Kinetic Analysis of the Lipase-Catalyzed Hydrolysis of Erythritol and Pentaerythritol Fatty Acid Esters: A Biotechnological Application for Making Low-Calorie Healthy Food Alternatives
Round 1
Reviewer 1 Report
This is an original and significant piece of work relevant to the Catalysts journal. The paper is studying the kinetics of two lipases applied for hydrolysis of erythritol and pentaerythritol fatty acid esters towards the development of applications that target the manufacture of low-calorie healthy food alternatives. The authors may improve the quality of the manuscript by considering the following minor comments.
Minor Comments
- The authors should briefly discuss similar applications of the two enzymes employed in the present study. This text should be added either in the Introduction or the Results and Discussion sections to guide the Reader.
- Although (B) and (C) have been indicated in Figure 1, (A) is missing from Sub-Figure 1A.
- The legend of Figure 1 repeats the rationale appearing at the last paragraph before the Figure (lines 72-77). The authors should amend the legend to include the title of the Figure as well as a description of Sub-Figures A-C.
- The data of Figure 1A represent a sigmoidal (or two-phase) response of reaction rate to increasing substrate concentrations. Thus, the term “awkward behavior” (page 4, line 86) should be eliminated and the authors should rephrase the sentence to include the term “sigmoidal” (or “two-phase”) to describe the pattern of experimental data.
- On page 7 (line 177) it is stated that “20 L of the appropriate lipase stock solution…” was applied. I think that the authors should have used “mL” instead of “L” here. Please check and correct in case there is an error.
- On page 7 (line 185) it is stated that 33 mL was the total reaction volume. However, by adding the volumes stated before at the same paragraph I am calculating a different value of mL as total reaction volume. Please check and correct in case there is an error in the 33 mL of total reaction volume stated.
- The Michaelis-Menten equation given on page 7 should be also numbered. Thus, the 3 equations presented at the manuscript should be numbered as equations 1-3 accordingly.
- All the symbols that appear in the equations should be defined in the text to guide the Reader.
Author Response
Response to Reviewer 1 : Authors would like to thank very much the Anonymous Reviewers of the Journal Catalysts for their constructive comments and suggestions. Reviewers’ comments/suggestions, and the responses of authors are as follows:
Reviewer 1:
This is an original and significant piece of work relevant to the Catalysts journal. The paper is studying the kinetics of two lipases applied for hydrolysis of erythritol and pentaerythritol fatty acid esters towards the development of applications that target the manufacture of low-calorie healthy food alternatives. The authors may improve the quality of the manuscript by considering the following minor comments. Minor Comments
R1: The authors should briefly discuss similar applications of the two enzymes employed in the present study. This text should be added either in the Introduction or the Results and Discussion sections to guide the Reader.{Authors’ response: A brief discuss on similar applications of the two enzymes employed has been incorporated in the Introduction, highlighted yellow, between lines 53-58. New references have been inserted i.e. 9-13, highlighted yellow}.
R1: Although (B) and (C) have been indicated in Figure 1, (A) is missing from Sub-Figure 1A.
{Authors’ response: Figure 1A has been thoroughly changed; its sub-graphs are referred as (1) and (2), in line 84}.
R1: The legend of Figure 1 repeats the rationale appearing at the last paragraph before the Figure (lines 72-77). The authors should amend the legend to include the title of the Figure as well as a description of Sub-Figures A-C.
{Authors’ response: The text, just before Figure 1, has been shortened, and contains only starting information, whereas the legend of Figure 1 contains only the necessary material. In both cases the corrections are highlighted by yellow color, between lines 80-83, and 85-91, respectively. There are not repeats any more}.
R1: The data of Figure 1A represent a sigmoidal (or two-phase) response of reaction rate to increasing substrate concentrations. Thus, the term “awkward behavior” (page 4, line 86) should be eliminated and the authors should rephrase the sentence to include the term “sigmoidal” (or “two-phase”) to describe the pattern of experimental data.
{Authors’ response: (a)The term “awkward behavior” has been replaced by the term “discontinuous”, and is highlighted yellow, in lines 86 and 92. Additionally, we added the expression “divided in two groups”, is highlighted by yellow color, in lines 81 and 86 / (b) authors kindly request Reviewer 1 to find a thoroughly response on the “sigmoidal” or not character of the graph in Figures 1A and B, in our relative response of Reviewer 3}.
