Reformulated Kinetics of Immobilized Enzymes in Non-Conventional Media: A Case of Lipase-Catalyzed Esterification

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

In this work, Papamichael and Stergiou proposed a novel model for the kinetics of esterification reactions catalyzed by lipases in non-anhydrous conditions. 

 

Although the topic is of interest to the biocatalysis community, the manuscript is very difficult to read especially for non-expert readers. The experimental data on which the model is based should be reported in the Supplement, at least citing the publications from which they are derived. 

 

Major points:

The introduction should benefit from an overview of the effects of organic solvents on free and immobilized enzymes.In fact, the organic solvents can inhibit, denature or activate the enzymes. In some cases, the organic solvents also improve the solubility of the substrate. See for example https://doi.org/10.1002/biot.202100712, https://doi.org/10.1021/acs.joc.1c01136, https://doi.org/10.1016/j.fuel.2019.116343, https://doi.org/10.1016/j.ijbiomac.2020.02.145.

How does this work fit into this complex scenario? The authors have modeled a reaction in which the solvent has no inhibitory or activating effect on the enzyme. Please clarify this point. 

 

I was wondering if the authors fit experimental data to the proposed model or simulated the Michaelis-Menten behavior. This should be crucial to validate the model and to improve the significance of the work.

Comments on the Quality of English Language

The quality of the English language is quite good.

Author Response

 

Response to Reviewer 1:

We thank Reviewer 1 for his/her constructive comments and suggestions.

A general authors’ response: The text has been thoroughly revised by editing existing English language issues.

The herein references, in [ ] brackets, are found at the end of this text.

 

1. In this work, Papamichael and Stergiou proposed a novel model for the kinetics of esterification reactions catalyzed by lipases in non-anhydrous conditions. Although the topic is of interest to the biocatalysis community, the manuscript is very difficult to read especially for non-expert readers. The experimental data on which the model is based should be reported in the Supplement, at least citing the publications from which they are derived.:

 

Authors’ response:

This study reports the development of simplified rate equations related to the kinetics of esterification reactions catalyzed by immobilized lipase, either in anhydrous aprotic and nonpolar organic solvents or in solvent-free systems. This concept, although derived from a previously published study by our research group [1], is however novel as it is based on experimentally validated results and verified mechanisms, also published by us, governing the aforementioned esterification reactions. Indeed, two different mechanisms were found describing esterifications catalyzed by immobilized lipase, either in anhydrous n-hexane (bi-bi ping-pong mechanism with double dead-end substrate inhibition*) [2] or in solvent-free conditions** (ordered bi-bi mechanism with single dead-end substrate inhibition) [3]. These findings necessitate a new in-depth reconsideration and simplification of the specific multivariate rate equations that describe the two different mechanisms that were revealed. Fitting such multiparametric equations of two independent variables to experimental responses is a difficult task that requires the application of surface-fitting methodologies, which are time-consuming and computationally demanding.

A thorough editing of all English language issues has been carried out, along with a highlighting of the references of our publications related to the experimental data and mechanisms on which the present study is based on. The text in the revised version is easily followed by the reader.

* Substrates: Anhydrous butyric acid as a short-chain fatty acid, and absolute ethanol as a short-chain alcohol.

** Absolute ethanol was both substrate and the mean where the esterification reaction was carried out.

 

 

2. The introduction should benefit from an overview of the effects of organic solvents on free and immobilized enzymes. In fact, the organic solvents can inhibit, denature or activate the enzymes. In some cases, the organic solvents also improve the solubility of the substrate. See for example https://doi.org/10.1002/biot.202100712, https://doi.org/10.1021/acs.joc.1c01136, https://doi.org/10.1016/j.fuel.2019.116343, https://doi.org/10.1016/j.ijbiomac.2020.02.145

 

Authors’ response:

In fact, this study focuses on the standardization of the kinetics of immobilized enzymes, according to all the objectives mentioned in 1, above. Therefore, we used the immobilized lipase-catalyzed esterifications (enzyme: CALB – immobilized Candida antarctica lipase B / Novozym 435®), as examples. This choice is due to sufficient knowledge of the mechanistic features of these reactions [1-3,4]. Nevertheless, the motivation was the need to provide fast and error-free estimates of important kinetic parameters that are useful in the productive areas of biotechnological and industrial applications. On the other hand, the investigation of whether certain organic solvents affect, either negatively or positively, free or immobilized enzymes constitutes a very interesting but very broad field in both experimental, theoretical, and computational enzymology.

