Recent Progress in Applications of Enzymatic Bioelectrocatalysis

Round 1

Reviewer 1 Report

I enjoyed reading this review by Adachi, Kitazumi, Shirai, Kano. This review is well written and provides a quick overview of important approaches taken over the last 5-10 years in the field of bioelectrocatalysis. The authors place significant emphasis on their own world-leading work, but also give adequate attention of colleagues across the world.

The structure of the review is good, including recent development in electrode materials (mesostructures and nanoparticles), enzyme engineering, integrated supercapacitors. Towards the end, the authors review a selection of applications in electrosynthesis, NAD(P)H regeneration and biophotoelectrocatalysis. 

The authors refrain from critically reviewing individual papers, but by nicely structuring the literature, they provide the community with a great overview of recent developments and efforts in the field.

I recommend publication as is. I found only few, very minor typo/grammar issues:

Line 64: psudo-DET
Line 224: In addition, further a downsized FDH without
Line 397: FNR shows the DET-type bioelectrocatalytic (i.e., remove 'the')

Author Response

I recommend publication as is. I found only few, very minor typo/grammar issues:

→Thank you very much for your positive and valuable comments.

Line 64: psudo-DET
Line 224: In addition, further a downsized FDH without
Line 397: FNR shows the DET-type bioelectrocatalytic (i.e., remove 'the')

→The sentences were revised.

Reviewer 2 Report

The paper proposes to present a brief review of "recent" developments on applications of enzymatic bioelectrocatalysis, focusing mainly on DET and MET systems. Although in general the contribute of the paper may be considered as reasonable, some aspects could be improved. In this reviewer opinion there is a certain "imbalance" on the described/cited previous studies. Too much studies of one or two research groups (for instance Armstrong group, ignoring several other studies of other groups with equivalent results). Also, there is too much examples for fructose dehydrogenase and hydrogenase enzymes comparing to other (also very much studied) enzymes.

I consider that at least some minor revisions are needed, listed below:

1. page 2, figure 1, caption: the authors claim that "Some redox enzymes that have the catalytic site alone (without any other redox site (as electron-donating site)) are also able to show DET-type bioelectrocatalysis". In my opinion there is quite significant and relevant number of redox enzymes with only one redox site (catalytic), showing DET-type bioelectrocatalysis and for that reason the expression "some" is not particular accurate.
2. page 2, line 57-58: again it is referred that "redox enzymes that are able to DET-type reactions are limited". This expression limited is not correct. On the contrary the number of redox enzymes that present direct electron transfer is continuously growing.
3. page 3, lines 72-73: I believe that the authors mean that overpotential has two components, thermodynamic and kinetics, maybe this sentence can be clarified.
4. page 3, line 92: there is a typo: "bioelectrocatalyses" instead of bioelectrocatalysis; this typo appears in other lines throughout the text.
5. page 3. second paragraph: The authors divide electrodes basically in carbon-based and metals. Although I agree that this is the majority of cases, there are exceptions that could be mentioned, such as the case of modified materials that act as the "electrode" themselves independently of the conductive support (e.g. several type of conducting hydrogels, etc). I believe that the paragraph introductory sentence could be improved/clarified.
6. page 4: the authors must be careful with the claim that "enzymes of which the electrode-active sites face away from the electrode are not included, because the long-range electron transfer kinetic constant exponentially decreases with an increase in the distance between the  electrode surface and the electrode-active site in an enzyme..." although once considered correct, this is an old idea that is being rethinking lately. There are enzymes (e.g. some hydrogenases that the authors refer often) that in spite the catalytic site might be far away from the electrode, the redox clusters relay make the enzyme communicate quite well (DET) with the electrode.
7. page 8 (but also in others): overall, the authors focus a lot on fructose dehydrogenase which hinders not giving a wide perspective. Maybe a comment on why focusing so much in this particular enzyme could be useful.
8. page 8, lines 291-293: EDLC, etc must be defined before using just the abbreviations.
9. page 9. Regarding bidireccional enzymes, more recent examples could be included.
10. Conclusions: these could be improved, the last sentence "Other perspectives will be also required for the improvement of present bioelectrochemical devices." should be clarified exactly what the authors mean or at least some examples could be described.

