Bienzymatic Cascade Combining a Peroxygenase with an Oxidase for the Synthesis of Aromatic Aldehydes from Benzyl Alcohols
, Frank Hollmann 3,* and Yonghua Wang 2,4,*Round 1
Reviewer 1 Report
The manuscript describes the enzymatic oxidation of benzyl alcohols to the corresponding aromatic aldehydes based on the concurrent use of an alcohol oxidase from P. eryngii and an unspecific peroxygenase from A. aegerita. Through this approach, the oxidase oxidises a molecule of the benzyl alcohol substrate, affording a molecule of product and hydrogen peroxide. The liberated hydrogen peroxide is used by the unspecific peroxygenase to oxidise another molecule of substrate. In this way, hydrogen peroxide, which is detrimental for the stability of the enzymes, does not accumulate in the reaction mixture, the entire process gains efficiency and virtually does not produce dangerous waste.
The work is interesting, but the paper deserves a deep revision and is not suitable for publication in the present form.
The title appears to be too generic. It should convey the idea underlying the work.
Experimental part is incomplete. The minor modification in the method employed for AnChOx production should be briefly explained.
The preparation of PeAAOx is severely deficient. Only the cultivation protocol of the E. coli cells is reported. Therefore, it seems that cells were already transformed with the plasmid containing the cloned gene. However, source and capture of the gene, cloning - including vector - and transformation of E. coli cells are missing.
The solvent for the standard solutions used for GC analyses should be specified.
Sentence in lines 204-209 seems to be rather confused: how can a poorly balanced activity ratio of PeAAOx and AaeUPO explain the low product formation rate of the reaction carried out in the presence of choline esterase?
In Figures 2 and 3 the two graphs, A and B, represent actually the same data from different points of view.
The discussion in lines 265-268 is not completely convincing: on the basis of the few examples reported, it is hard to draw a general behavior. It is proposed that electron-donating groups favor the reaction (btw, this is an already known notion and this behavior is shared by some other AAOs from fungi), but the example in entry 6 gave a result comparable to that of the unsubstituted benzyl alcohol (entry 2). Moreover, the orto-hydroxyl substituted aldehyde (entry 3) gave the worst result. Cinnamyl alcohol is reported to be converted with low efficiency, but its conversion is not so different with respect to entries 2 and 6. Authors should revise and better explain their conclusions, also in the light of the existing literature.
Other points:
Line 23 “was observed was achieved”
Sentence in lines 32-33 should be checked
Sentence in lines 133-34 should be rewritten.
Sentence in line 151 should be checked.
Line 213 “is”
Missing space in ref. 16
Defective reference 18
Check the correctness of ref. 22
Author Response
We thank the reviewers for their efforts and their constructive criticism that helped us to improve the quality of this manuscript. We have revised the manuscript following their suggestions. A detailed answer is appended below.
Reviewer: 1
- The manuscript describes the enzymatic oxidation of benzyl alcohols to the corresponding aromatic aldehydes based on the concurrent use of an alcohol oxidase from P. eryngii and an unspecific peroxygenase from A. aegerita. Through this approach, the oxidase oxidises a molecule of the benzyl alcohol substrate, affording a molecule of product and hydrogen peroxide. The liberated hydrogen peroxide is used by the unspecific peroxygenase to oxidise another molecule of substrate. In this way, hydrogen peroxide, which is detrimental for the stability of the enzymes, does not accumulate in the reaction mixture, the entire process gains efficiency and virtually does not produce dangerous waste.The work is interesting, but the paper deserves a deep revision and is not suitable for publication in the present form.
Answer: Thank you very much for sharing your enthusiasm about the reaction system. We sincerely appreciate the reviewer's suggestions and have done our best to revise the manuscript in your spirit.
- The title appears to be too generic. It should convey the idea underlying the work.
Answer: We agree with the reviewer and have changed the manuscript title to: ‘Bienzymatic cascade combining a peroxygenase with an oxidase for the synthesis of aromatic aldehydes from benzyl alcohols.’
- Experimental part is incomplete. The minor modification in the method employed for AnChOx production should be briefly explained.
Answer: We agree with the reviewer that the preparation on AnChOx should have been described in greater detail. However, also taking the reviewer’s later comments into account, we have decided to remove the experiments dealing with AnChOx from the revised manuscript. On the one hand, we have essentially performed only one experiment. Provided the system would have been further optimized, especially considering the relative ratio of oxidase and peroxidase, possibly similar productivities to the present system could have been achieved. On the other hand, this ‘benchmark system’ was never in focus of this study, particularly because of the additional starting material (choline) needed and the additional waste product (betaine) produced.
- The preparation of PeAAOx is severely deficient. Only the cultivation protocol of the E. coli cells is reported. Therefore, it seems that cells were already transformed with the plasmid containing the cloned gene. However, source and capture of the gene, cloning - including vector - and transformation of E. coli cells are missing.