In our studies, on which this manuscript under review is based, were reported either the choice of aprotic and nonpolar solvent (n-hexane) or solvent-free reaction systems. This solvent is an alkane that exhibits not detectable dipole and quadrupole moments, due to the homogeneous distribution of its polarization and the subsequent homogeneous solutions of any potentially soluble compound in this solvent [5]. Additionally, in those studies we also chosen an immobilized biocatalyst (CALB) in order to investigate the mechanisms of esterification and to formulate the appropriate equations. In the case of solvent-free conditions (in absolute ethanol), the esterification reaction was affected by the excess of substrate ethanol. Nevertheless, it seems more likely that ethanol's dipole and quadrupole moments potentially did not affect, at least to a significant degree, the esterification reaction, which was performed under anhydrous conditions by continuous removal of the formed H₂O molecules from the reaction medium [2,3]; under these anhydrous conditions only traces of ethanol could behave as protic solvent molecules [2,3].

We would like to thank Reviewer 1 for his/her kind suggestions regarding the four example references in order to help us in the enrichment of the section Introduction, of our manuscript. However, the objectives of all four suggested references focus on the effects that certain organic solvents exhibit on free and immobilized enzymes through different functions (e.g. inhibition, denaturation, activation, etc.). Furthermore, the esterification methodologies followed as well as other lipase-catalyzed reactions in the proposed reports were carried out under completely different experimental conditions, e.g. in binary mixtures of polar organic solvents and water, or during transesterification reactions, and in proportions known to be almost destructive to the enzyme structure due to various effects. Therefore, Abstract, Introduction and Conclusions were revised accordingly by adding also four more appropriate references in order to strength our methods, the results and the conclusions of our work.

In the reference https://doi.org/10.1021/acs.joc.1c01136, which was proposed by Reviewer 1, is mentioned that “Thus, the modulation of the hydrogen-bonding network at the active site by the different solvent environments is a key structural feature regulating enzyme activity”. To our knowledge [2,3, Papamichael et al., DOI: 10.1080/09593331003664136, Theodorou et al., doi:10.1016/j.biortech.2006.07.037] we agree to this sentence. The elucidation of the described therein mechanisms [2,3, Papamichael et al., DOI: 10.1080/09593331003664136, Theodorou et al., doi:10.1016/j.biortech.2006.07.037] mechanisms that are mentioned above (response to comment 1), were based also in the study of hydrogen bonding that occur in the anhydrous reaction systems of aprotic nonpolar solvent n-hexane or solvent-free systems, and/or in systems containing small percentages of organic solvents (our studies which are based on results from proton inventories and solvent isotope effects). These elucidated mechanisms are described by multiparametric and multivariable rate equations; and these equations need to be systematically simplified on the basis, as well as the  purposes, as those described in our manuscript under review.

 

 

3. How does this work fit into this complex scenario? The authors have modeled a reaction in which the solvent has no inhibitory or activating effect on the enzyme. Please clarify this point.

 

Authors’ response:

We would like to mention that this comment concerns more our previous studies on which this manuscript under investigation is based and much less this manuscript itself. We based on our previous published studies according to our detailed response in the comment (2). Our concern, in this manuscript under review, is the simplification of the previously found and published multiparametric and multivariable rate equations, and not how these equations were formulated. All these details are described in the related references.

 

 

4. I was wondering if the authors fit experimental data to the proposed model or simulated the Michaelis-Menten behavior. This should be crucial to validate the model and to improve the significance of the work.

 

Authors’ response:

We recall our previous responses (1-3, above) to cite the objectives of this manuscript under review, as well as the bases that inspired us to the idea of ​​its conseption and implementation. Accordingly, the answer to this comment of Reviewer 1 is yes. Nonetheless, we should provide more details. As a matter of fact, a successful fitting process like the one mentioned by Reviewer 1 has been carried out and has been previously reported by our research group (2019) [3]. We, the authors of this manuscript under review, decided not to use at all the data mentioned therein, i.e., in [3], as well as not to include them in this response to Reviewer 1. Therefore, Reviewer 1 is kindly requested to understand the reasons for this decision. Apart from a possible self-plagiarism, only two of the four authors of the report [3], namely the third and fourth, were involved in the conception and implementation of this study.

 

[1] [Foukis, A.; Stergiou,P-Y.; Filippou, M.; Koukouritaki, M.; Parapouli, M.; Theodorou, L.G.; E Hatziloukas, E.; Afendra, A.; Pandey, A; Papamichael, E.M. Kinetic constraints and features imposed by the immobilization of enzymes onto solid matrices: A key to advanced biotransformation, Indian J. Exp. Biol. 2014, 52, 1045-1051, and references therein]

[2] Foukis, A., Gkini, O.A., Stergiou, P-Y., Papamichael, E.M. New insights and tools for the elucidation of lipase catalyzed esterification reaction mechanism in n-hexane: The synthesis of ethyl butyrate, Mol. Catal. 2018, 455, 159-163.

