered mesoporous synthe-
sized aluminosilicate, obtaining acceptable kinetic values for the biocatalyst compared to
the free enzyme, remaining stable for more than 25 days [
140
]. Yu et al. immobilized CA in
silica functionalized with carboxylate groups, which provided a very high degree of enzyme
uptake, and, although the enzyme slightly changes its conformation with respect to the free
enzyme, their activities were almost equivalent (95.6% that of the free enzyme versus that of
the immobilized enzyme) [
141
]. Vinoba and coworkers adsorbed bovine carbonic anhydrase
(BCA) inside octa(aminophenyl)-silsesquioxane silica nanoparticles modified with silver or
gold, which continued to be active after 20 recycling runs [
142
]. This adsorption approach
has been developed extensively, a recent example being the fixation of CA together with
FDH to produce formate in silica nanoparticles modified by polydopamine and polyethy-
lamine [
143
]. Here, the production of formate was expedited up to 30-fold with respect to
the free enzyme and activity was retained at 86.7% after 10 cycles.
Colloids are another type of support used to adsorb CA [
138
]. Crummblis et al. im-
mobilized CA in gold sols, obtaining an enzyme with levels of activity comparable to
that of the native one [
164
]. Curiously, denatured CA was also immobilized in modified
Sepharose and subsequent enzyme renaturation using a cycle of heating and cooling, result-
ing in an active enzyme with elevated activity [
144
]. CA immobilization via electrostatic
adsorption has been studied with nanoparticles using different charges [
145
]. Positively
charged nanoparticles do not adsorb human CA II, while negatively charged ones do,
showing kinetic activity that depends on the degree of hydration of both the enzyme and
the particle surface. More recently, CA was fixed onto two different types of membrane via
layer-by-layer assembly: the first with a porous membrane and the second without [
146
].

Molecules 2023,28, 5520 19 of 52
The carbonation rate of the porous membrane was three times higher than that of the
enzyme alone. On the contrary, the nonporous membrane was less active (70–90%) than the
native non-immobilized enzyme. The adhesive properties of the polysaccharide chitosan
modified with different compounds have also been employed to immobilize CA [
153
,
165
].
Matrixes of chitosan with different coating methods and a given textile package have been
shown to adsorb CA in a “drop-in-ready” method, with high efficiency for CO
2
scrubbing.
The physical properties of these matrixes for CO
2
capture were maintained for more than
31 days, with high efficiency (>80%) at moderate temperatures.
4.2. Entrapment and Encapsulation
Immobilization by entrapment occurs when a polymer, gel, or metal organic frame-
work (MOF) is generated in the presence of an enzyme [
132
,
166
]. In such cases, the protein
can remain trapped within the hollows of the polymer. The nature of these interactions is
not chemical in origin, but rather physical. The proteins have free movement at a local level,
but the motion is highly restricted to the confined hollows, and most of the molecules are
isolated and interact only with the matrix. Drozdov’s group immobilized CA into the pores
of different sol–gel magnetite with singular magnetic properties using this method [
146
].
They studied the physical properties of the new material as well as the overall structure of
the enzyme, mainly using infrared spectroscopy, concluding that the protein maintains its
3D arrangement in the generated nanoparticles. The immobilized enzyme was stable and
catalytically active at 90
◦
C, which is the temperature at which the native free enzyme is
completely denatured.
Encapsulation is similar to entrapment in the sense that molecules are also free in
solution and their movements are restricted; however, molecules are captured in higher
bags where they can interact with each other. Sol–gel matrices have also been employed
for the encapsulation of CA with excellent results. Polyurethane foam has also been
employed to entrap not only the enzyme itself but also E. coli cells expressing CA [
148
].
Indeed, whole-cell catalyst CO
2
hydration activity was measured by comparing both sole
and whole-cell immobilized enzymes with respect to the free enzyme. The efficiency
of hydratase activity (Equation (4)) was 16-fold higher for the whole-cell immobilized
enzyme than for the free enzyme. Interestingly, the activity of the whole cell trapped in
the PUF was approximately 100% for at least nine cycles. MOFs are structurally ordered
materials formed from inorganic complexes bridged by organic ligands that are projected
in three dimensions [
167
]. Hollows of defined sizes are arranged monotonously in MOFs.
MOFs are employed in a multitude of applications, with the immobilization of proteins
being one of the most promising [
168
,
169
]. CA has been encapsulated in different MOFs
with different features, most of them being zeolites constituted by imidazolates, with
acceptable or excellent results [
149
,
154
,
170
]. The enzyme encapsulation within MOFs
generates enzyme diffusion through windows that have a smaller size than the cavity.
Whether or not the term encapsulation can be properly applied to the immobilization of
enzymes in MOFs depends on the ratio between the pore and the enzyme size. In any case,
an MOF based on Ni(II) showed a high degree of reusability for CA, retaining more than
65% of its activity after eight cycles [
149
]. Zinc has also been used as a base for MOFs to
immobilize CA. In this case, the Zn-OH groups of the hollow imitate the active site of the
enzyme, which permits high CO
2
capture efficiency [
171
,
172
]. MOFs containing several
lanthanides have also been employed. In this framework, the existence of a high level of
electrostatic interactions substantially increases capacity for CO
2
uptake [
173
]. Here, taking
advantage of the lanthanide contraction, the specific dimensions of the hollows can be
modulated, with the Eu(III) derivative having the highest affinity towards carbon dioxide.
In all these examples, infrared spectroscopy is one of the key techniques for characterizing
the degree of CO2capture, as well as the distortions of the framework.
Ionic liquids (ILs) have also been employed to immobilize CA, although to a lesser ex-
tent [
150
,
174
–
177
]. While CO
2
is nonpolar, owing to the difference in electronegativity of the
carbon and oxygen atoms, the charges of ILs can absorb CO
2
to a high degree; thus, this is a

Molecules 2023,28, 5520 20 of 52
field fertile for exploitation. Recently, CA was immobilized in poly(ionic liquids) (PILs) by
mixing the monomer hydrophobic IL 1-vinyl-3-hexylimidazolium bis(trifluoromethylsulfon
yl)imide with an ethylene glycol derivative that had previously been polymerized using
crosslinking [
133
]. After generating the PIL, CA was entrapped within the hollows of the
polymer. The yield of the resulting CA was highly dependent on the size of the porous
material and the degree of humidity (the dry PIL was less efficient). The authors also
tuned the degree of particle size using previous sonication and studied the kinetic param-
eters of CA-PIL. These values were comparable to that of the free enzyme, although the
entrapped CA was stable for a month without detectable loss of activity, while the free
enzyme decreased its activity by more than 30%. The CA-PIL was reused for five cycles
with 60% activity.
4.3. Covalent Binding and Crosslinking
Covalent binding implies the formation of bonds between the groups of adequately
functionalized supports and an enzyme. This is, by far, the most extended approach for
immobilizing enzymes, particularly for CA [
152
,
155
,
178
,
179
]. Several protein functional
groups can be used for this purpose. CA has been covalently bound to different supports by
its amine groups by reaction with glutaraldehyde [
156
,
180
]. Generally, mesoporous supports
containing hollows of controlled sizes are grafted with amine groups to obtain solid materials
that are prone to covalently binding to enzymes using glutaraldehyde. This was carried
out with the support SBA-15, in which three different amine compounds were inserted,
followed by covalent immobilization of HCA [
142
,
152
,
181
]. The resulting material was
morphologically characterized, and its activity, thermal stability, and reusability were also
determined, obtaining better results than those of the free enzyme. Kimmel et al. immobilized
CA on the surface of propylene fiber membranes. These membranes, commercially available,
were coated with a siloxane layer and functionalized with amine groups. Posteriorly, CA
was attached to these fibers via glutaraldehyde crosslinking under two conditions: with and
without chitosan tethering. Then, the authors applied these fibers to CO
2
removal, finding
enhancements of 115% and 37% versus the buffer and the blood controls, respectively [
157
].
Moreover, carboxylic groups activated by carbodiimide and N-hydroxysuccinimide agents
were used to covalently immobilize CA in microtubes [
182
]. The resulting immobilized
enzyme enhanced its ability to sequester CO2with respect to the free enzyme.
Finally, the crosslinking method is actually a special way of covalent binding. The
proteins are bound to other large proteins to form high molecular weight complexes
without any solid support or, properly, the enzymes themselves being a solid support.
Typically, the protein is precipitated with the appropriate agent and then crosslinked,
which can be performed with a purified protein or an extract of a still unpurified enzyme.
This method confers high stability and a high degree of enzyme recovery. CA has been
immobilized via crosslinking in numerous studies. Recently, Xu et al. encapsulated
crosslinked CA in alginate beads and confirmed that this crosslinked CA enhanced the
growth of microalgae cultures [
183
]. The crosslinked CA was stable during 10-cycle assays.
Magnetic nanoparticles aggregated with CA were obtained by crosslinking the enzyme
with glutaraldehyde, improving the yield of absorbing CO
2
up to 3.4-fold with respect to
the free enzyme and retaining 95% activity after five cycles of reuse [
158
]. These magnetic
nanoparticles are amply used because they allow for very simple separation and recovery
of the biocatalyst by using an external magnetic field. They are considered excellent carriers
and supporting matrices for enzyme immobilization, providing several advantages for
the design of biocatalytic processes (i.e., large surface area, large surface-to-volume ratio,
high mass transference, etc.). More recently, Chang et al. crosslinked CA and geopolymer
microspheres with glutaraldehyde and performed a detailed study on the morphology,
stability, and activity of the immobilized support [
159
]. After 60 days of storage at 25
◦
C,
immobilized CA still presented 28.9% activity, whereas free enzyme activity was less than
10%. CA and FDH have also been crosslinked to reduce CO
2
. Zhang et al. used microbial
transglutaminase (MTG) as the crosslinking medium for CA and FDH labeled with peptide

Molecules 2023,28, 5520 21 of 52
tags and previously expressed in E. coli [
160
]. MTG catalyzes the formation of an isopeptide
bond between the
ε
-amino group of lysine and a glutamine. The authors studied the activity
and reusability of several crosslinked particles with different tags at CA/FDH ratios of
1:1, 1:2, and 1:3. Because CA is much more active than FDH, it is expected that the lower
the CA/FDH ratio, the higher the formate yield obtained. However, the optimal found
CA/FDH ratio was 1:2. The authors attributed the lower yields obtained for a 1:3 ratio to
FDH steric hindrances in the crosslinked aggregates.
5. Carbon Capture Storage and Utilization: State-of-the-Art, Costs, and Perspectives
Methods for CO
2
capture are classified into precombustion, postcombustion, and
oxy-combustion processes [
12
,
184
]. The precombustion approach is related to hydrogen
gas production. This is obtained in numerous industrial processes such as electric power
generation, ammonia or fertilizer synthesis, and petroleum refinement. The precombustion
process refers to the conversion of the primary solid fuel (coal or biomass) by reforming
it into a mixture of CO and H
2
gas (syngas). This gas reacts with the water stream at
high temperatures and pressures to produce CO
2
and more H
2
(water–gas shift reaction).
Finally, CO
2
is captured using several methods. Postcombustion CCS methodology denotes
all the processes employed to capture CO
2
from exhaust gas (its major component being
nitrogen) resulting from industrial chemical processes. CO
2
gas is emitted at relatively low
temperature and pressure. Oxy-fuel combustion consists of the oxidation of fuel using
pure oxygen instead of air, obtaining an almost pure CO
2
atmosphere without nitrogen
gas. Most CCS methods have been developed for postcombustion gases and are referred to
here, except where otherwise indicated. In many cases, the methodology can be the same
for both post- and precombustion approaches, although with different designs depending
on the P/T conditions of the exhaust gases. Physical adsorption and absorption [
185
]
(geological storage [
186
] probably being the most relevant among absorption approaches)
and cryogenic distillation [187] are the main methods used for CO2CCS.
Global CO
2
emissions from combustion processes grew by 0.9% in 2022, reaching a total
of 36.8 Gt [
188
]. Energy used in industry, agriculture or land use, buildings, transport, direct
industrial processes, waste, and others with 37.8%, 18.4%, 17.5%, 16.2%, 5.2%, 3.2%, and 1.7%,
respectively, are the contributions to CO
2
emissions by the different sectors [
189
]. Only ca.
40 million Tm, i.e., 0.1%, was removed from the atmosphere using CCS methods in 2019. Thus,
we are still very far from being efficient in eliminating the CO
2
expulsed into the atmosphere.
The estimated present costs of CO
2
Tm removal vary nowadays from 40 to 80 USD
depending on the method [
190
]; however, the net contribution to CO
2
elimination from
the atmosphere is difficult to calculate, since net contributions in the whole process have
to be taken into account. Hepburn and coworkers analyzed the perspectives, including
cost, for different methods of CO
2
utilization [
190
]. They analyzed ten different methods
of CO
2
utilization. For instance, chemical production, particularly the generation of urea,
on one hand, and the production of polycarbonate polyols, on the other, are two fields in
which CO
2
capture can be exploited. They estimated that CO
2
utilization in chemicals in
2050 could be around 0.3–0.6 Gt CO
2
/yr with costs ranging from
−
80 to 320 USD per Tm
of CO
2
(a negative value would indicate an additional economic benefit, while a positive
value indicates that the cost of capturing and utilizing CO
2
would be higher than the
value generated from it). Fuels, that is, CO
2
-methanol plants, were also considered in their
study, although they stated that many different scenarios can vary their prospects from 1 to
4.2 Gt/yr in 2050 and, in terms of cost, from 0 to 670 USD per Tm of CO2.
6. Carbonic Anhydrase in Carbon Capture Storage
Although CA is not used in all previous technologies, its use is extended or has
a good perspective in others, mainly chemical adsorption and mineralization, whose
description is commented on below. Table 4describes relevant studies performed with CA
in CCS research.

Molecules 2023,28, 5520 22 of 52
Table 4. Relevant studies on CO2uptake processes performed using CA, and their main features.
Process Idea of the Study CO2Uptake Conditions Relevant Conclusions CA Activities Ref.
Chemical absorption
To examine the stability and activity of the enzyme
under different pH values and amine solvents.
Compare its performance to other
common solvents.
CA tested for pH [7–11]. Temperature stability
for up to 100 h of incubation. CA stability
tested for 7 capture solvents (1 M or 3 M,
150 days, 40 ◦C).
CA stable at 60 ◦C, pH range [7–11]: residual
activity at pH 5 or 12, ranging from 12 to 91%.
The enzyme enhances reaction rates. NaCl,
K2CO3, AMP, and MDEA show
additive effects.
After 100 h (25 ◦C), CA activity was kept higher
than 75% for 1M NaCl, AMP and MDEA and
125% in 1M K2CO3
[191]
To develop the CA-MOFs composite, with superior
catalytic performance and high stability to promote
CO2absorption into a tertiary amine solution. 0.05 g L−1and 40 ◦C, PCO2 15 kPa.
CA loading in ZIF-L-1 increased with the
added enzyme amount. CA/ZIF-L-1
composite has higher catalytic activity and
stability than the free CA. The immobilization
of CA on ZIF-L-1 improves the CA
conformational stability
CO2absorption rate of CA/ZIF-L-1 in 1 M MEA
and MDEA at 40 ◦C: 3.0 ×106kmol s−1m−2. CA
loading reached up to 87 mg g−1. The highest
immobilized CA activity was 1.5 times that of the
free CA
[192]
Pilot-scale experiments with CA-enhanced MDEA
for CC, bench-marking its mass transfer
performance against the industrial standard 30 wt%
MEA.
Experiments were done using 30 wt% MEA
and MDEA solvents varying CA
concentrations (0, 0.85, and 3.5 g/L) at
different column L/G ratios.
Enzyme-enhanced MDEA solutions exhibit
80% of the mass transfer performance at 30
wt% MEA and have the potential to reduce the
size and cost of absorbents in carbon capture.
CO2capture efficiency higher than 90% for high
L/G ratios. The mass transfer increased 20-fold
for 25 and 50 wt% MDEA solutions by adding
0.2 g/L CA.
[193]
Use of columns with membranes contractors with
polyionic liquids (PIL), amines and CA for CO2
uptake.
PIL blend (F9:1(M10)) with immobilized CA in
30 wt% MDEA at low feed gas (15% CO2in
N2.) pressure (1.3 bar)
Addition of the enzyme to the MDEA solution
with PILs significantly improves CO2uptake
rate and reduced the equilibration time.
CA addition to MDEA solution improved the
CO2uptake rate by 1.7 times. CA and membrane
improved the uptake rate by twice. [194]
Carbonation
To present the effects of adding small quantities of
immobilized CA on the absorption of CO2into
potassium carbonate.
CO2absorption (partial pressure 90 kPa) in a
30% K2CO3solution. A wet-walled column
(40–80 ◦C) with 38 g/L CA.
CA addition improves the CO2absorption in
K2CO3solvents. The rate of CA-catalyzed CO2
hydration increased with the CA concentration
Increasing CA concentration from 0.4 to 1.8 µM
increases the absorption rate by 34% (40 ◦C) [195]
To explore the potential of using enzymes to
catalyze the conversion of CO2into bicarbonate
and on the carbonation rate of brucite, Mg(OH)2.
Gas flow (CO2/N210%/90%) at 10 psig.
Experimental durations were 11, 7, and 3 days
(low, medium, and high flow experiments).
CA accelerates the carbonation of brucite.
Higher CO2gas flow rates results in faster
carbonation. Mineralogical compositions
depend on the CO2flow rate.
The carbonation rate of brucite using BCA was
accelerated by up to 240% compared to controls. [196]
To propose a novel method based on microbially
induced calcium precipitation to improve the
cementitious properties of steel slag.
Bacillus mucilaginosus placed in a sealed
container, the air pressure was reduced to
−0.05 MPa. CO2(99.99%) pumped to maintain
the gas pressure (0.25 MPa, 30 ◦C, 32 h).
Ca-silicate promotes the growth of bacteria,
enhances the production of polymers and
improves bacterial adhesion.
Ca-silicate-containing medium (CSCM) can be
used as a microbial carrier.
Maximum bacterial growth rate in CSCM was
1.5 times higher than that in the control medium
(CM). Bacterial activity in CSCM was 40% of that
in CM (30 h). At a dosage of bacterial powders of
1 wt.%, carbonation reached the maximum.
[197]
To develop and characterize a highly efficient and
stable biocatalyst for CO2sequestration using silica
nanocomposite with auto-encapsulated CA.
(CA)-based biocatalyst encapsulated in a biosilica
with a peptide R5.
CaCO3precipitation was carried out at 30 ◦C
and monitored turbidimetrically at 600 nm.
The final pH of the buffer was
approximately 9.3
The encapsulated CA was not leached from the
silica matrix. Encapsulation in silica effectively
improved the thermostability and activity of
the enzyme.
Encapsulation efficiency greater than 95%.
Residual activity of the self-encapsulated CA was
more than 50% (30 min, 80 ◦C). Activity of
ngCA-R5@silica decreased only after 2.5 days at
60 ◦C. Encapsulated CA: 98% and 80% of its
initial activity (1 day and 5 days of incubation,
50 ◦C). CaCO3precipitation is reduced by
5.5-fold when ngCA-R5@silica (30 µg/mL) is
present compared to the uncatalyzed reaction.
[198]

Molecules 2023,28, 5520 23 of 52
Table 4. Cont.
Process Idea of the Study CO2Uptake Conditions Relevant Conclusions CA Activities Ref.
Mineralizaton
Mineralization experiments were performed using
Curvibacter lanceolatus strain HJ-1, including its
secreted extracellular polymeric substances (EPS)
and CA (CA).
Three types of mineralization experiments:
with CA (duration 96 h), with EPS (96 h), and
with bacteria (50 days).
Strain HJ-1, EPS, and CA promote carbonate
precipitation. HJ-1 and EPS1 experiments
contained calcite and aragonite. CA formed
calcite only. HJ-1 and EPS are favorable for
aragonite precipitation.
The mass of precipitate (inorganic plus organic
substances) increases until ca. 55 mg. The
maximum degree of calcification was
approximately 6.6%. In the control groups
without CA no precipitate was formed. In the
absence of HCO3−, the optimized calcification
rate followed the order: HJ-1 (49.5%) >CA(6.6%)
>EPS2(4.1%).
[199]
Production of CA from the bacterium Aeribacillus
pallidus TSHB1, a thermostable and alkaline-stable
bacterium, highly effective in the formation of
CaCO3from aqueous CO2.
Tris buffer (15 mL, pH 7.4) containing 0.9 g
CaCl2-2H2O, CA 0.05 mg (37 ◦C).
A 3.8-fold higher CA production by A. pallidus
than that under unoptimized conditions.
Enzyme thermostable that retains activity at
alkaline pH: it is useful in carbon
sequestration.
The partially purified enzyme produces
precipitation of 42.5-mg CO32−mg−1protein.
CO2sequestration is efficient. [200]
Study of the biochemical properties,
thermostability, and inhibition of CA from
Sulfurihydrogenibium azorense, SazCA.
Hepes buffer 10 mM, NaBF4 20 mM, pH 7.5.
Phenol red (0.2 mM) as an indicator. SazCA is highly thermostable and can survive
incubation at 90–100 ◦C.
kcat value 4.40 ×106s−1,KMvalue 12.5 mM,
kcat/KM3.5 ×108M−1s−1, 5-fold faster than the
second CA. SazCA is the second faster enzyme,
after superoxide dismutase (SOD).
[201]
To explore wollastonite (calcium silicate)
carbonation for the removal of anthropogenic CO2
and evaluate the effectiveness of different natural
(CA) and CA biomimetic catalysts (Zr-based MOFs)
to enhance CO2capture.
Water 170 mL, the catalyst (30 ppm), adjusted
at pH 4 (25 ◦C). Wollastonite crystals (100 mg)
added to the solution. CA is immobilized on
the MOFs UiO-66 and MOF-808@Mg(OH)2by
an impregnation process.
CA accelerates carbonate precipitation but
hinders carbonation of wollastonite. Thick
carbonate coatings formed on the most
reactive surfaces of wollastonite act as
passivating layers leading to a reduction in the
dissolution and carbonation rates.
The passivating effect explains why the
conversion of wollastonite into calcite was so
limited (up to 14 mol%). Zr-based MOFs
accelerate the dissolution of wollastonite.
[202]

