An O-Demethylation Metabolite of Rabeprazole Sulfide by Cunninghamella blakesleeana 3.970 Biotransformation
Round 1
Reviewer 1 Report
The o-demethylation reaction of rabeprazole sulfide was optimized in this work. English should be checked by a native speaker. The paper is designed in a rather confusing way, and practically until the end of the paper it is not clear what its purpose is. This should be explained in the introduction. The discussion is quite brief, and in general some claims, such as the ranges used in optimizing individual parameters, should be supported by citations. The purpose of optimizing this biotransformation is also a key question. In particular, the potential of possible further applications of the biotransformation studied is not mentioned in the paper.
1. Does O-demethylrabeprazole sulfide have a specific pharmacological potential or was the work intended to show that it is possible to perform demethylation reactions with these fungi of the family Cunninghamellaceae with the Cunnihamella gene?
2. Figure S-1 - There are two lines for each strain and an explanation should be added for each. The name of the metabolite should also be provided.
3. Figure S-2 - The world "conditions" should be deleted from the title of the figure.
4. Page 2, line 64 - Rabeprazole sulfide should be a substrate in the reaction, so the goal is to metabolize it as much as possible, i.e., keep its concentration as low as possible.
5. Figures 1-5 - How the yield was calculated?
6. Figures 1-5 - Since the purpose of these measurements is to optimize individual parameters, it is necessary to indicate in title of each Figure the values of the parameters that were not optimized in a given set.
7. Chapters 2.6 and 2.7 These chapters should be transferred to the supplement or to the experimental part.
8. Page 8, line 18, Chapter 3.2 - It is not clear if the substrate was added to the biotransformation media.
9. Chapter 3.3 - It is not clear whether the metabolites are intracellular or extracellular.
10. Chapter 3.3. The HPLC column should be defined.
11. Chapter 3.2. More details should be given about the elution, the stationary phase should be defined.
12. Conclusion and Fig. 8 should be included in the introduction section. Introduction part should be expanded with the importance of the o-demethylation reaction.
Author Response
Dear editor and reviewers,
Thank you very much for your email and reviewer's comments on our manuscript entitled " An O-demethylation metabolite of rabeprazole sulfide by Cunninghamella blakesleeana 3.970 biotransformation". These comments are very constructive and helpful to improve our manuscript. After carefully studied the reviewers' comments, we have made corresponding corrections with the track mark in the manuscript. Also, we’ve listed our answers to the reviewer's comment as below in blue italic font. Hope these efforts will make the manuscript acceptable for publication.
The responses to reviewer’s comments and the adaptive corrections are as follows:
Comments to Author(s) of Manuscript :
Manuscript Number: catalysts-2063454
Title: An O-demethylation metabolite of rabeprazole sulfide by Cunninghamella blakesleeana 3.970 biotransformation
The o-demethylation reaction of rabeprazole sulfide was optimized in this work. English should be checked by a native speaker. The paper is designed in a rather confusing way, and practically until the end of the paper it is not clear what its purpose is. This should be explained in the introduction. The discussion is quite brief, and in general some claims, such as the ranges used in optimizing individual parameters, should be supported by citations. The purpose of optimizing this biotransformation is also a key question. In particular, the potential of possible further applications of the biotransformation studied is not mentioned in the paper.
We would like to show our gratitude and appreciation to our reviewer. The whole manuscript had been modified according to our review’s suggestions. The revised manuscript had been polished by the MDPI language services. The questions are answered as the following.
- Does O-demethylrabeprazole sulfide have a specific pharmacological potential or was the work intended to show that it is possible to perform demethylation reactions with these fungi of the family Cunninghamellaceae with the Cunnihamella gene?
Response to comment #1:
There are few reports on the pharmacological study of O-demethylrabeprazol sulfide until recently. Our work intention is to explore the biotransformation metabolites spectrum of rabeprazole, and the results indicated an O-dimethyl product obtained by Cunninghamella blakesleeana 3.970, which belongs to the Cunninghamellaceae genus. So, this kind of O-demethyl reaction could be documented and if there is any similar O-demethyl reaction would be needed in other scenario. This strain could be used as a candidate biocatalyst.
- Figure S-1 - There are two lines for each strain and an explanation should be added for each. The name of the metabolite should also be provided.
Response to comment #2:
The name of the metabolites had been supplemented. We’ve moved this figure to the main manuscript as Figure 2 due to its importance.
