A Novel Purification Process of Sardine Lipases Using Protein Ultrafiltration and Dye Ligand Affinity Chromatography

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Developing enzyme purification processes is essential to characterize the enzyme’s function and application. The authors reported a novel three-step process for purifying a lipase from whole sardine viscera. The authors' methods are simple, but they reduced the purification steps. The authors claimed they had overcome the challenges, such as lowering yield and increasing production costs. The data seemed to support the authors’ claim. However, it is not clear whether their methods might be better than previously developed methods. If possible, the authors should compare the current and previous reported results, showing the reduced time and increased efficiency. The authors might develop a method to purify the lipase in this work.

Minor: The authors used the abbreviations in the manuscript. You should check them: SPLSV, PLSV, or LPSV (page 6) was used.

Author Response

Developing enzyme purification processes is essential to characterize the enzyme’s function and application. The authors reported a novel three-step process for purifying a lipase from whole sardine viscera. The authors' methods are simple, but they reduced the purification steps.

Q1. The authors claimed they had overcome the challenges, such as lowering yield and increasing production costs. The data seemed to support the authors’ claim. However, it is not clear whether their methods might be better than previously developed methods. If possible, the authors should compare the current and previous reported results, showing the reduced time and increased efficiency.

R1. This is a pertinent suggestion of the reviewer, in the newly revised manuscript we have included in the discussion section, a comparison of the current and previous reported results.

Q2. The authors might develop a method to purify the lipase in this work.

R2. Yes, we developed a novel method to purify lipases without dialysis for desalting or ion exchange resins because lipases can be adsorbed at high salt concentration in dye affinity adsorbent and eluted with 0 M of NH₄ sulfate.

Q3. Minor: The authors used the abbreviations in the manuscript. You should check them: SPLSV, PLSV, or LPSV (page 6) was used. The authors might develop a method to purify the lipase in this work.

R3. Thanks for the observation, we have corrected the abbreviations.

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors studied a very intriguing method for purifying proteins from a complex sample. The method is novel. However, comparing with some existing methods may make the paper more convincing. I have following comments:

1. The cutoff of the molecular weight in the final quantification gel is not acceptable for protein purification. Please upload the full gel image.
2. The purified protein is not compared to a standard. So it's not clear what the identity of the protein. There is also no identification associated with it.  
3. Is there any previous trial on purifying the protein? If so, please make comparison. 
4. Have the author explored some more canonical column purification method such as hydrophobic interaction column or ion exchange?

Author Response

The authors studied a very intriguing method for purifying proteins from a complex sample. The method is novel. However, comparing with some existing methods may make the paper more convincing. I have the following comments:

Q1. The cutoff of the molecular weight in the final quantification gel is not acceptable for protein purification. Please upload the full gel image.

R1.  This is a relevant comment of the author. It was not necessary to have a broad or lower range MW less than 40 kDa because we used ultrafiltration with 30 kDa MWCO. The cutoff of b-galactosidase was corrected to correspond to the high range MWM provided by Biorad (https://www.bio-rad.com/es-mx/applications-technologies/introduction-western-blot-protein-standards?ID=LUSP9D7MU).

Q2. The purified protein is not compared to a standard.  So it's not clear what the identity of the protein. There is also no identification associated with it.

R2. Important observation of the reviewer. Unfortunately, there are no available standards for lipases purified from sardine or fish viscera. We tried to use commercial microbial lipases to compare activity and found that these commercial lipases are not provided totally purified in SDS_PAGE analysis.

Q3. Is there any previous trial on purifying the protein? If so, please make a comparison.

R3. This is a pertinent suggestion of the reviewer. In last paragraph of the Results and discussion section of the new manuscript, we have made this comparison.

Q4. Has the author explored some more canonical column purification method such as hydrophobic interaction column or ion exchange?

R4. We are studying the purification of microbial lipases using hydrophobic interaction adsorbents (Octyl Sepharose CL-4B).

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript describes a three-step purification protocol for isolating lipases from sardine viscera using ammonium sulfate precipitation, ultrafiltration, and dye-ligand affinity chromatography. While the authors report an increase in specific activity and a molecular mass determination by SDS-PAGE, the overall impact and novelty of the work are limited.