[3] Foukis, A., Gkini, O.A., Stergiou, P-Y., Papamichael, E.M. The mechanism of lipase-catalyzed synthesis of food flavoring ethyl butyrate in a solvent-free system, J. Food Nutrition and Metabolism, 2019, 2-7, DOI:10.31487/j.JFNM.2019.03.01.

[4] Stergiou, P-Y.; Foukis, A.; Filippou, M.; Koukouritaki, M.; Parapouli, M.; Theodorou, L.G.; Hatziloukas, E.; Amalia Afendra, A.; Pandey, A.; Papamichael, E.M. Advances in lipase-catalyzed esterification reactions, Biotechnol. Adv. 2013, 31, 1846-1859.

[5] Klamt, A. Fundamental criticism of the dielectric continuum approach. In COSMO-RS ch. 3, Ed. Klamt, A., Elsevier, New York, 2005, pp. 43-47.

Reviewer 2 Report

Comments and Suggestions for Authors

General comments:

- In the methods section, it is stated that the experimental data and mechanistic information are from novel published and/or unpublished results by the authors. If novel unpublished data are used, I believe they should be included in the paper. In this paper, there is no mention of specific experimental results in the “Results and Discussion” section, and there is no explanation of the experimental setup in the section “Methods and Tools”. That is acceptable for previously published data, but any novel data should be fully reported here. This represents my main concern about the manuscript. If the authors did not use novel experimental data, then only the sentence in lines 141-144 should be changed.

- Additionally, the authors refer to “lipase” throughout the whole manuscript but do not specify which particular enzymes from which organisms were employed. I think it is important to address that. As there are numerous known lipases from different sources, I believe that the specific enzymes used in this research need to be clearly identified. The same applies to the generalizations regarding LMW fatty acids and alcohols.

- Finally, could the authors comment more on the applicability of their study on different systems? In the “Conclusion” section, the authors have a good paragraph about the broader applicability of the study, but specifically I would like to know their opinion on whether their findings can be applied to different lipase enzymes, LMW fatty acids, and alcohols. It would be helpful to know if they think the results are specific to the lipases and substrates investigated here or if they can be generalized to other systems as well.

 

 

Minor specific comments:

12 – “Recently there are…”, “there” is missing

25 – “non-conventional”, space not needed

56 – “…Michaelis-Menten parameters based both on…”, “by” not needed

84 – “evidently apparent” sounds strange, I would recommend using only “evident” or “apparent”

181 – something is not right with this sentence. Maybe only the colon sign is not necessary here (it seems like it is not here on purpose), or maybe some part of the sentence is missing. Please check

193 – I think the sentence would be clearer if authors omitted “they”, only “values of both k-1_ppg and k-3_ppg approach zero”

228 – acid should not be in capital letters

238 – x not used in other parts of the text as multiplication sign, only here

Comments on the Quality of English Language

English quality is fine, only minor editing is needed in some parts as given above in "minor specific comments"

Author Response

 

Response to Reviewer 2:

We thank Reviewer 2 for his/her constructive comments and suggestions.

A general authors’ response: The text has been thoroughly revised by editing existing English language issues.

The herein references, in [ ] brackets, are found at the end of this text.

 

1. In the methods section, it is stated that the experimental data and mechanistic information are from novel published and/or unpublished results by the authors. If novel unpublished data are used, I believe they should be included in this paper. In this paper, there is no mention of specific experimental results in the “Results and Discussion” section, and there is no explanation of the experimental setup in the section “Methods and Tools”.. That is acceptable for previously published data, but any novel data should be fully reported here. This represents my main concern about the manuscript. If the authors did not use novel experimental data, then only the sentence in lines 141-144 should be changed.

 

Authors’ response:

This study focuses on the development of simplified rate equations related to the kinetics of esterification reactions catalyzed by immobilized lipase, either in anhydrous aprotic and nonpolar organic solvents (n-hexane) or in solvent-free systems. We were inspired by this new idea from previous published studies of our research group [1-4]. Subsequently, we (the authors of this manuscript under review) regarded it necessary to conduct a new in-depth reconsideration and simplification of the specific multivariable rate equations describing the two different revealed mechanisms. The fitting of such multiparametric equations of two independent variables to experimental responses is a difficult task requiring the application of surface-fitting methodologies, which are time-consuming and computationally demanding. Therefore, and although experimental data are not published in this manuscript under review, we relied on previous results both published and new unpublished results, which we will include in the revised version of this study, according to the comments and suggestions of the Reviewer 2 [5].