- Figure S-2 - The word "conditions" should be deleted from the title of the figure.
Response to comment #3:
The word “conditions” had been deleted.
- Page 2, line 64 - Rabeprazole sulfide should be a substrate in the reaction, so the goal is to metabolize it as much as possible, i.e., keep its concentration as low as possible.
Response to comment #4:
Your comments are very helpful to us. We’ll try this in the future experiments.
- Figures 1-5 - How the yield was calculated?
Response to comment #5:
The yield which should be modified as conversion rate is calculated as the following equation.
- Figures 1-5 - Since the purpose of these measurements is to optimize individual parameters, it is necessary to indicate in title of each Figure the values of the parameters that were not optimized in a given set.
Response to comment #6:
We’ve made the modifications in the revised manuscript.
- Chapters 2.6 and 2.7 These chapters should be transferred to the supplement or to the experimental part.
Response to comment #7:
Thank for your instruction, these two chapters had been moved to the supplement information.
- Page 8, line 18, Chapter 3.2 - It is not clear if the substrate was added to the biotransformation media.
Response to comment #8:
We’ve modified this sentence, and indicated that the substrate was added into the biotransformation media.
- Chapter 3.3 - It is not clear whether the metabolites are intracellular or extracellular.
Response to comment #9:
In this study, our primary focus is to explore the biotransformation metabolites spectrum. So, we did not pay major attention to whether the metabolites are intracellular or extracellular.
To avoid the metabolites are intracellular, when the extractions were performed, the transformation culture s were collected, ultrasonic treated for 30min, and centrifugated 3000rpm for 10 min to retrieve the supernatant. Pellets were washed and extracted three times using ethyl acetate. The combined water phase and organic phase were extracted three times with equal volume of ethyl acetate. The organic phase was then isolated, evaporated by reduced pressure distillation to obtain the raw metabolites. The raw metabolites were dissolved in the methanol for further use.
- Chapter 3.3. The HPLC column should be defined.
Response to comment #10:
The HPLC column is the Shimadzu WondaSil C18 Superb (250×4.6mm×5μm) column, which had been supplemented into the 3.3 chapter in the revised manuscript.
- Chapter 3.2. More details should be given about the elution, the stationary phase should be defined.
Response to comment #11:
This information had been supplemented into the 3.2 chapter in the revised manuscript.
- Conclusion and Fig. 8 should be included in the introduction section. Introduction part should be expanded with the importance of the o-demethylation reaction.
Response to comment #12:
We’ve moved this figure to be Figure 1 in the revised manuscript. Several literatures and the relative contents of importance of O-demethylation had been supplemented into the introduction part in the revised manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments to Author(s) of Manuscript :
Manuscript Number: catalysts-2063454
Title: An O-demethylation metabolite of rabeprazole sulfide by Cunninghamella blakesleeana 3.970 biotransformation
This manuscript describes finding and optimizing cell biocatalysts for O-demethylation of rabeprazole sulfide. The authors i) screened by conducting cell reactions using several strains using rabeprazole sulfide as a substrate, which was converted to unknown compounds. After deciding to study the unknown compounds from Cunninghamella blakesleeana 3.970, the authors further ii) optimized media or culture conditions to increase the conversion of rabeparazole sulfide. Finally, iii) the authors identified the unknown compound as o-demethylated metabolite of rabeprazole sulfide
The authors attempt to find a novel cell biocatalyst to convert rabeprazole sulfide to new derivatives. However, it is hard to say the manuscript will interest the readers of “catalysts” since the findings are minor compared to other publications on “catalysts” and there are lots of improvement to be made. The manuscript was difficult to follow and incomplete. Therefore, the reviewer suggests the editors to the manuscript.
1. H-NMR/ C-NMR results indicated in supplementary data and the numbers written in lines 155-159 do not match. Additionally, H-NMR does not show any splitting patterns, which is extremely suspicious. This data should be clarified to claim the novel finding of cell reactions.
2. The authors are using the word “yield” but they are showing “conversion.” Yield is defined as amount of the desired products per substrate in mol units. If the authors were referring to “yield” as the amount of decreased substrate, they should change all the words to “conversion.” If the authors claim that the word yield is correct, the flow of the manuscript should be changed in the order of identification of substrate and then optimization of the growing-cell reaction.
3. Introduction should be improved to explain previous reports related to the results rather than broad comments about rabeprazole sulfide. What is the importance of studying demethylation of the compound? Are there any other reports regarding demethylation using cells using the compound or similar compounds?