Molecules 2023,28, 5520 24 of 52
6.1. Chemical Absorption
Chemical absorption has been the most used CCS method for decades [
203
]. This
procedure involves the scrubbing of exhaust gas at low pressures and temperatures with al-
kaline solutions typically containing amines and/or carbonates or hydroxide solutions [
203
].
Amines are weak bases that can capture protons from Brönsted acid CO
2
. The reactions of
primary/secondary or tertiary amines produce carbamates or bicarbonate anions, respec-
tively, according to the reactions described below.
The amines typically used for these purposes are alkanolamines. An alcohol group
increases water solubility and decreases vapor pressure compared to analogous amines.
The main chemical solvent used as an absorber is monoethanolamine (MEA). The solutions
typically consist of an aqueous solution of 20–30 wt% MEA. CO
2
is captured at low pressure
(ca. 1 bar) and in a mixed gas containing other gases, such as N
2
, SO
x
, and NO
x
. Secondary
amine diethanolamine (DEA) and tertiary amine N-methyl diethanolamine (MDEA) are
also amply used amines.
Several points should be considered when choosing a suitable solvent for CO
2
se-
questration. First, the enthalpy of the reaction: the higher the enthalpy of the reaction, the
higher the cost of solvent regeneration. This is a crucial point because it is estimated that
60–80% of the costs of these processes arise from solvent regeneration [204]. The enthalpy
of (exothermic) reactions 6 and 7 increases from tertiary to secondary and primary amines;
thus, primary amines improve both energetic and economic costs. Moreover, the power of
corrosion also follows the same order (primary amines are the most corrosive). Another
decisive issue is their ability to load CO
2
. Tertiary amines have the highest capabilities
in this regard [
205
]. According to these thermodynamic aspects, tertiary amines are the
best ones to use. However, another crucial point is the kinetics of CO
2
sequestration;
indeed, tertiary amines have low reaction rates and are kinetically much more inert than
primary amines [
206
,
207
]. Owing to the slow kinetics of tertiary amines, primary amines
(or secondary amines) are currently preferred. However, the high costs of cooperation and
maintenance due to the ease of amine degradation and the formation of highly corrosive
salts are drawbacks when operating with amines.
Consequently, for kinetic reasons, primary amines, specifically MEA, are by far the
most widely used alkanolamines in the industry. Numerous plants have been developed
using MEA solutions. These systems are typically coupled with industrial processes and
CO
2
is captured with postcombustion gases. For instance, their use is extended to the iron
and steel industries (responsible for approximately 31% of all industrial CO
2
emissions).
These plants can recover 85–95% of the CO
2
in gas. As an example, a steel production
plant recently established by Emirate Steel Industries has a yield plant using CCS based on
amine absorption that captures 0.8 Mt CO2/year [208].
In the last decade, CA has been revealed as a tool for accelerating CO
2
uptake in
chemical absorption processes. Gundersen et al. studied CA stability and activity for
a long time (150 days) as a function of pH, temperature, and the solvent, combining
MEA and MDEA solutions, among others [
191
]. They concluded that CA was suitable
for these purposes; the biocatalyst was stable and active between pH 7 and 11, with
maximum activity at 40
◦
C. In addition, the enzyme preserved its activity between 12
and 91% of the original activity depending on the solvent employed. The absorption
of CA was also accomplished in MOF ZIF-L-1 in the presence of MDEA [
198
]. In ZIF-L
(a zeolitic imidazolate framework), the imidazolate groups enhance CA immobilization,
and CO
2
uptake is hence greatly increased. The authors highlighted that this new MOF
obtained excellent CO
2
absorption rates at 40
◦
C and a CO
2
partial pressure of 15 kPa,
while the activity was maintained for six reuse cycles [
192
]. Additionally, a pilot-scale
plant was set up with CA in solution in the presence of MDEA. The authors observed
an enhancement in CO
2
capture in the presence of the enzyme and demonstrated the
possibility of translating the laboratory results to higher scales [
193
]. However, because the
free enzyme is damaged by amines, immobilization is necessary. Kim et al. also studied the

Molecules 2023,28, 5520 25 of 52
effect of CA on CO
2
absorption rates in the presence of MEA and MDEA, although they
used a membrane contactor with hydrophobic and hydrophilic supports [
194
]. This system
allows an expanded contact surface to enhance CO2absorption.
6.2. Chemical Carbonation
Chemical carbonation is probably the most efficient method for capturing CO
2
. This
is performed when CO
2
is bubbled through an alkaline solution, typically consisting of
dissolved KOH or Ca(OH)
2
, where potassium or calcium carbonates precipitate. The
limiting step for capturing CO
2
in postcombustion processes is Equation (3). However,
due to Equation (4), CO
2
capture is much faster in alkaline media since carbonates are
formed, and so hydrogen carbonate concentration decreases, and Equation (3) is shifted
towards the consumption of CO
2
. Indeed, once bicarbonate anion (soluble) is formed,
reactions such as Equations (6) and (7) (amine formation, Scheme 2) occur much faster in
alkaline media. Even so, the limiting step, for kinetic reasons, continues to be the CO
2
gas uptake, as commented previously. Thus, the main challenge in applying alkaline
solutions, either amines or carbonates, to the CCS approach is speeding up CO
2
conversion
to bicarbonate [
195
]. Consequently, numerous studies on CA to increase the mass transfer
of CO
2
capture have been proven not only at the laboratory level, and its feasibility has
been demonstrated on an industrial scale [
15
,
205
]. Novozymes NS81239 CA (NCA) at 2
µ
M
increased the absorption rate of CO
2
into potassium carbonate by ca. 30%, augmenting
this uptake at temperatures in the range of 40–60
◦
C [
195
,
196
]. Power et al. demonstrated
that bovine CA accelerated the carbonation rate of brucite Mg(OH)
2
from CO
2
gas by up to
240% [
209
]. In these studies, CA was supplied as a free enzyme; therefore, its regeneration
was not studied. Biological tools have also been used to enhance carbonation. Jin et al.
accelerated calcium carbonate precipitation by employing Bacillus mucilaginosus on steel
slag powder [
197
], increasing the carbonation degree from 66.34 to 86.25%. Moreover, the
mechanical properties and durability of the treated steel slag were enhanced. The CA
immobilization, as described in the previous section, strongly improves the reusability of
the enzyme as well as the chemical carbonation. For instance, Jo and coworkers proved the
suitability of CA encapsulated in a biosilica matrix, obtaining good yields for carbonation
compared to the free enzyme [198].
CO2(aq)+2R1R2NH R1R2COO−(aq)+R1R2NH+
2(aq)(6)
CO2(aq)+R1R2NR3HCO−
3(aq)+R1R2R3NH+(aq)(7)
Scheme 2.
Reactions of CO
2
with primary, secondary (Equation (6)), and tertiary (Equation (7))
amines in water solutions.
6.3. Mineralization
Biomineralization is a very slow and exothermic process by which carbonate minerals
are formed from silicates and CO
2
under basic conditions [
210
]. The starting silicates
usually contain divalent metals such as Ca(II) and Mg(II) or trivalent metals such as
Fe(III). This event occurs in nature on a regular basis and is responsible for the formation
of inorganic structures in living organisms such as exoskeletons in protozoa, algae or
invertebrates, and shells or plant mineral structures. It is also responsible for the presence
of large amounts of limestone on the Earth’s surface [
211
,
212
]. When trying to emulate
biomineralization, which takes place over very large timescales, the main drawback is
speeding up the process. Artificial mineralization mimics nature, although in short periods.
It involves the injection of CO
2
directly into geological formations to promote a carbonate-
forming reaction with alkaline minerals [
213
]. This mineral sequestration would be a
viable alternative for subsequent storage because the carbonate products formed would not
require monitoring owing to their high stability and safety. On the other hand, in-ground
or ex situ mineralization is based on the exposure of crushed rock material in a processing
plant where CO
2
is introduced, facilitating the formation of carbonate minerals. Natural

Molecules 2023,28, 5520 26 of 52
minerals or alkaline solid waste can be used [
213
,
214
]. The use of natural silicates requires
a large amount of material, which implies a very large operational size and an unfeasible
economic mineral impact. Ex situ mineralization can also be carried out using alkaline
wastes containing divalent metals such as ash originating from the coal or metallurgical
industry, cement and concrete wastes, or iron and steel slag [
215
,
216
]. This method would
reduce not only environmental CO
2
but also the accumulation of waste from industrial
activities, although a major disadvantage in that its capacity is much smaller than that
of CO
2
mineralization from silicates. At laboratory scale, this mineralization has been
satisfactorily performed by directly extracting CO
2
from the air, and its direct extraction
by passing the air through cooling towers using NaOH solutions has also been proposed
for larger scales [
217
]. However, the same authors pointed out the elevated costs of this
approach on a large scale.
The efficiency of the biomineralization process can also be accelerated by modifying
certain parameters such as increasing the temperature, pressure, or retention time. The
biomineralization process is also favored by the presence of purines, NaCl, or CA. The
presence of CA accelerates the rate of hydration of CO
2
dissolved in water; therefore,
possible modifications to CA to support high pH and temperature conditions without
losing its advantageous functionality have been studied [
218
,
219
]. On the other hand,
some varieties of carbonic anhydrase are inhibited in the presence of high concentrations
of hydrogen carbonate, which becomes a problem for its use in industry. However, this
can be circumvented, at least partially, by increasing the pH to values equal to or higher
than 9.0, conditions under which some CAs are still stable and functional. Immobilization
improves the stability of CA at high temperatures or alkaline conditions, as confirmed
by
Arias et al.
when forming calcite
in vitro
by mineralization using CA immobilized in
eggshell membranes [
219
]. Recombinant CAs have also been used to accelerate mineraliza-
tion under extreme conditions. For instance, CA from the alkalistable Aeribacillus pallidus
was genetically modified, achieving acceptable yields in the presence of pollutants such as
NO
x
and SO
x
[
200
]. Similarly, CA from the thermophilic bacterium Sulfurihydrogenibium
azorense was modified, and its half-life was found to be 8 days when the biomineralization
process was carried out at 70
◦
C and 53 days at a reaction temperature of 50
◦
C [
201
].
Di Lorenzo et al.
studied the effect of CA and a Zr-based MOF in the carbonation process
of wollastonite (CaSiO
3
) to produce calcite (CaCO
3
) [
202
]. Although CA accelerated CO
2
uptake by the silicate, the total gas absorber quantity was lower than that of the MOF. Jin
et al. also took advantage of CA to accelerate the carbonation of
γ
-dicalcium silicate, which
is also present in steel slag. They used a powder containing alkali-resistant CA bacteria,
increasing the yield by 19.0% [220].
7. Biotechnological Aspects of CO2Reduction
7.1. Hindrances to Biochemically Reducing CO2
Forward Equation (5) presents several drawbacks that hinder its application outside
the natural living environment, that is, employing it with biotechnological aims. First,
owing to the low redox potential of the CO
2
/HCO
2−
pair (
−
430 mV), the reaction is highly
endergonic under physiological conditions. Three main strategies have been developed
to overcome this problem (Figure 8): coupling thermodynamically favorable reactions in
the presence of an excess concentration of the reducing agent [
136
,
221
–
224
]; coupling the
reaction to an electrochemical device that provides electrons from a battery anode (i.e., by
supplying electric energy) [
225
–
228
]; and using photosensitive molecules able to absorb
light and regenerate redox partners [31,229–233].

Molecules 2023,28, 5520 27 of 52
Molecules 2023, 28, x FOR PEER REVIEW 30 of 56
Figure 8. The three different (enzymatic, electrochemical, and photochemical) approaches to regen-
erating cofactor NADH and producing formate with FDH.
Regeneration of the cofactor NADH is crucial for obtaining formate with acceptable
yields [234–237]. As previously mentioned, metal-dependent FDHs do not necessarily ac-
cept electrons from NADH (see Figure 6); nevertheless, whatever the cofactor is, either in
nature or in biotechnological approaches, it has to be regenerated for adequate progress
of the reaction. NADH, the primary source of electrons, is an expensive reactant, and thus
economic aspects are also relevant in cofactor regeneration. The approaches to regenerat-
ing NADH are similar to those followed to facilitate the thermodynamics of the reaction.
Two additional issues related to the oxidation of this cofactor also have to be kept in mind:
NAD+ can strongly interact through its negative phosphate charges, with positive charges
on the active site of the biocatalyst, inhibiting the biocatalyst, particularly in nonmetal-
dependent FDHs [238]. Moreover, it is well known that NAD+ trends to form dimers (see
below), which makes cofactor regeneration impossible [221,234]. Carbon dioxide uptake
into the reaction medium is another crucial factor. As commented throughout this text,
since CO2 is a nonpolar gas, its solubility in water or similar solvents is low and, more
importantly, the kinetics of solubilization and mass transfer are very slow [204]. Hence,
the employment of systems (specific solvents or solutions) that can incorporate CO2 into
their structures is decisive. As discussed above (see Sections 6.1 and 6.2), amines have been
used extensively [204].
Ionic liquids, solvents in which positive cations can interact with the oxygen atoms
of a CO2 molecule through their sole pair of electrons, are also excellent media to solve, in
high quantities, carbon dioxide gas [214,239,240]. The selection of the appropriate condi-
tions for the reaction is also crucial. Dealing with a gas, high pressures and low tempera-
tures are ideal conditions for solubilizing CO2, while with respect to pH, carbon dioxide
solubilization is favored at high pH values (Equations (3) and (4)). However, optimal con-
ditions are determined by FDH stability and activity, and temperatures must thus be mod-
erated (lower than 50 °C), pressures cannot be high, and pH values must be between 6.0
and 7.5, the range at which FDH shows its highest yields. Finally, the use of the adequate
Figure 8.
The three different (enzymatic, electrochemical, and photochemical) approaches to regener-
ating cofactor NADH and producing formate with FDH.
Regeneration of the cofactor NADH is crucial for obtaining formate with acceptable
yields [
234
–
237
]. As previously mentioned, metal-dependent FDHs do not necessarily
accept electrons from NADH (see Figure 6); nevertheless, whatever the cofactor is, either in
nature or in biotechnological approaches, it has to be regenerated for adequate progress of
the reaction. NADH, the primary source of electrons, is an expensive reactant, and thus
economic aspects are also relevant in cofactor regeneration. The approaches to regenerating
NADH are similar to those followed to facilitate the thermodynamics of the reaction. Two
additional issues related to the oxidation of this cofactor also have to be kept in mind:
NAD
+
can strongly interact through its negative phosphate charges, with positive charges
on the active site of the biocatalyst, inhibiting the biocatalyst, particularly in nonmetal-
dependent FDHs [
238
]. Moreover, it is well known that NAD
+
trends to form dimers (see
below), which makes cofactor regeneration impossible [
221
,
234
]. Carbon dioxide uptake
into the reaction medium is another crucial factor. As commented throughout this text,
since CO
2
is a nonpolar gas, its solubility in water or similar solvents is low and, more
importantly, the kinetics of solubilization and mass transfer are very slow [
204
]. Hence, the
employment of systems (specific solvents or solutions) that can incorporate CO
2
into their
structures is decisive. As discussed above (see Sections 6.1 and 6.2), amines have been used
extensively [204].
Ionic liquids, solvents in which positive cations can interact with the oxygen atoms
of a CO
2
molecule through their sole pair of electrons, are also excellent media to solve,
in high quantities, carbon dioxide gas [
214
,
239
,
240
]. The selection of the appropriate
conditions for the reaction is also crucial. Dealing with a gas, high pressures and low
temperatures are ideal conditions for solubilizing CO
2
, while with respect to pH, carbon
dioxide solubilization is favored at high pH values (Equations (3) and (4)). However,
optimal conditions are determined by FDH stability and activity, and temperatures must
thus be moderated (lower than 50
◦
C), pressures cannot be high, and pH values must be
between 6.0 and 7.5, the range at which FDH shows its highest yields. Finally, the use of

Molecules 2023,28, 5520 28 of 52
the adequate enzyme, that is, the FDH of the appropriate organism, is also a key factor in
the success of the reaction.
Approaches to circumventing these problems and achieving significant formate yields as
a starting point for obtaining value-added chemicals are discussed in the following sections.
7.2. Coupled reactions: Enzymatic Multicascades
Equation (5) is reversible although under physiological conditions is highly shifted
towards the backward reaction. The first successful conversion of CO
2
from formate
using FDH in a laboratory system was reported by Hopner and collaborators [
100
]. They
used the metal-dependent FDH from Pseudomonas oxalaticus as biocatalyst and NADH
concentrations in such a way that reaction 5, still being thermodynamically unfavorable,
was not completely shifted towards the oxidation of formate. They devised a sealed
system containing
14
CO
2
and measured the radioactivity of the H
14
COOH formed. In
this pioneering study, the kinetic parameters (k
cat
/K
M
) of the enzyme and its pH activity
profile were determined. However, the cofactor was not regenerated and the rates between
the forward and reverse reactions in their working conditions were 1:30, with a turnover
number for CO2reduction as low as 3 s−1, which makes these results insufficient.
The simplest and most straightforward way to increase formic acid generation is to
eliminate the products from the reaction media, that is, regenerating the NADH cofactor
using a reducing chemical agent present in the medium. This approach was developed in
the 1980s with redox biocatalytic systems [
241
,
242
]. The reaction desired to take place is
coupled with another “inverse” reaction, in which the reactant is added at high concen-
trations. Thus, the reaction is thermodynamically favored, and the cofactor is regenerated
(Figure 9A). Chenault and Whitsides, pioneers in using this technique with formate dehy-
drogenase, employed CbFDH to regenerate NADH by coupling formate oxidation with
the reduction in lactate to pyruvate using D-lactate dehydrogenase and obtained accept-
able results, with nicotinamide residual activity of 55% after each run [
242
]. However,
in their reaction, FDH was used for formate oxidation (backward Equation (5)), and the
coupled reaction was used to regenerate NAD
+
. Since then, the reduction of CO
2
(forward
Equation (5)
) using FDHs in a conjugated oxidation–reduction reaction system for regen-
erating the NADH cofactor has been carried out with several enzymes. Yu et al. cloned
the FDH gene from Cupriavidus necator in E. coli and coupled the reduction of CO
2
with
the oxidation of D-glucose to D-
δ
-gluconolactone using glucose dehydrogenase (GcDH) to
regenerate NADH [
243
] (Figure 9A). The expressed enzyme, an O
2
-tolerant, Mo-dependent
FDH, was able to effectively reduce formic acid comparable to that of the nonrecombinant
protein. Glutamate dehydrogenase (GDH) is also used to regenerate NADH. GDH cat-
alyzes the oxidation of glutamate to
α
-ketoglutarate and ammonia, thereby reducing NAD
+
to NADH. GDH is highly stable over a wide range of pH values and at temperatures as
high as 85 ◦C, as well as being widely commercially available and inexpensive [236,244].
Nonetheless, the most extensive coupled reaction approach is the well-known and
widely employed enzymatic cascade reaction that drives from CO
2
to formic acid, catalyzed
using FDH, from this species to formaldehyde, i.e., formaldehyde dehydrogenase (FalDH),
the biocatalyst, and, lately, to reduce this molecule to methanol, by the action of the alcohol
dehydrogenase (ADH) (Figure 9B). The oxidases mentioned above are typically used for
NADH regeneration.
El-Zahab et al. co-immobilized FDH, FaldDH, and ADH together with GDH into
polystyrene particles to reduce CO
2
to methanol [
245
] (Figure 9B). The NADH cofactor
was also immobilized, although separately. The results obtained with the immobilized
enzymes were similar to those obtained for the free enzymes; however, importantly, the
yield of the reaction was maintained at over 80% after 11 cycles. Ji et al. coupled the same
reaction cascade [
246
], but in their study, the four redox enzymes were entrapped in hollow
nanofibers together with CA to facilitate CO
2
absorption. The methanol yield was 36.17%,
retaining ca. 80% of the productivity after 10 reuses, with an accumulative yield of more
than of more than 900% for NADH regeneration. In another study using the same cascade,

Molecules 2023,28, 5520 29 of 52
Ren and collaborators encapsulated the same biocatalysts in MOF ZIF-8 and investigated
the effect of polyethyleneimine (PEI) on anchoring the NADH cofactor and, hence, on the
yield of the reaction [
247
]. Compared to the free system, the yield of this reactor system
increased 4.6-fold and the activity after eight cycles was retained by 50%.
Ionic liquids are known to solubilize CO
2
[
248
,
249
]. Taking advantage of this, Pinelo’s
laboratory immobilized the four mentioned enzymes as well as the cofactor in a series of
modified ILs composed of choline (CH) and amino acids (CHGlu, CHPro, CHGLy, and
CHHis) [
250
]. They generated a membrane reactor in which the products were removed
in situ and the reaction was displaced towards the desired product. The yield of CHGlu
increased up to fivefold compared to that of with the control aqueous system when NADH
was regenerated. In another study, immobilization of the four enzymes was performed on
superparamagnetic nanoparticles. Here, the yield was low (2.3% of methanol per NADH
molecule); however, under CO
2
pressure (126 psi), the reaction yield increased 64-fold after
30 min of reaction [250].
Molecules 2023, 28, x FOR PEER REVIEW 32 of 56
36.17%, retaining ca. 80% of the productivity after 10 reuses, with an accumulative yield
of more than of more than 900% for NADH regeneration. In another study using the same
cascade, Ren and collaborators encapsulated the same biocatalysts in MOF ZIF-8 and in-
vestigated the effect of polyethyleneimine (PEI) on anchoring the NADH cofactor and,
hence, on the yield of the reaction [247]. Compared to the free system, the yield of this
reactor system increased 4.6-fold and the activity after eight cycles was retained by 50%.
Ionic liquids are known to solubilize CO2 [248,249]. Taking advantage of this, Pinelo’s
laboratory immobilized the four mentioned enzymes as well as the cofactor in a series of
modified ILs composed of choline (CH) and amino acids (CHGlu, CHPro, CHGLy, and
CHHis) [250]. They generated a membrane reactor in which the products were removed
in situ and the reaction was displaced towards the desired product. The yield of CHGlu
increased up to fivefold compared to that of with the control aqueous system when
NADH was regenerated. In another study, immobilization of the four enzymes was per-
formed on superparamagnetic nanoparticles. Here, the yield was low (2.3% of methanol
per NADH molecule); however, under CO2 pressure (126 psi), the reaction yield increased
64-fold after 30 min of reaction [250].
Figure 9. (A) Several enzymes whose oxidation reactions have been coupled to FDH CO2 reduction
for regenerating NADH. (B) A classical enzyme multicascade for the reduction of CO2 to methanol;
here, GDH is coupled for regenerating NADH. (C) Uses of methanol at the global level in the year
2020 shown in percentages according to their production and demand [251].
Figure 9.
(
A
) Several enzymes whose oxidation reactions have been coupled to FDH CO
2
reduction
for regenerating NADH. (
B
) A classical enzyme multicascade for the reduction of CO
2
to methanol;
here, GDH is coupled for regenerating NADH. (
C
) Uses of methanol at the global level in the year
2020 shown in percentages according to their production and demand [251].
Phosphite dehydrogenase (PTDH, Figure 10A) has also been used for NADH regener-
ation in a multicascade approach to obtain methanol from CO
2
. Cazelles et al. studied the