All referred esterification reactions were catalyzed by the immobilized enzyme Candida antarctica lipase B (Novozym 435® - CALB). The experimental setup of the present study included the manipulation of equation parameters and dependent variables attempting to simplify multiparametric and multivariate model-equations related to the aforementioned reaction conditions, on a new basis. Consequently, the revised version of the manuscript under review has been corrected accordingly, as well as the sentence in lines 141-144 has also been modified accordingly.

 

 

2. Additionally, the authors refer to “lipase” throughout the whole manuscript but do not specify which particular enzymes from which organisms were employed. I think it is important to address that. As there are numerous known lipases from different sources, I believe that the specific enzymes used in this research need to be clearly identified. The same applies to the generalizations regarding LMW fatty acids and alcohols.:

 

Authors’ response:

We would like to thank Reviewer 2 for his/her valuable comment and suggestions, and consequently we accordingly conformed. The text of the revised version has been corrected by referring all necessary information. The referred lipase in the initial version is the Candida antarctica lipase B / Novozym 435®, which is commonly referred as CALB. The low molecular fatty acid (concerning the published results) is the butanoic acid and the corresponding alcohol is the ethanol. In the unpublished results are included the fatty acids propanoic, butanoic and pentanoic, as well as the alcohols methanol, ethanol, n-butanol and n-pentanol.

 

 

3. Finally, could the authors comment more on the applicability of their study on different systems? In the “Conclusion” section, the authors have a good paragraph about the broader applicability of the study, but specifically I would like to know their opinion on whether their findings can be applied to different lipase enzymes, LMW fatty acids, and alcohols. It would be helpful to know if they think the results are specific to the lipases and substrates investigated here or if they can be generalized to other systems as well:

 

Authors’ response:

In our studies related to and preceding this manuscript under review, we reported results from esterification reactions catalyzed by the immobilized enzyme Candida antarctica lipase B (Novozym 435® - CALB), in anhydrous aprotic and nonpolar organic solvents (n-hexane) or in solvent-free systems. These esterifications were carried out between short-chain fatty acids and alcohols (please see the above 2), and for different purposes. Mixtures of fatty acids (propanoic, butanoic and pentanoic), generated from the acidogenesis of low-value liquid industrial waste biomass, were esterified with selected alcohols, either ethanol or n-butanol. Later, ethyl butyrate, a food-flavoring additive, was synthesized by esterification of butanoic acid and ethanol. In all these cases, the esters were synthesized under anhydrous conditions in either n-hexane or solvent-free systems, where the produced water was continuously removed from the reaction mixture. The mechanisms of these esterification reactions have been also elucidated. This knowledge was key to the choice and also to recommend this particular methodology, as: (a) the water molecules formed during the esterification reaction negatively affect the chemical equilibrium by reversing the direction of the reaction, due to hydrolysis of the ester, reducing both its % yield and its concentration in the reaction mixture, and (b) the presence of water in measurable concentrations, within the reaction mixture, would seriously affect the structural stability of the immobilized lipase and therefore its catalytic capacity through a possible denaturation. [https://doi.org/10.1186/1472-6807-8-9; https://doi.org/10.1016/j.ijbiomac.2020.02.145, and therein references]. In our view, taking also into account our unpublished data [5], we consider the aforementioned methodology as applicable to similar reaction systems.

Furthermore, various immobilized lipases having flexible lids can catalyze quite a few of the thermodynamically favored reactions under the aforementioned conditions. So far, we have used this methodology for the successful esterification of short-chain fatty acids (C₃ - C₅) and alcohols (C₁, C₂, C₄ and C₅) [3,4,5]. In upcoming future studies, we will be able to report on the esterification of medium- and long-chain fatty acids and alcohols.

CALB is not recognized as a highly specific enzyme like most of the hydrolases, however only a limited number of fatty acids and/or alcohols can be suitable substrates for it. Similarly, the applied methodologies vary occasionally. All of the above vary depending on the structure of each enzyme and much more so for lipases. The catalytic ability of lipases depends largely on their general structure, as in all enzymes, but much more on the existence and specific structure of the lid. So, a general and simple answer to this question of Reviewer 2 is no. However, a "yes" answer should be given if and only if molecular modeling methods (in-silico studies of ligand binding to lipase molecules), together with experimental data including among others molecular engineering studies, could be applied to produce appropriately modified lipases. Finally, the choice of the proper enzyme, its form either free-in-solution or immobilized, as well as the composition of the reaction medium depends on the desired product.

 

 

Minor specific comments:

12 – “Recently there are…”, “there” is missing // Authors’ response: It has been corrected in the text of the revised version.