4. For all the graphs, the y-axis should be identical.
5. For all the graphs, the titles should be removed. They are already in the captions.
6. The reviewer is uncertain why the orthogonal optimization was performed in the first place before testing several factors individually. The orthogonal optimization would reveal both significancy and optimal value of the factors. The section 2.1-2.3 is unnecessary if the orthogonal optimization would be introduced in this manuscript. Additionally, the optimization result does not seem to represent the result in section 2.1-2.3 where high substrate concentration seems to be very toxic to the reaction.
7. Additionally, the authors should fully explain how the orthogonal optimization was designed and analyzed in the method session. Otherwise, readers that are not familiar with this field will be unable to understand or reproduce the experiments.
8. In the orthogonal optimization, the initial concentration of the substrate was included as one of the factors, and the authors aim for the highest yield. It would be better to compare the final concentration of products rather than yield since the goal is to get the most products from the substrate.
9. Supplementary figure1 is the most important data since this is the start of seeking a novel compound and reaction from a scratch. Please move the figure to the main manuscript.
10. HPLC chromatography does not prove the purity of the fractionated compound. There are way more chemicals not detected through HPLC.
11. Conclusion and discussion are not relevant to what is shown in this manuscript. There are a number of reasons why oxidation did not work in this current system. For example, substrate specificity may not have matched. reason why reaction did not happen
Author Response
Dear editor and reviewers,
Thank you very much for your email and reviewer's comments on our manuscript entitled " An O-demethylation metabolite of rabeprazole sulfide by Cunninghamella blakesleeana 3.970 biotransformation". These comments are very constructive and helpful to improve our manuscript. After carefully studied the reviewers' comments, we have made corresponding corrections with the track mark in the manuscript. Also, we’ve listed our answers to the reviewer's comment as below in blue italic font. Hope these efforts will make the manuscript acceptable for publication.
The responses to reviewer’s comments and the adaptive corrections are as follows:
Comments to Author(s) of Manuscript :
Manuscript Number: catalysts-2063454
Title: An O-demethylation metabolite of rabeprazole sulfide by Cunninghamella blakesleeana 3.970 biotransformation
This manuscript describes finding and optimizing cell biocatalysts for O-demethylation of rabeprazole sulfide. The authors i) screened by conducting cell reactions using several strains using rabeprazole sulfide as a substrate, which was converted to unknown compounds. After deciding to study the unknown compounds from Cunninghamella blakesleeana 3.970, the authors further ii) optimized media or culture conditions to increase the conversion of rabeparazole sulfide. Finally, iii) the authors identified the unknown compound as o-demethylated metabolite of rabeprazole sulfide
The authors attempt to find a novel cell biocatalyst to convert rabeprazole sulfide to new derivatives. However, it is hard to say the manuscript will interest the readers of “catalysts” since the findings are minor compared to other publications on “catalysts” and there are lots of improvement to be made. The manuscript was difficult to follow and incomplete. Therefore, the reviewer suggests the editors to the manuscript.
We would like to show our gratitude and appreciation to our reviewer. The whole manuscript had been modified according to our review’s suggestions. The revised manuscript had been polished by the MDPI language services. Some specific questions are answered as the following.
- H-NMR/ C-NMR results indicated in supplementary data and the numbers written in lines 155-159 do not match. Additionally, H-NMR does not show any splitting patterns, which is extremely suspicious. This data should be clarified to claim the novel finding of cell reactions.
Response to comment #1:
Thanks for your instruction, we’ve restudied the original data, and modified the NMR description in the main manuscript, and updated the NMR spectrums in the supplementary information.
- The authors are using the word “yield” but they are showing “conversion.” Yield is defined as amount of the desired products per substrate in mol units. If the authors were referring to “yield” as the amount of decreased substrate, they should change all the words to “conversion.” If the authors claim that the word yield is correct, the flow of the manuscript should be changed in the order of identification of substrate and then optimization of the growing-cell reaction.
Response to comment #2:
Thank you for your instruction, we’ve changed all the yield into conversion rate.
- Introduction should be improved to explain previous reports related to the results rather than broad comments about rabeprazole sulfide. What is the importance of studying demethylation of the compound? Are there any other reports regarding demethylation using cells using the compound or similar compounds?
Response to comment #3:
The introduction section had been modified, in which several literatures involving the demethylation reactions are cited, and the importance of demethylation is described.