R1: On page 7 (line 177) it is stated that “20 L of the appropriate lipase stock solution…” was applied. I think that the authors should have used “mL” instead of “L” here. Please check and correct in case there is an error.
{Authors’ response: There was a typographic error of the system. In the submitted text several characters (e.g. m) were in SYMBOL script, but they were converted into Palatino Linotype script e.g. ), and they disappeared when this Word document (in Palatino Linotype script) was converted (automatically) in PDF document. It was stated: 20 mL. All these (three characters, i.e. m, D, and the newly added Û) have been corrected in the revised form, and are highlighted by green color}.
R1: On page 7 (line 185) it is stated that 33 mL was the total reaction volume. However, by adding the volumes stated before at the same paragraph I am calculating a different value of mL as total reaction volume. Please check and correct in case there is an error in the 33 mL of total reaction volume stated.
{Authors’ response: The text has been corrected accordingly, i.e. 27 mL (of bidistilled water) + 3 mL (of 5 % w/v gum Arabic stock solution) + 0.02 mL (of the appropriate lipase stock solution) + 2.98 mL (sum of volumes of DMSO and substrate) = 33 mL. The correction is highlighted by yellow color, between lines 183-186}.
R1: The Michaelis-Menten equation given on page 7 should be also numbered. Thus, the 3 equations presented at the manuscript should be numbered as equations 1-3 accordingly.
{Authors’ response: The Michaelis-Menten equation has been numbered as equation (1), whereas the next equations as (2) and (3), respectively. These equations are highlighted by yellow color, between lines 219-222}.
R1: All the symbols that appear in the equations should be defined in the text to guide the Reader.
{Authors’ response: Three appropriate lines of text has been provided just after equation (3), is highlighted by yellow color, between lines 223-225}.
Best Regards
Prof. Emmanuel M. Papamichael PhD
Professor Emeritus
Guest Editor of Special Issue Biocatalytic Applications in Biotechnology, of Catalysts
Reviewer 2 Report
Review comments on the manuscript "Kinetic analysis of the lipase-catalysed hydrolysis of erythritol and pentaerythritol fatty acid esters: A biotechnological application for making low-calory healthy food alternatives":
I thank the authors for the contribution. The manuscript is concise and may be published after some revision.
In the introdution, please state why these two specific lipases are used for the experiements here and why the pKa values are important.
In the results and discussion part: Please provide first the data and then the estimated parameters from the fit. Also provide the goodness of fit (r2 value) in order to get a feeling for the quality of fit. Include a table presenting the determined pKa values and within this a column for comparison to literature data. Concerning the diagrams (this is a major revision!) have all data plotted in the same style/layout, with a font size making sure that everything in these diagrams is well readable. In Fig. 3 the equivalent points are not well readable.
Caption to table 1, I presume you mean PPL in stead of PPE?
Talbe 1: The last line in the table (above the bottom rule) is not clearly placed.
Line 97: Closing parenthesis is missing.
Line 177: You mean 20 liter?
The equations: Although the Michaelis-Menten equation is well known, explain all parameters of the equations you apply.
Line 230: The sentence starting there "The incomplete ..." is very long and complex. Please divide the sentence into several shorter ones, with a clear and logical thread.
Conclusion: Please highlight clearly what one learns from your results.
Why is the data of Figure S1 in the supplementary information and not in the main manuscript?
Author Response
Response to Reviewer 2: Authors would like to thank very much the Anonymous Reviewers of the Journal Catalysts for their constructive comments and suggestions. Reviewers’ comments/suggestions, and the responses of authors are as follows:
Reviewer 2:
Review comments on the manuscript "Kinetic analysis of the lipase-catalysed hydrolysis of erythritol and pentaerythritol fatty acid esters: A biotechnological application for making low-calory healthy food alternatives": I thank the authors for the contribution. The manuscript is concise and may be published after some revision.
R2: In the introduction, please state why these two specific lipases are used for the experiments here and why the pKa values are important.
{Authors’ response: A relative sentence has been added in the Introduction and it is highlighted by yellow color, between lines 53-54, which is incorporated to the response of a relative comment of Reviewer 1. Furthermore, an as far as concerning the importance of pKa values another relative sentence has been added in the Introduction and it is highlighted by yellow color, between lines 63-64}.