25 – “non-conventional”, space not needed // Authors’ response: It has been corrected in the text of the revised version.

56 – “…Michaelis-Menten parameters based both on…”, “by” not needed // Authors’ response: It has been corrected in the text of the revised version.

84 – “evidently apparent” sounds strange, I would recommend using only “evident” or “apparent” // Authors’ response: It has been corrected in the text of the revised version.

181 – something is not right with this sentence. Maybe only the colon sign is not necessary here (it seems like it is not here on purpose), or maybe some part of the sentence is missing. Please check // Authors’ response: The sentence was omitted in the revised version. it was considered that it contributed nothing to the text..

193 – I think the sentence would be clearer if authors omitted “they”, only “values of both k-1_ppg and k-3_ppg approach zero” // Authors’ response: It has been corrected in the text of the revised version.

228 – acid should not be in capital letters // Authors’ response: It has been corrected in the text of the revised version.

238 – x not used in other parts of the text as multiplication sign, only here // Authors’ response: It has been corrected in the text of the revised version.

 

[1] Stergiou, P-Y.; Foukis, A.; Filippou, M.; Koukouritaki, M.; Parapouli, M.; Theodorou, L.G.; Hatziloukas, E.; Amalia Afendra, A.; Pandey, A.; Papamichael, E.M. Advances in lipase-catalyzed esterification reactions, Biotechnol. Adv. 2013, 31, 1846-1859.

[2] [Foukis, A.; Stergiou,P-Y.; Filippou, M.; Koukouritaki, M.; Parapouli, M.; Theodorou, L.G.; E Hatziloukas, E.; Afendra, A.; Pandey, A; Papamichael, E.M. Kinetic constraints and features imposed by the immobilization of enzymes onto solid matrices: A key to advanced biotransformation, Indian J. Exp. Biol. 2014, 52, 1045-1051, and references therein]

[3] Foukis, A., Gkini, O.A., Stergiou, P-Y., Papamichael, E.M. New insights and tools for the elucidation of lipase catalyzed esterification reaction mechanism in n-hexane: The synthesis of ethyl butyrate, Mol. Catal. 2018, 455, 159-163.

[4] Foukis, A., Gkini, O.A., Stergiou, P-Y., Papamichael, E.M. The mechanism of lipase-catalyzed synthesis of food flavoring ethyl butyrate in a solvent-free system, J. Food Nutrition and Metabolism, 2019, 2-7, DOI:10.31487/j.JFNM.2019.03.01.

[5] Papamichael, E.M.; Stergiou, P-Y. Mechanistic interpretation of esterification reactions catalyzed by immobilized lipase in nonconventional reaction media (tentative). ] Papamichael, E.M., University of Ioannina, Department of Chemistry, Enzyme Biotechnology and Genetic engineering group, Ioannina, Greece. 2025, (manuscript in preparation).

Reviewer 3 Report

Comments and Suggestions for Authors

This work reports reforms kinetics of immobilized enzymes in non-conventional media: A case of lipase catalyzed esterification. However, the novelty of this work is not clear. The authors should clearly and comprehensively explain the novelty of their work. Also, there are many clear English mistakes.

Despite that there are dozens of published works about the kinetics of the enzymatic esterification, however the authors have included are only 15 references. No paper from 2021, 2022, 2023, ad 2024 were cited.

Comments on the Quality of English Language

Extensive editing of English language required.

Author Response

Response to Reviewer 3:

We thank Reviewer 3 for his/her constructive comments and suggestions.

A general authors’ response: The text has been thoroughly revised by editing existing English language issues.

 

1. This work reports reforms kinetics of immobilized enzymes in non-conventional media: A case of lipase catalyzed esterification. However, the novelty of this work is not clear. The authors should clearly and comprehensively explain the novelty of their work. Also, there are many clear English mistakes.

Despite that there are dozens of published works about the kinetics of the enzymatic esterification, however the authors have included are only 15 references. No paper from 2021, 2022, 2023, ad 2024 were cited.:

 

Authors’ response:

