Enzymatic Synthesis of Thymol Octanoate, a Promising Hybrid Molecule

Round 1

Reviewer 1 Report

Page 3: It would be useful to know if the esterification reaction of thymol and octanoic acid is reversible?

Page 4, Line 144: The term bio-imprinting should be defined.

Page 5, line 158: Please define immobilization efficiency. Is this expressed in terms of total protein loaded or in terms of post-immobilization activity?

Page 5, lines 168-169: What is the basis of the activity comparison. Is it expressed in terms of activity per unit volume, or as a fraction of total protein??

Page 6, line 209: The hindrance of mass transfer is referred to here but is unclear which region in the reacting continuum is relevant? Is the hindrance to mass transfer across the boundary layer between liquid and support surface or perhaps within the particle?

Page 7, line 228-230: Certainly enzyme inhibition is a possibility and it should be possible to independently verify this. On the other hand is 100% conversion theoretically possible or is there a thermodynamic limit to the final conversion, less than 100%.

Page 7, Figure 5: What is the sensitivity of the enzyme to acidity. Many lipase exhibit a significant pH dependence with respect to activity. Is this relevant and if so perhaps the authors could comment?

Page 8, Figure 7: The shape of the curve shown here contrasts with the classic enzyme kinetic behavior in which one sees a relatively flat slope at the outset of the reaction followed by a steeper gradient before tailing off? Was any attempt made to model the kinetics and determine rate equations and kinetic constants?

Page 9, line 277: See my earlier comment regarding reversibility of the reaction (page 3). Does this behavior not suggest that this is an equilibrium reaction?

Page 9, line 296: Reference to thymol caprylate is made here. Is this an error or was synthesis of thymol caprylate investigated in this study? Perhaps some additional context could be provided?

Page 11, line 352: Was the reaction "killed" at this point to prevent further conversion prior to analysis. Does the addition of n-heptane affect enzyme activity with regards to further use?

 

 

 

 

 

 

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

 

The manuscript submitted by Ferreira and coworkers describes the esterification of octanoic acid with thymol under enzymatic conditions.

The authors described the isolation and immobilization of some enzymes and the study of the reactions and their optimization.

The reaction showed acceptable yields and conversions, and the conclusions are adequate for preliminary work.

It seems like this research may have good scope and be used for future developments.

I can recommend its publication after some changes described above:

 

Mayor changes:

1.     Figures 6 and 7. Figure 7 shows a maximum conversion of close to 94%. However, the chromatogram in figure 6 shows a significant amount of residual thymol. It is necessary to establish the areas of chromatograms used to generate figure 7. The authors should check if the red chromatogram was indeed obtained after 24h or if it was maybe from a different time.

2.     The hypothesis of inhibition must be carefully checked and appropriately discussed. I think a control experiment using the ester in the initial reaction mixture is necessary to support or reject the hypothesis.

 

Minor changes

1.     Page 6, line 206: Please change figure 2 for figure 3

2.     Figure 5 and related discussion. Please use the same proportion units as before. So 0.5:1 must be 1:2, and so on.

3.     Page 10, lines 345-346. The reaction is not performed solvent-free since the enzyme is in solution. Please change.

4.     Page 12, line 406. A conclusion saying “conversion close to 94%” is unacceptable. Please use the exact number. If that was calculated using chromatography, a more accurate value is certainly possible.

 

In summary, this work deserves publication in Catlysts, but a revision is needed

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

This study consists in the enzymatic production of a valuable ester via enzymatic esterification reaction in solvente-free systems. The effect of lipase source and its form (free or immobilized), alcohol/acid molar ratio, and reaction time on the reaction was examined. The purpose and methods of the study are carefully presented and the results support the purpose of the study in part. However, the following questions should be answered.

1. The authors should report in Abstract that immobilized biocatalysts were also used to produce ester, however free lipase B from Candida antarctica was the most active biocatalyst. I recommend report that the reactions were conducted in solvente-free systems. The authors should remove “possible reusability of the enzyme”. This assay was not performed in this study.

2. The authors should better expalin why octanoic acid was used as carboxylic acid model. 3. Explain breifly why Immobead 150 was used as enzyme support and immobilization technique utilized for preparing heterogeneous biocatalysts.

4. Bioimprinting strategy could also be bruefly discussed.

5. Lines 74/78: Put references.

6. I recommend emphasize the positive performance of lipases in solvent-free reaction systems. Cite some references

https://doi.org/10.1039/D1CY00696G

https://doi.org/10.1016/J.JECE.2021.107062

https://doi.org/10.1016/J.ENZMICTEC.2022.110019

https://doi.org/10.3390/molecules27092692

7. How is this system different to other reports to merit publication? Please, report.

8. Lines 158/161: The authors should better explain why an improvement on the immobilization procedure using bioimprinting strategies were obtained. Briefly explain why such chemicals, including thymol, were tested in this study. I recommend cite some recent studies. It is very interesting to the readers. Moreover, I recommend a brief explanation for a decrease of catalytic performance of the heterogeneous biocatalyst when compared with its soluble form. The authors should replace “free lipase” by “soluble lipase”.

9. The authors should check all references – 70 references were cited in this manuscript. However, in Lines 318 and 325 has been cited reference [83].

Author Response

"Please see the attachment"

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The authors corrected the manuscript, as suggested. Therefore, I recommend its acceptance as it is.