The Studies of Sepharose-Immobilized Lipases: Combining Techniques for the Enhancement of Activity and Thermal Stability

Round 1

Reviewer 1 Report

The paper shows some studies of immobilized lipases in liquid forms as an atempt to unify the storage stability assessment. Although the topic of lipase immobilization is interesting and has great potential for application, this study does not present a great contribution to the literature, as the CALB and CRL system immobilized on octyl agarose has already been explored in many articles. Furthermore, the stability data of the immobilized enzymes, mainly in different buffers and conditions, are not accompanied by the respective soluble enzymes, which is very important to verify whether the immobilization was successful or not, in economic terms. Another important point is that the reusability of the biocatalyst was not tested.

Other observations from the text are mentioned below for better presentation of the theme and results.

Title: I suggest changes in the Title, since the term “studies of immobilized lipases in liquid forms” do not seem adequate. The immobilization process on octyl-agarose generates solid biocatalysts.

Please explain why the ionic force 100 mM was chosen to immobilize CALB. Many papers in literature suggest that immobilizations at low ionic force (5 mM) favor the hydrophobic interactions between enzyme and support in interfacial activation process of lipases.

Line 179: The authors says that the immobilization was performed at three different temperatures, 4°C, 22°C, and 37°C. Where are the results of immobilization efficiency at these immobilization conditions? The free lipase remains 100 % stable at 37 °C during the immobilization process? Please, clarify these points.

Line 227: The authors says that the application of the developed procedure allowed us to obtain the activity of immobilized CRL-OF 37.65± 2.84 U. Please, show all results of immobilization efficiency with incubation time. The 14h incubation time will not necessarily be the most efficient for CRL.

Table 3: I suggest the separation of CALB and CRL data in two Tables. Many data in only one Table seems disorganized and difficult to read and identify the data.

Table 3 – The authors should perform the experiment using soluble lipases to compare with the immobilized lipases data.

The authors measured na storage stability at 65º C, but in my opinion, this is not storage stability, as commercial immobilized enzymes are stored at room temperature or refrigerated at 4º C. In my view, these are thermal stability data

Author Response

Dear Reviewer,

thank you for your comments.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript 2348356 reports CALB and CRL-OF immobilized on the Octyl-sepharose CL-4B matrix and analyze stability in the climatic chambers in presence of various solvents. However, the full text doesn’t organize logically and methods and results need to be revised before acceptance. This manuscript can be considered after major corrections. The following comments are in detail for the authors to consider.

1.      Title: I do not agree with the storage stability only measuring the activity observation only seven days periods. It may belong to others’ stability like authors Ref 65 Climatic Chamber stability. The stability analysis doesn’t belong to the practical application of enzyme storage. For instance, no one stores enzymes at 65 °C or visible spectral conditions.  

2.      Introduction, line 56, the activity of free enzymes may unsatisfactory which is very misleading statement.

3.      Introduction, Table 1, Ref. 20, doesn’t discuss isoelectric point. Please check this ref. Please check other references also.

4.      Introduction, line 96, authors claim storage stability in the liquid phase is a new method. They already published similar work in the Catalysts journal (Ref. 65). In line 364, Ref 66, Chiaradia et al. analyzed storage stability by keeping CALB in phosphate buffer.

5.      The introduction should revise to focus on some stability analysis and then why this work is important.    

6.      Most of the methods section is copied from authors’ past work (Ref 65) which is one kind of plagiarism. These sections need to be rewritten.

7.      Line 468-471 are unnecessary because this information is provided in the following sections.

8.      Line 480, for stability analysis, why lipase immobilization in citrate buffer? Why not use phosphate buffer immobilized lipase?

9.      In overall, please concise method sections.

10.  In the result and discussion, there are a lot of reference works reported that unnecessarily enlarge this section and it is difficult to find out this article’s work. It seems the discussion is like a review paper. I would suggest extensive revision here. Keep only relevant references and highlights your works.

11.  If possible compare stability analysis using Table 1.

12.  You can consider to circular dichroism (CD) analysis for enzyme secondary structure analysis.

13.  For more authenticity, you should consider FTIR spectrum/XPS surface analysis spectrum data that some data comes from machines.