As a matter of fact, a sufficient number of articles on topics related to enzyme kinetics have been published in the last four years. However, not all of them could be mentioned in the present manuscript. In this study, we developed simplified rate equations describing frequently encountered kinetic mechanisms of esterification reactions, which are catalyzed by immobilized lipase. We chose to simplify the rate equations related to esterifications that take place either in anhydrous aprotic and nonpolar organic solvents or in solvent-free systems. The reason for this choice was based on our experimental knowledge and on previously published results that are cited in this study. These aforementioned results and the resulting experience led us to reasonable and valid approximations toward the development of simplified rate equations, which are formulated in the present work. Of course, we conformed with the comments and suggestions of Reviewer 3, and four important papers reporting data from esterifications catalyzed by immobilized lipases are cited in the revised version of this manuscript. Two of them were published within years 2018-2019, whereas two others were published within years 2021-2023. In the earlier papers, the authors fitted their experimental data with simplified equations corresponding to either a reversible second-order kinetic model that includes apparent kinetic constants or a Michaelis-Menten equation that includes apparent parameters (maximum velocity and Michaelis constant). In the later works, the authors fitted their experimental data either with simplified equations corresponding to ping-pong bi-bi, ordered bi-bi and random bi-bi mechanisms (with double substrate inhibition) accepting approximations or with simplified equations corresponding to the ping-pong bi-bi mechanism (with triple dead-end substrate inhibition). In all four articles, the immobilized lipase-catalyzed esterifications were performed under anhydrous conditions in aprotic solvent and/or solvent-free systems. Nevertheless, in all these cases the simplifications of the rate equations were not systematic as in our study under review, and no experimental work is described to demonstrate which of the known mechanisms best fitted the experimental data, in contrast to the present study which is based on experimentally proven mechanisms. This latter, does not add value to this work under consideration, at first glance, however it contributes significantly to its novelty which is based not only on reasonable approximations but on proven approximations which give the added value to the present study as well. Furthermore, our methodological approach developed in this study can be used in a wider range of reactions catalyzed by immobilized enzymes under conditions that favor the formation of the desired product, being at the same time well informative for those scientists who are interested in having a simple and logical basis to control and compare their results leading to the evolution and progress of their work.

The text has undergone an extensive editing of English language. The novelty of the work is distinguishable in the Introduction, as well as within the whole text. However, it is better explained in the Conclusions of the revised version of this manuscript under review.

 

Comments on the Quality of English Language

Extensive editing of English language required.

Submission Date 11 September 2024

Date of this review 24 Sep 2024 17:05:41

Reviewer 4 Report

Comments and Suggestions for Authors

The paper “ Reformed kinetics of immobilized enzymes in non-conventional media: A case of lipase-catalyzed esterification ” presents the kinetics, mechanism, andsimplified equations. There are a few concerns that the author needs to incorporate.

1. In the Abstract the authors must mention what it meansto use non-conventional media to ease the understanding of the reader 2. In the introduction, the authors mention “environmentally friendly” building blocks….”. In brief mentions does it follow sustainable development goals SDGs? You may refer to the following literature.  3. In brief explain what is Michaelis Menten's kinetics and parameters in the introduction. 4. The authors must highlight the research gap in the introduction. 5. If authors can mention step-by-step schematic of the work as a methodology to ease readers' understanding. 6. What were the assumptions made during this study?  7. Some references are outdated please cite the latest references. 8. The authors must support results with appropriate references. 9. Kindly check for grammatical errors. 10. The discussion could be elaborated to highlight the main contribution of this study as it is a little bit difficult to capture what exactly is the intended objective.

 

Author Response

 

Response to Reviewer 4:

We thank Reviewer 4 for his/her constructive comments and suggestions.

A general authors’ response: The text has been thoroughly revised by editing existing English language issues.

The herein references, in [ ] brackets, are found at the end of this text.

 

1. In the Abstract the authors must mention what it means to use non-conventional media to ease the understanding of the reader

 

Authors’ response:

It is a formal term used in synthetic chemistry and biochemistry. As non-conventional media or non-conventional solvents are referred, among others, the non-aqueous and/or the solvent-free reaction media, as well as the solvents derived from biomass treatments and/or the ionic solvents, the eutectic solvents, etc. In this study under review the term non-conventional media is referred to either non-aqueous, non-polar and aprotic solvents or to solvent-free reaction media. We have made use of this term in earlier works of ours [1-3].

 

 

2. In the introduction, the authors mention “environmentally friendly” building blocks….”. In brief mentions does it follow sustainable development goals SDGs? You may refer to the following literature.

 

Authors’ response:

The referred “Subsequently, a remarkable economically and environmentally friendly manufacturing of building blocks was achieved providing esters (food additives) of high purity through the potential exploitation of various wastes, to a large extent, from food industries”, in the Introduction is related to the: (a) methods of esterification [1-3] providing the wasteless synthesis of esters (recyclable biocatalyst, solvent n-hexane, pure water), and (b) potential use of industrial wastes, where through acidogenesis were recovered useful short chain fatty acids and alcohols (e.g., n-butanol). To our knowledge at least the SDGs No. 3,7,9,and 13 are followed.

