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Article
Peer-Review Record

Recombinant C-Terminal Catalytic Domain of Rat L-Gulono Lactone Oxidase Produced in Bacterial Cells Is Enzymatically Active

Curr. Issues Mol. Biol. 2024, 46(8), 8958-8968; https://doi.org/10.3390/cimb46080529
by Abdul Aziz M. Gad 1,2,*, Anna Gora-Sochacka 1 and Agnieszka Sirko 1
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Curr. Issues Mol. Biol. 2024, 46(8), 8958-8968; https://doi.org/10.3390/cimb46080529
Submission received: 20 June 2024 / Revised: 5 August 2024 / Accepted: 13 August 2024 / Published: 16 August 2024

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript: Recombinant C-terminal Catalytic Domain of Rat L-gulono Lactone Oxidase Produced in Bacterial Cells is Enzymatically Active

The manuscript by Gad et al. demonstrated the expression and characterization of rat GULO in E. coli for its biological activity. Overall, the manuscript is interesting and requires revision as follows:

Comments

1. Introduction, the commercial production of L-ascorbic acids should be stated.

2. Subsections 2.1-2.3 can be minimized as many procedures are well-known for expression, purification and SDS pages analysis details, i.e., https://doi.org/10.3390/ijms25052746.

3. The catalytic mechanism or reaction details can be provided.

4. Please provided the kinetics parameters of purified enzyme. Also, characterize enzyme for optimum pH, temeratures and stability data.

5. Discussion is weak, it can be minor polished with recent updates.

Author Response

Comment1: Introduction, the commercial production of L-ascorbic acids should be stated.
Response: I fully agree with you, and this part was already added to the introduction section  (line 27 to 38 in page No. 1) which highlighted in yellow in the attached manuscript.
 Comment 2: subsections 2.1-2.3 can be minimized as many procedures are well-known for expression, purification and SDS pages analysis details, i.e., https://doi.org/10.3390/ijms25052746.
Response: thank you for these notes and I already reduced them (lines 146-169 in page No. 4) and highlighted in yellow in the attached manuscript.
Comment 3: the catalytic mechanism or reaction details can be provided.
Response: I wrote the mechanism in details in page 2 (line 45-49) and highlighted in yellow in the attached manuscript.
Comment 4: Please provided the kinetics parameters of purified enzyme. Also, characterize enzyme for optimum pH, temperatures and stability data.
Response: I totally agree with you, I carried out all these experiments (page 4, line: 177-191/ page 7,8, line: 309-356) and highlighted in yellow in the attached manuscript.
Comment 5: discussion is weak, it can be minor polished with recent updates.
Response: It was done through all manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, rat L-gulonolactone oxidase enzyme was expressed in E. coli and analyzed. The function of C-terminal catalytic domain was explored. However, the introduction and discussion sections were insufficient. The manuscript could be accepted for be published If the authors considered the suggestion below.

 

1. Detailed introduction of why rat GULO was chosen should be provided. More introduction about advantages of bacterial expression system should be included in the manuscript. The reason why bacterial expression system (E. coli) was chosen must be provided.

 

2. The protein size and activity of fGULO and cGULO should be compared with the ones expressed in plants (tobacco, lettuce, potato and Arabidopsis). Additional discussion about these differences would help the elongation of the conclusion. 

Author Response

Comment 1: Detailed introduction of why rat GULO was chosen should be provided. More introduction about advantages of bacterial expression system should be included in the manuscript. The reason why bacterial expression system (E. coli) was chosen must be provided.

Response: thank you for pointing this out, I totally agree with you. This part was provided and highlighted in yellow in the attached manuscript (page 1, line41-44 and page 2, line58-63).

Comment 2: the protein size and activity of fGULO and cGULO should be compared with the ones expressed in plants (tobacco, lettuce, potato and Arabidopsis). Additional discussion about these differences would help the elongation of the conclusion. 

Response: thanks a lot for this observation. The additional data were added and discussed in page 5, line: 231-237 and page 9, line: 371-379. All changes are highlighted in yellow in the attached manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

L-gulonolactone oxidase (GULO) is crucial for the final step of L-ascorbic acid (vitamin C) synthesis. While many species can synthesize vitamin C, humans, guinea pigs, bats, and other primates cannot due to the loss of functional GULO genes, now present as pseudogenes with numerous mutations. This study explored producing full-length and truncated C-terminal domain GULO in E. coli, with both forms purified via a C-terminal His-tag and demonstrated for biological activity.

 

Comments:

Formatting mistake in Line 46, cOmplete.

 

Why text in Line 129 is a different font?

 

Figure 3 data is not clear and why it is not in supplementary figures.

Comments on the Quality of English Language

Formatting of paper is not even

Author Response

Comment 1: formatting mistake in Line 46, cOmplete.

Response: I agree with you and I already corrected it in page 2, line 74, which is highlighted in yellow in the attached manuscript.

Comment 2: why text in Line 129 is a different font?

Response: thank you for this observation, it is a mistake during writing and I also corrected it in page 4, line 158-159, which is highlighted in yellow in the attached manuscript.

Comment 3: figure 3 data is not clear and why it is not in supplementary figures.

Response: because I run my sample in the same gel with another samples from the lab . The desired samples have been sliced and presented as in figures 1 and 2.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept

Comments on the Quality of English Language

fine

Author Response

Comment: Accept and English language is fine

Response: Thank you so much for your effort and valuable comments to improve this manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Could be considered to be punblished.

Author Response

Comment: Could be considered to be published

Response: Thank you so much for your precise reviewing and valuable advises to improve the quality of this manuscript.

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