14.  Authurs used a total of 10 self-citation and used many unnecessary ref to avoid self-citation. For instance, Line 518 and in the conclusion.       

The manuscript 2348356 reports CALB and CRL-OF immobilized on the Octyl-sepharose CL-4B matrix and analyze stability in the climatic chambers in presence of various solvents. However, the full text doesn’t organize logically and methods and results need to be revised before acceptance. This manuscript can be considered after major corrections. The following comments are in detail for the authors to consider.

1.      Title: I do not agree with the storage stability only measuring the activity observation only seven days periods. It may belong to others’ stability like authors Ref 65 Climatic Chamber stability. The stability analysis doesn’t belong to the practical application of enzyme storage. For instance, no one stores enzymes at 65 °C or visible spectral conditions.  

2.      Introduction, line 56, the activity of free enzymes may unsatisfactory which is very misleading statement.

3.      Introduction, Table 1, Ref. 20, doesn’t discuss isoelectric point. Please check this ref. Please check other references also.

4.      Introduction, line 96, authors claim storage stability in the liquid phase is a new method. They already published similar work in the Catalysts journal (Ref. 65). In line 364, Ref 66, Chiaradia et al. analyzed storage stability by keeping CALB in phosphate buffer.

5.      The introduction should revise to focus on some stability analysis and then why this work is important.    

6.      Most of the methods section is copied from authors’ past work (Ref 65) which is one kind of plagiarism. These sections need to be rewritten.

7.      Line 468-471 are unnecessary because this information is provided in the following sections.

8.      Line 480, for stability analysis, why lipase immobilization in citrate buffer? Why not use phosphate buffer immobilized lipase?

9.      In overall, please concise method sections.

10.  In the result and discussion, there are a lot of reference works reported that unnecessarily enlarge this section and it is difficult to find out this article’s work. It seems the discussion is like a review paper. I would suggest extensive revision here. Keep only relevant references and highlights your works.

11.  If possible compare stability analysis using Table 1.

12.  You can consider to circular dichroism (CD) analysis for enzyme secondary structure analysis.

13.  For more authenticity, you should consider FTIR spectrum/XPS surface analysis spectrum data that some data comes from machines.

14.  Authurs used a total of 10 self-citation and used many unnecessary ref to avoid self-citation. For instance, Line 518 and in the conclusion.        

Author Response

Dear Reviewer,

thank you for your comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

The article highlights the importance to evaluate the storage stability of two well-known lipases such as CRL and CAL-B by. In this case, the proposed methodology is based on the use of a climatic chamber after enzyme immobilization. Thus, the immobilizations of both lipases were developed onto an Octyl-Sepharose carrier, and the immobilization parameters were correctly displayed and analyzed. The discussion is described in many details since the article focus is the evaluation of the enzyme stabilities, but it is fair that a good methodology assure that the latest results will have strong basis.

1) Line 273: The concept “immobilized lipases in liquid form” is misleading. This need to be clarified, also attempts appear in line 290.

2) The selection of CAL-B and CRL need to be justified in the aims of the work, and the advantages of this methodology to be applied for other hydrolases (not only lipases) can be addressed in the conclusion section

3) The abbreviation DCP (1,2-dichloropropane) must appear before line 306, possibly in page 7

4) Full details and picture of the climatic chamber needs to be provided in the manuscript.

5) Lipase-catalyzed reaction can be performed in aqueous, organic and neoteric solvents. I have missed a description of lipase stabilities in green solvents such as deep eutectic solvents, 2-methyltetrahydrofuran or cyclopentylmethyl ether, rather than focusing on the use of a few buffers (pH 4 and 7.5).

6) The reference section needs to be carefully revised since

6.1.) There are many citations that are superfluous, especially in the introduction see some examples were perennial reviews can be included instead of :

a) Lines 37-38: “Despite the powerful position of chemical synthesis, alternative reactions using enzymatic catalysts are earning more interest from researchers [3].

[3] Panic, M.; Radovic, M.; Maros, I.; Tusek, A.J.; Bubalo, M.C.; Redovnikovic, I.R. Development of environmentally friendly lipase catalysed kinetic resolution of (R,S)-1-phenylethyl acetate using aqueous natural deep eutectic solvents, Process Biochem. 2021, 599 102, 1-9.