Molecules 2023,28, 5520 30 of 52
effect of regenerating NADH using three different coupled reactions, namely phosphite ox-
idation, catalyzed using phosphite reductase; glycerol oxidation to dihydroxyacetone, per-
formed using glycerol dehydrogenase; and a natural photosystem (chloroplasts) extracted
from spinach leaves that oxidize water to molecular oxygen [
252
]. They encapsulated the
three enzymes in phospholipid–silica nanocapsules and obtained excellent activities with
PTDH with respect to the free enzymes in solution (55 times higher activities) under 5 bar
of CO
2
pressure for 3 h, although the other two systems were not so efficient. Singh et al.
also employed PTDH for regenerating NADH [
253
]. They expressed recombinant proteins
(FDH, FalDH from different bacteria, and ADH from yeast) in E. coli and performed assays
with free enzymes in water solution and in the presence of many different cosolvents. IL
1-ethyl-3-methylimidazolium acetate (EMIM-Ac) was found to be the most effective in
increasing methanol production. Indeed, the yield was enhanced more than twofold (from
3.28 mM of methanol to 7.86 mM, 6 h of reaction) in the presence of 1% EMIM-Ac because
of the ability of EMIM cations to interact with CO
2
, increasing solubility. Finally, lactate
dehydrogenase (LDH) was also used to regenerate NADH in a multicascade reaction [
254
].
This enzyme was immobilized in a sol–gel matrix, and CO
2
reduction was acceptable after
1 h of reaction, as indicated by the authors.
Using CO
2
as a substrate for the generation of methanol is an attractive process
because of its potential use as a fuel and the multitude of products obtained from it at the
industrial level. Figure 9C shows the chemicals produced from methanol at a global level
in 2020 [
251
]. Methanol is a precursor of numerous compounds such as olefines (25.9%
of the demand for methanol, the year 2020), formaldehyde (25.0%), gasoline blending
(13.1%), biodiesel, and so on. Therefore, it is of great commercial interest due to its potential
application both in the energy industry as a fuel and in environmental CO
2
sequestration
to mitigate high atmospheric CO
2
levels. The methanol production in the year 2023
was
98.9 ×106Tm
and it is projected that by the year 2027, more than 8
×
10
6
Tm will
be obtained from e-methanol (produced from captured carbon dioxide and hydrogen
produced from renewable electricity, ca 5
×
10
6
Tm) and biomethanol (produced from
sustainable biomass, ca 3
×
10
6
Tm). For example, currently, 4000 Tm of methanol is
produced annually using biocatalysts based on copper and zinc oxide in Iceland, recycling
some 5500 tons of CO
2
annually. Other approaches to achieving the same effect have also
been undertaken in Germany and China [
222
]. The use of enzymes is an advantage for the
conversion of CO
2
to methanol because of the high selectivity of the catalyzed reaction.
This reduction of CO
2
to methanol is considered a green chemical process and occurs at
atmospheric temperature and pressure [253].
7.3. Electrochemical Regeneration of NADH Cofactor
NADH can be regenerated at the cathode of an electrolytic cell by applying adequate
electric voltage (Figure 10A). This method can be extensively applied and permits the easy
separation of products [
225
–
228
]. In principle, as the supported potential difference can be
as high as desired within technical constraints, the cofactor regeneration under appropriate
conditions could be high, although the complete energy cycle is probably not. If a cell
is supported by renewable energy, this approach can also be considered green. In the
electrolytic cell, the cofactor can be the primary acceptor of electrons from the electrode
(direct electrochemical regeneration, Figure 10B) or, in contrast, other molecules can accept
the electrons, the cofactor being reduced by these mediators (indirect mode Figure 10B).

Molecules 2023,28, 5520 31 of 52
Molecules 2023, 28, x FOR PEER REVIEW 34 of 56
Figure 10. (A) Schematic view of an electrochemical device for obtaining formic acid from CO2. (B)
Scheme of three possible mechanisms for transferring electrons from the cathode to the cofactor and
finally to FDH: without any mediator species, with a mediator (typically a photosynthesizer mole-
cule), and also with an enzyme intercalated between the mediator and the cofactor.
The direct mode has two intrinsic disadvantages that are very difficult to overcome:
the formation of (NAD)2-inactive dimers and the necessity of using high overpotentials.
The electrochemical reduction of NAD+ molecule proceeds in two stages (Figure 11). In
the first step, NAD+ captures an electron and an NAD* radical is formed. In the second
step, an additional electron and a proton are accepted by the NAD* radical. However,
NAD* can dimerize and, furthermore, due to the adsorption of NAD+ onto the electrode,
this dimerization process is favored over the uptake of the second electron. The need to
use high overpotentials for direct NADH reduction is another limitation of this method.
Several studies modifying the electrode nature concluded that mass transfer between the
Figure 10.
(
A
) Schematic view of an electrochemical device for obtaining formic acid from CO
2
.
(
B
) Scheme of three possible mechanisms for transferring electrons from the cathode to the cofactor
and finally to FDH: without any mediator species, with a mediator (typically a photosynthesizer
molecule), and also with an enzyme intercalated between the mediator and the cofactor.
The direct mode has two intrinsic disadvantages that are very difficult to overcome:
the formation of (NAD)
2
-inactive dimers and the necessity of using high overpotentials.
The electrochemical reduction of NAD
+
molecule proceeds in two stages (Figure 11). In
the first step, NAD
+
captures an electron and an NAD* radical is formed. In the second
step, an additional electron and a proton are accepted by the NAD* radical. However,
NAD* can dimerize and, furthermore, due to the adsorption of NAD
+
onto the electrode,
this dimerization process is favored over the uptake of the second electron. The need to
use high overpotentials for direct NADH reduction is another limitation of this method.
Several studies modifying the electrode nature concluded that mass transfer between the
cathode and the cofactor was a crucial step for favoring reduction versus dimer formation.
These problems can be partially circumvented by selecting an appropriate electrode [
255
].

Molecules 2023,28, 5520 32 of 52
For instance, Ag or Pt electrodes coated on Cu foams were successfully employed for
NADH regeneration [
256
]. The existence of a mediator on the electrode surface is another
key point for avoiding dimer formation. Mediators such as (2,2-bipyridyl) (pentamethyl-
cyclopentadienyl)rhodium, [Cp*Rh(bpy)], and methylviologen (MV) have been shown
to decrease the overpotential for NADH regeneration, facilitating electron transfer and
NADH recovery [221].
Molecules 2023, 28, x FOR PEER REVIEW 35 of 56
cathode and the cofactor was a crucial step for favoring reduction versus dimer formation.
These problems can be partially circumvented by selecting an appropriate electrode [255].
For instance, Ag or Pt electrodes coated on Cu foams were successfully employed for
NADH regeneration [256]. The existence of a mediator on the electrode surface is another
key point for avoiding dimer formation. Mediators such as (2,2-bipyridyl) (pentamethyl-
cyclopentadienyl)rhodium, [Cp*Rh(bpy)], and methylviologen (MV) have been shown to
decrease the overpotential for NADH regeneration, facilitating electron transfer and
NADH recovery [221].
Figure 11. The two reduction steps of NAD+. The addition of one electron produces the radical
NAD*. This radical can either be reduced by the addition of an additional electron and a proton
(up), to produce 1,4-NADH (up) or combine with another radical to generate the 4,4′-dimer (NAD)2.
In an elegant experiment, Song et al. generated a cysteine residue in FDH from Thio-
bacillus sp. KNK65MA and aached the mutated enzyme to copper nanoparticles (CuNPs)
deposited on the electrode surface [257]. Polyethylene glycol (PEG) was then used to cross-
link the FDH-NAD+ system so that the cofactor could swing from CuNP to the enzyme
and vice versa. No mediator was used. This system produced 8.5 mM formate, several
times that achieved with a free enzyme.
The enzyme cascade for methanol production from CO2 (Figure 10B) has also been
widely performed using an electrochemical approach. Addo et al. coupled the multien-
zyme cascade for producing methanol from CO2 to a polyneutral red electrode to regen-
erate NADH [258]. Electroenzymatic reduction in CO2 was also achieved with maximum
Faradaic efficiency (99 + 5%) by the immobilization of Mo-dependent FDH from E. coli at
the surface of a carbon electrode [259]. Here, reduction was achieved through the mediator
cobaltocene being covalently bound to the linear polymer poly(allylamine), which trans-
ferred electrons from the cathode to the enzyme. In another interesting study, CO2 reduc-
tion was electrochemically achieved by using copper deposited in a glassy carbon elec-
trode, the Rh(III) complex [Cp*Rh(bpy)Cl]+ (Cp* = pentamethylcyclopentadienyl; bipy =
Figure 11.
The two reduction steps of NAD
+
. The addition of one electron produces the radical
NAD*. This radical can either be reduced by the addition of an additional electron and a proton (up),
to produce 1,4-NADH (up) or combine with another radical to generate the 4,40-dimer (NAD)2.
In an elegant experiment, Song et al. generated a cysteine residue in FDH from
Thiobacillus sp. KNK65MA and attached the mutated enzyme to copper nanoparticles
(CuNPs) deposited on the electrode surface [
257
]. Polyethylene glycol (PEG) was then used
to crosslink the FDH-NAD
+
system so that the cofactor could swing from CuNP to the
enzyme and vice versa. No mediator was used. This system produced 8.5 mM formate,
several times that achieved with a free enzyme.
The enzyme cascade for methanol production from CO
2
(Figure 10B) has also been
widely performed using an electrochemical approach. Addo et al. coupled the mul-
tienzyme cascade for producing methanol from CO
2
to a polyneutral red electrode to
regenerate NADH [
258
]. Electroenzymatic reduction in CO
2
was also achieved with max-
imum Faradaic efficiency (99
±
5%) by the immobilization of Mo-dependent FDH from
E. coli
at the surface of a carbon electrode [
259
]. Here, reduction was achieved through the
mediator cobaltocene being covalently bound to the linear polymer poly(allylamine), which
transferred electrons from the cathode to the enzyme. In another interesting study, CO
2
reduction was electrochemically achieved by using copper deposited in a glassy carbon
electrode, the Rh(III) complex [Cp*Rh(bpy)Cl]
+
(Cp* = pentamethylcyclopentadienyl; bipy
= bipyridine) as a mediator and CbFDH as a biocatalyst, with yields threefold higher than
those of previous analogous works with copper foil electrodes [
260
]. Chen et al. also used

Molecules 2023,28, 5520 33 of 52
an Rh(III) complex as a mediator for the electrochemical regeneration of NADH [
261
]. MOF
NU-1006 containing CbFDH was deposited on the electrode surface of fluorine-dopped tin
oxide glass. NU-1006 is a mesoporous material with a channel size that can accommodate
CbFDH (6
×
4
×
11 nm). The electrode was obtained using a multilayer system in which
the entrapped enzyme was the latter and was in contact with the solution containing
CO
2
. Regeneration of the cofactor was optimum because of the modified electrode, with a
formate generation rate of 79
±
3.4 mM h
−1
. All these experiments have the advantage of
easy regeneration of NADH, due to the possibility of creating a sufficient negative redox
potential. However, its translation towards more complex systems, such as a multicascade
system coupled to other oxidoreductases, no longer seems biotechnologically accessible.
Barin et al. immobilized CbFDH on polystyrene nanofibers and NADH in a copper
foam electrode [
262
]. Although the activity of this system was inferior to that of the
free enzyme, the immobilized enzyme was stable for a long period (41% of the initial
yield after 20 days) and had acceptable reusability after eight cycles (53% of the initial
activity). The authors also observed an inhibitory effect of NADH at concentrations
higher than 0.51 mM. Indeed, several studies have demonstrated the inhibition of FDH
by the cofactor at concentrations higher than millimolar. On the other hand, Zhang et al.
encapsulated FDH, FaldDH, and ADH in MOF ZIF-8 and used the Rh complex (Cp*Rh(2,2
0
-
bipyridyl-5,5
0
-dicarboxylic acid)Cl
2
) grafted on the cathode as a mediator to perform
NADH regeneration [
263
]. The concentration of methanol obtained was fivefold (from
0.061 to 0.320 mM) with the encapsulated enzyme compared to the free enzyme. Moreover,
when NADH was electrochemically regenerated, an increase of 0.742 mM was observed.
This again demonstrated that electrochemical NADH regeneration is probably the best
approach to achieving this goal.
7.4. Photochemical NADH Regeneration
Photochemical reactions are another suitable approach to reducing CO
2
[
31
,
229
–
232
].
The energy arises from light, which is an inexpensive, renewable, and clean source. This
requires a supply of electrons and a photosensitizer mediator scheme (Figure 12). The
electron donor, D, also called the sacrificial agent, is typically a stable solute present in large
concentrations that can be easily oxidized using a photosynthesizer in its excited state [
223
].
Amines such as triethanolamine (TEOA), triethylamine (TEA), and ethylenediaminete-
traacetic acid (EDTA) are the most commonly employed, although many other species, such
as 3-(N-morpholino) propanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid, 2-(N-morpholino)ethanesulfonic acid mercaptoethanol, phosphite, propanol, and
even molecular hydrogen gas have also been used [
30
]. The redox potentials of these
systems must be low enough to be oxidized using the photosynthesizer so that a pool of
electrons is always present in the solution. This electron donor supplier must be present
at high concentrations, typically not lower than 100 mM, for the system to be effective. A
water molecule has also been proposed, imitating natural photosynthesis, as a sacrificial
agent; however, the redox potential of H2/H2O is too low to produce an efficient system.
The key to these reactions is photosensitizers, molecules that are excited by light
[31,232]
.
These species act as electron mediators, capturing electrons from the sacrificial agent and
transferring them to the cofactor (Figure 12). Then, the cofactor is regenerated, and CO
2
can
be reduced using FDH. The mediator is a molecule that is stable in its oxidized state and
can be photoactivated, passing from the ground to an excited state in which this molecule
has a much higher redox potential, being able to oxidize the sacrificial agent and becoming
reduced. In this reduced state, the mediator is highly reducing and immediately relin-
quishes the electrons to the cofactor. This transfer proceeds with a proton cession, such
that the cofactor in its oxidation state (NAD
+
or others) is reduced (NADH or analogous).
The photosensitizers must fulfill three requirements [
30
,
264
]: First, the band gap between
the HOMO and LUMO must be low enough to accept an electron from visible light (see
Figure 3). This is normally satisfied in extended
π
–
π
-conjugated systems or semiconductors.
Second, in the excited state, its redox potential must be sufficiently high to oxidize the

Molecules 2023,28, 5520 34 of 52
sacrificial agent under solution conditions. Finally, the ground state redox potential must be
sufficiently low to reduce the cofactor. Other chemical (stability), economical (inexpensive),
and environmental (clean) requirements must also be satisfied.
Molecules 2023, 28, x FOR PEER REVIEW 37 of 56
(NADH or analogous). The photosensitizers must fulfill three requirements [30,264]: First,
the band gap between the HOMO and LUMO must be low enough to accept an electron
from visible light (see Figure 3). This is normally satisfied in extended π–π-conjugated
systems or semiconductors. Second, in the excited state, its redox potential must be suffi-
ciently high to oxidize the sacrificial agent under solution conditions. Finally, the ground
state redox potential must be sufficiently low to reduce the cofactor. Other chemical (sta-
bility), economical (inexpensive), and environmental (clean) requirements must also be
satisfied.
Figure 12. Scheme of the photochemical reduction of CO2 using FDH.
Various photosensitizers have also been used. Rh(III) and Ru(III) bipyridyl com-
plexes are well known for their photosensitivity; consequently, their use has been ex-
tended [264–269]. Guo et al. employed Cp*Rh(bpy)(H2O)]2+ (Cp = cyclopentadienyl; bpy =
2,2′-bipyridyl) as a synthesizer in a system where FDH and FalDH were immobilized on
polyethylene membranes doped with the widely used semiconductor TiO2 [270]. A com-
parison between the results using water or EDTA as a sacrificial agent and as a function
of pH was described, the laer being much more efficient for formaldehyde production.
The optimal FDH:FalDH ratio was 1:0.3, reaching up to 6.5% formaldehyde production
after 4 h of reaction. Photosynthesizers composed of xanthene dyes have also been used.
Kim and col. combined eosin Y with cobaloxime complexes for regenerating NADH using
TEOA as the sacrificial electron donor [271]. The system exhibited an acceptable turnover
number for formate generation (ca. 1.6) and an optimal NADH production (0.038 mM/h).
In situ changes in eosin Y infrared and UV-visible spectroscopy properties eosin Y was
also used to follow in situ formic acid generation using infrared and UV-visible absorption
spectroscopies [272]. Interestingly, EDTA was used not only as an electron sacrificial agent
but also as a source of CO2, without any additional electron carriers.
Nanomaterials, which act as porphyrin-based photosensitizers, are another set of
well-developed approaches for acting in these photochemical reactions. Ji and coworkers
designed a biomimetic chlorosome by combining porphyrin, eosin Y, and [Cp*RhCl2]2 to
generate supramolecular assemblies [273]. Using TEOA as the ultimate electron donor
agent, an enzyme cascade was coupled, obtaining 38 µM methanol from CO2 after 2 h of
reaction. MOFs have revolutionized many technochemical applications [157]. The MOFs
used for CO2 reduction are also basic pivots in this respect. MOFs contain molecules and
holes with different chemical and physical features (hydrophilicity, hydrophobicity, acid-
base properties, photochemical features, etc.) that make them ideal for catalysis in general,
and photobiocatalysis in particular. An excellent recent example is Xing’s work [229],
where a porphyrin ligand was covalently bound to a Zr-based MOF and, posteriorly, the
complex Cp*Rh(bpydc)Cl2 (bpydc = 2,2′-bipyridine-5,5′-dicarboxylic acid) was
Figure 12. Scheme of the photochemical reduction of CO2using FDH.
Various photosensitizers have also been used. Rh(III) and Ru(III) bipyridyl com-
plexes are well known for their photosensitivity; consequently, their use has been ex-
tended [
264
–
269
]. Guo et al. employed Cp*Rh(bpy)(H
2
O)]
2+
(Cp = cyclopentadienyl;
bpy = 2,20-bipyridyl
) as a synthesizer in a system where FDH and FalDH were immobi-
lized on polyethylene membranes doped with the widely used semiconductor TiO
2
[
270
]. A
comparison between the results using water or EDTA as a sacrificial agent and as a function
of pH was described, the latter being much more efficient for formaldehyde production.
The optimal FDH:FalDH ratio was 1:0.3, reaching up to 6.5% formaldehyde production
after 4 h of reaction. Photosynthesizers composed of xanthene dyes have also been used.
Kim and col. combined eosin Y with cobaloxime complexes for regenerating NADH using
TEOA as the sacrificial electron donor [
271
]. The system exhibited an acceptable turnover
number for formate generation (ca. 1.6) and an optimal NADH production (0.038 mM/h).
In situ changes in eosin Y infrared and UV-visible spectroscopy properties eosin Y was
also used to follow in situ formic acid generation using infrared and UV-visible absorption
spectroscopies [
272
]. Interestingly, EDTA was used not only as an electron sacrificial agent
but also as a source of CO2, without any additional electron carriers.
Nanomaterials, which act as porphyrin-based photosensitizers, are another set of
well-developed approaches for acting in these photochemical reactions. Ji and coworkers
designed a biomimetic chlorosome by combining porphyrin, eosin Y, and [Cp*RhCl
2
]
2
to
generate supramolecular assemblies [
273
]. Using TEOA as the ultimate electron donor
agent, an enzyme cascade was coupled, obtaining 38
µ
M methanol from CO
2
after 2 h of
reaction. MOFs have revolutionized many technochemical applications [
157
]. The MOFs
used for CO
2
reduction are also basic pivots in this respect. MOFs contain molecules and
holes with different chemical and physical features (hydrophilicity, hydrophobicity, acid-
base properties, photochemical features, etc.) that make them ideal for catalysis in general,
and photobiocatalysis in particular. An excellent recent example is Xing’s work [
229
], where
a porphyrin ligand was covalently bound to a Zr-based MOF and, posteriorly, the complex
Cp*Rh(bpydc)Cl
2
(bpydc = 2,2
0
-bipyridine-5,5
0
-dicarboxylic acid) was incorporated into the
organic frame, generating a system that can be activated by light owing to the porphyrin,
the Rh(III) complex, and original aromatic ligands of the MOF. FDH was then immobilized
via electrostatic entrapment. Using TEOA, up to 244
µ
g/mL of formic acid was formed
after 4 h of reaction. A similar approach employing an MOF based on pyrane skeleton
linkers, NU-1006, has also been developed [
261
]. In this case, the Rh(III) complex attached
to the MOF was used as an electron mediator, reducing NADH and being reduced by a
pyrene photosynthesizer. FDH was also trapped in the MOF and the reaction took place