- For all the graphs, the y-axis should be identical.
Response to comment #4:
All the graphs had been modified with the identical y-axis.
- For all the graphs, the titles should be removed. They are already in the captions.
Response to comment #5:
All the titles in the graphs had been deleted.
- The reviewer is uncertain why the orthogonal optimization was performed in the first place before testing several factors individually. The orthogonal optimization would reveal both significancy and optimal value of the factors. The section 2.1-2.3 is unnecessary if the orthogonal optimization would be introduced in this manuscript. Additionally, the optimization result does not seem to represent the result in section 2.1-2.3 where high substrate concentration seems to be very toxic to the reaction.
Response to comment #6:
Thanks for the instruction. There are lots of factors which may contribute to the biotransformation results. In this experiment, we firstly performed the screen for the four different culture media. Because the culture media is the prerequisite for growth state of the strains. So, it should be determined at the first place. Then we could perform the orthogonal optimization for other factors, which may affect the biotransformation results.
It is true that high substrate concentration seems to be toxic to the strains. This is usual because the majority substrates used for biotransformation are not the natural metabolic substances for the strains. Even if we would like to anticipate that a high substrate loading may generate high concentration of metabolite, it could not be achieved for most of the time.
- Additionally, the authors should fully explain how the orthogonal optimization was designed and analyzed in the method session. Otherwise, readers that are not familiar with this field will be unable to understand or reproduce the experiments.
Response to comment #7:
In the experiment, there are multiple factors which may contribute to the biotransformation results. Among these factors, some may play essential roles and some may no. In this experiment, crucial factors could be attributed to the culture time, media starting pH, culture volume, inoculation volume and the substrate concentration. The aim of orthogonal optimization is a method for studying multi-factor and multi-level experiments. It involves the selection of a few representative points from a comprehensive test according to orthogonality. This method ensures good experiment results and reduces the test times. Therefore, in this experiments, orthogonal optimization was designed to analyze the influence of the factors just mentioned above, to finally get a better biotransformation results.
- In the orthogonal optimization, the initial concentration of the substrate was included as one of the factors, and the authors aim for the highest yield. It would be better to compare the final concentration of products rather than yield since the goal is to get the most products from the substrate.
Response to comment #8:
Thank for your instruction, we would like to pay more attention on the final concentration of product in the future experiment.
- Supplementary figure1 is the most important data since this is the start of seeking a novel compound and reaction from a scratch. Please move the figure to the main manuscript.
Response to comment #9:
This figure had been moved into the main manuscript as Figure 1.
- HPLC chromatography does not prove the purity of the fractionated compound. There are way more chemicals not detected through HPLC.
Response to comment #10:
Thanks for your instruction. It is true that the HPLC chromatography could not prove the purity of the fractionated compound. What we performed is to exhibit a major metabolite is detected by HPLC, and it could be identified by further MS and NMR.
- Conclusion and discussion are not relevant to what is shown in this manuscript. There are a number of reasons why oxidation did not work in this current system. For example, substrate specificity may not have matched. reason why reaction did not happen
Response to comment #11:
The conclusion and discussion part had been supplemented and modified to focus on the demethylation reaction, not the oxidation.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The authors have responded to the comments very well but a few things remain not fixed.
1. The authors are still using "yield" rather than "conversion" in the manuscript. This should be corrected.
2. The authors should annotate an initial substrate concentration when talking about yield. Please correct the abstract by adding [S]0.
Author Response
Dear editor and reviewer,
Thanks again for your careful reviewing of our manuscript entitled " An O-demethylation metabolite of rabeprazole sulfide by Cunninghamella blakesleeana 3.970 biotransformation". We’ve made the modifications as our reviewer suggested.
The authors have responded to the comments very well but a few things remain not fixed.
- The authors are still using "yield" rather than "conversion" in the manuscript. This should be corrected.
Response to comment #1:
Thanks for your instruction, it is our carelessness. All the word “yield” in the manuscripts had been corrected into the word “conversion’. These corrections as listed in the revised manuscripts in line 124, 132, 164, 180, 181, 263, and in Table 2.
- The authors should annotate an initial substrate concentration when talking about yield. Please correct the abstract by adding [S]0.
Response to comment #2:
Thanks for your instruction, we’ve supplemented a sentence in the abstract in line 20, i.e. “when the substrate concentration is 3g/L”.
Author Response File: 
Author Response.pdf