This an specific example of the use of DES in lipase-catalyzed kintetic resolution transformations.

b) Lines 38-40: “Due to the milder reaction conditions, the possibility of reuse, and environmental friendliness, biocatalysis has advantages over chemical reactions [4, 5]. “

[5] Kourist, R.; de Maria, P.D.; Miyamoto, K. Biocatalytic strategies for the asymmetric synthesis of profens - recent trends and developments, Green Chem. 2011, 13, 2607-2618.

This a review regarding enzyme-catalyzed profen synthesis.

c) Lines 40-42: It should be noted that the efficiency of reactions catalyzed by enzymes differs and requires many improvements and modifications [6].

[6] Yan, H.D.; Guo, B.H.; Wang, Z.; Qian, J.Q. Surfactant-modified Aspergillus oryzae lipase as a highly active and enantioselective catalyst for the kinetic resolution of (RS)-1-phenylethanol, 3 Biotech, 2019, 9, 9.

This is a specific example dealing with the use of surfactants to modify a determined lipase behaviour.

d) Lines 43-44: Lipases, belonging to hydrolases, are a meaningful group of enzymes used in reactions of pharmaceutical importance [1, 7, 8].

[7] Siódmiak, T.; Ziegler-Borowska, M.; Marszałł, M.P. Lipase-immobilized magnetic chitosan nanoparticles for kinetic resolution of(R,S)-ibuprofen. J. Mol. Catal. B Enzym. 2013, 94, 7–14.

Unnecessary self-citation is not needed, there are so many reviews in the field dealing with the use of lipases for the synthesis of pharmaceuticals.

e) Lines 49-50: One of the best-known and most widely applied lipases are lipase B from Candida antarctica (CALB) and lipase from Candida rugosa (CRL) [12, 13].

[13] Cen, Y.X.; Li, D.Y.; Xu, J.; Wu, Q.S.; Wu, Q.; Lin, X.F. Highly Focused Library-Based Engineering of Candida antarctica Lipase B 619 with (S)-Selectivity Towards sec-Alcohols, Adv. Synth. Catal. 2019, 361, 126-134.

There have been published many reviews in the last two decades highlighting the importance of CALB, the reference 13 does not make any sense here.

…and I can continue with many other items. The authors must made efforts to collect important reviews in the introduction that are of interest for the audience, and focus on specific examples in the results and discussion section that would help to interpretate some achievements. Also, the authors must avoid superfluous self-citation in some points.

6.2.) There are many references wrongly cited, especially from MDPI journals because they have article numbers instead of page numbers, see for instance:

[18] Catalysts, 2020, 10, 876 instead of Catalysts, 2020, 10, 876-896

[38] Molecules, 2016, 21, 1577 instead of Molecules, 2016, 21, 1577–1601

Same for references: 39, 40, 41. 65 and 75 among others

[22] Biotechnol. Bioeng. instead of Biotechnology and Bioengineering

[62 and 70] Avoid the use of capital letters for article title.

Overall, I can recommend publication in Catalysts although I do not find many novelty on the evaluation of enzyme stability without providing new items such as the use of these enzymes in determined reactions or their application in green solvents.

The article reachs the standards for publication in terms of English language.

Author Response

Dear Reviewer,

thank you for your comments.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors properly revised the manuscript. Before the paper publication, some points need to clarify:

The authors studied the influence of the various solvents (water, buffer, 1,2- 100 dichloropropane), temperature, light in the visible spectral range (400-800 nm), and addition of calcium ions on the storage stability of biocatalysts. In this context, the authors need to justify why these solvents, temperatures were chosen for the study.

In addition, it is importante to mention that the influence of calcium ions in the immobilization media has already been discussed in other papers (see the references below) and the calcium ions influence depend on the enzyme tested.

Yuliya Lokha, Sara Arana-Peña, Nathalia S. Rios, Carmen Mendez-Sanchez, Luciana R.B. Gonçalves, Fernando Lopez-Gallego, Roberto Fernandez-Lafuente, Modulating the properties of the lipase from Thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions, Enzyme and Microbial Technology, 133, 2020, 109461, ISSN 0141-0229, https://doi.org/10.1016/j.enzmictec.2019.109461.