Molecules 2023,28, 5520 35 of 52
with a yield of formic acid production of 0.144
±
0.003 mM after 24 h, while the cofactor
was regenerated at a rate of 28 mMh
−1
. In another recent study, MOF Mil-125-NH
2
was
functionalized with the Rh complex in such a way that it was covalently fixed to the
secondary sphere of the MOF and FDH was subsequently immobilized [
274
]. The system
obtained a formic acid yield of 9.5 mM in 24 h, whereas the NADH regeneration was 64%.
Graphitic carbon nitride (g-C
3
N
4
)-based materials are other fascinating agents with
extremely interesting properties, such as photocatalysts [
275
]. These materials have also
been used in biocatalysis and to generate formate from CO
2
. Zeng et al. used g-C
3
N
4
doped with the metal dichalcogenide WS
2
, which provides g-C
3
N
4
with specific semicon-
ductor properties that can be used as photosensitizers [
276
]. Using [Cp*Rh(phen)H
2
O]
2+
(
phen = 1,10-phenanthroline
) as a mediator and TEOA as the sacrificial agent, a multi-
cascade system for obtaining methanol from CO
2
was designed with acceptable results
(methanol productivity 372.1
µ
mol h
−1
gcat
−1
). In a similar approach, Meng and col.
designed nanospheres with thiophene incorporated into hollows in a double shell that
acted as the photosynthesizer [
277
]. This nanomaterial was coupled to [Cp*Rh(bpy)H
2
O]
2+
,
which in turn was coupled to the cofactor to reduce CO
2
. Optimal NADH yield regen-
eration was obtained (74%). Silver nanoclusters combined with TiO
2
and g-C
3
N
4
have
also been employed in efficient devices for formate production [
278
]. These nanoclusters
are good light sensors, making it possible to obtain good yields in CO
2
uptake with the
metal-dependent FDH from Clostridium ljungdahliia. Carbon nitride has also been used in
microbial electrosynthesis. Here, the photoanode chamber was formed using an activated
carbon fiber (ACF) supported by NiCoWO
4
in g-C
3
N
4
[
279
]. The oxidation of water by
light in this chamber provides the electrons for reducing CO
2
in the cathode chamber,
formed by a g-C
3
N
4
/ACF (without NiCoWO
4
), in which a culture of E. coli was adhered as
a biofilm. FDH from E. coli produced the reduction of CO
2
due to the electrons arriving
from the biocathode and the protons, also produced in the cathode by the photochemical
decomposition of water, which crossed a cation exchange membrane. The witty system, first
applied in CO2reduction, provided highly efficient formate synthesis (12.8 mM per day).
When dealing with photochemical reactions, a critical point is the reactive oxygen
species (ROS) that can be generated by photoexcitation processes. To avoid this, sys-
tems mimicking chloroplasts have been developed, creating divided compartments where
reactions occur separately. The photoactivation process was achieved by combining a
Rh(III) complex conjugated onto g-C
3
N
4
previously modified using thiophene, with tri-
ethanolamine used as the electron sacrificial agent [
280
]. The key to this approach arises
from the encapsulation of FDH into a MOF (MAF-7), which protects the enzyme from pho-
toactivation reactions. NADH shuttled electrons from the reduction compartment towards
FDH, obtaining 16.75 mM of formic acid after 9 h of illumination. Immobilization of FDH
together with CA in the TOF ZIF-8 was also carried out by Yu et al. in another system
imitating photosynthesis and with g-C
3
N
4
as the photosynthesizer [
281
]. CA accelerated
the interconversion of CO
2
/HCO
3−
, allowing for an effective mass transfer rate between
the gas and liquid phases. The authors reported production of 243
µ
M formic acid with
excellent system stability since the yield production was above 80% after 10 batches.
Graphene has also been employed as a photocatalyst in systems that imitate photo-
synthesis and produce formic acid from CO
2
[
210
]. Ji’s group conceived and developed a
nanofiber polyurethane system as a support where the cationic electrolyte, a polyalanine,
was deposited onto graphene oxide, while the cascade of proteins FDH, FalDH, and ADH
was entrapped in the hollows of the nanofiber to generate methanol [
246
,
282
,
283
]. Finally,
polymers that mimic photosystems in CO
2
reduction have also been candidates for biotech-
nological applications. Kim et al. reported a photosystem based on polydiacetylenes and
covalently attached (phen)Ru(bpy)
2
as the mediator [
284
]. The system exhibited acceptable
regeneration values for NADH.
It should also be noted that semiartificial photocatalysis centers have been also de-
signed to reduce CO
2
. Sokol et al. generated a sophisticated electrocell in which the
photoanode was the photosystem II from Thermosynechococcus elongatus embedded in a

Molecules 2023,28, 5520 36 of 52
redox polymer composed of the complex [Os(bipy)
2
Cl]Cl bound to a polymer of a deriva-
tive of allylamine was, in turn, deposited on TiO
2
[
285
]. Here, light (680 nm) oxidized
water to O
2
, generating electrons that were shuttled towards the cathode, where FDH
from Desulfovibrio vulgaris had adhered to TiO2but, in this case, coated with a fluorine tin
oxide. Excellent Faraday efficiencies (>70%) and acceptable yields (0.185
µ
mol/cm
2
) were
obtained, although the system exhibited the progressive photodegradation of PSII.
Due to its high substrate and product selectivity as a biocatalyst, FDH has been used
in recent years to obtain chemical products from reduced forms of CO
2
, such as formic
acid. In addition to its own applicability, this acid is used as a springboard product to
obtain a wide variety of derivatives. For example, CO
2
can be converted to oxalic acid
through the coupling of two formate molecules [
286
]. This can occur via a metal–formate
intermediate (typically sodium or potassium alkaline metals), obtained from the electro-
or photocatalytic reduction of CO
2
coupled to FDH. After obtaining this metal–formate,
two molecules are coupled to give oxalate, which, after acidification, gives rise to oxalic
acid. Oxalic acid produced through this coupling route can be used in various industries,
including pharmaceuticals, as a component of some antibiotics, or textiles and in other
industries such as food (beer or wine production) or chemicals [
286
]. In addition, from the
reduction of this oxalic acid, a wide variety of derivatives are obtained. The first oxalic acid
reduction product is glyoxylic acid, which, after reduction, produces glycolic acid and also
ethylene glycol. These chemicals are the starting points in the production of agrochemicals,
flavors, cosmetics, and polymers [287].
7.5. CO2Reduction by Whole-Cell Bacteria
Finally, it should be pointed out that recent studies focused on CO
2
reduction encom-
pass an even more open overview of what nature provides us in this area. Indeed, whole
cells can also be used for fixing CO
2
, using H
2
as the reductant agent [
288
–
290
], electro-
chemical reduction [
291
], or even a combination of H
2
and photocatalysis [
74
]. Whole-cell
biocatalysis is a promising method developed in the last few years that shows high efficacy
and selectivity and can be used in soft conditions. Moreover, since there is no requirement
for purifying enzymes, the most expensive step in biocatalytic processes, the whole-cell
approach is a hopeful methodology that will probably have the highest efficiency/cost
ratio. It is also relatively easy to adjust the experimental conditions to be developed if the
suitable organism or correct molecular biology tools in them are adequately employed.
Table 5reports the most relevant results obtained in the CO
2
reduction to formate using
the whole-cell approach. The culture of these microorganisms supplied by H
2
gas reduces
CO
2
obtaining extraordinarily high concentrations of formate. Methylobacteria species are
satisfactory in this aspect [290].

Molecules 2023,28, 5520 37 of 52
Table 5. CO2reduction to obtain formate using whole-cell approaches.
Reduction Organism Significant Assay Features/Conditions Formate Production Ref
Hydrogenation
Overexpressed genes of FDH from
E. coli, Clostridium carboxidi-vorans,
Pyrococcus furiosus and
Methanobacterium thermos-formicicum
in E. coli JM109(DE3)
H2atmosphere. Wet cell pellet (0.5 g wet cells/mL)
resuspended in 50 mM sodium phosphate buffer, pH
7.0, containing 0.25 M sodium bicarbonate as a source
of CO2. Incubation 37 ◦C.
The highest formic yield (FDH from
P. furiosus) was more than 1 g L−1h−1[292]
Hydrogenation Escherichia coli
CO
2
and H
2
placed under pressure (up to 10 bar). First,
pressurized the system to rapidly convert 100%
conversion of gaseous CO2to formic acid. Next,
NaOH addition to the E. coli cell suspension (pH > 8).
Formate concentration to 150 (4 bar) and
200 (6 bar) mmol L−1. Increasing the
pressure to 10 bar (122.88 mmol L−1CO2
and 3.61 mmol L−1H2): > 0.5 mol L−1
formate at 23 h of reaction.
[293]
Hydrogenation Acetobacterium woodii
and Thermoanaerobacter kivui
Cells grown with 28 mM glucose or 0.1 M pyruvate (50
mM imidazole, 20 mM KCl, 20 mM MgSO4, 2 mM
DTE, 4 µM resazurin, pH 7.0). 1 mg/mL in an anoxic
medium. Shaking for 10 min (60 ◦C), with additional
300 mM KHCO
3
. The experiment started by replacing
the gas phase with H2+ CO2(80:20%, 2 ×105Pa).
Optimal formate production rates of
234 mmol g−1protein h−1[270]
Hydrogenation H2-dependent CO2reductase from
Acetobacterium woodie expressed in E.
coli JM109
Whole-cell E. coli in presence of formate (10 mM) and
methylviologen (10 mM) as electron acceptor. E. coli
cells incubated with H2+ CO2(80:20%, 1.1 ×105Pa).
6 mM formic acid in 60 h. Addition of
2.5 mM HCO3−increased 4-fold the
formate generation. [294]
Hydrogenation Acetobacterium woodii and
Thermoanaerobacter kivui
50 mM imidazole, 20 mM MgSO
4
, 20 mM KCl, 20 mM
NaCl, 2 mM DTE, pH 7.0; or 50 mM K-phosphate,
20 mM KCl, 20 mM NaCl, 2 mM DTE, pH 7.0) was
maintained at 30 and 60 ◦C for A. woodii and T. kivui,
respectively. Gas flow rate was maintained at a value
of 25 mL/min
60 mM and 80 formic acid generation after
5 h of reaction for A. woodii and T. kivui,
respectively. [295]
Electrochemical Methylobacterium extorquens AM1,
System: 1 mM H2SO4with a platinum electrode
placed in the anode; protons supplied to the cathode
through a proton-exchange membrane. 1.9 g wet cells,
10 mM methyl viologen (MV) added to the cathode
reactor as an electron mediator
Formate concentrations of up to 60 mM,
80 h (1.9 g wet cells, 10 mM MV, pH 7.0) [290]

Molecules 2023,28, 5520 38 of 52
Table 5. Cont.
Reduction Organism Significant Assay Features/Conditions Formate Production Ref
Electrochemical Shewanella oneidensis MR-1
The electrochemical cell with two compartments
divided by a proton-exchange memberane. Copper
plates and Ag/AgCl electrodes. Whole-cell
S. oneidensis MR-1 (wet-cell, 0.5 g) and MV, 10 mM). RT,
anaerobic conditions.
Formic acid at 0.59 mM h−1for 24 h.
Medium supplemented with fumarate and
nitrate: 1.9 mM h−1for 72 h. LB
supplemented with 40 mM fumarate, 1mM
nitrate and 20 mM DL-lactate: 136.84 mM
formic acid at 72 h. (3.8 mM h
−1
g
−1wet-cell
)
[296]
Electrochemical E. coli
NaHCO3electrolyte saturated with N2or CO2media
at three different poised potentials, i.e., 0.4, 0.8 and
1.0 V vs. Ag/AgCl. E. coli-immobilized
FePc-CDC/ACF and ACF electrodes in the presence of
the NR mediator (FePc: iron pfthalocyanine; CDC:
carbide-derived carbon; ACF: activated carbon fiber;
NR: Neutral Red).
Maximum formate production rate of
~30 mg/L-h under CO2flow (120 mg/L-h)
with NR mediator [297]
Electrochemical Methylorubrum extorquens AM1
Nafion 115 (proton permeable) membrane. Cathode:
0.6 g cell pellet (potassium phosphate 200 mM, pH 7.0)
as catholyte. Electron mediator: ethyl viologen 10 mM.
0.6 g cell pellet suspended into catholyte
(200 mM-potassium phosphate at pH 7) saturated by
CO2purging (30 min). Water splitting reaction in
100 mM-H2SO4.
Formate production: 6 mM h−1[298]
Electrochemical Shewanella loihica PV-4 Cathode: biohydrogel formed by Shewanella loihica
PV-4 immobilized in graphene oxide.
High Faradaic efficiency (~99.5%) and
46-fold increase of formate titer without
exogenous mediator (4.2 mM formate. 36 h)
[299]
Hydrogen/car-bohydrate
fermentation Saccharomyces cerevisiae
Two phases:
Glucose fermentation for generating CO2
CO2Ru catalysis hydrogenation
26% of the CO2was hydrogenated.
Addition of His 150 mM: 128 mM in formic
acid at 48 h. [300]
Photocatalytic
hydrogenation Shewanella oneidensis MR-1
Anaerobic conditions: N2-filled chamber and samples
irradiated from outside the chamber by a KL5125 Cold
150W light source. Irradiation into MV, with
triethanolamine (TEOA) as sacrificial agent in 50 mM
HEPES, 50 mM NaCl, pH 7, 23 ◦C.
Formate (
∼
1500 nmol) was produced when
MR-1 was incubated with CO2
(∼5000 nmol) after 48 h incubation [301]

Molecules 2023,28, 5520 39 of 52
Leo et al. genetically modified E. coli cells to express the hydrogen-dependent CO
2
re-
ductase from Acetobacterium woodie [
294
]. In this bacterial culture, increasing the cell density
up to 30 mg/mL resulted in a formate production yield of 6 mM/h, whereas the addition
of 2.5 mM HCO
3−
increased the formate generation rate fourfold. In this way, the authors
confirmed that E. coli CA also played an important role in providing the correct substrate to
FDH. Müller’s laboratory took advantage of the metabolic machinery of the acetogenic bac-
teria Acetobacterium woodii and Thermoanaerobacter kivui to convert H
2
and CO
2
from syngas
into formic acid, obtaining production rates of
234 mmol g−1protein h−1[288]
.E. coli has
been also used for formate generation with and without overexpression of exogenous FDH
genes. Indeed, as commented previously (see Section 3.2), E. coli has two metal-dependent
FDHs capable of reducing CO
2
to formate. This bacterium has been used in the whole-cell
strategy with extraordinarily high formate generation. Indeed, more than 0.5 molL
−1
of
formate concentration was achieved when E. coli cells were grown under CO
2
and H
2
gas
at 10 bar pressure for 23 h [
293
]. E. coli JM109(DE3) strain has also been used as a host
bacterium where to overexpress FDHs from Clostridium carboxidivorans, Pyrococcus furiosus
(Pf) and Methanobacterium thermosformicicum, also obtaining excellent yields [
292
]. Indeed,
the highest formate generation yield of this study, obtained with Pf FDH, was more than
1 gL−1h−1.
Electrochemical reduction is another approach for obtaining high yields of formate
within the whole-cell frame. An excellent and very recently released review describes
in detail this methodology [
288
]. Table 5highlights the remarkable features of the—up
to now—few articles published in this area concerning formic acid generation. In these
studies, yields are as good as for those using the H
2
reduction strategy. For instance,
Le et al.
achieved to obtain 3.8 mM h
−1
g
−1wet-cell
of formic acid in the cathode electrode at 72 h
when Shewanella oneidensis MR-1 was grown in LB media supplemented with nitrate 1 mM
and DL-lactate 20 mM [296].
It is also remarkable that two other different approaches to applying whole-cell have
been developed. Guntermann and col. devised a two-phase system where Saccharomyces
cerevisiae D-glucose fermentation produces ethanol and CO
2
in the water phase. This
was coupled to a hydrogen gas source that, in the presence of a Ru catalyst solved in
the tetradecane phase, generated formic acid at concentrations of 128 mM after 48 h of
reaction [
301
]. On the other hand, light-driven photocatalytic hydrogenation has been
applied to a culture of Shewanella oneidensis MR-1 in anaerobic conditions [
282
]. Using
methyl viologen as a photoactivated molecule and triethanolamine (TEOA) as the sacrificial
agent, Rowe et al. obtained formic acid at concentrations higher than 1.5 mmol for 48 h
of reaction.
Finally, it is worth remarking that the use of microorganisms that incorporate formic
acid into their metabolic routes has also been another object of research in the last years.
Formatotrophic organisms (capable of assimilating formate for use as a carbon source)
offer a new approach to formate utilization, although they are difficult to cultivate, which
partially restricts their applicability [
302
,
303
]. One strategy is to biotechnologically adapt
microorganisms to assimilate formate by adapting their metabolism to the formatotrophic
growth model through metabolic engineering tools. Both methanol and formate can
be assimilated in the central metabolism through various metabolic pathways, and the
bio-production of other compounds such as ethanol, acetone, isopropanol, or short- and
medium-chain fatty acids and alcohols from these compounds is very promising [
304
,
305
].
To this end, the introduction of pathways, such as the Calvin cycle, the serine cycle, the
acetyl-CoA reductive pathway or the glycine pathway in hosts, or the design of new
pathways, is proposed [
304
,
305
]. The most commonly used hosts are E. coli,S. cerevisiae
and Cupriavidus necator, as they are easy to modify with genetic engineering tools and are
industrially applicable [306].
Synthetic pathways, such as the reductive glycine pathway (rGly, pathway for for-
mate assimilation), can support higher biological yields than natural pathways and could
therefore be implemented in a variety of microorganisms. The rGly pathway has been

Molecules 2023,28, 5520 40 of 52
introduced into the E. coli host by redesigning its central metabolism to be able to assimilate
formate. In terms of strategy, the pathway to be integrated is divided into modules and
introduced together into the host bacteria to express the enzymes necessary to form the
complete metabolic pathway [
304
,
305
]. With this methodology, a strain capable of growing
from formate with a doubling time of ~70 h and a growth yield of ~1.5 g cell dry weight
(gCDW) per mole of formate was achieved [
303
]. In this way, products such as lactate
or isobutanol, both pyruvate derivatives, were obtained. Cotton et al. used C. necator to
modify it with rGly, so that it was able to grow under formate-rich conditions. In this
respect, the production of methylketones, isoprenoids and terpenes, isobutanol, alkanes,
and alkenes from CO2 using C. necator seems particularly promising. With the same mi-
croorganism, the Calvin cycle was also used for the assimilation of formate or methanol,
although with low energy efficiencies (20–35%) [
307
]. On the other hand, Collas et al. very
recently devised an intelligent approach in which they engineered a crotonate biosynthetic
pathway in C. necator [
306
]. This mechanism permits the conversion of formic acid in
crotonate with extraordinary yields in a continuous process. Indeed, using this approach,
they obtained 148 mg/L of product. This is a new starting point for the generation of new
value-added chemicals.
Although whole-cell technology is still incipient, new opportunities that open a win-
dow to capture CO2under standard conditions, where nature develops, are enormous.
8. Conclusions and Perspectives
A panoramic overview of the state-of-the-art on carbon capture and its reduction in
C
1
forms by employing the biocatalysts CA and FDH was presented. CO
2
uptake is one of
the main challenges that science, in general, and chemistry, in particular, face today. Nature
offers appropriate tools and clues for finding approaches to solve this huge problem posed
to mankind. CCSU techniques are well developed in the laboratory and, to a lesser extent,
at a large scale, although their application is still far from being appropriately adapted for
obtaining high yields without great energetic costs. While CA immobilization is currently a
reality and it stabilizes the enzyme allowing its reuse, the employment of recombinant CAs
with higher resistance to temperature and pressure is a field that still remains to be fully
developed. These molecular biology tools will help speed up CO
2
/HCO
3−
conversion at
an industrial scale in the next few years.
Carbon dioxide reduction is also a reality at the laboratory scale, and research lines
are well established, although they are still far from showing high yields. The formic acid
molecule is a rich form for transporting hydrogen in an efficient way and is the primary
step for producing many more reduced molecules in such a way that carbon is recycled
and reused as an energy H
2
vector. Mass transfer from the gas to the solution is a limiting
step in reaching the substrate to the biocatalyst. On the other hand, improvements in
the stability and high performance of FDHs, as well as in their stability and reuse via
immobilization, have been developed, with a significant explosion of this research in the
last decade, despite still being a relatively virgin area. However, its application to larger
scales has not been established.
Measures to mitigate climate change are urgently needed. CAs and FDHs are excellent
devices for capturing CO
2
and transforming it into fuel, which is an interesting way to
reduce two problems into one. According to the exponential progress existing in the
research in this area nowadays, surprising approaches and solutions to these problems will
probably be found not in the next decades but in years.
Funding:
This work was partially supported by MICINN-FEDER-AEI 10.13039/501100011033
(PID2021-124695OB-C21/C22 and PDC2022-133313-C21/C22), MICINN—European Union Next
Generation EU-PRTR (TED2021-129626B-C21/C22), and SENECA (21884/PI/22) grants.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Molecules 2023,28, 5520 41 of 52
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
National Center for Environmental Information. Annual 2021 Global Climate Report. Available online: https://www.ncei.noaa.
gov/access/monitoring/monthly-report/global/202113 (accessed on 24 February 2023).
2.
Intergovernmental Panel on Climate Change (IPCC). Climate Change 2022, Mitigation of Climate Change. Available online:
https://www.ipcc.ch/report/ar6/wg3/downloads/report/IPCC_AR6_WGIII_FullReport.pdf (accessed on 24 February 2023).
3.
Fellowes, T.E.; Anggadi, F.; Byrne, M.; Vila-Concejo, A.; Bruce, E.; Baker, E. Stability of Coral Reef Islands and Associated Legal
Maritime Zones in a Changing Ocean. Environ. Res. Lett. 2022,17, 093003. [CrossRef]
4.
Borges, F.O.; Sampaio, E.; Santos, C.P.; Rosa, R. Impacts of Low Oxygen on Marine Life: Neglected, but a Crucial Priority for
Research. Biol. Bull. 2022,243, 104–119. [CrossRef] [PubMed]
5.
Arrhenius, S.; Holden, E.S. On the Influence of Carbonic Acid in the Air Upon the Temperature of the Earth. Publ. Astron. Soc.
Pac. 1897,9, 14–24. [CrossRef]
6.
Arrhenius, S. The Celestial Bodies, In Particular the Earth, as Abodes of Living Beings. In Worlds in the Making: The Evolution of the
Universe; Harper: New York, NY, USA, 1908; pp. 39–63.
7.
Friedlingstein, P.; Andrew, R.M.; Rogelj, J.; Peters, G.P.; Canadell, J.G.; Knutti, R.; Luderer, G.; Raupach, M.R.; Schaeffer, M.; van
Vuuren, D.P.; et al. Persistent Growth of CO
2
Emissions and Implications for Reaching Climate Targets. Nat. Geosci.
2014
,7,
709–715. [CrossRef]
8.
Lindsey, R. Climate Change: Atmospheric Carbon Dioxide. Science and Information for a Climate-Smart Nation. Available online:
https://www.climate.gov/news-features/understanding-climate/climate-change-atmospheric-carbon-dioxide (accessed on 24
February 2023).
9.
IPCC. Climate Change 2007: Synthesis Report. Summary for Policymakers. Valencia (Spain) 2007. Available online: https://www.
ctc-n.org/resources/ipcc-fourth-assessment-report-climate-change-2007-synthesis-report-summary-policymakers (accessed on
24 February 2023).
10.
EPA. United States Environmental Protection Agency. Overview of Greenhouse Gases. Available online: https://www.epa.gov/
ghgemissions/overview-greenhouse-gases (accessed on 24 February 2023).
11.
Hoegh-Guldberg, O.; Jacob, D.; Taylor, M.; Bolanos, T.G.; Bindi, M.; Brown, S.; Camilloni, I.A.; Diedhiou, A.; Djalante, R.;
Ebi, K.; et al.
The Human Imperative of Stabilizing Global Climate Change at 1.5 Degrees C. Science
2019
,365, eaaw6974.
[CrossRef]
12.
Rajabloo, T.; Valee, J.; Marenne, Y.; Coppens, L.; De Ceuninck, W. Carbon Capture and Utilization for Industrial Applications.
Energy Rep. 2023,9, 111–116. [CrossRef]
13.
Kamkeng, A.D.N.; Wang, M.; Hu, J.; Du, W.; Qian, F. Transformation technologies for CO
2
utilisation: Current status, challenges
and future prospects. Chem. Eng. J. 2021,409, 128138. [CrossRef]
14.
Villa, R.; Porcar, R.; Nieto, S.; Donaire, A.; García-Verdugo, E.; Luis, S.V.; Lozano-Rodríguez, P. Sustainable chemo-enzymatic
synthesis of glycerol carbonate (meth)acrylate from glycidol and carbon dioxide enabled by ionic liquid technologies. Green Chem.
2021,11, 4191–4200. [CrossRef]
15.
Knoche, W. Chemical Reactions of CO
2
in Water. In Biophysics and Physiology of Carbon Dioxide, Proceedings in Life Sciences; Bauer,
C., Gros, G., Bartels, H., Eds.; Springer: Berlin/Heidelberg, Germany, 1980. [CrossRef]
16.
Greenwood, N.N.; Earnshow, A. Chemistry of the Elements, 2nd ed.; Butterworth-Heinemann, Elsevier: Oxford, UK, 1997.
[CrossRef]
17.
Talekar, S.; Jo, B.H.; Dordick, J.S.; Kim, J. Carbonic Anhydrase for CO
2
Capture, Conversion and Utilization. Curr. Opin. Biotechnol.
2022,74, 230–240. [CrossRef]
18. Lindskog, S. Structure and Mechanism of Carbonic Anhydrase. Pharmacol. Ther. 1997,74, 1–20. [CrossRef]
19.
De Oliveira Maciel, A.; Christakopoulos, P.; Rova, U.; Antonopoulou, I. Carbonic Anhydrase to Boost CO
2
Sequestration:
Improving Carbon Capture Utilization and Storage (CCUS). Chemosphere 2022,299, 134419. [CrossRef]
20.
Yoshimoto, M.; Walde, P. Immobilized Carbonic Anhydrase: Preparation, Characteristics and Biotechnological Applications.
World J. Microbiol. Biotechnol. 2018,34, 151. [CrossRef]
21.
Appel, A.M.; Bercaw, J.E.; Bocarsly, A.B.; Dobbek, H.; DuBois, D.L.; Dupuis, M.; Ferry, J.G.; Fujita, E.; Hille, R.;
Kenis, P.J.A.; et al.
Frontiers, Opportunities, and Challenges in Biochemical and Chemical Catalysis of CO
2
Fixation. Chem. Rev.
2013
,113, 6621–6658.
[CrossRef]
22.
Bierbaumer, S.; Nattermann, M.; Schulz, L.; Zschoche, R.; Erb, T.J.; Winkler, C.K.; Tinzl, M.; Glueck, S.M. Enzymatic Conversion of
CO2: From Natural to Artificial Utilization. Chem. Rev. 2023,123, 5702–5754. [CrossRef]
23. Badger, M.R.; Sharwood, R.E. Rubisco, the Imperfect Winner: It’s All about the Base. J. Exp. Bot. 2023,74, 562–580. [CrossRef]
24. Bauwe, H. Photorespiration—Rubisco’s Repair Crew. J. Plant. Physiol. 2023,280, 153899. [CrossRef]
25.
Alpdagtas, S.; Binay, B. Nadp
+
-Dependent Formate Dehydrogenase: A Review. Biocatal. Biotransform.
2021
,39, 260–268.
[CrossRef]
26.
Alekseeva, A.A.; Savin, S.S.; Tishkov, V.I. NAD
+
-Dependent Formate Dehydrogenase from Plants. Acta Nat.
2011
,3, 38–54.
[CrossRef]

