Review Reports

Round 1

Reviewer 1 Report

Dear authors, a manuscript entitled “Enzymatic Poly(octamethylene suberate) Synthesis in Two-step Polymerization Based on the New Greener Polymer-5B Technology describe developing a new concept strategy of a greener method for polymers synthesis. The work has the potential to be published in Processes. However, in its present form is hard to read and some doubts need clarification.

 

• Line 15-16. The clear aim of study without experimental details, pH concentration will be a better introduction to our abstract.
• Line 20 (bulk polymerysation) instead of (Bulk polymerisation).
• Line 22-34. The abstract shouldn’t contain experimental details. The highlighted part is chaotic and should be rewritten with focusing on the main findings without experimental details. The end of the abstract should be finished with a short conclusion.
• Line 38; The keywords contain typos.
• Line 45; Does only hydrolases are able to degrade chemical bonds in synthetic polymers?
• Line 113-128; The aim of the study should be possible short and enough informative for readers to understand the needs of our research. The experimental details are in materials and methods sections that are enough and correct.
• Line 143; was dissolved…. in buffer water?
• In materials and methods, the schema that will shortly show the general concept of the reported study should be added.
• Line 177; Authors performed reactions at45oC sometimes 60-80oC. Why any results for optimal temperature or pH aren’t presented. The free and immobilized catalyst can exhibit different resistance to higher temperatures and of course temperature optima. Moreover, some of the used preparation are enzymes immobilized by simple adsorption and can be liberated from the carrier by higher temperatures or other properties of reaction media (pH, ionic strange). Does the residual activity after filtration was checked?
• Line 309-311; Authors state a very original hypothesis that could be confirmed by a simple desorption study with the use of some ionic agent and then check activity in obtained medium.
• Line 330-331; Authors should not speculate without clear reference or results. This kind of discussion should be avoided. The enzyme molecular structure in active form is usually optimal to work in an environment that contains water. However, some lipases work very well in deep eutectic solvents or anionic liquids but in this case, catalyze transesterification reaction. The substrate solubility and occurrence of nonionic detergents can be also factors for the opening of its active site and higher activity.

Stauch B, Fisher SJ, Cianci M. Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation. J Lipid Res. 2015 Dec;56(12):2348-5

Magalhães RP, Cunha JM, Sousa SF. Perspectives on the Role of Enzymatic Biocatalysis for the Degradation of Plastic PET. Int J Mol Sci. 2021 Oct 19;22(20):11257

 

• The manuscript needs an English native speaker intervention. The style also should be corrected.

Author Response

Answer to Review 1:

Dear authors, a manuscript entitled "Enzymatic Poly(octamethylene suberate) Synthesis in Two-step Polymerization based on the New Greener Polymer-5B Technology describes developing a new concept strategy of a greener method for polymer synthesis. The work has the potential to be published in Processes. However, in its present form is hard to read and some doubts need clarification.

• Line 15-16. The clear aim of study without experimental details, pH concentration will be a better introduction to our abstract.

 

Answer: The abstract was corrected to focus on the new concept of the two-step polymerization strategy.

 

• Line 20 (bulk polymerysation) instead of (Bulk polymerisation).

 

Answer: The correction was performed in the reviewed manuscript.

 

• Line 22-34. The abstract shouldn't contain experimental details. The highlighted part is chaotic and should be rewritten with focusing on the main findings without experimental details. The end of the abstract should be finished with a short conclusion.

 

Answer: The abstract was modified according to the reviewer´s suggestion, and a short conclusion was included at the end about the novelty and benefits that the two-step polymerization can bring to the polyester synthesis.

 

• Line 38; The keywords contain typos.

 

Answer: The keywords were corrected.

 

• Line 45; Does only hydrolases are able to degrade chemical bonds in synthetic polymers?

 

Answer: In the original manuscript (line 45) the authors did not say that only hydrolases are able to degrade chemical bonds. Of course, these bonds can be hydrolyzed by chemical catalysts and other enzymes.

 

• Line 113-128; The aim of the study should be possible short and enough informative for readers to understand the needs of our research. The experimental details are in materials and methods sections that are enough and correct.

 

Answer: The aim of the study was shorted, and special attention was gaven to the innovative concept of the two-step polymerization strategy. Additionally, for a better understanding of the readers was also established links with the patent of Polymer-5B technology ([18]) and previous work published by authors in this journal (Processes 2021, 9(2), 365) [17]).