Sara Arana-Peña, Nathalia S. Rios, Diego Carballares, Luciana R.B. Gonçalves, Roberto Fernandez-Lafuente, Immobilization of lipases via interfacial activation on hydrophobic supports: Production of biocatalysts libraries by altering the immobilization conditions, Catalysis Today, 362, 2021, Pages 130-140, ISSN 0920-5861, https://doi.org/10.1016/j.cattod.2020.03.059.

L. Fernandez-Lopez, N. Rueda, R. Bartolome-Cabrero, M.D. Rodriguez, T.L. Albuquerque, J.C.S. Dos Santos, O. Barbosa, R. Fernandez-Lafuente Improved immobilization and stabilization of lipase from Rhizomucor miehei on octyl-glyoxyl agarose beads by using CaCl2 Process Biochem., 51 (2016), pp. 48-52

Author Response

Dear Reviewer 1

I would like to thank you very much for thoroughly reading the manuscript, issuing an opinion, and presenting valuable insights on quality.

The authors properly revised the manuscript.

Thank you for your comment.

Before the paper publication, some points need to clarify:

The authors studied the influence of the various solvents (water, buffer, 1,2- 100 dichloropropane), temperature, light in the visible spectral range (400-800 nm), and addition of calcium ions on the storage stability of biocatalysts. In this context, the authors need to justify why these solvents, temperatures were chosen for the study.

Thank you for your comment.

The justification for the choice of solvents

In the previous paper (Climatic Chamber Stability Tests of Lipase-Catalytic Octyl-Sepharose Systems) we described immobilized lipases stored in a dry form in a climatic chamber. While, in the currently reviewed work, we wanted to present how the thermal stability and activity of immobilized lipases in aqueous solutions (buffers) will be affected by the storage them in the climatic chamber. Water was chosen as the reference in the study, while citrate buffer (pH 4, 500 mM) was selected based on our preliminary study exhibiting that this buffer is optimal for lipase and octyl-sepharose (results of the study of the effect of buffer and ionic strength used under immobilization were described in paper Climatic Chamber Stability Tests of Lipase-Catalytic Octyl-Sepharose Systems). Whereas, by adding 1,2-dichloropropane (DCP), we wanted to examine how resistant the system (immobilized lipase) is to the influence of an organic solvent. In the next step, we will test the enantioselective activity of the lipase in organic solvents, and this study (described in this paper) is a preliminary assessment of whether the immobilized lipase will be catalytic active in a non-aqueous environment (organic solvents).

The justification for the choice of light in the visible spectral range (400-800 nm)

The paper attempts to standardize the conditions of immobilized lipase testing. Studies on the influence of light (photostability) in this range were supposed to show whether the created catalytic system (immobilized lipase) is resistant to visible light, whether it should be protected from light during storage, or whether it would be necessary to protect it from light when performing in reaction (e.g. kinetic resolution). On the other hand, another assumption was to see if exposure to light would affect lipase activity. It has been shown that the VIS range has no negative impact on the stability of the tested CALB. Photostability tests were performed based on the guidelines of the International Conference on Harmonization (ICH): ICH Topic Q 1 A (R2) Stability Testing of new Drug Substances and Products; ICH Topic Q1B Photostability Testing of New Active Substances and Medicinal Products.

The justification for the choice of temperature

Thermal stability tests were carried out in accordance with ICH guidelines under stress conditions - stress testing (ICH Topic Q 1 A (R2) Stability Testing of new Drug Substances and Products). A high temperature of 65°C was chosen to evaluate how it affects the activity and stability of immobilized lipases. A temperature of 4°C was selected to compare the results. This low temperature creates reference conditions for the results obtained in the climatic chamber.

The justification for the choice of Ca²⁺

One of the assumptions in the studies was to enhance the activity and stability of the immobilized lipase. Based on our preliminary research and literature data, calcium ions were selected as having a potentially positive effect. The aim was to check two issues: first, whether the tested ions would have a positive effect when a citrate buffer with a low pH - 4 and a high ionic strength - 500mM is used. The influence of calcium ions on the activity of CALB (immobilized) suspended in a buffer with a low pH and high ionic strength is insufficiently described in the literature. The second issue was to check whether using the substrate - olive oil, we will get an enhancement of the catalytic activity. Literature data indicate that activity may alter depending on the substrate. We indicated a significant enhancement of activity when these ions were added to the citrate buffer.