Molecules 2023,28, 5520 42 of 52
27.
Maia, L.B.; Moura, I.; Moura, J.J.G. Molybdenum and Tungsten-Containing Formate Dehydrogenases: Aiming to Inspire a
Catalyst for Carbon Dioxide Utilization. Inorganica Chim. Acta 2017,455, 350–363. [CrossRef]
28.
Jormakka, M.; Byrne, B.; Iwata, S. Formate Dehydrogenase—A Versatile Enzyme in Changing Environments. Curr. Opin. Struct.
Biol. 2003,13, 418–423. [CrossRef]
29.
Yang, J.Y.; Kerr, T.A.; Wang, X.S.; Barlow, J.M. Reducing CO
2
to HCO
2
- at Mild Potentials: Lessons from Formate Dehydrogenase.
J. Am. Chem. Soc. 2020,142, 19438–19445. [CrossRef] [PubMed]
30.
Wang, Y.; Li, M.; Zhao, Z.; Liu, W. Effect of Carbonic Anhydrase on Enzymatic Conversion of CO
2
to Formic Acid and
Optimization of Reaction Conditions. J. Mol. Catal. B Enzym. 2015,116, 89–94. [CrossRef]
31.
Thauer, R.K.; Jungermann, K.; Decker, K. Energy-Conservation in Chemotropic Anaerobic Bacteria. Bacteriol. Rev.
1977
,41,
100–180. [CrossRef]
32.
Zhang, Y.; Zhao, Y.; Li, R.; Liu, J. Bioinspired NADH Regeneration Based on Conjugated Photocatalytic Systems. Solar RRL
2021
,
5, 2000339. [CrossRef]
33.
Lee, S.H.; Choi, D.S.; Kuk, S.K.; Park, C.B. Photobiocatalysis: Activating Redox Enzymes by Direct or Indirect Transfer of
Photoinduced Electrons. Angew. Chem. Int. Ed. 2018,57, 7958–7985. [CrossRef]
34. Supuran, C.T.; Capasso, C. An Overview of the Bacterial Carbonic Anhydrases. Metabolites 2017,7, 56. [CrossRef]
35.
DiMario, R.J.; Machingura, M.C.; Waldrop, G.L.; Moroney, J.V. The Many Types of Carbonic Anhydrases in Photosynthetic
Organisms. Plant Sci. 2018,268, 11–17. [CrossRef]
36.
Kupriyanova, E.; Pronina, N.; Los, D. Carbonic Anhydrase—A Universal Enzyme of the Carbon-Based Life. Photosynthetica
2017
,
55, 3–19. [CrossRef]
37.
Zimmerman, S.A.; Ferry, J.G. The Beta and Gamma Classes of Carbonic Anhydrase. Curr. Pharm. Des.
2008
,14, 716–721.
[CrossRef]
38.
McGinn, P.J.; Morel, F.M.M. Expression and Regulation of Carbonic Anhydrases in the Marine Diatom Thalassiosira pseudonana
and in Natural Phytoplankton Assemblages from Great Bay, New Jersey. Physiol. Plant. 2008,133, 78–91. [CrossRef]
39.
Roberts, S.B.; Lane, T.W.; Morel, F.M.M. Carbonic Anhydrase in the Marine Diatom Thalassiosira weissflogii (Bacillariophyceae).
J. Phycol. 1997,33, 845–850. [CrossRef]
40.
Beauchemin, M.; Morse, D.
δ
-Carbonic Anhydrases: Structure, Distribution, and Potential Roles. In Carbonic Anhydrases as
Biocatalysts: From Theory to Medical and Industrial Applications; Universitéde Montréal: Montréal, QC, Canada, 2015; pp. 337–349.
[CrossRef]
41.
Dou, Z.; Heinhorst, S.; Williams, E.B.; Murin, C.D.; Shively, J.M.; Cannon, G.C. CO
2
Fixation Kinetics of Halothiobacillus neapolitanus
Mutant Carboxysomes Lacking Carbonic Anhydrase Suggest the Shell Acts as a Diffusional Barrier for CO2.J. Biol. Chem. 2008,
283, 10377–10384. [CrossRef]
42.
Alterio, V.; Langella, E.; Buonanno, M.; Esposito, D.; Nocentini, A.; Berrino, E.; Bua, S.; Polentarutti, M.; Supuran, C.T.; Monti,
S.M.; et al. Zeta-Carbonic Anhydrases Show CS
2
Hydrolase Activity: A New Metabolic Carbon Acquisition Pathway in Diatoms?
Comput. Struct. Biotechnol. J. 2021,19, 3427–3436. [CrossRef]
43.
Del Prete, S.; Vullo, D.; Fisher, G.M.; Andrews, K.T.; Poulsen, S.-A.; Capasso, C.; Supuran, C.T. Discovery of a New Family of
Carbonic Anhydrases in the Malaria Pathogen Plasmodium falciparum—The
η
-Carbonic Anhydrases. Bioorg. Med. Chem. Lett.
2014,24, 4389–4396. [CrossRef]
44.
Nawaly, H.; Tanaka, A.; Toyoshima, Y.; Tsuji, Y.; Matsuda, Y. Localization and Characterization Theta Carbonic Anhydrases in
Thalassiosira pseudonana.Photosynth. Res. 2023,156, 217–229. [CrossRef]
45.
Nocentini, A.; Supuran, C.T.; Capasso, C. An Overview on the Recently Discovered Iota-Carbonic Anhydrases. J. Enzym. Inhib.
Med. Chem. 2021,36, 1988–1995. [CrossRef]
46.
Del Prete, S.; Nocentini, A.; Supuran, C.T.; Capasso, C. Bacterial Iota-Carbonic Anhydrase: A New Active Class of Carbonic
Anhydrase Identified in the Genome of the Gram-Negative Bacterium Burkholderia Territorii. J. Enzym. Inhib. Med. Chem.
2020
,
35, 1060–1068. [CrossRef]
47.
Kikutani, S.; Nakajima, K.; Nagasato, C.; Tsuji, Y.; Miyatake, A.; Matsuda, Y. Thylakoid Luminal Theta-Carbonic Anhydrase
Critical for Growth and Photosynthesis in the Marine Diatom Phaeodactylum Tricornutum. Proc. Natl. Acad. Sci. USA
2016
,113,
9828–9833. [CrossRef]
48.
Alterio, V.; Langella, E.; De Simone, G.; Monti, S.M. Cadmium-Containing Carbonic Anhydrase CDCA1 in Marine Diatom
Thalassiosira weissflogii.Mar. Drugs 2015,13, 1688–1697. [CrossRef]
49.
Angeli, A.; Buonanno, M.; Donald, W.A.; Monti, S.M.; Supuran, C.T. The Zinc–but Not Cadmium–Containing
ζ
-Carbonic from
the Diatom Thalassiosira weissflogii Is Potently Activated by Amines and Amino Acids. Bioorg. Chem.
2018
,80, 261–265. [CrossRef]
50.
Nathan, V.K.; Ammini, P. Carbon Dioxide Sequestering Ability of Bacterial Carbonic Anhydrase in a Mangrove Soil Microcosm
and Its Bio-Mineralization Properties. Water Air Soil Pollut. 2019,230, 192. [CrossRef]
51.
Banci, L.; Bertini, I.; Luchinat, C.; Donaire, A.; Martinez, M.-J. The Factors Governing the Coordination Number in the Anion
Derivatives of Carbonic Anhydrase. Comments Inorg. Chem. 1990,9, 245–261. [CrossRef]
52.
Hirakawa, Y.; Senda, M.; Fukuda, K.; Yu, H.Y.; Ishida, M.; Taira, M.; Kinbara, K.; Senda, T. Characterization of a Novel Type of
Carbonic Anhydrase That Acts without Metal Cofactors. BMC Biol. 2021,19, 105. [CrossRef] [PubMed]
53.
Jo, B.H.; Kim, I.G.; Seo, J.H.; Kang, D.G.; Cha, H.J. Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a
Biocatalyst for CO2Sequestration. Appl. Environ. Microbiol. 2013,79, 6697–6705. [CrossRef]

Molecules 2023,28, 5520 43 of 52
54.
Ekinci, D.; Beydemir, S.; Alim, Z. Some Drugs Inhibit
in vitro
Hydratase and Esterase Activities of Human Carbonic Anhydrase-I
and II. Pharmacol. Rep. 2007,59, 580–587. [PubMed]
55.
Steger, F.; Reich, J.; Fuchs, W.; Rittmann, S.K.-M.R.; Gübitz, G.M.; Ribitsch, D.; Bochmann, G. Comparison ofCarbonic Anhydrases
for CO2Sequestration. Int. J. Mol. Sci. 2022,23, 957. [CrossRef]
56.
Faridi, S.; Satyanarayana, T. Novel Alkalistable
α
-Carbonic Anhydrase from the Polyextremophilic Bacterium Bacillus Halodurans:
Characteristics and Applicability in Flue Gas CO2Sequestration. Environ. Sci. Pollut. Res. 2016,23, 15236–15249. [CrossRef]
57.
Capasso, C.; De Luca, V.; Carginale, V.; Cannio, R.; Rossi, M. Biochemical Properties of a Novel and Highly Thermostable Bacterial
Alpha-Carbonic Anhydrase from Sulfurihydrogenibium yellowstonense YO3AOP1. J. Enzym. Inhib. Med. Chem.
2012
,27, 892–897.
[CrossRef]
58.
Ramanan, R.; Kannan, K.; Vinayagamoorthy, N.; Ramkumar, K.M.; Sivanesan, S.D.; Chakrabarti, T. Purification and Characteriza-
tion of a Novel Plant-Type Carbonic Anhydrase from Bacillus subtilis.Biotechnol. Bioprocess Eng. 2009,14, 32–37. [CrossRef]
59.
Kim, S.S.; Kim, N.J.; Hong, S.; Kim, S.S.; Sung, J.; Jin, M.S. The Structural Basis of the Low Catalytic Activities of the Two Minor
Beta-Carbonic Anhydrases of the Filamentous Fungus Aspergillus fumigatus.J. Struct. Biol. 2019,208, 61–68. [CrossRef]
60.
Sridharan, U.; Ragunathan, P.; Kuramitsu, S.; Yokoyama, S.; Kumarevel, T.; Ponnuraj, K. Structural and Functional Characteriza-
tion of a Putative Carbonic Anhydrase from Geobacillus kaustophilus Reveals Its Cambialistic Function. Biochem. Biophys. Res.
Commun. 2021,547, 96–101. [CrossRef]
61.
Wang, W.; Zhang, Y.; Wang, L.; Jing, Q.; Wang, X.; Xi, X.; Zhao, X.; Wang, H. Molecular Structure of Thermostable and
Zinc-Ion-Binding Gamma-Class Carbonic Anhydrases. Biometals 2019,32, 317–328. [CrossRef]
62.
Alber, B.E.; Ferry, J.G. A Carbonic Anhydrase from the Archaeon Methanosarcina thermophila.Proc. Natl. Acad. Sci. USA
1994
,91,
6909–6913. [CrossRef]
63.
Del Prete, S.; Vullo, D.; Di Fonzo, P.; Osman, S.M.; AlOthman, Z.; Donald, W.A.; Supuran, C.T.; Capasso, C. Sulfonamide Inhibition
Profile of the Gamma-Carbonic Anhydrase Identified in the Genome of the Pathogenic Bacterium Burkholderia pseudomallei the
Etiological Agent Responsible of Melioidosis. Bioorg. Med. Chem. Lett. 2017,27, 490–495. [CrossRef]
64.
Del Prete, S.; Vullo, D.; De Luca, V.; Supuran, C.T.; Capasso, C. Biochemical Characterization of the Delta-Carbonic Anhydrase
from the Marine Diatom Thalassiosira weissflogii, TweCA. J. Enzym. Inhib. Med. Chem. 2014,29, 906–911. [CrossRef]
65.
Zhang, H.; Blanco-Ameijeiras, S.; Hopkinson, B.M.; Bernasconi, S.M.; Mejia, L.M.; Liu, C.; Stoll, H. An Isotope Label Method
for Empirical Detection of Carbonic Anhydrase in the Calcification Pathway of the Coccolithophore Emiliania huxleyi.Geochim.
Cosmochim. Acta 2021,292, 78–93. [CrossRef]
66.
Xu, Y.; Feng, L.; Jeffrey, P.D.; Shi, Y.; Morel, F.M.M. Structure and Metal Exchange in the Cadmium Carbonic Anhydrase of Marine
Diatoms. Nature 2008,452, 56–61. [CrossRef]
67.
Supuran, C.T.; Capasso, C. The
η
-Class Carbonic Anhydrases as Drug Targets for Antimalarial Agents. Expert Opin. Ther. Targets
2015,19, 551–563. [CrossRef]
68.
Kitahara, M.; Fudo, S.; Yoneda, T.; Nukaga, M.; Hoshino, T. Anisotropic Distribution of Ammonium Sulfate Ions in Protein
Crystallization. Cryst. Growth Des. 2019,19, 6004–6010. [CrossRef]
69.
Woods, L.A.; Dolezal, O.; Ren, B.; Ryan, J.H.; Peat, T.S.; Poulsen, S.-A. Native State Mass Spectrometry, Surface Plasmon
Resonance, and X-Ray Crystallography Correlate Strongly as a Fragment Screening Combination. J. Med. Chem.
2016
,59,
2192–2204. [CrossRef]
70.
Avvaru, B.S.; Busby, S.A.; Chalmers, M.J.; Griffin, P.R.; Venkatakrishnan, B.; Agbandje-McKenna, M.; Silverman, D.N.; McKenna,
R. Apo-Human Carbonic Anhydrase II Revisited: Implications of the Loss of a Metal in Protein Structure, Stability, and Solvent
Network. Biochemistry 2009,48, 7365–7372. [CrossRef] [PubMed]
71.
Bergenhem, N.C.; Hallberg, M.; Wisén, S. Molecular Characterization of the Human Carbonic Anhydrase-Related Protein
(HCA-RP VIII). Biochim. Biophys. Acta 1998,1384, 294–298. [CrossRef] [PubMed]
72.
Picaud, S.S.; Muniz, J.R.; Kramm, A.; Pilka, E.S.; Kochan, G.; Oppermann, U.; Yue, W.W. Crystal Structure of Human Carbonic
Anhydrase-Related Protein VIII Reveals the Basis for Catalytic Silencing. Proteins 2009,76, 507–511. [CrossRef] [PubMed]
73.
Lakkis, M.M.; Bergenhem, N.C.; O’Shea, K.S.; Tashian, R.E. Expression of the Acatalytic Carbonic Anhydrase VIII Gene, Car8,
during Mouse Embryonic Development. Histochem. J. 1997,29, 135–141. [CrossRef] [PubMed]
74.
Hirota, J.; Ando, H.; Hamada, K.; Mikoshiba, K. Carbonic Anhydrase-Related Protein is a Novel Binding Protein for Inositol
1,4,5-Trisphosphate Receptor Ttype 1. Biochem. J. 2003,372, 435–441. [CrossRef]
75.
Borén, K.; Andersson, P.; Larsson, M.; Carlsson, U. Characterization of a Molten Globule State of Bovine Carbonic Anhydrase III:
Loss of Asymmetrical Environment of the Aromatic Residues Has a Profound Effect on Both the Near- and Far-UV CD Spectrum.
Biochim. Biophys. Acta 1999,1430, 111–118. [CrossRef]
76.
Capasso, C.; Supuran, C.T. Inhibition of Bacterial Carbonic Anhydrases as a Novel Approach to Escape Drug Resistance. Curr.
Top. Med. Chem. 2017,17, 1237–1248. [CrossRef]
77.
Wilbur, K.M.; Anderson, N.G. Electrometric and Colorimetric Determination of Carbonic Anhydrase. J. Biol. Chem.
1948
,176,
147–154. [CrossRef]
78.
Fuchs, W.; Steger, F.; Reich, J.; Ribitsch, D.; Rittmann, S.K.-M.R.; Bochmann, G. A Simple and Straightforward Method for Activity
Measurement of Carbonic Anhydrases. Catalysis 2021,11, 819. [CrossRef]
79.
Verpoorte, J.A.; Mehta, S.; Edsall, J.T. Esterase Activities of Human Carbonic Anhydrases B and C. J. Biol. Chem.
1967
,242,
4221–4229. [CrossRef]

