faster (with a current density of % 200 lAcm
−2 [232]). S. fumaroxidans expresses
another very fast CO 2 reducer SeCys-W-FDH, with a rate of % 200 s
−1 (reported
as 90Umg
−1 ) [229–231], but its CO 2 reduction activity cannot kinetically compete
with its highly efficient formate oxidation, rate of % 5.6 Â 10
3 s
−1 (value reported
as 2700Umg
−1 ) and K m
HCOO− of 10 lM. Unfortunately, these enzymes are
extremely oxygen-sensitive, and no further studies towards a biotechnological
application were pursuit, as far as we know.
Several other FDHs have been described to be able to reduce CO 2 , but at
considerably lower rates. Numerous studies have been conducted with
metal-independent FDHs, many of which relying on sacrificial electron donors
[233–248]. This is the case of the C. boidinii NAD-dependent metal-independent
FDH, that, in spite of its considerably low k cat
HCO3− value of only 0.009 s
−1 (K m
HCO3−
%
27.3 mM [240]; k
HCO3−
% 0.3 M
−1 s
−1 ; k cat
HCOO−
% 5.0 s
−1 ; K m
HCOO
−
% 5.0 mM; k
HCOO−
% 1.0 Â 10
3 M
-1
s
-1 [109]), has been largely exploited for its
ability to reduce CO 2 . To push the reaction in the desired, but thermodynamically
unfavourable, direction
14 is important to remove NAD
+
/regenerate NADH (also
essential for the process to become cost-effective, since NADH is a very expensive
reducing agent). Four selected examples of different strategies to force the reaction
towards the CO 2 reduction are: (a) an electroenzymatic cell where NADH is
electrochemically regenerated through a rhodium complex, with which a formate
formation rate of % 3.2 Â 10
−4 lmolmin
−1 mg
−1 was achieved [240]; (b) electrochemical NADH regeneration, but with an electropolymerised mediatorregenerator (neutral red) in a novel cathode with immobilised FDH, which is
able to produce formate at a rate of % 60 lMmin
−1 ) [249]; (c) photocatalytical
NADH regeneration using a rhodium complex and a visible light-active photocatalyst) that enabled a formate formation rate of % 1 lmolmin
−1 [250]; (d) and
enzymatic regeneration, using glutamate dehydrogenase with NAD(H) being
covalently attached to micro-particles, to be easily recovered and reused, in an
approach that allowed to improve the reaction yield from 0.12 to 1.27 methanol
formed/NADH consumed (in this study, formate was further reduced to methanol)
[251]. The Thiobacillus sp KNK65MA NAD-dependent metal-independent FDH
exhibit an as well low k cat value (k cat
HCO3−
% 0.32 s
−1 ; K m
HCO3−
% 9.2 mM; k
HCO3
−
% 35 M
−1 s
−1 ), but its specificity for formate is only 3 times superior (k cat
HCOO
−
% 1.8 s
−1 ; K m
HCOO−
% 16 mM; k
HCOO−
% 110 M
.1 s
.1 ) [252]. This Thiobacillus
enzyme was successfully used to reduce CO 2 by coupling it with a NADH photoelectrochemical regeneration system (Fig. 14), with a formate production rate of 2
lMmin
−1 (current density % 3.5mAcm
−2 ) [245].
The metal-dependent FDHs display a wide range of CO 2 reduction rates. The
Clostridium carboxidivorans NAD-dependent SeCys-W-FDH exhibits a considerably low k cat
CO
2 value (only 0.08 s
−1 ; K m
HCO3−
% 50 lM) [144, 145, 147].
14 The reduction potential values of the NAD(P)
+ /NAD(P)H (−0.32 V) and CO 2 /HCOO
−
(−0.43 V) pairs indicate that the NADH-dependent CO 2 reduction (Eq. 11) is thermodynamically
highly unfavourable. To force the reaction towards the CO 2 reduction is important to remove the
product (NAD
+ ) and maintain (regenerate) the substrate (NADH) concentration.
