Study on Optimal Production Conditions of Fibrinolytic Kinase Derived from the Nereid Worm, Perinereis aibuhitensis Grub
, Jun Cheng 2,†, Xiaozhen Diao 1, Yang Man 1, Boyu Chen 1, Haixing Zhang 1, Jeevithan Elango 1,3 and Wenhui Wu 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsCOMMENTS TO THE AUTHORS
I have read carefully the paper entitled “Study on optimal production conditions of fibrinolytic kinase of the nereid worm, Perinereis aibuhitensis Grub, in E. coli.” This is an apparently interesting and comprehensive study since it covers the actual topic. The experimental design is well established and the results adequately commented.
However, there are some issues through the text that need revision. My suggestion is that the paper should be accepted with major revisions to make them more readable and easy to follow.
GENERAL COMMENTS
- Firstly, the paper is not written according to the Instructions for Manuscript preparation. Authors should read the Instructions carefully.
- Authors should pay attention on: general format of manuscript (order of chapters: Introduction, Material and Methods, Results, Discussion). Also, citing references in reference list is not adequate as required in the Instructions (for example, Journal titles should be in italics and abbreviations should be used; between the two authors' names authors should put “;” instead of “,”, etc.). Authors should check all references and make corrections.
- The name of the bacteria E. coli should be in italics (check in all text)
- Authors should avoid writing in the first person (us, we); they should use the passive instead.
- The order of listing the tables is not traceable in the paper. The authors arranged the tables in the following order: Table 6, 2, 3, 4, 8 5, 1, 7. As a result, the text of the work is difficult to follow. Authors should check throughout the text whether the references to the tables in the text are appropriate, and also adjust the order of the tables to the text.
SPECIFIC COMMENTS:
- Subsection 3.3.3. Was a commercial enzyme assay used? If so, state which one the manufacturer is, and in that case the protocol does not need to be listed in the table.
- Discussion section:
In the discussion you explained in detail and connected your results and drew adequate conclusions. However, you need to expand the discussion with the results of other authors who dealt with similar problems, and comment on and relate them to your results.
Comments for author File: 
Comments.pdf
Author Response
GENERAL COMMENTS
- Firstly, the paper is not written according to the Instructions for Manuscript preparation. Authors should read the Instructions carefully.
- Authors should pay attention on: general format of manuscript (order of chapters: Introduction, Material and Methods, Results, Discussion). Also, citing references in reference list is not adequate as required in the Instructions (for example, Journal titles should be in italics and abbreviations should be used; between the two authors' names authors should put “;” instead of “,”, etc.). Authors should check all references and make corrections.
- The name of the bacteria E. coli should be in italics (check in all text)
- Authors should avoid writing in the first person (us, we); they should use the passive instead.
- The order of listing the tables is not traceable in the paper. The authors arranged the tables in the following order: Table 6, 2, 3, 4, 8 5, 1, 7. As a result, the text of the work is difficult to follow. Authors should check throughout the text whether the references to the tables in the text are appropriate, and also adjust the order of the tables to the text.
Response: Many thanks for your great effort to improvise our manuscript standard, we critically consider all your comments and revised our manuscript accordingly. All the above issues are revised now.
SPECIFIC COMMENTS:
- Subsection 3.3.3. Was a commercial enzyme assay used? If so, state which one the manufacturer is, and in that case the protocol does not need to be listed in the table.
Response: Based on the reviewer’s advice, we have provided the informations of commercial enzyme in 2.1. Raw materials and reagents.
- Discussion section:
In the discussion you explained in detail and connected your results and drew adequate conclusions. However, you need to expand the discussion with the results of other authors who dealt with similar problems, and comment on and relate them to your results.
Response: Yes, more information is added in the discussion section.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript fermentation-3169496 is devoted to the optimization of conditions for the biosynthesis of fibrinolytic kinase using genetically modified E. coli. The work is done on a very relevant topic. The merit of the work is a detailed methodological presentation of the material, the reliability of the results is beyond doubt.
Disadvantages of the work:
1) It is recommended to change the title, since a ready-made genetically engineered strain of E. coli was used in the work.
2) The last sentence in the Abstract needs to be corrected. The reliability of the model is reflected not by the graph, but by the convergence of the model and experimental data.
3) It is necessary to expand the discussion and compare the obtained results with other E. coli strains, into which fibrinolytic kinase genes from other sources were introduced.
4) The composition of the fermentation medium and the seed medium are very different, it is necessary to explain why. Provide links to where the compositions of the media were taken from?