 

 

3. In brief explain what is Michaelis Menten's kinetics and parameters in the introduction

 

Authors’ response:

To our knowledge, Michaelis Menten kinetics is an elementary issue for biochemists and enzymologists. Similarly, the parameters of the Michaelis Menten equation are well-known and exhaustively investigated entities. We considered it would be at least boring for someone to read explanations in the introduction about these two very well-known topics.

 

 

4. The authors must highlight the research gap in the introduction.

 

Authors’ response:

In preparing this study under review, we did not intend to fill a research gap in this area. Our motivation was the increasing use of immobilized enzymes, both in academic research and industry. We intended to provide easily calculated and error-free estimated values of significant kinetic parameters, useful in biotechnological and industrial applications where the rapid acquisition of useful and important information is required. Therefore, we chose to develop this concept of simplification of multiparametric and multivariable model-equations in the area of esterification reactions catalyzed by immobilized lipase, having quite enough published results [1-3]. Attempts to simplify similar multiparametric and multivariable model-equations have been published by others [4-6]. However, in our study under review, a methodological approach is developed regarding the aim and scope of the concept of simplification. Appropriate simplifications should rely on reasonable approximations in order to be functional, adequate, appropriate and applicable according to the experimental conditions under consideration. These latter can be fulfilled if and only if they are based on experimentally documented results, in order to avoid as much as possible errors that might be included in the approximations.

 

 

5. If authors can mention step-by-step schematic of the work as a methodology to ease readers' understanding.

 

Authors’ response:

This study is the top of an applied methodology relying on a systematic approach. We carried out a modular research plan that included the combination of several analytical methods such as proton inventories, solvent isotopic effects, factorial experimental designs, surface fitting, and statistical evaluation of experimental results. These findings were then combined and constituted the basis for the development of simplifying assumptions and reasonable approximations, as well as for the implementation of this study under review.

 

 

6. What were the assumptions made during this study?

 

Authors’ response:

All assumptions are mentioned within the text in the place where are firstly referred. In summary, the assumptions are related to our concept to bypass complicated procedures, such as the surface fitting of experimental kinetic data. A methodologic approach like that applied in this study needs the evaluation of the functionality and adequacy of any suggested approximation to meet accuracy, validity and suitability, depending on the cases of its applicability. All the specific equations formulated to describe various cases of esterification processes (Appendix: Equations A1-A4) were successfully transformed into simplified forms involving only two parameters, namely k_cat and . All transformations correspond to the reaction conditions and were found to be successful regardless of the physical significance of .

All assumptions are described systematically within the subsection “3.4. The development of simplified rate equations”. General assumptions are based on the commonly accepted experimental logic to consider which relation holds true in the examined case, either [S] << K_m (any K_m) or [S] >> K_m. Next, for reaction comprising two substrates, assumptions on the relation of their total concentrations in the reaction should also be taken into account (e.g., [S₁]_t ≈ [S₂]_t, or [S₁]_t ≠ [S₂]_t). Moreover, relations as for example, [S₁]_t ≈ [S₂]_t << K_mS1, K_mS2 and/or [S₁]_t ≠ [S₂]_t >> K_mS1, K_mS2 should be considered too. Further assumptions should be based on the experientially estimated values of all parameters of the multiparametric and multivariable equations, by surface fitting [2.3]. These are the reasons that we have introduced in the subsection 3.4. the terms of “cases” and “subcases”, in order to cover all possible situations.

 

 

7. Some references are outdated please cite the latest references

 

Authors’ response:

In our view the results of pioneer studies are never become outdated. More likely Reviewer 4 is referred to King and Altman, J. Phys. Chem., 1956, 60, 1375-1378, and Cleland, W.W., Biochim. Biophys. Acta, Spec. Sect. Enzymol. Subj. 1963, 67, 104-137. Nevertheless, these two works are classic in the field of enzyme kinetics and they should be referred if their concept and results are used. On the other hand, we have incorporated in the revised version of this work under review, four new important papers.

 

 

8. The authors must support results with appropriate references.

 

Authors’ response:

All results of this study under review are supported by relevant references (6,9,10,12 in the revised version)

 

 

 

9. Kindly check for grammatical errors.

 

Authors’ response:

The text has been thoroughly edited and all existing English language issues have been corrected.

 

 

10. The discussion could be elaborated to highlight the main contribution of this study as it is a little bit difficult to capture what exactly is the intended objective.

 

Authors’ response:

The section Results and Discussion has been edited in the revised version of this work under review. We consider that the main contribution of this study, as well as its objectives were highlighted in the section Results and Discussion. For this reason, we incorporated the subsection “2.3. Validation of the applied approximations”

 

 

[1] Foukis, A., Gkini, O.A., Stergiou, P-Y., Sakkas, V.A, Dima, A., Boura, K., Koutinas, A., Papamichael, E.M. Sustainable production of a new generation biofuel by lipase-catalyzed esterification of fatty acids from liquid industrial waste biomass, Bioresour. Technol. 2017, 238, 122-128.