R2. In the results and discussion part: Please provide first the data and then the estimated parameters from the fit
{Authors’ response: It would occupy a useless extra space in the Journal. All the data can be easily retrieved by simple click on each graph, provided that your computer is equipped by an OriginPro trial version; then, double click on any point, select “Data1_B”, and click on the “Workbook”}.
R2. Also provide the goodness of fit (r2 value) in order to get a feeling for the quality of fit.
{Authors’ response: An extra footnote of Table 1 has been provided accordingly, where the Goodness of fit R2 are referred, and it is highlighted by yellow color, in line 77. In all cases of fitting, the goodness of fit (R2 value) was estimated ≤ 0.998 ± 0.010}.
R2. Include a table presenting the determined pKa values and within this a column for comparison to literature data.
{Authors’ response: This is a good idea; however, we think that another Table could causes an extra space in the Journal. On the other hand, all the requested information by the Reviewer 2 is given in the text, Section 2.2, just before and after Figure 2, in lines107-113 and 119-123}.
R2. Concerning the diagrams (this is a major revision!) have all data plotted in the same style/layout, with a font size making sure that everything in these diagrams is well readable. In Fig. 3 the equivalent points are not well readable.
{Authors’ response: In the text, all figures are appeared in reduced size, however in the Word document, it is easy to magnify the figures by right-clicking, choosing the “size” and proceeding accordingly. In the PDF form, you can just magnify the entire document easily. On the other hand, all the figures have been submitted in the Journal in dimensions over than 1000 x 1000 pixels, where all details on the graphs are well readable}.
R2. Caption to table 1, I presume you mean PPL instead of PPE?
{Authors’ response: It has been corrected accordingly, in all situations; all these occurrences are highlighted by yellow color, in lines 75 and 182}.
R2. Talbe 1: The last line in the table (above the bottom rule) is not clearly placed
{Authors’ response: It has been corrected accordingly, and it is highlighted by yellow color, in the last line of Table 1, just over the line 77}.
R2. Line 97: Closing parenthesis is missing.
{Authors’ response: It has been corrected accordingly, and it is highlighted by yellow color, in line104 }.
R2. Line 177: You mean 20 liter?
{Authors’ response: Please, consider the response given for the same comment of Reviewer 1}.
R2. The equations: Although the Michaelis-Menten equation is well known, explain all parameters of the equations you apply.
{Authors’ response: A relative text of three lines has been provided just after equation (3) in lines 224-226, where the parameters of all three equations are explained in details. This text is highlighted by yellow color, whereas one specific character is highlighted by green color}.
R2. Line 230: The sentence starting there "The incomplete ..." is very long and complex. Please divide the sentence into several shorter ones, with a clear and logical thread.
{Authors’ response: A thoroughly revised text (three sentences) replaces the one very long and complex sentence. This text is highlighted by yellow color, in lines 242-249}.
R2. Conclusion: Please highlight clearly what one learns from your results.
{Authors’ response: Please, find within the above mentioned three-sentences text, that it has been provided clearly what one learns from our results}.
R2. Why is the data of Figure S1 in the supplementary information and not in the main manuscript?
{Authors’ response: Usually, simple Michaelis-Menten curves, although contributing in the conclusions, however they are not of high interest, as in other cases}.
Best Regards
Prof. Emmanuel M. Papamichael PhD
Professor Emeritus
Guest Editor of Special Issue Biocatalytic Applications in Biotechnology, of Catalysts
Reviewer 3 Report
In this work the authors present the kinetic analysis of the hydrolysis of erythritol and pentaerythritol fatty acids.
The main issue with this manuscript is the interpretation of kinetic data. The authors believe that the sigmoidal behavior observed in the fitting of the experimental data is due to the substrate concentration, but they should demonstrate that this hypothesis is valid with proper evidences. Is it possible to measure the dimension of the substrate as a function of concentration? If not, this hypothesis cannot be proved.
Moreover, the behavior observed is very typical of cooperative binding. Such mechanism has been vastly covered in literature. See https://doi.org/10.1371/journal.pcbi.1003106 for instance. The authors should investigate also this mechanism to that is enzyme-related and not substrate related. If cooperative binding is confirmed, it would also better explain the differences observed between PPL and CALB).
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Round 2
Reviewer 3 Report
The authors replied to all the comments providing valid explanations.