Carbon Dioxide Utilisation—The Formate Route
59
−2 [232]). S. fumaroxidans expresses
another very fast CO 2 reducer SeCys-W-FDH, with a rate of % 200 s
−1 (reported
as 90Umg
−1 ) [229–231], but its CO 2 reduction activity cannot kinetically compete
with its highly efficient formate oxidation, rate of % 5.6 Â 10
3 s
−1 (value reported
as 2700Umg
−1 ) and K m
HCOO− of 10 lM. Unfortunately, these enzymes are
extremely oxygen-sensitive, and no further studies towards a biotechnological
application were pursuit, as far as we know.
Several other FDHs have been described to be able to reduce CO 2 , but at
considerably lower rates. Numerous studies have been conducted with
metal-independent FDHs, many of which relying on sacrificial electron donors
[233–248]. This is the case of the C. boidinii NAD-dependent metal-independent
FDH, that, in spite of its considerably low k cat
HCO3− value of only 0.009 s
−1 (K m
HCO3−
%
27.3 mM [240]; k
HCO3−
% 0.3 M
−1 s
−1 ; k cat
HCOO−
% 5.0 s
−1 ; K m
HCOO
−
% 5.0 mM; k
HCOO−
% 1.0 Â 10
3 M
-1
s
-1 [109]), has been largely exploited for its
ability to reduce CO 2 . To push the reaction in the desired, but thermodynamically
unfavourable, direction
14 is important to remove NAD
+
/regenerate NADH (also
essential for the process to become cost-effective, since NADH is a very expensive
reducing agent). Four selected examples of different strategies to force the reaction
towards the CO 2 reduction are: (a) an electroenzymatic cell where NADH is
electrochemically regenerated through a rhodium complex, with which a formate
formation rate of % 3.2 Â 10
−4 lmolmin
−1 mg
−1 was achieved [240]; (b) electrochemical NADH regeneration, but with an electropolymerised mediatorregenerator (neutral red) in a novel cathode with immobilised FDH, which is
able to produce formate at a rate of % 60 lMmin
−1 ) [249]; (c) photocatalytical
NADH regeneration using a rhodium complex and a visible light-active photocatalyst) that enabled a formate formation rate of % 1 lmolmin
−1 [250]; (d) and
enzymatic regeneration, using glutamate dehydrogenase with NAD(H) being
covalently attached to micro-particles, to be easily recovered and reused, in an
approach that allowed to improve the reaction yield from 0.12 to 1.27 methanol
formed/NADH consumed (in this study, formate was further reduced to methanol)
[251]. The Thiobacillus sp KNK65MA NAD-dependent metal-independent FDH
exhibit an as well low k cat value (k cat
HCO3−
% 0.32 s
−1 ; K m
HCO3−
% 9.2 mM; k
HCO3
−
% 35 M
−1 s
−1 ), but its specificity for formate is only 3 times superior (k cat
HCOO
−
% 1.8 s
−1 ; K m
HCOO−
% 16 mM; k
HCOO−
% 110 M
.1 s
.1 ) [252]. This Thiobacillus
enzyme was successfully used to reduce CO 2 by coupling it with a NADH photoelectrochemical regeneration system (Fig. 14), with a formate production rate of 2
lMmin
−1 (current density % 3.5mAcm
−2 ) [245].
The metal-dependent FDHs display a wide range of CO 2 reduction rates. The
Clostridium carboxidivorans NAD-dependent SeCys-W-FDH exhibits a considerably low k cat
CO
2 value (only 0.08 s
−1 ; K m
HCO3−
% 50 lM) [144, 145, 147].
14 The reduction potential values of the NAD(P)
+ /NAD(P)H (−0.32 V) and CO 2 /HCOO
−
(−0.43 V) pairs indicate that the NADH-dependent CO 2 reduction (Eq. 11) is thermodynamically
highly unfavourable. To force the reaction towards the CO 2 reduction is important to remove the
product (NAD
+ ) and maintain (regenerate) the substrate (NADH) concentration.
Carbon Dioxide Utilisation—The Formate Route
59