1. The techniques used are well-established and commonly applied in enzyme purification. The manuscript does not offer a novel methodology or discovery that would justify publication in a journal with a biophysical or molecular focus.
2. The conclusion of successful purification is based solely on SDS-PAGE. This is insufficient. Additional analytical methods—such as negative-stain electron microscopy or mass spectrometry—should be used to validate purity, particularly given the importance of homogeneity in enzymology and biocatalysis research. Alternatively, the authors should justify why the observed purity is adequate for the intended downstream applications.
3. The study lacks mechanistic insight or functional characterization beyond basic activity assays. There is no kinetic analysis, substrate specificity study, or structural information provided, making the manuscript scientifically thin and not sufficiently rigorous.
4. The manuscript does not align well with the typical scope of Biophysica, which prioritizes studies offering deeper mechanistic, structural, or computational insights into biomolecular systems. The manuscript reads more as a technical note suited for a lower-impact or methods-focused outlet.

Comments on the Quality of English Language

The manuscript contains pervasive grammatical errors, poor sentence structure, and formatting inconsistencies. This significantly hinders readability and detracts from the professionalism expected in a peer-reviewed journal. Substantial editing would be required for even basic clarity.

Author Response

Q1. This manuscript describes a three-step purification protocol for isolating lipases from sardine viscera using ammonium sulfate precipitation, ultrafiltration, and dye-ligand affinity chromatography. While the authors report an increase in specific activity and a molecular mass determination by SDS-PAGE, the overall impact and novelty of the work are limited.

R1. We appreciate the reviewer’s feedback and the opportunity to clarify the contributions of our work. While we acknowledge that lipase purification has been previously reported, this study presents a tailored and efficient three-step protocol specifically optimized for sardine (Sardinella spp.) viscera, a low-cost and underutilized biological resource. The increase in specific activity and the clear banding pattern observed in SDS-PAGE highlights the effectiveness of the proposed method.

Moreover, the combination of ammonium sulfate precipitation, ultrafiltration, and dye-ligand affinity chromatography provides a scalable and reproducible approach with potential industrial relevance, particularly in biocatalysis and waste valorization contexts. We believe this work adds value by contributing to a practical purification strategy and by promoting the use of marine by-products in enzyme biotechnology. We have revised the discussion to better emphasize these aspects and to clarify the novelty and relevance of our findings.

Q2. The techniques used are well-established and commonly applied in enzyme purification. The manuscript does not offer a novel methodology or discovery that would justify publication in a journal with a biophysical or molecular focus.

R2. We understand the reviewer’s perspective. While the techniques employed are indeed established, their strategic combination and optimization for sardine viscera—a low-cost and underexploited biological source—represent a practical contribution to enzyme recovery from marine by-products. Our aim was not to develop new methodologies, but to demonstrate the applicability and efficiency of this protocol for obtaining active, purified lipases with potential industrial relevance. Nevertheless, we acknowledge the journal’s scope and have revised the discussion to better clarify the significance and intended application of our findings.

Q3. The conclusion of successful purification is based solely on SDS-PAGE. This is insufficient. Additional analytical methods—such as negative-stain electron microscopy or mass spectrometry—should be used to validate purity, particularly given the importance of homogeneity in enzymology and biocatalysis research. Alternatively, the authors should justify why the observed purity is adequate for the intended downstream applications.

R3. We thank the reviewer for this important observation. We agree that comprehensive purity assessment is essential, particularly in mechanistic enzymology. However, the primary objective of our work was to develop a cost-effective and scalable purification protocol for sardine lipases with sufficient activity and purity for applied biocatalytic processes, rather than for structural or mechanistic studies. SDS-PAGE was used here as a standard and practical indicator of enrichment and molecular size consistency. While advanced techniques such as mass spectrometry or EM would provide further resolution, they were beyond the scope of our current study focused on process development and valorization of marine by-products.

Q4. The study lacks mechanistic insight or functional characterization beyond basic activity assays. There is no kinetic analysis, substrate specificity study, or structural information provided, making the manuscript scientifically thin and not sufficiently rigorous.

R4. We appreciate the reviewer’s comment and agree that mechanistic and functional characterization would significantly enrich the scientific depth of the study. However, the primary focus of this work was to establish a straightforward and effective protocol for partial purification of lipases from sardine viscera, with the goal of enabling their future application in biocatalysis.

Q5. The manuscript does not align well with the typical scope of Biophysica, which prioritizes studies offering deeper mechanistic, structural, or computational insights into biomolecular systems. The manuscript reads more as a technical note suited for a lower-impact or methods-focused outlet.

R5. We thank the reviewer for this clear assessment. We understand that Biophysica emphasizes studies with strong mechanistic or structural insights. As mentioned above, our manuscript is intended to present a practical and efficient protocol for lipase recovery from sardine viscera, with potential relevance to biocatalysis and waste valorization. While we acknowledge the methodological nature of the study, we hoped its applicability and optimization for a novel biological source might be of interest to the journal’s readers.