 

• Line 143; was dissolved.... in buffer water?

 

Answer: Yes, the PBL enzyme was solubilized entirely in the polymerization media composed of water with pH corrected previously to 5.0 with NaOH solution. The information was included in the manuscript.

 

• In materials and methods, the schema that will shortly show the general concept of the reported study should be added.

 

Answer: A new schema (Figure 1) was added to the reviewed manuscript to illustrate the two-step polymerization strategy general concept.

 

• Line 177; Authors performed reactions at45oC sometimes 60-80oC. Why any results for optimal temperature or pH aren't presented. The free and immobilized catalyst can exhibit different resistance to higher temperatures and of course temperature optima. Moreover, some of the used preparation are enzymes immobilized by simple adsorption and can be liberated from the carrier by higher temperatures or other properties of reaction media (pH, ionic strange). Does the residual activity after filtration was checked?

 

Answer: The temperature used in the polymer synthesis was studied and optimized in previous work published in (Processes 2021, 9(2), 365) [17]) and associated with the stability of the biocatalyst (IMM-PBLI) in the synthesis of polyesters in miniemulsiom, water, and the organic solvent. Statements for selected temperatures were added in the Results and discussion of 45ºC in 1^st step (lines 379 – 381) and 60 or 80ºC in 2^nd step (lines 391-393). Additionally, it was not observed activity in the filtrates collected after the polymerization at 65ºC in miniemulsion, water, and organic solvent.

 

• Line 309-311; Authors state a very original hypothesis that could be confirmed by a simple desorption study with the use of some ionic agent and then check activity in obtained medium.

 

Answer: In previous work (Processes 2021, 9(2), 365) [17]), the results proved that did not occur linkage of the adsorbed enzyme from solid support in the filtrates collected after the polymerization at 65ºC confirmed by ¹H NMR inclusively for the experiments using organic solvent and miniemulsion which polymerization media contain a 1.6% of surfactant (Triton- X100).

 

• Line 330-331; Authors should not speculate without clear reference or results. This kind of discussion should be avoided. The enzyme molecular structure in active form is usually optimal to work in an environment that contains water. However, some lipases work very well in deep eutectic solvents or anionic liquids but in this case, catalyze transesterification reaction. The substrate solubility and occurrence of nonionic detergents can be also factors for the opening of its active site and higher activity.

 

Answer: In previous work (Processes 2021, 9(2), 365) [17]), it was shown that polyester synthesis was not performed by common free lipases like Candida rugosa lipase and Burkholderia cepacian in water. However, these enzymes function and synthesize polyesters in miniemulsion, but this ability was lost when those common lipases were immobilized in solid supports. In opposition, high efficiency of polyester synthesis in water and miniemulsion was observed for PBL (previously named CAL B) in both the free form and immobilized enzyme preparations.

This work extended the efficient polyester synthesis in water of other commercial Cal B immobilized preparations and one commercial immobilized esterase (Table 2 of this work). The high efficiency of PBL and the esterase in the polyester synthesis in water is due to these enzymes are not characterized by the need for interfacial activation mechanism proposed by other authors as observed by us for the common lipases like Candida rugosa lipase and Burkholderia cepacian and previously published (Processes 2021, 9(2), 365) [17]).

 

 

• The manuscript needs an English native speaker intervention. The style also should be corrected.

 

The English language and style were edited and reviewed by English native speakers as identified in the Acknowledgments.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors in their original research article presented the possibility of the enzymatic synthesis of poly(octamethylene suberate). Three different media, i.e., water, miniemulsion or mixture of organic solvents were compared. Moreover, the polymerization was carried out in a two-step process with direct polycondensation and incubation of wet prepolymers. In my opinion, the manuscript fits the aim and scope of the Processes journal. Some minor suggestions are presented below.

Minor suggestions:

1. The abstract is too long, and according to the instruction for authors, it should be no longer than 200 words.
2. I propose to add a statement of the novelty to the manuscript. The aim of the work may be enriched with a few sentences showing the novelty of this approach and this work.
3. In the second formula, the multiplication sign should be corrected.
4. The reference list should be improved according to the instruction for authors, as well as, in some parts of the manuscript citation style should be corrected, e.g., lines 114, 303.