In addition, it is importante to mention that the influence of calcium ions in the immobilization media has already been discussed in other papers (see the references below) and the calcium ions influence depend on the enzyme tested.

Yuliya Lokha, Sara Arana-Peña, Nathalia S. Rios, Carmen Mendez-Sanchez, Luciana R.B. Gonçalves, Fernando Lopez-Gallego, Roberto Fernandez-Lafuente, Modulating the properties of the lipase from Thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions, Enzyme and Microbial Technology, 133, 2020, 109461, ISSN 0141-0229, https://doi.org/10.1016/j.enzmictec.2019.109461.

Sara Arana-Peña, Nathalia S. Rios, Diego Carballares, Luciana R.B. Gonçalves, Roberto Fernandez-Lafuente, Immobilization of lipases via interfacial activation on hydrophobic supports: Production of biocatalysts libraries by altering the immobilization conditions, Catalysis Today, 362, 2021, Pages 130-140, ISSN 0920-5861, https://doi.org/10.1016/j.cattod.2020.03.059.

L. Fernandez-Lopez, N. Rueda, R. Bartolome-Cabrero, M.D. Rodriguez, T.L. Albuquerque, J.C.S. Dos Santos, O. Barbosa, R. Fernandez-Lafuente Improved immobilization and stabilization of lipase from Rhizomucor miehei on octyl-glyoxyl agarose beads by using CaCl2 Process Biochem., 51 (2016), pp. 48-52

Thank you for your comment. We included these publications in the discussion. Literature data are not consistent as to whether calcium ions have an undoubtedly positive, neutral, or negative effect on CALB. Therefore, we wanted to test lipase in our catalytic reaction. We suspended immobilized lipase (CALB) in citrate buffer with pH 4 and high ionic strength with the addition of calcium ions. The purpose was to check if we could obtain enhancement of activity and stability. Thanks to the use of calcium ions, we achieved an increase in the activity and stability of the tested lipase, which was our intention. Studies at a low pH and a high ionic strength are not sufficiently described in the literature.

Changes in the manuscript are color-coded:

yellow - corrections after the 1st round

green - corrections after the 2nd round

Author Response File: Author Response.pdf

Reviewer 2 Report

Revised manuscript is more richer than version 1 where author included free enzymes activity, immobilization efficiency and revised part of the manuscript. However, it still need to rigorous revision to accept it. The title still does not fully present the outcome of this study. Although the novelty of this study is not convincing, the content may somehow create interest to reader. But it requires extensive revision specially Results and Discussion to readable to reader. This section still has superfluous citation that ultimately hide the study outcome. I don't recommend to accept this manuscript in current format.        

Extensive revision requires to readability of this manuscript.

Author Response

Dear Reviewer 2,

I would like to thank you very much for thoroughly reading the manuscript, issuing an opinion, and presenting valuable insights on quality. The suggested changes were included in the revised version of the manuscript.

1. Revised manuscript is more richer than version 1 where author included free enzymes activity, immobilization efficiency and revised part of the manuscript. However, it still need to rigorous revision to accept it.

Thank you for your comment.

2. The title still does not fully present the outcome of this study.

Thank you for your comment. The title has been modified.

Previous title:

The studies of sepharose-immobilized lipases as an attempt to unify the thermal stability assessment

Modified (current) title:

The studies of sepharose-immobilized lipases: combining techniques for the enhancement of activity and thermal stability

3. Although the novelty of this study is not convincing, the content may somehow create interest to reader. But it requires extensive revision specially Results and Discussion to readable to reader. This section still has superfluous citation that ultimately hide the study outcome. I don't recommend to accept this manuscript in current format.   

Thank you for your comment. The sections: Introduction and Results and Discussion have been corrected/ revised. The superfluous citations were deleted.

Changes in the manuscript are color-coded:

yellow - corrections after the 1st round

green - corrections after the 2nd round

4. Comments on the Quality of English Language: Extensive revision requires to readability of this manuscript.

The revision of the manuscript has been made.

Author Response File: Author Response.pdf