Nevertheless, it enabled the photoelectrochemical CO 2 reduction to formate within
an enzyme cascade that led to the methanol production at % 4 lMmin
−1
) [253].
The Methylobacterium extorquens AM1 NAD-dependent Cys-W-FDH has also
been exploited with different approaches to drive the electrochemical CO 2 reduction
[242, 254–258]. Using mediated enzymatic bioelectrocatalysis with gas diffusion
electrodes
15 , current densities of 15–20mAcm
−2 were attained [254, 255]; in a
whole-cell catalyst, M. extorquens was able to electrochemically produced formate
concentrations of up to 60 mM [242].
The R. capsulatus [182] and Cupriavidus oxalaticus [259] NAD-dependent Cys–
Mo–FDH enzymes, on the other hand, have k cat
CO
2 values, of 1.5 s
−1 and % 3 s
−1
,
respectively, but % 25 and % 30 times (respectively) lower than the one for formate
oxidation (R. capsulatus K m
HCOO−
% 280 lM and K m
CO
2 not determined, assays with
100 mM hydrogencarbonate; C. oxalaticus K m
HCOO−
% 100 lM and
K m
HCO3−
% 40 mM). The C. necator NAD-dependent Cys–Mo–FDH, on the contrary, catalyses the reduction of CO 2 with a k cat
CO
2 % 11 s
−1 (K m
CO
2 % 2.7 mM;
K m
NADH
% 45 lM) [260, 261]. To fulfil the potential industry application of this
oxygen-tolerant and robust enzyme, it is necessary to implement a NADH regenerating system that pushes the reaction towards CO 2 reduction (as discussed above; see
Footnote 14) [262]. With the C. necator FDH, this was successfully achieved with
the inclusion of glucose dehydrogenase in the system (Fig. 15), which, while catalysing the re-reduction of NAD
+ to NADH, enabled the continuous electron delivery
to drive the CO 2 reduction and, therefore, improved the reaction yield from 0.2 to 1.8
formate formed/NADH consumed [262].
The E. coli SeCys-Mo-FDH H was also shown to be able to reduce CO 2 [263],
but at rates considerably lower than the ones of formate oxidation, < 1 versus
160 s
−1 [263]. Interestingly, when the reaction is driven electrochemically (protein
film voltammetry), the formate oxidation was only two times higher than the CO 2
reduction, with current densities of 180 versus 80 lAcm
−2 , respectively [263]. This
E. coli enzyme feature has been exploited in fuel cell devices (FDH immobilised in
redox mediators-functionalised redox polymers) [15, 16], where CO 2 could be
reduced with a very high Faradaic efficiency (99%) and a current density of % 60
lAcm
−2
(K m
HCO3−
% 2.5 mM) [16]. Thanks to the FDH H-containing
formate-hydrogen lyase system (Sect. 4.2.2.)
16 , engineered E. coli whole cells
were also used as a “cell factory” to very efficiently produce formate from a gaseous
mixture of CO 2 and dihydrogen (56:44; up to 10 bar) (Fig. 16); an 100% of CO 2
conversion was achieved, with formate (more than 500 mM) being accumulated
outside the bacterial cells [264]. Intact E. coli cells were also used in a microbial
15 Gas diffusion electrodes (GDE) promote electrochemical reactions between the liquid and the
gaseous phase, thus eliminating the limitations arising from slow mass transport when
hydrogencarbonate/carbonate is used as CO 2 source.
16 Under physiological conditions the E. coli formate-hydrogen lyase system catalyses the
formate oxidation to CO 2 coupled to the reduction of protons to dihydrogen (Sect. 4.2.2.); yet,
under a pressurised (up to 10 bar) atmosphere of CO 2 and dihydrogen, engineered E. coli (with
abolished “respiratory” FDH, pyruvate-formate lyase and all major hydrogenases) whole cells
efficiently catalyse the reverse reaction of formate formation.