Author Response

Answer to Review 2:

 

The authors in their original research article presented the possibility of the enzymatic synthesis of poly(octamethylene suberate). There different media, i.e., water, miniemulsion or mixture of organic solvents were compared. Moreover, the polymerization was carried out in a two-step process with direct polycondensation and incubation of the wet prepolymers. In my opinion, the manuscript fits the aim and scope of the Processes Journal. Some minor suggestions are presented below.

 

Minor suggestions:

• The abstract is too long, and according to the instruction for authors, it should be no longer than 200 words.

 

Answer: The abstract was reduced according to review´s suggestion and rules of the journal.

 

• I propose to add a statement of the novelty to the manuscript. The aim of the work may be enriched with a few sentences showing the novelty of this approach and this work.

 

Answer: Statements about the novelty concept of the two-step polymerization developed in this work were added to the Abstract (lines 31-32), to Introduction , and Conclusions .

 

• In the second formula, the multiplication sign should be corrected.

 

Answer: The correction was performed.

 

• The reference list should be improved according to the instruction for authors, as well as, in some parts of the manuscript citation style should be corrected, e.g., lines 114, 303.

 

Answer: The reference list was improved according to the instruction for authors.

Author Response File: Author Response.docx

Reviewer 3 Report

This paper developed a greener polymerization process by enzymatic poly(octamethylene suberate synthesis. Overall, the results of tests are reasonable and correct, which can be published after a minor revision. The problems with the current paper is commented below:

1. Introduction could be improved to add some background and literature reviews, and there are too many paragraphs in introduction.
2.  In a typical drying process, the 2nd step of enzymatic polymerization of these wet prepolymers was carried out without stirring in an oven, under a moderate temperature range between 60 and 80 ℃ needed for the fusion of POS synthesized in 1st step. Please explain why choose the temperature range between 60 and 80 ℃.
3. It seems that Fig. 2 can be changed into a bar chart.
4. All figures should be mentioned with legends individually.
5. Please ensure high quality of the figures.

      6. Units and References should be arranged according to rules of the Journal. 

 

Author Response

Answer to Review 3:

 

This paper developed a greener polymerization process by enzymatic poly(octamethylene suberate synthesis. Overall, the results of tests are reasonable and correct, which can be published after a minor revision. The problems with the current paper is commented below:

 

• Introduction could be improved to add some background and literature reviews, and there are too many paragraphs in introduction.

 

Answer: Some literature reviews were added to the Introduction improving the background in the field of polyester synthesis and, consequently, the number of references increased from 30 to 39.

 

• In a typical drying process, the 2nd step of enzymatic polymerization of these wet prepolymers was carried out without stirring in an oven, under a moderate temperature range between 60 and 80 °C needed for the fusion of POS synthesized in 1st step. Please explain why choose the temperature range between 60 and 80 °C.

 

Answer: The temperature range between 60 and 80^oC was selected as the authors previously observed high stability of the IMM-PBLI biocatalyst when POS synthesis was performed at 65^oC in miniemulsion and water compared with lower stability in the organic solvent. This statement was added to the Results and discussion (lines 391 - 393), including the reference (Processes 2021, 9(2), 365) [17]) where this observation was published.

 

• It seems that Fig. 2 can be changed into a bar chart.

 

Answer: We do not understand this comment as the figure is already a bar chart and, for this reason, the figure was kept without modification.

 

• All figures should be mentioned with legends individually.

 

Answer: According to the reviewer's suggestion, a file with individual figures and respective legends was loaded with the reviewed manuscript.

 

• Please ensure high quality of the figures

 

Answer: The figures seem to have enough quality, but they were improved.

 

• Units and References should be arranged according to rules of the Journal.

 

Answer: The units and references presented along the text were reviewed and modified according to the journal's rules.

The English language and style were edited and reviewed by English native speakers as identified in the Acknowledgments.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear authors, the manuscript was significantly improved.  The quality of some figures e.g Fig1 is poor and before publication should be improved.

Author Response

Figure 1, Figure 6, and Figure 8 were improved. The new versions were incorporated into the manuscript. Please find in the annexed Reply to Review 1.pdf file old and new versions of Figure 1, Figure 6, and Figure 8.

Author Response File: Author Response.pdf