Q6. The manuscript contains pervasive grammatical errors, poor sentence structure, and formatting inconsistencies. This significantly hinders readability and detracts from the professionalism expected in a peer-reviewed journal. Substantial editing would be required for even basic clarity.

R6. We thank the reviewer for this important feedback. We regret that the current version did not meet the expected standards of clarity and presentation. In response, we have undertaken a thorough revision of the manuscript, with careful attention to grammar, sentence structure, and formatting. We hope that these changes now allow the content to be more clearly and effectively communicated.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank the authors for addressing the comments. Please see my updated comments:

Q1: If the ultracentrifugation with 30KD MWCO was used in the method, SDS page lower than 40KD should be very clean. In that case, the authors should have no trouble showing the full gel image.

Q2: Do the authors have access to mass spectrometry? If so, the identity of the protein can be confirmed. 

Author Response

Thank the authors for addressing the comments. Please see my updated comments:

Q1: If the ultracentrifugation with 30KD MWCO was used in the method, SDS page lower than 40KD should be very clean. In that case, the authors should have no trouble showing the full gel image.

R1: The reviewer's comment is completely correct and that is why we eliminated that part of the gel from the original image. Unfortunately, we do not save the image of the complete gel.

 

Q2: Do the authors have access to mass spectrometry? If so, the identity of the protein can be confirmed.

R2. We understand the reviewer's comment that it is possible to confirm the identity of the protein through mass spectroscopy, however we do not have this type of analysis in our laboratory.

 

P.D. We appreciate the reviewers' comments which have significantly improved the presentation of this manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. The language has improved significantly compared to the previous version. The manuscript is now more readable and professional. Sentence structure is clearer, and technical terms are used more appropriately throughout the text.
2. In Section 3.3, please clarify the buffer assignments and salt concentrations. The sentence currently reads: “Peaks 1 and 2 were eluted in the wash with the loading buffer A solution (1.0 M ammonium sulfate), so it is clear that they do not show affinity for the adsorbent under the run conditions. Peak 3 was eluted at a concentration of 0.5 M ammonium sulfate, while peak number four was eluted at 0.0 M ammonium sulfate.” Please revise to explicitly state this and include the concentration of buffer B in the results section, so readers can clearly follow the gradient and elution conditions.
3. Consider including the initial weight of the raw sardine material. This would help readers evaluate the overall recovery yield and practical scalability of the purification process. Even an approximate value would be useful.

Author Response

Q1. The language has improved significantly compared to the previous version. The manuscript is now more readable and professional. Sentence structure is clearer, and technical terms are used more appropriately throughout the text.

R1. We greatly appreciate your comments, which have significantly improved the presentation of this manuscript, which was reviewed by collaborators who are experts on the topic and in the English language.

 

Q2. In Section 3.3, please clarify the buffer assignments and salt concentrations. The sentence currently reads: “Peaks 1 and 2 were eluted in the wash with the loading buffer A solution (1.0 M ammonium sulfate), so it is clear that they do not show affinity for the adsorbent under the run conditions. Peak 3 was eluted at a concentration of 0.5 M ammonium sulfate, while peak number four was eluted at 0.0 M ammonium sulfate.” Please revise to explicitly state this and include the concentration of buffer B in the results section, so readers can clearly follow the gradient and elution conditions.

R2. We thank the observation of the reviewer. We change the paragraph as follow in order to be more explicitly and clarify the buffer assignements.

“Peaks 1 and 2 were eluted in the wash with the loading buffer A solution (1.0 M ammonium sulfate), so it is clear that they do not show affinity for the adsorbent under this run condition. Peak 3 was eluted at a 50% of buffer B, that correspond to 0.5 M ammonium sulfate concentration, while peak number four was eluted at 100% of buffer B, that correspond to 0.0 M ammonium sulfate.”

Additionally, we add the definition of buffer b in section 2.5: “The adsorbed proteins were eluted at a flow rate of 1.0 mL/min using two linear gradients with buffer B (50 mM potassium phosphate pH 7.0 )”

 

Q3. Consider including the initial weight of the raw sardine material. This would help readers evaluate the overall recovery yield and practical scalability of the purification process. Even an approximate value would be useful.

R3. We appreciate the reviewer recommendation. We included the initial weight of viscera 186 g in each experiment in section 2.2.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The author answered all my questions. I have no further comments.