Molecules 2023,28, 5520 44 of 52
80.
Effendi, S.S.W.; Ng, I.-S. The Prospective and Potential of Carbonic Anhydrase for Carbon Dioxide Sequestration: A Critical
Review. Process Biochem. 2019,87, 55–65. [CrossRef]
81.
Supuran, C.T.; De Simone, G. Carbonic Anhydrases: An Overview. In Carbonic Anhydrases as Biocatalysts: From Theory to Medical
and Industrial Applications; Elsevier: Florence, Italy, 2015; pp. 3–13. [CrossRef]
82.
Niks, D.; Hille, R. Reductive Activation of CO
2
by Formate Dehydrogenases. In Enzymes of Energy Technology; Methods in
Enzymology; Armstrong, F., Ed.; Elsevier Acad. Press. Inc.: San Diego, CA, USA, 2018; Volume 613, pp. 277–295. [CrossRef]
83.
Nielsen, C.F.; Lange, L.; Meyer, A.S. Classification and Enzyme Kinetics of Formate Dehydrogenases for Biomanufacturing via
CO2Utilization. Biotechnol. Adv. 2019,37, 107408. [CrossRef] [PubMed]
84.
Maia, L.B.; Moura, I.; Moura, J.J.G. Molybdenum and Tungsten-Containing Enzymes: An Overview. In Molibdenum and Tungsten
Enzymes: Biochemistry; Hille, R., Schulzke, C., Kirk, M.L., Eds.; RSC Metallobiology Series; Royal Society of Chemistry: Cambridge,
UK, 2017; Volume 5, pp. 1–80.
85.
Lemaire, O.N.; Jespersen, M.; Wagner, T. CO
2
-Fixation Strategies in Energy Extremophiles: What Can We Learn From Acetogens?
Front. Microbiol. 2020,11, 486. [CrossRef] [PubMed]
86.
Moon, M.; Park, G.W.; Lee, J.; Lee, J.-S.; Min, K. Recent Progress in Formate Dehydrogenase (FDH) as a Non-Photosynthetic CO
2
Utilizing Enzyme: A Short Review. J. CO2 Util. 2020,42, 101353. [CrossRef]
87.
Alpda˘gta¸s, S.; Turunen, O.; Valjakka, J.; Binay, B. The Challenges of Using NAD
+
-Dependent Formate Dehydrogenases for CO
2
Conversion. Crit. Rev. Biotechnol. 2022,42, 953–972. [CrossRef]
88.
Sakai, Y.; Murdanoto, A.P.; Konishi, T.; Iwamatsu, A.; Kato, N. Regulation of the Formate Dehydrogenase Gene, FDH1, in the
Methylotrophic Yeast Candida boidinii and Growth Characteristics of an FDH1-Disrupted Strain on Methanol, Methylamine, and
Choline. J. Bacteriol. 1997,179, 4480–4485. [CrossRef]
89. Tishkov, V.I.; Popov, V.O. Protein Engineering of Formate Dehydrogenase. Biomol. Eng. 2006,23, 89–110. [CrossRef]
90.
Pagano, P.; Guo, Q.; Ranasinghe, C.; Schroeder, E.; Robben, K.; Hase, F.; Ye, H.; Wickersham, K.; Aspuru-Guzik, A.; Major, D.T.;
et al. Oscillatory Active-Site Motions Correlate with Kinetic Isotope Effects in Formate Dehydrogenase. ACS Catal.
2019
,9,
11199–11206. [CrossRef]
91.
Yilmazer, B.; Isupov, M.N.; De Rose, S.A.; Bulut, H.; Benninghoff, J.C.; Binay, B.; Littlechild, J.A. Structural insights into the
NAD
+
-dependent Formate Dehydrogenase Mechanism Revealed from the NADH Complex and the Formate NAD
+
-Ternary
Complex of the Chaetomium thermophilum Enzyme J.Struct. Biol. 2020,212, 107657. [CrossRef]
92.
Pala, U.; Yelmazer, B.; Corbacioglu, M.; Ruupunen, J.; Valjakka, J.; Turunen, O.; Binay, B. Functional Effects of Active Site
Mutations in NAD
+
-Dependent Formate Dehydrogenases on Transformation of Hydrogen Carbonate to Formate. Protein Eng.
Des. Sel. 2018,31, 327–335. [CrossRef]
93. Cakar, M.M.; Ruupunen, J.; Mangas-Sanchez, J.; Birmingham, W.R.; Yildirim, D.; Turunen, O.; Turner, N.J.; Valjakka, J.; Binay, B.
Engineered Formate Dehydrogenase from Chaetomium Thermophilum, a Promising Enzymatic Solution for Biotechnical CO
2
Fixation. Biotechnol. Lett. 2020,42, 2251–2262. [CrossRef]
94.
Guo, Q.; Gakhar, L.; Wickersham, K.; Francis, K.; Vardi-Kilshtain, A.; Major, D.T.; Cheatum, C.M.; Kohen, A. Structural and
Kinetic Studies of Formate Dehydrogenase from Candida boidinii.Biochemistry 2016,55, 2760–2771. [CrossRef]
95.
Sato, R.; Amao, Y. Studies on the Catalytic Mechanism of Formate Dehydrogenase from Candida boidinii Using Isotope-Labelled
Substrate and Co-Enzyme. Catal. Today 2023,411–412, 113796. [CrossRef]
96.
Jiang, W.; Lin, P.; Yang, R.; Fang, B. Identification of Catalysis, Substrate, and Coenzyme Binding Sites and Improvement Catalytic
Efficiency of Formate Dehydrogenase from Candida boidinii.Appl. Microbiol. Biotechnol. 2016,100, 8425–8437. [CrossRef]
97.
Altas, N.; Aslan, A.S.; Karatas, E.; Chronopoulou, E.; Labrou, N.E.; Binay, B. Heterologous Production of Extreme Alkaline
Thermostable NAD
+
-Dependent Formate Dehydrogenase with Wide-Range pH Activity from Myceliophthora thermophila.Process
Biochem. 2017,61, 110–118. [CrossRef]
98.
Choe, H.; Joo, J.C.; Cho, D.H.; Kim, M.H.; Lee, S.H.; Jung, K.D.; Kim, Y.H. Efficient CO
2
-Reducing Activity of NAD-Dependent
Formate Dehydrogenase from Thiobacillus sp. KNK65MA for Formate Production from CO
2
Gas. PLoS ONE
2014
,9, e103111.
[CrossRef]
99.
Aslan, A.S.; Valjakka, J.; Ruupunen, J.; Yildirim, D.; Turner, N.J.; Turunen, O.; Binay, B. Chaetomium thermophilum Formate
Dehydrogenase Has High Activity in the Reduction of Hydrogen Carbonate (HCO
3−
) to Formate. Protein Eng. Des. Sel.
2017
,30,
47–55. [CrossRef]
100.
de Bok, F.A.M.; Hagedoorn, P.L.; Silva, P.J.; Hagen, W.R.; Schiltz, E.; Fritsche, K.; Stams, A.J.M. Two W-Containing Formate
Dehydrogenases (CO
2
-Reductases) Involved in Syntrophic Propionate Oxidation by Syntrophobacter fumaroxidans.Eur. J. Biochem.
2003,270, 2476–2485. [CrossRef]
101.
Maia, L.B.; Fonseca, L.; Moura, I.; Moura, J.J.G. Reduction of Carbon Dioxide by a Molybdenum-Containing Formate Dehydroge-
nase: A Kinetic and Mechanistic Study. J. Am. Chem. Soc. 2016,138, 8834–8846. [CrossRef]
102.
Bassegoda, A.; Madden, C.; Wakerley, D.W.; Reisner, E.; Hirst, J. Reversible Interconversion ofCO
2
and Formate by a Molybdenum-
Containing Formate Dehydrogenase. J. Am. Chem. Soc. 2014,136, 15473–15476. [CrossRef]
103.
Axley, M.J.; Grahame, D.A.; Stadtman, T.C. Escherichia coli Formate-Hydrogen Lyase. Purification and Properties of the Selenium-
Dependent Formate Dehydrogenase Component. J. Biol. Chem. 1990,265, 18213–18218. [CrossRef] [PubMed]

Molecules 2023,28, 5520 45 of 52
104.
Oliveira, A.R.; Mota, C.; Mourato, C.; Domingos, R.M.; Santos, M.F.A.; Gesto, D.; Guigliarelli, B.; Santos-Silva, T.; Romao, M.J.;
Cardoso Pereira, I.A. Toward the Mechanistic Understanding of Enzymatic CO
2
Reduction. ACS Catal.
2020
,10, 3844–3856.
[CrossRef]
105.
da Silva, S.M.; Pimentel, C.; Valente, F.M.A.; Rodrigues-Pousada, C.; Pereira, I.A.C. Tungsten and Molybdenum Regulation
of Formate Dehydrogenase Expression in Desulfovibrio Vulgaris Hildenborough. J. Bacteriol.
2011
,193, 2909–2916. [CrossRef]
[PubMed]
106. Schuchmann, K.; Mueller, V. Direct and Reversible Hydrogenation of CO2to Formate by a Bacterial Carbon Dioxide Reductase.
Science 2013,342, 1382–1385. [CrossRef]
107.
Yu, X.; Niks, D.; Mulchandani, A.; Hille, R. Efficient Reduction of CO
2
by the Molybdenum-Containing Formate Dehydrogenase
from Cupriavidus necator (Ralstonia eutropha). J. Biol. Chem. 2017,292, 16872–16879. [CrossRef]
108.
Radon, C.; Mittelstadt, G.; Duffus, B.R.; Burger, J.; Hartmann, T.; Mielke, T.; Teutloff, C.; Leimkuhler, S.; Wendler, P. Cryo-EM
Structures Reveal Intricate Fe-S Cluster Arrangement and Charging in Rhodobacter capsulatus Formate Dehydrogenase. Nat.
Commun. 2020,11, 1912. [CrossRef]
109.
Hartmann, T.; Leimkuehler, S. The Oxygen-Tolerant and NAD
+
-Dependent Formate Dehydrogenase from Rhodobacter capsulatus
Is Able to Catalyze the Reduction of CO2to Formate. FEBS J. 2013,280, 6083–6096. [CrossRef]
110.
Ruschig, U.; Muller, U.; Willnow, P.; Hopner, T. CO
2
Recution to Formate by NADH Catalyzed by Formate Dehydrogenase from
Pseudomonas oxalaticus.Eur. J. Biochem. 1976,70, 325–330. [CrossRef]
111.
Müller, U.; Willnow, P.; Ruschig, U.; Höpner, T. Formate Dehydrogenase from Pseudomonas oxalaticus.Eur. J. Biochem.
1978
,83,
485–498. [CrossRef]
112.
Cakar, M.M.; Mangas-Sanchez, J.; Birmingham, W.R.; Turner, N.J.; Binay, B. Discovery of a New Metal and NAD
+
-Dependent
Formate Dehydrogenase from Clostridium ljungdahlii.Prep. Biochem. Biotechnol. 2018,48, 327–334. [CrossRef]
113.
Min, K.; Moon, M.; Park, G.W.; Lee, J.-P.; Kim, S.J.; Lee, J.-S. Newly Explored Formate Dehydrogenases from Clostridium Species
Catalyze Carbon Dioxide to Formate. Bioresour. Technol. 2022,348, 126832. [CrossRef]
114.
Almendra, M.J.; Brondino, C.D.; Gavel, O.; Pereira, A.S.; Tavares, P.; Bursakov, S.; Duarte, R.; Caldeira, J.; Moura, J.J.G.; Moura, I.
Purification and Characterization of a Tungsten-Containing Formate Dehydrogenase from Desulfovibrio gigas.Biochemistry
1999
,
38, 16366–16372. [CrossRef]
115.
Yamamoto, I.; Saiki, T.; Liu, S.M.; Ljundgdahl, L.G. Purification and Properties of NADP-Dependent Formate Dehydrogenase
from Clostridium thermoaceticum, a Tungsten Selenium Iron Protein. J. Biol. Chem. 1983,258, 1826–1832. [CrossRef]
116.
Cordas, C.M.; Moura, J.J.G. Molybdenum and Tungsten Enzymes Redox Properties—A Brief Overview. Coord. Chem. Rev.
2019
,
394, 53–64. [CrossRef]
117.
Hartmann, T.; Schwanhold, N.; Leimkuehler, S. Assembly and Catalysis of Molybdenum or Tungsten-Containing Formate
Dehydrogenases from Bacteria. Biochim. Biophys. Acta Proteins Proteom. 2015,1854, 1090–1100. [CrossRef]
118. Kirk, M.L.; Hille, R. Spectroscopic Studies of Mononuclear Molybdenum Enzyme Centers. Molecules 2022,27, 4802. [CrossRef]
119.
Jormakka, M.; Tornroth, S.; Byrne, B.; Iwata, S. Molecular Basis of Proton Motive Force Generation: Structure of Formate
Dehydrogenase-N. Science 2002,295, 1863–1868. [CrossRef]
120.
Niks, D.; Hille, R. Molybdenum- and Tungsten-Containing Formate Dehydrogenases and Formylmethanofuran Dehydrogenases:
Structure, Mechanism, and Cofactor Insertion. Protein Sci. 2019,28, 111–122. [CrossRef]
121.
Robinson, W.E.; Bassegoda, A.; Reisner, E.; Hirst, J. Oxidation-State-Dependent Binding Properties of the Active Site in a
Mo-Containing Formate Dehydrogenase. J. Am. Chem. Soc. 2017,139, 9927–9936. [CrossRef]
122.
Meneghello, M.; Oliveira, A.R.; Jacq-Bailly, A.; Pereira, I.A.C.; Leger, C.; Fourmond, V. Formate Dehydrogenases Reduce CO
2
Rather than HCO3−: An Electrochemical Demonstration. Angew. Chem. Int. Ed. 2021,60, 9964–9967. [CrossRef]
123.
Tiberti, M.; Papaleo, E.; Russo, N.; De Gioia, L.; Zampella, G. Evidence for the Formation of a Mo-H Intermediate in the Catalytic
Cycle of Formate Dehydrogenase. Inorg. Chem. 2012,51, 8331–8339. [CrossRef]
124. Khan, M.R. Immobilized Enzymes: A Comprehensive Review. Bull. Natl. Res. Cent. 2021,45, 207. [CrossRef]
125.
Bie, J.; Sepodes, B.; Fernandes, P.C.B.; Ribeiro, M.H.L. Enzyme Immobilization and Co-Immobilization: Main Framework,
Advances and Some Applications. Processes 2022,10, 494. [CrossRef]
126.
Porcar, R.; Lavandera, I.; Lozano, P.; Altava, B.; Luis, S.V.; Gotor-Fernandez, V.; Garcia-Verdugo, E. Supported Ionic Liquid-
like Phases as Efficient Solid Ionic Solvents for the Immobilisation of Alcohol Dehydrogenases towards the Development of
Stereoselective Bioreductions. Green Chem. 2021,23, 5609–5617. [CrossRef]
127.
Sheldon, R.A.; Basso, A.; Brady, D. New Frontiers in Enzyme Immobilisation: Robust Biocatalysts for a Circular Bio-Based
Economy. Chem. Soc. Rev. 2021,50, 5850–5862. [CrossRef] [PubMed]
128.
Lozano, P.; Garcia-Verdugo, E.; Bernal, J.M.; Izquierdo, D.F.; Isabel Burguete, M.; Sanchez-Gomez, G.; Luis, S.V. Immobilised
Lipase on Structured Supports Containing Covalently Attached Ionic Liquids for the Continuous Synthesis of Biodiesel in ScCO
2
.
ChemSusChem 2012,5, 790–798. [CrossRef]
129.
Ren, S.; Jiang, S.; Yan, X.; Chen, R.; Cui, H. Challenges and Opportunities: Porous Supports in Carbonic Anhydrase Immobilization.
J. CO2 Util. 2020,42, 101305. [CrossRef]
130.
Molina-Fernandez, C.; Luis, P. Immobilization of Carbonic Anhydrase for CO
2
Capture and Its Industrial Implementation: A
Review. J. CO2 Util. 2021,47, 101475. [CrossRef]

Molecules 2023,28, 5520 46 of 52
131.
Rasouli, H.; Nguyen, K.; Iliuta, M.C. Recent Advancements in Carbonic Anhydrase Immobilization and Its Implementation in
CO2Capture Technologies: A Review. Sep. Purif. Technol. 2022,296, 121299. [CrossRef]
132.
Yuan, Y.; Wang, F.; Li, H.; Su, S.; Gao, H.; Han, X.; Ren, S. Potential Application of the Immobilization of Carbonic Anhydrase
Based on Metal Organic Framework Supports. Process Biochem. 2022,122, 214–223. [CrossRef]
133.
Molina-Fernandez, C.; Peters, A.; Debecker, D.P.; Luis, P. Immobilization of Carbonic Anhydrase in a Hydrophobic Poly(Ionic
Liquid): A New Functional Solid for CO2Capture. Biochem. Eng. J. 2022,187, 108639. [CrossRef]
134.
Rouf, S.; Greish, Y.E.; Al-Zuhair, S. Immobilization of Formate Dehydrogenase in Metal Organic Frameworks for Enhanced
Conversion of Carbon Dioxide to Formate. Chemosphere 2021,267, 128921. [CrossRef]
135.
Singh, R.K.R.; Tiwari, M.K.; Singh, R.K.R.; Lee, J.-K. From Protein Engineering to Immobilization: Promising Strategies for the
Upgrade of Industrial Enzymes. Molecules 2013,18, 1232–1277. [CrossRef]
136.
Di Spiridione, C.; Aresta, M.; Dibenedetto, A. Improving the Enzymatic Cascade of Reactions for the Reduction of CO
2
to CH
3
OH
in Water: From Enzymes Immobilization Strategies to Cofactor Regeneration and Cofactor Suppression. Molecules
2022
,27, 4913.
[CrossRef]
137.
Zhu, X.; Du, C.; Gao, B.; He, B. Strategies to Improve the Mass Transfer in the CO
2
Capture Process Using Immobilized Carbonic
Anhydrase. J. Environ. Manag. 2023,332, 117370. [CrossRef]
138.
Ren, S.; Chen, R.; Wu, Z.; Su, S.; Hou, J.; Yuan, Y. Enzymatic Characteristics of Immobilized Carbonic Anhydrase and Its
Applications in CO2Conversion. Colloid. Surface B 2021,204, 111779. [CrossRef]
139. Shen, J.; Salmon, S. Biocatalytic Membranes for Carbon Capture and Utilization. Membranes 2023,13, 367. [CrossRef] [PubMed]
140.
Wanjari, S.; Prabhu, C.; Satyanarayana, T.; Vinu, A.; Rayalu, S. Immobilization of Carbonic Anhydrase on Mesoporous Alumi-
nosilicate for Carbonation Reaction. Micropor. Mesopor. Mat. 2012,160, 151–158. [CrossRef]
141.
Yu, Y.; Chen, B.; Qi, W.; Li, X.; Shin, Y.; Lei, C.; Liu, J. Enzymatic Conversion of CO
2
to Bicarbonate in Functionalized Mesoporous
Silica. Micropor. Mesopor. Mat. 2012,153, 166–170. [CrossRef]
142. Vinoba, M.; Bhagiyalakshmi, M.; Jeong, S.K.; Yoon, Y.I.; Nam, S.C. Carbonic Anhydrase Conjugated to Nanosilver Immobilized
onto Mesoporous SBA-15 for Sequestration of CO2.J. Mol. Catal. B Enzym. 2012,75, 60–67. [CrossRef]
143.
Mao, M.; Zhai, T.; Meng, L.; Meng, Z.; Liu, W. Controllable Preparation of Mesoporous Silica and Its Application in Enzyme-
Catalyzed CO2Reduction. Chem. Eng. J. 2022,437, 135479. [CrossRef]
144.
Azari, F.N.-G.M. Reversible Denaturation of Carbonic Anhydrase Provides a Method for Its Adsorptive Immobilization. Biotechnol.
Bioeng. 1999,62, 193–199. [CrossRef]
145.
Kim, J.K.; Abdelhamid, M.A.A.; Pack, S.P. Direct Immobilization and Recovery of Recombinant Proteins from Cell Lysates by
Using EctP1-Peptide as a Short Fusion Tag for Silica and Titania Supports. Int. J. Biol. Macromol. 2019,135, 969–977. [CrossRef]
146.
Yong, J.K.J.; Stevens, G.W.; Caruso, F.; Kentish, S.E. In Situ Layer-by-Layer Assembled Carbonic Anhydrase-Coated Hollow Fiber
Membrane Contactor for Rapid CO2Absorption. J. Memb. Sci. 2016,514, 556–565. [CrossRef]
147.
Ivanovski, V.; Shapovalova, O.E.; Drozdov, A.S. Structural Rearrangements of Carbonic Anhydrase Entrapped in Sol-Gel
Magnetite Determined by ATR-FTIR Spectroscopy. Int. J. Mol. Sci. 2022,23, 5975. [CrossRef]
148.
Moon, H.; Kim, S.; Jo, B.H.; Cha, H.J. Immobilization of Genetically Engineered Whole-Cell Biocatalysts with Periplasmic
Carbonic Anhydrase in Polyurethane Foam for Enzymatic CO
2
Capture and Utilization. J. CO2 Util.
2020
,39, 101172. [CrossRef]
149.
Jiao, M.; He, J.; Sun, S.; Vriesekoop, F.; Yuan, Q.; Liu, Y.; Liang, H. Fast Immobilization of Human Carbonic Anhydrase II on
Ni-Based Metal-Organic Framework Nanorods with High Catalytic Performance. Catalysts 2020,10, 401. [CrossRef]
150.
Abdelrahim, M.Y.; Martins, C.F.; Neves, L.A.; Capasso, C.; Supuran, C.T.; Coelhoso, I.M.; Crespo, J.G.; Barboiu, M. Supported
Ionic Liquid Membranes Immobilized with Carbonic Anhydrases for CO
2
Transport at High Temperatures. J. Memb. Sci.
2017
,
528, 225–230. [CrossRef]
151.
Martins, C.F.; Neves, L.A.M.; Estevão, L.A.; Rosatella, A.; Alves, V.D.; Afonso, C.A.M.; Crespo, J.G.; Coelhoso, I.M. Effect of Water
Activity on Carbon Dioxide Transport in Cholinium-Based Ionic Liquids with Carbonic Anhydrase. Sep. Purif. Technol.
2016
,168,
74–82. [CrossRef]
152.
Vinoba, M.; Bhagiyalakshmi, M.; Jeong, S.K.; Yoon, Y.I.; Nam, S.C. Capture and Sequestration of CO
2
by Human Carbonic
Anhydrase Covalently Immobilized onto Amine-Functionalized SBA-15. J. Phys. Chem. C 2011,115, 20209–20216. [CrossRef]
153.
Shen, J.; Yuan, Y.; Salmon, S. Durable and Versatile Immobilized Carbonic Anhydrase on Textile Structured Packing for CO
2
Capture. Catalysts 2022,12, 1108. [CrossRef]
154.
Xv, J.; Zhang, Z.; Pang, S.; Jia, J.; Geng, Z.; Wang, R.; Li, P.; Bilal, M.; Cui, J.; Jia, S. Accelerated CO
2
Capture Using Immobilized
Carbonic Anhydrase on Polyethyleneimine/Dopamine Co-Deposited MOFs. Biochem. Eng. J. 2022,189, 108719. [CrossRef]
155.
Shamna, I.; Jeong, S.K.; Margandan, B. Covalent Immobilization of Carbonic Anhydrase on Amine Functionalized Alumino-
Siloxane Aerogel Beads for Biomimetic Sequestration of CO2.J. Ind. Eng. Chem. 2021,100, 288–295. [CrossRef]
156.
Iliuta, I.; Rasouli, H.; Iliuta, M.C. Intensified CO
2
Capture in Wall-Coated Microreactors with Immobilized Carbonic Anhydrase:
Experimental and Modeling. Sep. Purif. Technol. 2023,307, 122590. [CrossRef]
157.
Kimmel, J.D.; Arazawa, D.T.; Ye, S.-H.; Shankarraman, V.; Wagner, W.R.; Federspiel, W.J. Carbonic Anhydrase Immobilized on
Hollow Fiber Membranes Using Glutaraldehyde Activated Chitosan for Artificial Lung Applications. J. Mater. Sci. Mater. Med.
2013,24, 2611–2621. [CrossRef]

