Unfolded Lipase at Interfaces Studied via Interfacial Dilational Rheology: The Impact of Urea

Round 1

Reviewer 1 Report

The interfacial behavior of lipase is of great interest. In the present, the authors studied the interfacial dilational rheological properties of lipase in the absence and presence of urea, respectively. The results were well presented and analyzed. However, there are still several problems in the writing and analysis. A minor revision should be made before the article can be accepted.

1.     According to the values of IFTs, the authors studied the interfacial behaviors of the lipase at gas-water interface. However, lipase catalyzes lipid hydrolysis at the oil-water interface. Why the authors did not study its interfacial behaviors at oil-water interface?

2.     I suggest that the authors include the interfacial properties of lipase in the absence of urea in the Fig.2-4 for better comparison.

3.     In Fig.3b, the vertical axis should be viscosity rather than elasticity.

4.     The Fig.7 is not clear. The text and icons in the figure are illegible.

5.     The last sentence above Fig.7, “Without urea, the elasticity decreases, and the viscosity increases with the increasing amplitude of the oscillations.” According to the Fig.7, the viscosity increased first and then decreased which is not consistent with the description of the original text.

6.     The conclusion is too long. A more concise conclusion is recommended.

7.     Author should expand the literature by including potent articles such as

Journal of Oleo Science, https://doi.org/10.5650/jos.ess21081

Journal of Molecular Liquids, https://doi.org/10.1016/j.molliq.2020.114823

Journal of Molecular Liquids,, https://doi.org/10.1016/j.molliq.2022.120198

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The work is clearly performed in a professional and serious manner, the controls placed on the experiments are sufficient to trust the results, the interpretations of the data are in general appropriate, the list of references is broad, appropriate and up-to-date, and the manuscript is written clearly with an excellent standard of prose. I therefore recommend this paper to be published after indicated minor revisions.

Q1. Authors are suggested to improve the quality of English of the manuscript

Author Response

Thank you for your kind and considerate review. The English quality of the manuscript was revisited, and some typos and inaccuracies were corrected. It was also handed in to an expert colleague for proofreading, and some revisions were applied accordingly. The changes are shown in the manuscript by “Track Changes”.

Reviewer 3 Report

Manuscript clearly states the problem to be investigated, properly describes experiments (but see below) and their results. The interpretation of the measured data is adequate. I believe that this work can be interesting not only for those working in the interfacial science but also for experts from the field of enzymatic reactions, especially triglyceride transformations which are important also industrially. I can endorse publication after considering following minor comments:

·         lines 96-97: to which interface exactly does this refer?

·         part 2.2 – was a drop of lipase solution formed in the urea solution or was the arrangement different?

·         Some statistical information like reproducibility, deviations etc. would be nice.

Author Response

Thank you for your kind and careful comments. We really appreciate your accurate and in-detail review. Our responses to all the mentioned points are listed below, respectively.

• The interface’s name is now mentioned in the manuscript in lines 96–97.
• To investigate the effect of urea on the lipase structure, lipase is dissolved into the aqueous urea solution. So, all the tests are conducted at the air–water interface. The procedure is detailed in section 2.1.
• The following text was added to the manuscript to accentuate the reproducibility and accuracy of the data: “All measurements have been carried out after calibration of the setup by measuring water surface tension to obtain ~72.0 mNm^-1 at 24 °C. Some errors in the range of ±0.2 mN·m^-1 are inevitable owing to the changes in the ambient conditions. After proper calibration, the accuracy in measuring the IFT is limited only to the setup resolution, ±0.1 mN·m^-1.”