Study on the Synthesis of Theaflavin-3,3′-Digallate Catalyzed by Escherichia coli Expressing Tea Tree Polyphenol Oxidase Isozymes and Its Enzymatic Solution
Round 1
Reviewer 1 Report
Comments for the paper titled “Study on the synthesis of theaflavin-3,3'-digallate catalyzed by Escherichia coli expressing tea tree polyphenol oxidase iso-3 zymes and its enzymatic solution”.
The manuscript is clearly written, well-structured and of high importance to the field of food technology, it providing a new method to synthesize TFs by reconstitution polyphenol oxidase. It describes the difference for recombinant enzyme proteins of tea tree polyphenol oxidase through the Escherichia coli expression system. Below you can find some remarks that could be considered by Authors.
Line 40, TFs also can be found the content increase during withering, so not always after cellular fragmentation….
Line 50-51, this sentence should be corrected.
Line 62, please correct the molecular structure of theaflavins (TFs), the ring should be a regular hexagon.
Line 66-69, you need look for a published paper to support your appoint.
Line 131, is EGCG a excrescent word?
Line 200, please correct writing the author name of the cited work.
Line 281, the difference for the sequence and structure that may induce the difference in catalytic conditions, so when you compare PPO activities their best catalytic conditions should be known.
Line 463-467, please correct writing author’s name, specific in capital letter.
Line 479, please keep same format in all references, author’s name, journal name in full name or abbreviation, capital and lower-case letter.
This work is novelty, and writing smooth.
Author Response
The manuscript is clearly written, well-structured and of high importance to the field of food technology, it providing a new method to synthesize TFs by reconstitution polyphenol oxidase. It describes the difference for recombinant enzyme proteins of tea tree polyphenol oxidase through the Escherichia coli expression system. Below you can find some remarks that could be considered by Authors.
Line 40, TFs also can be found the content increase during withering, so not always after cellular fragmentation….
Answer: Thank you for your review. We have made the necessary modifications. Theaflavins (TFs) are a class of compounds with benzophenone structure formed by oxidative polymerization of cats and their derivatives under the catalyst of tea Polyphenol oxidation (PPO) during the processing of black tea
Line 50-51, this sentence should be corrected.
Answer: Thank you for your review. We have made the necessary modifications. Thereforee, Theaflavins have broad application prospects
Line 62, please correct the molecular structure of theaflavins (TFs), the ring should be a regular hexagon.
Answer: Thank you for your review. We have reviewed literature and books, and found that theaflavin rings belong to the seven circular rings formed by the polymerization of catechin monomers. So I don't quite understand what you mean. In addition, there are too many images in this manuscript, so we have deleted this image.
Line 66-69, you need look for a published paper to support your appoint.
Answer: Thank you for your review. We have added it.
Line 131, is EGCG a excrescent word?
Answer: Thank you for your review and we apologize for our mistake. EGCG is redundant and we have deleted it.
Line 200, please correct writing the author name of the cited work.
Answer: Thank you for your review and we apologize for our mistake. We have made modifications.
Line 281, the difference for the sequence and structure that may induce the difference in catalytic conditions, so when you compare PPO activities their best catalytic conditions should be known.
Answer: Thank you for your review. We have made the necessary modifications.
Line 463-467, please correct writing author’s name, specific in capital letter.
Answer: Thank you for your review and we apologize for our mistake. We have made modifications.
Line 479, please keep same format in all references, author’s name, journal name in full name or abbreviation, capital and lower-case letter.
Answer: Thank you for your review and we apologize for our mistake. We have made modifications.
Reviewer 2 Report
The work is a very interesting one, but I find some descriptions of the results are superficial and some conclusions are not supported. There is an English problem and a format problem. Scientific names are not wri tten in italics, the references cited in the text do not correspond to the list at the end.
The main area of improvement is a more detailed description of the results presented both in figures and tables. For example, you can state the molecular mass of the expressed proteins on figure 3. In some bands a double band can be seen, and different patterns of intensity are found, do you have any explanation? Did you recover the inclusion bodies with a specific protocol? It may not be the only explanation for the lack of solubility, as the truncated fragment expression is still not good.
