Comparative Analysis of Universal Protein Extraction Methodologies for Screening of Lipase Activity from Agricultural Products

Round 1

Reviewer 1 Report

The authors in their original research article entitled: "Comparative analysis of universal protein extraction methodologies for screening of lipase activity from agricultural products" presented very interesting results regarding the use of 4 different protein extraction methods and evaluation of their lipase activities. Although this article presents an interesting approach to obtaining enzymes, the article needs to be improved, and below I present my comments and suggestions.

Suggestions:

• There is no information about replicates of the presented results. Moreover, the statistical analysis was omitted in this study, which should be definitely improved. In such a type of article, the statistical analysis of obtained results is an inherent part of the paper.
• The article lacks in-depth discussion.
• Supplementary Data S3, S4 & S5 – In my opinion, it is not necessary to provide the standard curves in the paper, but if you decided to do that you should also add the function formula and r² coefficient.

Minor suggestions:

• Lines 19, 24, 25, 28 - unnecessary hyphens
• Line 33 - Lipase is just one enzyme, lipases may be a group of enzymes.
• Line 88 – “9th revision of Korean National Standard Food Composition Table (Republic of Korea)” – it should be added to the Reference List
• Table 1, 2, and 3 – The “Source” should be changed to the names of used plants.
• Supplementary Data S1 & S2 – in my humble opinion Latin names of the used plants should be provided.

Author Response

We appreciate your review. Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper deals with the results by some extraction methods such as ammonium sulfate precipitation, acetone precipitation and two commercially available protein extraction kit.  The authors employed specific enzyme extraction case, and the results were not generalized: fully discussion by the obtained data was not shown, so that it would be unknown that the best results can be obtained to apply the other protein case.  Additionally, the separation methods would be common methods: the authors should show the novelty points clearly.  The contents would be involved in the purification and separation analysis area, so that the paper should be submitted to the analytical chemistry journals.  So the paper should be rejected.

Author Response

We appreciate your review. Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear authors,

I congratulate you on the nice picture illustrating the extraction methodologies of proteins with the scope of identifying the lipase activity of some particular products from agriculture. I believe that your work can be accepted for publishing in the Catalysts journal after you include in the manuscript some discussions where you should discuss/compare in detail your results and the methods used for your protocols with the literature reports. Otherwise, the paper is well written and provides sufficient data to sustain the results and the protocols involved.

Author Response

We appreciate your review. Please see the attachment. 

Author Response File: Author Response.pdf

Reviewer 4 Report

The authors analysed four methods for evaluating the lipase activity presents in the crude extracts of several raw materials. The methods are wide knew and contributing with low novelty in this field. For improving this work the authors could consider the below comments.

1.- The authors must include in the manuscript figures where summarize the data obtained.

2.- The authors must explain with detail the analytical methods and the equipment used.

3.- The authors must indicate how many replicas were made of each sample with the corresponding deviation.

Author Response

We appreciate your review. Please see the attachment. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors referred to the comments and revised the manuscript as suggested by me. I still feel that the discussion in this article should be more extensive.

Author Response

Point 1: The authors referred to the comments and revised the manuscript as suggested by me. I still feel that the discussion in this article should be more extensive.

: We appreciate the reviewer’s comment and considered it with meticulous care. As the reviewer commented, we strengthen our discussion in the sections of Results & Discussions and Conclusions in the revised manuscript (line 128-142, 226-242, 243-257, and 382-400).

: We also modified several descriptions for polishing our manuscript. Once again, we truly appreciate the reviewer’s constructive suggestions.

Reviewer 2 Report

This paper deals with the extraction methods of lipase.  The paper has following problems so that the paper should be rejected.

• As mentioned by the previous reviewers' comments, the authors should describe the generalized results. If it is impossible, the authors should show the reason, and should discuss why such results were obtained.  Because in this manuscript, the authors only show the experimental results, and the manuscript did not contain novel concept.

Author Response

Point 1: As mentioned by the previous reviewers' comments, the authors should describe the generalized results. If it is impossible, the authors should show the reason and should discuss why such results were obtained. Because in this manuscript, the authors only show the experimental results, and the manuscript did not contain a novel concept.

: We appreciate the reviewer’s comment and considered it with meticulous care. Indeed, we conducted an enzymatic reaction once per agricultural product in the present study because of the following reason. The present study focused on figuring out the differences between protein extraction processes and providing practical information for screening lipase activity from agricultural products. For this, it was more critical to reveal a global tendency of each protein extraction methodology on various plant samples than to conduct replication to derive precise results of each experiment. Hence, we used fully 24 kinds of agricultural products and compared the average value of both protein yield and specific activity in accordance with protein extraction methodologies. Additionally, there was a practical limitation to conduct replication in our experimental platform. We screened 87 kinds of agricultural products simultaneously at preliminary screening (Supplementary Data S1) and also comparatively analyzed four kinds of protein extraction methodologies using 24 kinds of samples in terms of both protein extraction yield (Table 1) and specific activity (Table 2 and 3). For these reasons, it was hard to conduct all replication exactly the same conditions. Of course, we certainly revealed a global tendency and provided practical information based on our present results for the first time.

: As already mentioned in the previous manuscript, there was no comparative or practical information covering protein extractions for the high-throughput screening of lipases as well as other enzymes from agricultural products. Our study did not aim to develop or optimize protein extraction methodologies at a broad range of proteins or for experimental purposes. Instead, we focused on providing several helpful, practical, and comprehensive results of protein extraction using commonly used methodologies in order to apply to the high-throughput screening of enzymes, especially lipases. Therefore, despite inadequate novelty or uniqueness, we believe that our results would be helpful for subscribers of Catalysts, who want to discover a novel and unique lipase from agricultural products and attentively consider that there was no crucial defect in the experimental design, derived results, and following discussion.

: Finally, our research team is actively conducting a further study for finding a stereospecific lipase from the agricultural products selected in this study. We are preparing novel and unique results compared with previous reports using our original determination methodologies for stereospecificity of lipase. Please anticipate these results. Once again, we really appreciate the reviewer’s perspicacious comments on our manuscript.

Reviewer 4 Report

The authors have adequately revised the manuscript according to reviewer comments and given appropriate explanation for reviewer questions

Author Response

We appreciate the reviewer's comments.