[2] Foukis, A., Gkini, O.A., Stergiou, P-Y., Papamichael, E.M. New insights and tools for the elucidation of lipase catalyzed esterification reaction mechanism in n-hexane: The synthesis of ethyl butyrate, Mol. Catal. 2018, 455, 159-163.

[3] Foukis, A., Gkini, O.A., Stergiou, P-Y., Papamichael, E.M. The mechanism of lipase-catalyzed synthesis of food flavoring ethyl butyrate in a solvent-free system, J. Food Nutrition and Metabolism, 2019, 2.

[4] Matsumoto, M.; Hasegawa, Y. Enzymatic Kinetics of Solvent-free Esterification with Bio-imprinted Lipase, Chem. Biochem. Eng. Q., 2019, 33, 495-499.

[5] Lorenzo, B.; Fernández, L.; Ortega, J.; Domínguez, L. Improvements in the Modeling and Kinetics Processes of the Enzymatic Synthesis of Pentyl Acetate, Processes 2023, 11, 1640.

[6] Dalla Costa, K.A.; Weschenfelder, T.A.; Steffens, C.; de Oliveira, D.; Cansian, R.L.; Dallago, R.M.; Zeni, J.; Paroul, N. Kinetic Study on Esterification of Ascorbyl Oleate Catalyzed by Lipase NS 88011, Biointerface Res. Appl. Chem. 2021, 11, 8374-8388.

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Comments and Suggestions for Authors

The paper “ Reformed kinetics of immobilized enzymes in non-conventional media: A case of lipase-catalyzed esterification ” presents the kinetics, mechanism, and simplified equations. There are a few concerns that the author needs to incorporate.

 

1. In the Abstract the authors must mention what it means to use non-conventional media to ease the understanding of the reader.
2. In the introduction, the authors mention “environmentally friendly” building blocks….”. In brief mentions does it follow sustainable development goals SDGs? You may refer to the following literature.
3. In brief explain what is Michaelis Menten's kinetics and parameters in the introduction.
4. The authors must highlight the research gap in the introduction.
5. If authors can mention step-by-step schematic of the work as a methodology to ease readers' understanding.
6. What were the assumptions made during this study?
7. Some references are outdated please cite the latest references.
8. The authors must support results with appropriate references.
9. Kindly check for grammatical errors.
10. The discussion could be elaborated to highlight the main contribution of this study as it is a little bit difficult to capture what exactly is the intended objective.

 

Submission Date 11 September 2024

Date of this review 16 Oct 2024 08:15:55

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

the Authors have responded properly to all comments.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Response to Reviewer 1:

We thank Reviewer 1 for his/her constructive comments and suggestions.

 

1. Dear Editor,

the Authors have responded properly to all comments.

Comments on the Quality of English Language

Minor editing of English language required.

 

Authors’ response:

The required English language editing of the manuscript has been carried out.

In addition, the section “Conclusions” has been thoroughly revised.

Reviewer 3 Report

Comments and Suggestions for Authors

1. Generally, conclusion should be provided without citations. You can refer to those cited papers in the discussion section. 

2. The following paper may be useful to be cited after the first sentence in the introduction section, or whenever possible "Has the time finally come for green oleochemicals and biodiesel production using large-scale enzyme technologies? Current status and new developments"

Comments on the Quality of English Language

a clean version of the manuscript should be provided, it is not easy to follow the quality of English in the current tracked version. 

Author Response

Response to Reviewer 3:

We thank Reviewer 3 for his/her constructive comments and suggestions.

 

1. Generally, conclusion should be provided without citations. You can refer to those cited papers in the discussion section

 

Authors’ response:

The “Conclusions” section has been thoroughly revised. In addition, all references have been deleted in accordance with the comments and suggestions of the Reviewer 3.

 

 

2. The following paper may be useful to be cited after the first sentence in the introduction section, or whenever possible "Has the time finally come for green oleochemicals and biodiesel production using large-scale enzyme technologies? Current status and new developments"

 

Authors’ response:

In accordance with the comments and suggestions of the Reviewer 3, the paper entitled "Has the time finally come for green oleochemicals and biodiesel production using large-scale enzyme technologies? Current status and new developments", has been cited after the first sentence, in the section Introduction.

 

 

3. A clean version of the manuscript should be provided, it is not easy to follow the quality of English in the current tracked version

 

Authors’ response:

A thorough English language editing of the manuscript was carried out.

Author Response File: Author Response.pdf