60
L. B. Maia et al.
an enzyme cascade that led to the methanol production at % 4 lMmin
−1
) [253].
The Methylobacterium extorquens AM1 NAD-dependent Cys-W-FDH has also
been exploited with different approaches to drive the electrochemical CO 2 reduction
[242, 254–258]. Using mediated enzymatic bioelectrocatalysis with gas diffusion
electrodes
15 , current densities of 15–20mAcm
−2 were attained [254, 255]; in a
whole-cell catalyst, M. extorquens was able to electrochemically produced formate
concentrations of up to 60 mM [242].
The R. capsulatus [182] and Cupriavidus oxalaticus [259] NAD-dependent Cys–
Mo–FDH enzymes, on the other hand, have k cat
CO
2 values, of 1.5 s
−1 and % 3 s
−1
,
respectively, but % 25 and % 30 times (respectively) lower than the one for formate
oxidation (R. capsulatus K m
HCOO−
% 280 lM and K m
CO
2 not determined, assays with
100 mM hydrogencarbonate; C. oxalaticus K m
HCOO−
% 100 lM and
K m
HCO3−
% 40 mM). The C. necator NAD-dependent Cys–Mo–FDH, on the contrary, catalyses the reduction of CO 2 with a k cat
CO
2 % 11 s
−1 (K m
CO
2 % 2.7 mM;
K m
NADH
% 45 lM) [260, 261]. To fulfil the potential industry application of this
oxygen-tolerant and robust enzyme, it is necessary to implement a NADH regenerating system that pushes the reaction towards CO 2 reduction (as discussed above; see
Footnote 14) [262]. With the C. necator FDH, this was successfully achieved with
the inclusion of glucose dehydrogenase in the system (Fig. 15), which, while catalysing the re-reduction of NAD
+ to NADH, enabled the continuous electron delivery
to drive the CO 2 reduction and, therefore, improved the reaction yield from 0.2 to 1.8
formate formed/NADH consumed [262].
The E. coli SeCys-Mo-FDH H was also shown to be able to reduce CO 2 [263],
but at rates considerably lower than the ones of formate oxidation, < 1 versus
160 s
−1 [263]. Interestingly, when the reaction is driven electrochemically (protein
film voltammetry), the formate oxidation was only two times higher than the CO 2
reduction, with current densities of 180 versus 80 lAcm
−2 , respectively [263]. This
E. coli enzyme feature has been exploited in fuel cell devices (FDH immobilised in
redox mediators-functionalised redox polymers) [15, 16], where CO 2 could be
reduced with a very high Faradaic efficiency (99%) and a current density of % 60
lAcm
−2
(K m
HCO3−
% 2.5 mM) [16]. Thanks to the FDH H-containing
formate-hydrogen lyase system (Sect. 4.2.2.)
16 , engineered E. coli whole cells
were also used as a “cell factory” to very efficiently produce formate from a gaseous
mixture of CO 2 and dihydrogen (56:44; up to 10 bar) (Fig. 16); an 100% of CO 2
conversion was achieved, with formate (more than 500 mM) being accumulated
outside the bacterial cells [264]. Intact E. coli cells were also used in a microbial
15 Gas diffusion electrodes (GDE) promote electrochemical reactions between the liquid and the
gaseous phase, thus eliminating the limitations arising from slow mass transport when
hydrogencarbonate/carbonate is used as CO 2 source.
16 Under physiological conditions the E. coli formate-hydrogen lyase system catalyses the
formate oxidation to CO 2 coupled to the reduction of protons to dihydrogen (Sect. 4.2.2.); yet,
under a pressurised (up to 10 bar) atmosphere of CO 2 and dihydrogen, engineered E. coli (with
abolished “respiratory” FDH, pyruvate-formate lyase and all major hydrogenases) whole cells
efficiently catalyse the reverse reaction of formate formation.
60
L. B. Maia et al.