Author Response

The paper proposes to present a brief review of "recent" developments on applications of enzymatic bioelectrocatalysis, focusing mainly on DET and MET systems. Although in general the contribute of the paper may be considered as reasonable, some aspects could be improved. In this reviewer opinion there is a certain "imbalance" on the described/cited previous studies. Too much studies of one or two research groups (for instance Armstrong group, ignoring several other studies of other groups with equivalent results). Also, there is too much examples for fructose dehydrogenase and hydrogenase enzymes comparing to other (also very much studied) enzymes

→Thank you very much for the valuable comments.

We think that FDH and hydrogenase are good examples for explaining DET mechanisms and systems. On the other hand, we comprehensively described other DET enzymes.

I consider that at least some minor revisions are needed, listed below:

1. page 2, figure 1, caption: the authors claim that "Some redox enzymes that have the catalytic site alone (without any other redox site (as electron-donating site)) are also able to show DET-type bioelectrocatalysis". In my opinion there is quite significant and relevant number of redox enzymes with only one redox site (catalytic), showing DET-type bioelectrocatalysis and for that reason the expression "some" is not particular accurate.

→The explanation and examples of enzymes containing single redox cofactors were added into the text.

2. page 2, line 57-58: again it is referred that "redox enzymes that are able to DET-type reactions are limited". This expression limited is not correct. On the contrary the number of redox enzymes that present direct electron transfer is continuously growing.

→The expression was revised.

3. page 3, lines 72-73: I believe that the authors mean that overpotential has two components, thermodynamic and kinetics, maybe this sentence can be clarified.

→The sentences were clarified.

4. page 3, line 92: there is a typo: "bioelectrocatalyses" instead of bioelectrocatalysis; this typo appears in other lines throughout the text.

→The typo was revised.

5. page 3. second paragraph: The authors divide electrodes basically in carbon-based and metals. Although I agree that this is the majority of cases, there are exceptions that could be mentioned, such as the case of modified materials that act as the "electrode" themselves independently of the conductive support (e.g. several type of conducting hydrogels, etc). I believe that the paragraph introductory sentence could be improved/clarified.

→The issues of other materials were added into the text.

6. page 4: the authors must be careful with the claim that "enzymes of which the electrode-active sites face away from the electrode are not included, because the long-range electron transfer kinetic constant exponentially decreases with an increase in the distance between the  electrode surface and the electrode-active site in an enzyme..." although once considered correct, this is an old idea that is being rethinking lately. There are enzymes (e.g. some hydrogenases that the authors refer often) that in spite the catalytic site might be far away from the electrode, the redox clusters relay make the enzyme communicate quite well (DET) with the electrode.

→We thank the point of the reviewer. The term "electrode-active site" means a catalytic site or a redox-active center which constitutes the electron-transfer pathway in the redox enzyme. To avoid misunderstanding, this explanation is added into the text.

page 8 (but also in others): overall, the authors focus a lot on fructose dehydrogenase which hinders not giving a wide perspective. Maybe a comment on why focusing so much in this particular enzyme could be useful.

→FDH is a good model enzyme for understanding protein-engineering effects on DET-type bioelectrocatalysis. This explanation was added into the text.

7. page 8, lines 291-293: EDLC, etc must be defined before using just the abbreviations.

→We are afraid that we properly defined EDLC and EPC in the above section of the old version.

8. page 9. Regarding bidirectional enzymes, more recent examples could be included.

→The sentence was revised in part.

9. Conclusions: these could be improved, the last sentence "Other perspectives will be also required for the improvement of present bioelectrochemical devices." should be clarified exactly what the authors mean or at least some examples could be described

→The specific issues are added into the text.