Molecules 2023,28, 5520 47 of 52
158.
Peirce, S.; Russo, M.E.; Isticato, R.; Lafuente, R.F.; Salatino, P.; Marzocchella, A. Structure and Activity of Magnetic Cross-Linked
Enzyme Aggregates of Bovine Carbonic Anhydrase as Promoters of Enzymatic CO
2
Capture. Biochem. Eng. J.
2017
,127, 188–195.
[CrossRef]
159.
Chang, S.; He, Y.; Li, Y.; Cui, X. Study on the Immobilization of Carbonic Anhydrases on Geopolymer Microspheres for CO
2
Capture. J. Clean. Prod. 2021,316, 128163. [CrossRef]
160.
Zhang, X.; Shao, W.; Chen, B.; Wang, M. Cross-Linking of Carbonic Anhydrase and Formate Dehydrogenase Based on Amino
Acid Specific Recognition: Conversion of Carbon Dioxide to Formic Acid. Enzym. Microb. Technol.
2021
,146, 109763. [CrossRef]
161. Nelson, J.M.; Griffin, E.G. Adsorption of Invertase. J. Am. Chem. Soc. 1916,38, 1109–1115. [CrossRef]
162.
Jesionowski, T.; Zdarta, J.; Krajewska, B. Enzyme immobilization by adsorption: A review. Adsorption
2014
,20, 801–821. [CrossRef]
163.
Nabavi Zadeh, P.S.; Åkerman, B. Immobilization of Enzymes in Mesoporous Silica Particles: Protein Concentration and Rotational
Mobility in the Pores. J. Phys. Chem. B 2017,121, 2575–2583. [CrossRef] [PubMed]
164.
Crumbliss, A.L.; Perine, S.C.; Stonehuerner, J.; Tubergen, K.R.; Zhao, J.; Henkens, R.W.; O’Daly, J.P. Colloidal Gold as a
Biocompatible Immobilization Matrix Suitable for the Fabrication of Enzyme Electrodes by Electrodeposition. Biotechnol. Bioeng.
1992,40, 483–490. [CrossRef]
165.
Suvannasara, P.; Juntapram, K.; Praphairaksit, N.; Siralertmukul, K.; Muangsin, N. Mucoadhesive 4-Carboxybenzenesulfonamide-
Chitosan with Antibacterial Properties. Carbohydr. Polym. 2013,94, 244–252. [CrossRef] [PubMed]
166.
Li, J.-J.; Yin, L.; Wang, Z.-F.; Jing, Y.-C.; Jiang, Z.-L.; Ding, Y.; Wang, H.-S. Enzyme-Immobilized Metal-Organic Frameworks: From
Preparation to Application. Chem. Asian J. 2022,17, e202200751. [CrossRef]
167. Zhou, H.-C.; Long, J.R.; Yaghi, O.M. Introduction to Metal–Organic Frameworks. Chem. Rev. 2012,112, 673–674. [CrossRef]
168.
Zhang, W.; Taheri-Ledari, R.; Saeidirad, M.; Qazi, F.S.; Kashtiaray, A.; Ganjali, F.; Tian, Y.; Maleki, A. Regulation of Porosity in
MOFs: A Review on Tunable Scaffolds and Related Effects and Advances in Different Applications. J. Environ. Chem. Eng.
2022
,
10, 108836. [CrossRef]
169.
Silva, A.R.M.; Alexandre, J.Y.N.H.; Souza, J.E.S.; Lima Neto, J.G.; de Sousa Junior, P.G.; Rocha, M.V.P.; dos Santos, J.C.S. The
Chemistry and Applications of Metal-Organic Frameworks (MOFs) as Industrial Enzyme Immobilization Systems. Molecules
2022,27, 4529. [CrossRef]
170.
Liu, Q.; Bai, X.; Pham, H.; Hu, J.; Dinu, C.Z. Active Nanointerfaces Based on Enzyme Carbonic Anhydrase and Metal-Organic
Framework for Carbon Dioxide Reduction. Nanomaterials 2021,11, 1008. [CrossRef]
171.
Huang, Y.; Zhang, S.; Chen, H.; Zhao, L.; Zhang, Z.; Cheng, P.; Chen, Y. A Zinc Coordination Complex Mimicking Carbonic
Anhydrase for CO2Hydrolysis and Sequestration. Inorg. Chem. 2019,58, 9916–9921. [CrossRef]
172.
Bien, C.E.; Chen, K.K.; Chien, S.-C.; Reiner, B.R.; Lin, L.-C.; Wade, C.R.; Ho, W.S.W. Bioinspired Metal-Organic Framework for
Trace CO2Capture. J. Am. Chem. Soc. 2018,140, 12662–12666. [CrossRef]
173.
Han, L.; Pham, T.; Zhuo, M.; Forrest, K.A.; Suepaul, S.; Space, B.; Zaworotko, M.J.; Shi, W.; Chen, Y.; Cheng, P.; et al. Molecular
Sieving and Direct Visualization of CO
2
in Binding Pockets of an Ultramicroporous Lanthanide Metal-Organic Framework
Platform. ACS Appl. Mater. Interfaces 2019,11, 23192–23197. [CrossRef] [PubMed]
174.
Wu, Z.; Nan, Y.; Zhao, Y.; Wang, X.; Huang, S.; Shi, J. Immobilization of Carbonic Anhydrase for Facilitated CO
2
Capture and
Separation. Chin. J. Chem. Eng. 2020,28, 2817–2831. [CrossRef]
175.
Neves, L.A.; Afonso, C.; Coelhoso, I.M.; Crespo, J.G. Integrated CO
2
Capture and Enzymatic Bioconversion in Supported Ionic
Liquid Membranes. Sep. Purif. Technol. 2012,97, 34–41. [CrossRef]
176.
Bednár, A.; Nemestóthy, N.; Bakonyi, P.; Fülöp, L.; Zhen, G.; Lu, X.; Kobayashi, T.; Kumar, G.; Xu, K.; Bélafi-Bakó, K. Enzymatically-
Boosted Ionic Liquid Gas Separation Membranes Using Carbonic Anhydrase of Biomass Origin. Chem. Eng. J.
2016
,303, 621–626.
[CrossRef]
177.
de Castro, A.M.; Neves, L.A.; Corvo, M.C.; Cabrita, E.J.; Crespo, J.G. Effect of Carbonic Anhydrase on CO
2
Absorption Promoted
by Choline Hydroxide Using Supported Liquid Membranes. Sep. Purif. Technol. 2022,280, 119921. [CrossRef]
178.
Migliardini, F.; De Luca, V.; Carginale, V.; Rossi, M.; Corbo, P.; Supuran, C.T.; Capasso, C. Biomimetic CO
2
Capture Using a
Highly Thermostable Bacterial
α
-Carbonic Anhydrase Immobilized on a Polyurethane Foam. J. Enzym. Inhib. Med. Chem.
2014
,
29, 146–150. [CrossRef]
179.
Zaidi, S.; Srivastava, N.; Khare, S.K. Microbial Carbonic Anhydrase Mediated Carbon Capture, Sequestration & Utilization: A
Sustainable Approach to Delivering Bio-Renewables. Bioresour. Technol. 2022,365, 128174. [CrossRef]
180.
Rasouli, H.; Iliuta, I.; Bougie, F.; Garnier, A.; Iliuta, M.C. Enhanced CO
2
Capture in Packed-Bed Column Bioreactors with
Immobilized Carbonic Anhydrase. Chem. Eng. J. 2022,432, 134029. [CrossRef]
181.
Vinoba, M.; Bhagiyalakshmi, M.; Jeong, S.K.; Yoon, Y.I.I.; Nam, S.C. Immobilization of Carbonic Anhydrase on Spherical SBA-15
for Hydration and Sequestration of CO2.Colloids Surf. B Biointerfaces 2012,90, 91–96. [CrossRef]
182.
Uygun, M.; Singh, V.V.; Kaufmann, K.; Uygun, D.A.; De Oliveira, S.D.S.; Wang, J. Micromotor-Based Biomimetic Carbon Dioxide
Sequestration: Towards Mobile Microscrubbers. Angew. Chem.-Int. Ed. 2015,54, 12900–12904. [CrossRef]
183.
Xu, X.; Kentish, S.E.; Martin, G.J.O. Direct Air Capture of CO
2
by Microalgae with Buoyant Beads Encapsulating Carbonic
Anhydrase. ACS Sustain. Chem. Eng. 2021,9, 9698–9706. [CrossRef]
184.
Sifat, N.S.; Haseli, Y. A Critical Review of CO
2
Capture Technologies and Prospects for Clean Power Generation. Energies
2019
,
12, 4143. [CrossRef]

Molecules 2023,28, 5520 48 of 52
185.
Fu, L.; Ren, Z.; Si, W.; Ma, Q.; Huang, W.; Liao, K.; Huang, Z.; Wang, Y.; Li, J.; Xu, P. Research Progress on CO
2
Capture and
Utilization Technology. J. CO2 Util. 2022,66, 102260. [CrossRef]
186.
Fagorite, I.V.; Chijioke, C.F.; Opara, I.A.; Onyekuru, S.O.; Oguzie, E.E. Environmental and Safety Issues Associated with Geological
Carbon Storage: A Review. Euro-Mediterr. J. Environ. Integr. 2022,7, 445–461. [CrossRef]
187.
Farrukh, S.; Wu, D.; Al-Dadah, R.; Gao, W.; Wang, Z. A Review of Integrated Cryogenic Energy Assisted Power Generation
Systems and Desalination Technologies. Appl. Therm. Eng. 2023,221, 119836. [CrossRef]
188.
International Energy Agency. CO2 Emissions in 2022. Available online: https://iea.blob.core.windows.net/assets/3c8fa115-35c4
-4474-b237-1b00424c8844/CO2Emissionsin2022.pdf (accessed on 30 June 2023).
189.
International Energy Agency. Global Energy-Related CO2 Emissions by Sector. Available online: https://www.iea.org/data-and-
statistics/charts/global-energy-related-co2-emissions-by-sector (accessed on 30 June 2023).
190.
Hepburn, C.; Adlen, E.; Beddington, J.; Carter, E.A.; Fuss, S.; Mac Dowell, N.; Minx, J.C.; Smith, P.; Williams, C.K. The
Technological and Economic Prospects for CO2Utilization and Removal. Nature 2019,575, 87–97. [CrossRef]
191.
Gundersen, M.T.; Von Solms, N.; Woodley, J.M. Enzymatically Assisted CO
2
Removal from Flue-Gas. In Energy Procedia;
Department of Chemical and Biochemical Engineering, Technical University of Denmark: Lyngby, Denmark, 2014; Volume 63,
pp. 624–632. [CrossRef]
192.
Zhang, S.; Du, M.; Shao, P.; Wang, L.; Ye, J.; Chen, J.; Chen, J. Carbonic Anhydrase Enzyme-MOFs Composite with a Superior
Catalytic Performance to Promote CO
2
Absorption into Tertiary Amine Solution. Environ. Sci. Technol.
2018
,52, 12708–12716.
[CrossRef]
193.
Gladis, A.; Lomholdt, N.F.; Fosbøl, P.L.; Woodley, J.M.; von Solms, N. Pilot Scale Absorption Experiments with Carbonic
Anhydrase-Enhanced MDEA-Benchmarking with 30 wt% MEA. Int. J. Greenh. Gas Control. 2019,82, 69–85. [CrossRef]
194.
Kim, T.-J.; Lang, A.; Chikukwa, A.; Sheridan, E.; Dahl, P.I.; Leimbrink, M.; Skiborowski, M.; Roubroeks, J. Enzyme Carbonic
Anhydrase Accelerated CO
2
Absorption in Membrane Contactor. In Energy Procedia; SINTEF Materials and Chemistry: Trondheim,
Norway, 2017; Volume 114, pp. 17–24. [CrossRef]
195.
Thee, H.; Smith, K.H.; Da Silva, G.; Kentish, S.E.; Stevens, G.W. Carbonic Anhydrase Promoted Absorption of CO
2
into Potassium
Carbonate Solutions. Greenh. Gases Sci. Technol. 2015,5, 108–114. [CrossRef]
196.
Hu, G.; Nicholas, N.J.; Smith, K.H.; Mumford, K.A.; Kentish, S.E.; Stevens, G.W. Carbon Dioxide Absorption into Promoted
Potassium Carbonate Solutions: A Review. Int. J. Greenh. Gas Control 2016,53, 28–40. [CrossRef]
197.
Jin, P.; Zhang, S.; Liu, Y.; Zhang, W.; Wang, R. Application of Bacillus mucilaginosus in the Carbonation of Steel Slag. Appl. Microbiol.
Biotechnol. 2021,105, 8663–8674. [CrossRef] [PubMed]
198.
Jo, B.H.; Seo, J.H.; Yang, Y.J.; Baek, K.; Choi, Y.S.; Pack, S.P.; Oh, S.H.; Cha, H.J. Bioinspired Silica Nanocomposite with
Autoencapsulated Carbonic Anhydrase as a Robust Biocatalyst for CO
2
Sequestration. ACS Catal.
2014
,4, 4332–4340. [CrossRef]
199.
Yang, G.; Li, L.; Li, F.; Zhang, C.; Lyu, J. Mechanism of Carbonate Mineralization Induced by Microbes: Taking Curvibacter
lanceolatus Strain HJ-1 as an Example. Micron 2021,140, 102980. [CrossRef]
200.
Bose, H.; Satyanarayana, T. Suitability of the Alkalistable Carbonic Anhydrase from a Polyextremophilic Bacterium Aeribacillus
pallidus TSHB1 in Biomimetic Carbon Sequestration. Bioprocess Biosyst. Eng. 2016,39, 1515–1525. [CrossRef]
201.
De Luca, V.; Vullo, D.; Scozzafava, A.; Carginale, V.; Rossi, M.; Supuran, C.T.; Capasso, C. An Alpha-Carbonic Anhydrase from
the Thermophilic Bacterium Sulphurihydrogenibium azorense Is the Fastest Enzyme Known for the CO
2
Hydration Reaction. Bioorg.
Med. Chem. 2013,21, 1465–1469. [CrossRef]
202.
Di Lorenzo, F.; Ruiz-Agudo, C.; Ibañez-Velasco, A.; Gil-San Millán, R.; Navarro, J.A.R.; Ruiz-Agudo, E.; Rodriguez-Navarro, C.
The Carbonation of Wollastonite: A Model Reaction to Test Natural and Biomimetic Catalysts for Enhanced CO
2
Sequestration.
Minerals 2018,8, 209. [CrossRef]
203.
Wang, M.; Lawal, A.; Stephenson, P.; Sidders, J.; Ramshaw, C. Post-Combustion CO
2
Capture with Chemical Absorption: A
State-of-the-Art Review. Chem. Eng. Res. Des. 2011,89, 1609–1624. [CrossRef]
204.
Heldebrant, D.J.; Koech, P.K.; Glezakou, V.-A.; Rousseau, R.; Malhotra, D.; Cantu, D.C. Water-Lean Solvents for Post-Combustion
CO2Capture: Fundamentals, Uncertainties, Opportunities, and Outlook. Chem. Rev. 2017,117, 9594–9624. [CrossRef]
205.
Sharif, M.; Zhang, T.; Wu, X.; Yu, Y.; Zhang, Z. Evaluation of CO
2
Absorption Performance by Molecular Dynamic Simulation for
Mixed Secondary and Tertiary Amines. Int. J. Greenh. Gas. Control 2020,97, 103059. [CrossRef]
206. Bernhardsen, I.M.; Krokvik, I.R.T.; Jens, K.-J.; Knuutila, H.K. Performance of MAPA Promoted Tertiary Amine Systems for CO2
Absorption: Influence of Alkyl Chain Length and Hydroxyl Groups. Energy Procedia 2017,114, 1682–1688. [CrossRef]
207.
Liu, B.; Cui, Z.; Tian, W. The Kinetics Investigation of CO
2
Absorption into TEA and DEEA Amine Solutions Containing Carbonic
Anhydrase. Processes 2021,9, 2140. [CrossRef]
208.
Bui, M.; Adjiman, C.S.; Bardow, A.; Anthony, E.J.; Boston, A.; Brown, S.; Fennell, P.S.; Fuss, S.; Galindo, A.; Hackett, L.A.; et al.
Carbon Capture and Storage (CCS): The Way Forward. Energy Environ. Sci. 2018,11, 1062–1176. [CrossRef]
209.
Power, I.M.; Harrison, A.L.; Dipple, G.M. Accelerating Mineral Carbonation Using Carbonic Anhydrase. Environ. Sci. Technol.
2016,50, 2610–2618. [CrossRef] [PubMed]
210. Ehrlich, H.; Bailey, E.; Wysokowski, M.; Jesionowski, T. Forced Biomineralization: A Review. Biomimetics 2021,6, 46. [CrossRef]
211.
Bose, H.; Satyanarayana, T. Microbial Carbonic Anhydrases in Biomimetic Carbon Sequestration for Mitigating Global Warming:
Prospects and Perspectives. Front. Microbiol. 2017,8, 1615. [CrossRef]

Molecules 2023,28, 5520 49 of 52
212.
Wang, F.; Dreisinger, D.B.; Jarvis, M.; Hitchins, T. The Technology of CO
2
Sequestration by Mineral Carbonation: Current Status
and Future Prospects. Can. Metall. Q. 2018,57, 46–58. [CrossRef]
213.
Hills, C.D.; Tripathi, N.; Carey, P.J. Mineralization Technology for Carbon Capture, Utilization, and Storage. Front. Energy Res.
2020,8, 142. [CrossRef]
214.
Bhatia, S.K.; Bhatia, R.K.; Jeon, J.-M.; Kumar, G.; Yang, Y.-H. Carbon Dioxide Capture and Bioenergy Production Using Biological
System–A Review. Renew. Sust. Energy Rev. 2019,110, 143–158. [CrossRef]
215.
Zajac, M.; Krol, M.; Bullerjahn, F.; Deja, J. Effect of Temperature on Carbon Dioxide Mineralisation in Recycled Cement Paste. Adv.
Cem. Res. 2023,35, 1–12. [CrossRef]
216.
Yin, B.; Xu, H.; Fan, F.; Qi, D.; Hua, X.; Xu, T.; Liu, C.; Hou, D. Superhydrophobic Coatings Based on Bionic Mineralization for
Improving the Durability of Marine Concrete. Constr. Build. Mater. 2023,362, 129705. [CrossRef]
217.
Heldebrant, D.J.; Kothandaraman, J.; Mac Dowell, N.; Brickett, L. Next Steps for Solvent-Based CO
2
Capture; Integration of
Capture, Conversion, and Mineralisation. Chem. Sci. 2022,13, 6445–6456. [CrossRef]
218.
Rodriguez-Navarro, C.; Cizer, O.; Kudlacz, K.; Ibanez-Velasco, A.; Ruiz-Agudo, C.; Elert, K.; Burgos-Cara, A.; Ruiz-Agudo, E. The
Multiple Roles of Carbonic Anhydrase in Calcium Carbonate Mineralization. CrystEngComm 2019,21, 7407–7423. [CrossRef]
219.
Fernández, M.S.; Montt, B.; Ortiz, L.; Neira-Carrillo, A.; Arias, J.L. Effect of Carbonic Anhydrase Immobilized on Eggshell
Membranes on Calcium Carbonate Crystallization In Vitro. In Biomineralization; Endo, K., Kogure, T., Nagasawa, H., Eds.;
Springer: Singapore, 2018; pp. 31–37.
220.
Jin, P.; Wang, R.; Zhang, S.; Chen, Y. Effect of Carbonic Anhydrase Bacteria on the Carbonation Process of
γ
-C2S. Adv. Cem. Res.
2021,34, 15–27. [CrossRef]
221.
Sharma, V.K.; Hutchison, J.M.; Allgeier, A.M. Redox Biocatalysis: Quantitative Comparisons of Nicotinamide Cofactor Regenera-
tion Methods. ChemSusChem 2022,15, e2022008. [CrossRef] [PubMed]
222.
Navarro-Jaen, S.; Virginie, M.; Bonin, J.; Robert, M.; Wojcieszak, R.; Khodakov, A.Y. Highlights and Challenges in the Selective
Reduction of Carbon Dioxide to Methanol. Nat. Rev. Chem. 2021,5, 564–579. [CrossRef]
223.
Corrado, M.L.; Knaus, T.; Schwaneberg, U.; Mutti, F.G. High-Yield Synthesis of Enantiopure 1,2-Amino Alcohols from L-
Phenylalanine via Linear and Divergent Enzymatic Cascades. Org. Process Res. Dev. 2022,26, 2085–2095. [CrossRef]
224.
Singh, P.; Srivastava, R. Utilization of Bio-Inspired Catalyst for CO
2
Reduction into Green Fuels: Recent Advancement and Future
Perspectives. J. CO2 Util. 2021,53, 101748. [CrossRef]
225.
Armstrong, F.A.; Cheng, B.; Herold, R.A.; Megarity, C.F.; Siritanaratkul, B. From Protein Film Electrochemistry to Nanoconfined
Enzyme Cascades and the Electrochemical Leaf. Chem. Rev. 2023,123, 5421–5458. [CrossRef]
226.
Meneghello, M.; Leger, C.; Fourmond, V. Electrochemical Studies of CO
2
-Reducing Metalloenzymes. Chem. Eur. J.
2021
,27,
17542–17553. [CrossRef]
227.
Rasheed, T.; Shafi, S.; Anwar, M.T.; Rizwan, K.; Ahmad, T.; Bilal, M. Revisiting Photo and Electro-Catalytic Modalities for
Sustainable Conversion of CO2.Appl. Catal. A-Gen. 2021,623, 118248. [CrossRef]
228.
Cadoux, C.; Milton, R.D. Recent Enzymatic Electrochemistry for Reductive Reactions. ChemElectroChem
2020
,7, 1974–1986.
[CrossRef]
229.
Xing, X.; Liu, Y.; Lin, R.-D.; Zhang, Y.; Wu, Z.-L.; Yu, X.-Q.; Li, K.; Wang, N. Development of an Integrated System for Highly
Selective Photoenzymatic Synthesis of Formic Acid from CO2.ChemSusChem 2023,16, e2022019. [CrossRef]
230.
Wang, Q.; Pan, Z. Advances and Challenges in Developing Cocatalysts for Photocatalytic Conversion of Carbon Dioxide to Fuels.
Nano Res. 2022,15, 10090–10109. [CrossRef]
231.
Fang, X.; Kalathil, S.; Reisner, E. Semi-Biological Approaches to Solar-to-Chemical Conversion. Chem. Soc. Rev.
2020
,49, 4926–4952.
[CrossRef]
232.
Ozgen, F.F.; Runda, M.E.; Schmidt, S. Photo-Biocatalytic Cascades: Combining Chemical and Enzymatic Transformations Fueled
by Light. ChemBioChem 2021,22, 790–806. [CrossRef]
233.
Chen, H.; Huang, Y.; Sha, C.; Moradian, J.M.; Yong, Y.-C.; Fang, Z. Enzymatic Carbon Dioxide to Formate: Mechanisms,
Challenges and Opportunities. Renew. Sust. Energy Rev. 2023,178, 113271. [CrossRef]
234.
Immanuel, S.; Sivasubramanian, R.; Gul, R.; Dar, M.A. Recent Progress and Perspectives on Electrochemical Regeneration of
Reduced Nicotinamide Adenine Dinucleotide (NADH). Chem. Asian J. 2020,15, 4256–4270. [CrossRef]
235.
Lee, Y.S.; Gerulskis, R.; Minteer, S.D. Advances in Electrochemical Cofactor Regeneration: Enzymatic and Non-Enzymatic
Approaches. Curr. Opin. Biotechnol. 2022,73, 14–21. [CrossRef]
236.
Zhang, Z.; Zhang, X.; Ji, X. Developing and Regenerating Cofactors for Sustainable Enzymatic CO
2
Conversion. Processes
2022
,
10, 230. [CrossRef]
237.
Bachosz, K.; Zdarta, J.; Bilal, M.; Meyer, A.S.; Jesionowski, T. Enzymatic Cofactor Regeneration Systems: A New Perspective on
Efficiency Assessment. Sci. Total Environ. 2023,868, 161630. [CrossRef]
238. Findrik, Z.; Vasic-Racki, D. Overview on Reactions with Multi-Enzyme Systems. Chem. Biochem. Eng. Q. 2009,23, 545–553.
239.
Neves, L.A.; Afonso, C.A.M.; Coelhoso, L.M.; Crespo, J.G. CO
2
Capture by Enzymatic Bioconversion in a Membrane Contactor
with Task Specific Ionic Liquids. In Procedia Engineering; Universidade Nova de Lisboa: Lisbon, Portugal, 2012; Volume 44,
pp. 557–558. [CrossRef]
240.
Qadir, M.I.; Dupont, J. Thermo- and Photocatalytic Activation of CO
2
in Ionic Liquids. Angew. Chem. Int. Ed.
2023
, e202301497.
[CrossRef]