5) If the composition of the medium has such a strong effect on the result, then why was the composition of the medium not optimized?
6) According to Figure 1 b, 1% inoculum is equivalent to 3% and 5% inoculum. Obviously, the authors added an increased dosage of inoculum for insurance, and not because it is the optimum.
7) According to the data in Section 2.6 and Figure 6 b, the maximum is observed at 20 hours of induction, but the authors choose 16 hours. Why? Later, in a three-factor experiment, the authors still obtained the optimum at 20 hours.
8) The numbering of tables in the work is chaotic and references to tables do not always correspond to reality (for example, Table 3 and Table 4).
Author Response
Disadvantages of the work:
1) It is recommended to change the title, since a ready-made genetically engineered strain of E. coli was used in the work.
Response: We thank the reviewer 2 for their extensive revision on our manuscript. We have revised the manuscript based on reviewer’s comment. The title has been revised.
2) The last sentence in the Abstract needs to be corrected. The reliability of the model is reflected not by the graph, but by the convergence of the model and experimental data.
Response: Many thanks. The last sentence in the Abstract has been corrected.
3) It is necessary to expand the discussion and compare the obtained results with other E. coli strains, into which fibrinolytic kinase genes from other sources were introduced.
Response: The discussion section is expanded as advised by the reviewer.
4) The composition of the fermentation medium and the seed medium are very different, it is necessary to explain why. Provide links to where the compositions of the media were taken from?
Response: According to literature reports, LB medium and TB medium have different effects on different host cells, one can increase protein expression, and the other can increase cell proliferation rate. In order to comprehensively consider the influence of different media on protein expression and cell proliferation, two media were used in the single-factor experiment, and the final result mainly referred to protein expression.
5) If the composition of the medium has such a strong effect on the result, then why was the composition of the medium not optimized?
Response: The expression results of the two media were so different that at that time, when the experiments were designed, we felt that it was not necessary to carry out the interaction experiments of the two media at the same time. In addition, the cost of the culture medium was one of the factors considered.
6) According to Figure 1 b, 1% inoculum is equivalent to 3% and 5% inoculum. Obviously, the authors added an increased dosage of inoculum for insurance, and not because it is the optimum.
Response: As can be seen from the results in Table 6, the gray value of 5% inoculum is indeed higher than 1% and 3%, so 5% inoculum is selected.
7) According to the data in Section 2.6 and Figure 6 b, the maximum is observed at 20 hours of induction, but the authors choose 16 hours. Why? Later, in a three-factor experiment, the authors still obtained the optimum at 20 hours.
Response: From the one-factor data in Table 6, it can be seen that the protein expression was higher at 16h than 20h although the dry weight of the cells was greater at 20h than at 16h. The final optimal fermentation time was still predicted by the results of the interaction (response surface experiment) and the results of the response surface indicated that 20h was optimal. This contradictory concept is possible that the response surface predictions may not agree with the results of the single factor. The final result is based on the results of the response surface.
8) The numbering of tables in the work is chaotic and references to tables do not always correspond to reality (for example, Table 3 and Table 4).
Response: The order of the table has been corrected.
Reviewer 3 Report
Comments and Suggestions for AuthorsAuthors evaluated the effect of inoculum, pH, OD, Temperature, Inducer concentration, and Time with different levels by a Plackett-Brugman design on protein production of a firbinolytic kinase (E coli), then the most significant factors and the levels for higher production were used for surface respond optimization. However the way the author present the results is very confusing, Figures have a sequence and clearly show by image analysis the level of protein produced, but tables start with table 6, followed by table 2, some times is difficult to understand if they present statistical results or the experimental design, thus I can not continue with the discussion and conclusion
Author Response
Authors evaluated the effect of inoculum, pH, OD, Temperature, Inducer concentration, and Time with different levels by a Plackett-Brugman design on protein production of a firbinolytic kinase (E coli), then the most significant factors and the levels for higher production were used for surface respond optimization. However the way the author present the results is very confusing, Figures have a sequence and clearly show by image analysis the level of protein produced, but tables start with table 6, followed by table 2, some times is difficult to understand if they present statistical results or the experimental design, thus I can not continue with the discussion and conclusion
Response: Accept our apologies for incorrect format. These issues have now been fixed, Wish the reviewer can consider for further process.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Editor, dear authors,
I read the responses to my comments from the original review. The authors responded carefully and made all the necessary changes to the text, therefore I suggest that the paper be accepted in its current form.
Reviewer 3 Report
Comments and Suggestions for AuthorsAuthors attended the suggestions, it has the quality to be published in Fermentation