The protein models can be better described and a more detailed catalytic mechanism for each enzyme could be proposed. The sentence used in the abstract to describe it (l. 29) could be further explored in the text
There is an English problem and a format problem. Scientific names are not written in italics, not all the references cited in the text correspond to the list at the end. The references have different formats.
After the results heading, the sentence in the model text was left (l. 272)
Some terms are incorrect. Cells are lysed or disrupted, not broken, for example. PCR equipment, not instrument (l. 140).
l. 63-64 the sentence is incomplete. I suppose they mean "as theaflavins constitute only 0.5-2% of black tea dry weight, their structures are difficult to isolate and characterize"
l.92-94 Sentence is too long... remove "it"
l 306 failed to achieve expression
Author Response
The work is a very interesting one, but I find some descriptions of the results are superficial and some conclusions are not supported. There is an English problem and a format problem. Scientific names are not wri tten in italics, the references cited in the text do not correspond to the list at the end.
Answer: Thank you for your review and we apologize for our mistake. We have made modifications.
The main area of improvement is a more detailed description of the results presented both in figures and tables. For example, you can state the molecular mass of the expressed proteins on figure 3. In some bands a double band can be seen, and different patterns of intensity are found, do you have any explanation? Did you recover the inclusion bodies with a specific protocol? It may not be the only explanation for the lack of solubility, as the truncated fragment expression is still not good.
Answer: Thank you for your review. We have made the necessary modifications. We have added a description of the results. In some bands, dual bands can be seen, and different intensity patterns have been found. This may be due to the expression of multiple proteins, but our target protein peak is much higher than other proteins in terms of purity, so it can be ignored. CsPPO2-N45 TrxA had the largest compatible gene fragment (2013 bp) with a protein size of 71.76KD
We have not yet performed inclusion body renaturation, as this method is complex and does not significantly improve protein activity after inclusion body recovery, making it difficult to apply in production. Therefore, we are considering inclusion body renaturation.
The protein models can be better described and a more detailed catalytic mechanism for each enzyme could be proposed. The sentence used in the abstract to describe it (l. 29) could be further explored in the text
Answer: Thank you for your review. We have made the necessary modifications. The amino acid residues of the active centers of Csppo1, CsPPO2, and CsPPO3 are all histidine, and all three exhibit different configurations. Further analysis, as shown in Figure 6, the active groups of CsPPO1 are located at positions 171, 192, 201, 357, 323, 356, and 327 of histidine in the protein sequence. The active groups of CsPPO2 are located at positions 183, 204, 213, 370, 369, and 340 of histidine in the protein sequence. The active groups of CsPPO3 are located at positions 181, 202, 211, 337, 333, and 366 of histidine in the protein sequence. The different positions of amino acids lead to differences in the spatial structure of enzyme activity centers, resulting in differences in enzyme catalytic activity towards substrates.
Comments on the Quality of English Language
There is an English problem and a format problem. Scientific names are not written in italics, not all the references cited in the text correspond to the list at the end. The references have different formats.
Answer: Thank you for your review and we apologize for our mistake. We have made modifications. We have polished the manuscript with language.
After the results heading, the sentence in the model text was left (l. 272)
Answer: Thank you for your review and we apologize for our mistake. We have deleted it.
Some terms are incorrect. Cells are lysed or disrupted, not broken, for example. PCR equipment, not instrument (l. 140).
Answer: Thank you for your review and we apologize for our mistake. We have made modifications.
- 63-64 the sentence is incomplete. I suppose they mean "as theaflavins constitute only 0.5-2% of black tea dry weight, their structures are difficult to isolate and characterize"
l.92-94 Sentence is too long... remove "it"
Answer: Thank you for your review and we apologize for our mistake. We have made modifications.
l 306 failed to achieve expression
Answer: Thank you for your review and we apologize for our mistake. We have made modifications. The TFs content in black tea is relatively low, accounting for only 0.5-2% of the dry weight. Consequently, directly isolating TFs monomers from black tea is chal-lenging, inefficient, and costly.