Answer: We apologize for the floppy description in the first version of the manuscript. The description of PeAAOx has been improved following the reviewer’s suggestion.
- The solvent for the standard solutions used for GC analyses should be specified.
Answer: Added as requested by the reviewer.
- Sentence in lines 204-209 seems to be rather confused: how can a poorly balanced activity ratio of PeAAOx and AaeUPO explain the low product formation rate of the reaction carried out in the presence of choline esterase?
Answer: We apologize for the typo in the original manuscript causing the confusion. Instead of ‘PeAAOx’ it should have read ‘AnChOx’. We have corrected this error and have also removed the single experiment with AnChOx (please see our comments above).
- In Figures 2 and 3 the two graphs, A and B, represent actually the same data from different points of view.
Answer: Corrected as implied by the reviewer.
- The discussion in lines 265-268 is not completely convincing: on the basis of the few examples reported, it is hard to draw a general behavior. It is proposed that electron-donating groups favor the reaction (btw, this is an already known notion and this behavior is shared by some other AAOs from fungi), but the example in entry 6 gave a result comparable to that of the unsubstituted benzyl alcohol (entry 2). Moreover, the orto-hydroxyl substituted aldehyde (entry 3) gave the worst result. Cinnamyl alcohol is reported to be converted with low efficiency, but its conversion is not so different with respect to entries 2 and 6. Authors should revise and better explain their conclusions, also in the light of the existing literature.
Answer: We agree with the reviewer that the initial discussion was too superficial. Actually, based on the limited selection of substrates tested we refrain from making general conclusions and only point out the apparent differences observed here with the previous literature.
- Other points: Line 23 “was observed was achieved”; Sentence in lines 32-33 should be checked; Sentence in lines 133-34 should be rewritten.; Sentence in line 151 should be checked.; Line 213 “is”
Answer: Corrected following the reviewer’s suggestions.
- Missing space in ref. 16; Defective reference 18; Check the correctness of ref. 22
Answer: Corrected following the reviewer’s suggestions.
Reviewer 2 Report
Comments for the Authors
In this manuscript, the authors Enzymatic synthesis of aromatic aldehydes from aromatic primary alcohols, Also, enzyme dosage, pH, and temperature, were investigated.
1) Abstract and introduction are clear and easy to understand. Overall well-structured manuscript.
2) Sufficient information about the previous study findings is presented for readers to follow the present study and procedures.
3) It would be nice if the authors expand the substrate scope to secondary alcohols, as well as selective oxidation of racemic mixtures of alcohols in their future studies.
4) Enzymatic synthesis is environmentally friendly and reduce the generation of waste compared to conventional chemical synthesis.
I believe this manuscript is suitable for publication in Catalysts.
Author Response
We thank the reviewers for their efforts and their constructive criticism that helped us to improve the quality of this manuscript. We have revised the manuscript following their suggestions. A detailed answer is appended below.
Reviewer: 2
- In this manuscript, the authors Enzymatic synthesis of aromatic aldehydes from aromatic primary alcohols, Also, enzyme dosage, pH, and temperature, were investigated.
Answer: Thank you very much for sharing your enthusiasm about the reaction system. We sincerely appreciate the reviewer's suggestions and have done our best to revise the manuscript in your spirit.
- 1) Abstract and introduction are clear and easy to understand. Overall well-structured manuscript.
2) Sufficient information about the previous study findings is presented for readers to follow the present study and procedures.
Answer: Thank you very much for sharing your enthusiasm about the reaction system.
- 3) It would be nice if the authors expand the substrate scope to secondary alcohols, as well as selective oxidation of racemic mixtures of alcohols in their future studies.
Answer: Thanks to the reviewer for his/her comments. Reviewer comments are well worth considering! We will further explore more substrates and valuable products in future work.
- 4) Enzymatic synthesis is environmentally friendly and reduce the generation of waste compared to conventional chemical synthesis.
Answer: Thank you very much for sharing your enthusiasm about the reaction system. As you said, enzymatic synthesis is indeed environmentally friendly and reduce the generation of waste compared to conventional chemical synthesis. We will also continue to carry out relevant work in the future.
- I believe this manuscript is suitable for publication in Catalysts.
Answer: Thank you very much for sharing your enthusiasm about the reaction system.
Round 2
Reviewer 1 Report
The manuscript has been amended by Authors and it is in my opinion now suitable for publication. Only two minor typos have been detected in the present version.
In line 204 “…than using is in combination with…” should perhaps be “…than using it in combination with…”; in line 365 subscripts are missing (H2O2).
Author Response
Thank you very much for the careful check! We have implemented the suggested changes (and also corrected some other minor issues in the reference list such as missing italics etc).