Molecules 2023,28, 5520 50 of 52
241.
Chenault, H.K.; Whitesides, G.M. Regeneration of Nicotinamide Cofactors for Use in Organic Synthesis. Appl. Biochem. Biotechnol.
1987,14, 147–197. [CrossRef] [PubMed]
242.
Chenault, H.K.; Simon, E.S.; Whitesides, G.M. Cofactor Regeneration for Enzyme-Catalysed Synthesis. Biotechnol. Genet. Eng. Rev.
1988,6, 221–270. [CrossRef]
243.
Yu, X.; Niks, D.; Ge, X.; Liu, H.; Hille, R.; Mulchandani, A. Synthesis of Formate from CO
2
Gas Catalyzed by an O
2
-Tolerant
NAD-Dependent Formate Dehydrogenase and Glucose Dehydrogenase. Biochemistry 2019,58, 1861–1868. [CrossRef]
244.
Liao, Q.; Liu, W.; Meng, Z. Strategies for Overcoming the Limitations of Enzymatic Carbon Dioxide Reduction. Biotechnol. Adv.
2022,60, 108024. [CrossRef]
245.
El-Zahab, B.; Donnelly, D.; Wang, P. Particle-Tethered NADH for Production of Methanol from CO
2
Catalyzed by Coimmobilized
Enzymes. Biotechnol. Bioeng. 2008,99, 508–514. [CrossRef]
246.
Ji, X.; Su, Z.; Wang, P.; Ma, G.; Zhang, S. Tethering of Nicotinamide Adenine Dinucleotide inside Hollow Nanofibers for High-
Yield Synthesis of Methanol from Carbon Dioxide Catalyzed by Coencapsulated Multienzymes. ACS Nano
2015
,9, 4600–4610.
[CrossRef]
247.
Ren, S.; Wang, Z.; Bilal, M.; Feng, Y.; Jiang, Y.; Jia, S.; Cui, J. Co-Immobilization Multienzyme Nanoreactor with Co-Factor
Regeneration for Conversion of CO2.Int. J. Biol. Macromol. 2020,155, 110–118. [CrossRef]
248.
Shukia, S.K.; Khokarale, S.G.; Bui, T.Q.; Mikkola, J.-P.T. Ionic Liquids: Potential Materials for Carbon Dioxide Capture and
Utilization. Front. Mater. 2019,6, 42. [CrossRef]
249.
Zeng, S.; Zhang, X.X.; Bai, L.; Zhang, X.X.; Wang, H.; Wang, J.; Bao, D.; Li, M.; Liu, X.; Zhang, S. Ionic-Liquid-Based CO
2
Capture
Systems: Structure, Interaction and Process. Chem. Rev. 2017,117, 9625–9673. [CrossRef]
250.
Zhang, Z.; Muschiol, J.; Huang, Y.; Sigurdardottir, S.B.; von Solms, N.; Daugaard, A.E.; Wei, J.; Luo, J.; Xu, B.-H.; Zhang, S.; et al.
Efficient Ionic Liquid-Based Platform for Multi-Enzymatic Conversion of Carbon Dioxide to Methanol. Green Chem.
2018
,20,
4339–4348. [CrossRef]
251. The Methanol Institute. Available online: https://www.methanol.org/applications/ (accessed on 7 July 2023).
252.
Cazelles, R.; Drone, J.; Fajula, F.; Ersen, O.; Moldovan, S.; Galarneau, A. Reduction of CO
2
to Methanol by a Polyenzymatic
System Encapsulated in Phospholipids-Silica Nanocapsules. New J. Chem. 2013,37, 3721–3730. [CrossRef]
253.
Singh, R.K.R.; Singh, R.K.R.; Sivakumar, D.; Kondaveeti, S.; Kim, T.; Li, J.; Sung, B.H.; Cho, B.-K.; Kim, D.R.; Kim, S.C.; et al.
Insights into Cell-Free Conversion of CO
2
to Chemicals by a Multienzyme Cascade Reaction. ACS Catal.
2018
,8, 11085–11093.
[CrossRef]
254.
Dave, B.C.; Rao, M.S.; Burt, M.C. Converting Carbon Dioxide to Methanol Comprises Serial Reduction of the Carbon Dioxide to
Methanol By Dehydrogenase Enzymes in the Presence of Reduced Nicotinamide Adenine Dinucleotide. US20070042479(A1), 18
August 2005.
255.
Alvarez-Malmagro, J.; Oliveira, A.R.; Gutierrez-Sanchez, C.; Villajos, B.; Pereira, I.A.C.; Velez, M.; Pita, M.; De Lacey, A.L. Bioelec-
trocatalytic Activity of W-Formate Dehydrogenase Covalently Immobilized on Functionalized Gold and Graphite Electrodes.
ACS Appl. Mater. Interfaces 2021,13, 11891–11900. [CrossRef]
256.
Barin, R.; Rashid-Nadimi, S.; Biria, D.; Asadollahi, M.A. Direct Electrochemical Regeneration of 1,4-NADH at the Copper Foam
and Bimetallic Copper Foam. Electrochim. Acta 2017,247, 1095–1102. [CrossRef]
257.
Song, H.; Ma, C.; Liu, P.; You, C.; Lin, J.; Zhu, Z. A Hybrid CO
2
Electroreduction System Mediated by Enzyme-Cofactor Conjugates
Coupled with Cu Nanoparticle-Catalyzed Cofactor Regeneration. J. CO2 Util. 2019,34, 568–575. [CrossRef]
258.
Addo, P.K.; Arechederra, R.L.; Waheed, A.; Shoemaker, J.D.; Sly, W.S.; Minteer, S.D. Methanol Production via Bioelectrocatalytic
Reduction of Carbon Dioxide: Role of Carbonic Anhydrase in Improving Electrode Performance. Electrochem. Solid-State Lett.
2011,14, E9–E13. [CrossRef]
259.
Yuan, M.; Sahin, S.; Cai, R.; Abdellaoui, S.; Hickey, D.P.; Minteer, S.D.; Milton, R.D. Creating a Low-Potential Redox Polymer for
Efficient Electroenzymatic CO2Reduction. Angew. Chem. Int. Ed. 2018,57, 6582–6586. [CrossRef]
260.
Kim, S.-H.; Chung, G.-Y.; Kim, S.-H.; Vinothkumar, G.; Yoon, S.-H.; Jung, K.-D. Electrochemical NADH Regeneration and
Electroenzymatic CO
2
Reduction on Cu Nanorods/Glassy Carbon Electrode Prepared by Cyclic Deposition. Electrochim. Acta
2016,210, 837–845. [CrossRef]
261.
Chen, Y.; Li, P.; Zhou, J.; Buru, C.T.; Dordevic, L.; Li, P.; Zhang, X.; Cetin, M.M.; Stoddart, J.F.; Stupp, S.I.; et al. Integration of
Enzymes and Photosensitizers in a Hierarchical Mesoporous Metal–Organic Framework for Light-Driven CO
2
Reduction. J. Am.
Chem. Soc. 2020,142, 1768–1773. [CrossRef]
262.
Barin, R.; Biria, D.; Rashid-Nadimi, S.; Asadollahi, M.A. Enzymatic CO
2
Reduction to Formate by Formate Dehydrogenase from
Candida Boidinii Coupling with Direct Electrochemical Regeneration of NADH. J. CO2 Util. 2018,28, 117–125. [CrossRef]
263.
Zhang, Z.; Li, J.; Ji, M.; Liu, Y.; Wang, N.; Zhang, X.; Zhang, S.; Ji, X. Encapsulation of Multiple Enzymes in a Metal-Organic
Framework with Enhanced Electro-Enzymatic Reduction of CO2to Methanol. Green Chem. 2021,23, 2362–2371. [CrossRef]
264.
Zhang, T.R.; Lu, S.Y. Sacrificial Agents for Photocatalytic Hydrogen Production: Effects, Cost, and Development. Chem. Catalysis
2022,2, 1502–1505. [CrossRef]
265.
Hernandez-Ibanez, N.; Gomis-Berenguer, A.; Montiel, V.; Ania, C.O.; Iniesta, J. Fabrication of a Biocathode for Formic Acid
Production upon the Immobilization of Formate Dehydrogenase from Candida boidinii on a Nanoporous Carbon. Chemosphere
2022,291, 133117. [CrossRef]

Molecules 2023,28, 5520 51 of 52
266.
Cheng, Y.; Shi, J.; Wu, Y.; Wang, X.; Sun, Y.; Cai, Z.; Chen, Y.; Jiang, Z. Intensifying Electron Utilization by Surface-Anchored Rh
Complex for Enhanced Nicotinamide Cofactor Regeneration and Photoenzymatic CO2Reduction. Research 2021,2021, 8175709.
[CrossRef]
267.
Ottone, C.; Pugliese, D.; Laurenti, M.; Hernandez, S.; Cauda, V.; Grez, P.; Wilson, L. ZnO Materials as Effective Anodes for the
Photoelectrochemical Regeneration of Enzymatically Active NAD
+
.ACS Appl. Mater. Interfaces
2021
,13, 10719–10727. [CrossRef]
268.
Tensi, L.; Macchioni, A. Extremely Fast NADH-Regeneration Using Phosphonic Acid as Hydride Source and Iridium-Pyridine-2-
Sulfonamidate Catalysts. ACS Catal. 2020,10, 7945–7949. [CrossRef]
269.
Liao, Q.; Guo, M.; Mao, M.; Gao, R.; Meng, Z.; Fan, X.; Liu, W. Construction and Optimization of a Photo-Enzyme Coupled
System for Sustainable CO2Conversion to Methanol. Process Biochem. 2023,129, 44–55. [CrossRef]
270.
Guo, M.; Gu, F.; Meng, L.; Liao, Q.; Meng, Z.; Liu, W. Synthesis of Formaldehyde from CO
2
Catalyzed by the Coupled
Photo-Enzyme System. Sep. Purif. Technol. 2022,286, 120480. [CrossRef]
271.
Kim, J.A.; Kim, S.; Lee, J.; Baeg, J.-O.; Kim, J.A. Photochemical Production of NADH Using Cobaloxime Catalysts and Visible-Light
Energy. Inorg. Chem. 2012,51, 8057–8063. [CrossRef] [PubMed]
272.
Laun, K.; Duffus, B.R.; Kumar, H.; Oudsen, J.-P.H.; Karafoulidi-Retsou, C.; Waffo, A.T.; Hildebrandt, P.; Ly, K.H.; Leimkuehler, S.;
Katz, S.; et al. A Minimal Light-Driven System to Study the Enzymatic CO
2
Reduction of Formate Dehydrogenase. ChemCatChem
2022,14, e202201067. [CrossRef]
273.
Ji, X.; Wang, J.; Kang, Y.; Mei, L.; Su, Z.; Wang, S.; Ma, G.; Shi, J.; Zhang, S. Enhanced Solar Energy Harvest and Electron Transfer
through Intra- and Intermolecular Dual Channels in Chlorosome-Mimicking Supramolecular Self-Assemblies. ACS Catal.
2018
,8,
10732–10745. [CrossRef]
274.
Lin, G.; Zhang, Y.; Hua, Y.; Zhang, C.; Jia, C.; Ju, D.; Yu, C.; Li, P.; Liu, J. Bioinspired Metalation of the Metal-Organic Framework
MIL-125-NH
2
for Photocatalytic NADH Regeneration and Gas-Liquid-Solid Three-Phase Enzymatic CO
2
Reduction. Angew.
Chem. Int. Ed. 2022,61, e202206283. [CrossRef] [PubMed]
275.
Ong, W.-J.; Tan, L.-L.; Ng, Y.H.; Yong, S.-T.; Chai, S.-P. Graphitic Carbon Nitride (g-C
3
N
4
)-Based Photocatalysts for Artificial
Photosynthesis and Environmental Remediation: Are We a Step Closer To Achieving Sustainability? Chem. Rev.
2016
,116,
7159–7329. [CrossRef]
276.
Zeng, P.; Ji, X.; Su, Z.; Zhang, S. WS
2
/g-C
3
N
4
Composite as an Efficient Heterojunction Photocatalyst for Biocatalyzed Artificial
Photosynthesis. RSC Adv. 2018,8, 20557–20567. [CrossRef]
277.
Meng, J.; Tian, Y.; Li, C.; Lin, X.; Wang, Z.; Sun, L.; Zhou, Y.; Li, J.; Yang, N.; Zong, Y.; et al. A Thiophene-Modified Doubleshell
Hollow g- C
3
N
4
Nanosphere Boosts NADH Regeneration via Synergistic Enhancement of Charge Excitation and Separation.
Catal. Sci. Technol. 2019,9, 1911–1921. [CrossRef]
278.
Gao, Y.; Li, W.; Sun, X.; Zhao, Y.; Ji, H.; Luo, H.; Wu, G.; Wan, L.; Zhang, L. Boosting the Performance of Formate Dehydrogenase
by Silver Nanoclusters for Photoreduction of CO2to Formate. ACS Sustain. Chem. Eng. 2022,10, 14888–14896. [CrossRef]
279.
Gupta, P.; Verma, N. Conversion of CO
2
to Formate Using Activated Carbon Fiber-Supported g-C
3
N
4
-NiCoWO4 Photoanode in
a Microbial Electrosynthesis System. Chem. Eng. J. 2022,446, 137029. [CrossRef]
280.
Tian, Y.; Zhou, Y.; Zong, Y.; Li, J.; Yang, N.; Zhang, M.; Guo, Z.; Song, H. Construction of Functionally Compartmental Inorganic
Photocatalyst-Enzyme System via Imitating Chloroplast for Efficient Photoreduction of CO
2
to Formic Acid. ACS Appl. Mater.
Interfaces 2020,12, 34795–34805. [CrossRef]
281.
Yu, S.; Lv, P.; Xue, P.; Wang, K.; Yang, Q.; Zhou, J.; Wang, M.; Wang, L.; Chen, B.; Tan, T. Light-Driven Enzymatic Nanosystem for
Highly Selective Production of Formic Acid from CO2.Chem. Eng. J. 2021,420, 127649. [CrossRef]
282.
Ji, X.; Kang, Y.; Su, Z.; Wang, P.; Ma, G.; Zhang, S. Graphene Oxide and Polyelectrolyte Composed One-Way Expressway for
Guiding Electron Transfer of Integrated Artificial Photosynthesis. ACS Sustain. Chem. Eng. 2018,6, 3060–3069. [CrossRef]
283.
Ji, X.; Su, Z.; Wang, P.; Ma, G.; Zhang, S. Integration of Artificial Photosynthesis System for Enhanced Electronic Energy-Transfer
Efficacy: A Case Study for Solar-Energy Driven Bioconversion of Carbon Dioxide to Methanol. Small
2016
,12, 4753–4762.
[CrossRef] [PubMed]
284.
Kim, S.; Lee, S.; Anjong, T.F.; Jang, H.Y.; Kim, J.-Y.; Lee, C.; Park, S.; Lee, H.J.; Yoon, J.; Kim, J. Artificial PhotocatalyticSystem Using
Polydiacetylene-(-NH-Phen)Ru(Bpy)
2
for Cofactor Regeneration and CO
2
Reduction. J. Phys. Chem. C
2016
,120, 28407–28414.
[CrossRef]
285.
Sokol, K.P.; Robinson, W.E.; Oliveira, A.R.; Warnan, J.; Nowaczyk, M.M.; Ruff, A.; Pereira, I.A.C.; Reisner, E. Photoreduction of
CO
2
with a Formate Dehydrogenase Driven by Photosystem II Using a Semi-Artificial Z-Scheme Architecture. J. Am. Chem. Soc.
2018,140, 16418–16422. [CrossRef] [PubMed]
286.
Schuler, E.; Ermolich, P.A.; Shiju, N.R.; Gruter, G.-J. Monomers from CO
2
: Superbases as Catalysts for Formate-to-Oxalate
Coupling. ChemSusChem 2021,14, 1517–1523. [CrossRef]
287.
Murcia-Valderrama, M.A.; van Putten, R.-J.; Gruter, G.-J.M. The Potential of Oxalic—And Glycolic Acid Based Polyesters (Review).
Towards CO2as a Feedstock (Carbon Capture and Utilization–CCU). Eur. Polym. J. 2019,119, 445–468. [CrossRef]
288.
Schwarz, F.M.; Mueller, V. Whole-Cell Biocatalysis for Hydrogen Storage and Syngas Conversion to Formate Using a Thermophilic
Acetogen. Biotechnol. Biofuels 2020,13, 32. [CrossRef]
289.
Luxuan, L.; Zhijun, X.; Huang, X. Whole-Cell-Based Photosynthetic Biohybrid Systems for Energy and Environmental Applica-
tions. ChemPlusChem 2021,86, 1021–1036.

Molecules 2023,28, 5520 52 of 52
290.
Hwang, H.; Yeon, Y.; Lee, S.; Choe, H.; Cho, D.; Park, S.; Kim, Y. Electro-Biocatalytic Production of Formate from Carbon Dioxide
using an Oxygen-Stable Whole-Cell Biocatalyst.Bioresour. Technol. 2015,185, 35–39. [CrossRef]
291.
Le, T.Q.A. Recent Applications and Strategies to Enhance Performance of Electrochemical Reduction of CO
2
Gas into Value-Added
Chemicals Catalyzed by Whole-Cell Biocatalysts. Processes 2023,11, 766. [CrossRef]
292.
Alissandratos, A.; Kim, H.-K.; Easton, C.J. Formate Production through Carbon Dioxide Hydrogenation with Recombinant Whole
Cell Biocatalysts. Bioresour. Technol. 2014,164, 7–11. [CrossRef]
293.
Roger, M.; Brown, F.; Gabrielli, W.; Sargent, F. Efficient Hydrogen-Dependent Carbon Dioxide Rreduction by Escherichia coli.Curr.
Biol. 2018,28, 140–145.e2. [CrossRef]
294.
Leo, F.; Schwarz, F.M.; Schuchmann, K.; Mueller, V. Capture of Carbon Dioxide and Hydrogen by Engineered Escherichia coli:
Hydrogen-Dependent CO2Reduction to Formate. Appl. Microbiol. Biotechnol. 2021,105, 5861–5872. [CrossRef]
295.
Schwarz, F.M.; Oswald, F.; Müller, V. Acetogenic Conversion of H
2
and CO
2
into Formic Acid and Vice Versa in a Fed-Batch-
Operated Stirred-Tank Bioreactor. ACS Sustain. Chem. Eng. 2021,9, 6810–6820. [CrossRef]
296.
Le, Q.A.T.; Kim, H.G.; Kim, Y.H. Electrochemical Synthesis of Formic Acid from CO
2
Catalyzed by Shewanella oneidensis MR-1
Whole-Cell Biocatalyst. Enzym. Microb. Technol. 2018,116, 1–5. [CrossRef]
297.
Singh, S.; Noori, M.T.; Verma, N. Efficient Bio-Electroreduction of CO
2
to Formate on a Iron Phthalocyanine-Dispersed CDC in
Microbial Electrolysis System. Electrochim. Acta 2020,338, 135887. [CrossRef]
298.
Phan, U.T.; Jeon, B.W.; Kim, Y.H. Microbial Engineering of Methylorubrum extorquens AM1 to Enhance CO
2
Conversion into
Formate. Enzym. Microb. Technol. 2023,168, 110264. [CrossRef]
299.
Chen, H.; Li, J.; Fan, Q.; Zheng, T.; Zhang, Y.; Yong, Y.-C.; Fang, Z. A feasible strategy for microbial electrocatalytic CO
2
reduction
via whole-cell-packed and exogenous-mediator-free rGO/Shewanella biohydrogel. Chem. Eng. J. 2023,460, 141863. [CrossRef]
300.
Guntermann, N.; Mengers, H.G.; Franciò, G.; Blank, L.M.; Leitner, W. Bio-energy Conversion with Carbon Capture and
Utilization (BECCU): Integrated Biomass Fermentation and Chemo-Catalytic CO
2
Hydrogenation for Bioethanol and Formic
Acid Coproduction. Green Chem. 2021,23, 9860–9864. [CrossRef]
301.
Rowe, S.F.; Le Gall, G.; Ainsworth, E.V.; Davies, J.A.; Lockwood, C.W.J.; Shi, L.; Elliston, A.; Roberts, I.N.; Waldron, K.W.;
Richardson, D.J.; et al. Light-Driven H
2
Evolution and C=C or C=O Bond Hydrogenation by Shewanella oneidensis: A Versatile
Strategy for Photocatalysis by Nonphotosynthetic Microorganisms. ACS Catal. 2017,7, 7558–7566. [CrossRef]
302.
Tuyishime, P.; Sinumvayo, J.P. Novel Outlook in Engineering Synthetic Methylotrophs and Formatotrophs: A Course for
Advancing C1-Based Chemicals Production. World J. Microbiol. Biotechnol. 2020,36, 118. [CrossRef] [PubMed]
303.
Yishai, O.; Lindner, S.N.; Gonzalez de la Cruz, J.; Tenenboim, H.; Bar-Even, A. The formate bio-economy. Curr. Opin. Chem. Biol.
2016,35, 1–9. [CrossRef] [PubMed]
304.
Shengyuan Guo, S.; Asset, T.; Atanassov, P. Catalytic Hybrid Electrocatalytic/Biocatalytic Cascades for Carbon Dioxide Reduction
and Valorization. ACS Catalysis 2021,11, 5172–5188. [CrossRef]
305.
Liang, B.; Zhao, Y.; Yang, J. Recent Advances in Developing Artificial Autotrophic Microorganism for Reinforcing CO
2
Fixation.
Front. Microbiol. 2020,11, 592631. [CrossRef]
306.
Collas, F.; Dronsella, B.B.; Kubis, A.; Schann, K.; Binder, S.; Arto, N.; Claassens, N.J.; Kensy, F.; Orsi, E. Engineering the Biological
Conversion of Formate into Crotonate in Cupriavidus necator.Metab. Eng. 2023,in press. [CrossRef]
307.
Cotton, C.A.; Claassens, N.J.; Benito-Vaquerizo, S.; Bar-Even, A. Renewable Methanol and Formate as Microbial Feedstocks. Curr.
Opin. Biotechnol. 2020,62, 168–180. [CrossRef]
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