Round 1

Reviewer 1 Report

Specific comments are listed as follows.

1. P2, line 53: the abbreviation word “CH” in first appearance should be provided the full name.

2. P3, line 94: “bioprinted LBC” should be “bioprinted BCL”.

3. Figure 1: The productivity of unprinted lipases (including BCL, CRL and PPL) should be provided as blank control.

4. Figures 2~4; The productivity of unprinted BCL should be provided as blank control.

5. P5, line 154: What is “the bBCL” referring to?

6. In case of 50 W, the printed BCL showed higher productivity with decreasing of carbon length of fatty acid within 25 min, while in case of 100 W and 200 W, the oppose results were found for printed BCL. Why?

7. Table 1~3: please provide the intensity of MW irradiation for the secondary structure by FTIR of lipases.

8. Table 3: As seen in table 3, the change of α-helix of lipases is irregular. Please clarify the interrelation ship between productivity and secondary structure of BCL printed with palmitic acid at different time.

9. Figures 5 and 6: How about the stability of unprinted BCL?

Author Response

Aracaju, January 18, 2023

 

 

Dear Editor of Catalysts,

 

Enclosed please find the revised version of the manuscript entitled “Use of bioprinted lipases in Microwave-assisted Esterification Reactions” by Melo et al. to be considered for publication in your journal as original article. The changes were introduced in the manuscript, considering the comments and suggestions of the reviewers. The English correction was performed by https://www.proof-reading-service.com/en/. The correction certificate is attached. We hope that all clarifications and changes made to the manuscript will satisfy you and the reviewers and meet the standards required for publication in your journal.

 

 

Sincerely yours,

Cleide M. F. Soares

Email: cleide18@yahoo.com.br

 

 

 

 

 

 

 

 

 

 

 

Reviewer #1

Suggestion 1:
- P2, line 53: the abbreviation word “CH” in first appearance should be provided the full name.

Answer 1: We agree with the suggestions, and the necessary modification is described in P2, line 54: “conventional heating (CH)”.

Suggestion 2:

- P3, line 94: “bioprinted LBC” should be “bioprinted BCL”.

Answer 2:  We agree with the suggestion, and the modification was made in the text.

 

Suggestion 3:

- Figure 1: The productivity of unprinted lipases (including BCL, CRL and PPL) should be provided as blank control.

Answer 3: We appreciate your suggestion. The blank control was calculated in previous studies by our research group [1] through the catalytic efficiency of unprinted lipases (BCL, CRL and PPL) in esterification reactions under conventional heating from the enzymatic productivity in the esterification reaction of fatty acid (lauric acid, myristic acid, palmitic acid, stearic acid, and oleic acid) and alcohol (consisting of a mixture of ethanol and butanol). BCL showed higher productivity for fatty acids in the following order: palmitic acid (16:0) – 4,029.95 µmol/h.mg > stearic acid (18:0) – 1,662.27 µmol/h.mg) > myristic acid (14:0) – 1,560.64 µmol/h.mg. CRL showed productivity for oleic acid (18:1) - 380.80 µmol/h.mg > stearic acid (18:0) - 346.60 µmol/h.mg > palmitic acid (16:0) - 336.11 µmol / h.mg > myristic acid (14:0) - 322.19 µmol/h.mg. And PPL achieved productivity for lauric acid (12:0) - 71.27 µmol/h.mg > myristic acid (14:0) - 66.52 µmol/h.mg > oleic acid – (18:1) - 64, 86 µmol/h.mg > stearic acid (18:0) - 64.40 µmol/h.mg > palmitic acid (16:0) - 62.46 µmol/h.mg. The proposal from this study was to reduce the reaction time and reuse the biocatalyst, which does not occur with the free enzyme.

1. Melo, J.J.C.; Gonçalves, J.R.; Brandão, L.M. de S.; Souza, R.L.; Pereira, M.M.; Lima, Á.S.; Soares, C.M.F. Evaluation of Lipase Access Tunnels and Analysis of Substance Transport in Comparison with Experimental Data. Bioprocess Biosyst Eng 2022, 45, 1149–1162, doi:10.1007/s00449-022-02731-x.

 

Suggestion 4:

- Figures 2~4; The productivity of unprinted BCL should be provided as blank control.

Answer 4: We appreciate your suggestion. The productivity of the unprinted BCL in the reactions assisted by microwave irradiation was calculated after the determination of the equilibrium in the time of 24 h in conventional heating, verifying that the BCL presented greater productivity for ester synthesis in the reaction with palmitic acid (productivity of 4,029.95 µmol/h.mg). Thus, esterification reactions assisted by microwave irradiation mediated by unprinted BCL were carried out with palmitic acid. The data referring to figures 2~4 show the comparison between the productivity of bioprinted lipases in reactions under conventional heating and the productivity of bioprinted lipases in reactions assisted by microwave irradiation (50 W~200 W). The proposal from this study was to reduce the reaction time and reuse the biocatalyst, which does not occur with the free enzyme.

 

Suggestion 5:

- P5, line 154: What is “the bBCL” referring to?

Answer 5: the abbreviated word “bBCL” appears in P1, line 20, and P3, line 98 with the full name: “Bioprinted BCL (bBCL)”

 

Suggestion 6:

- In case of 50 W, the printed BCL showed higher productivity with decreasing of carbon length of fatty acid within 25 min, while in case of 100 W and 200 W, the oppose results were found for printed BCL. Why?

Answer 6: BCL showed the highest productivity for lauric acid in the reaction time of 25 min (198,661.73 µmol/h.mg). Then, the highest productivity was for myristic acid with 25 min of reaction (123,614.43 µmol/h.mg). Productivity increases with decreasing lengths of fatty acid carbon chains. The effects on esters of different chain lengths as acyl donors in microwave assisted synthesis using BCL indicate that fatty acid polarity increases as chain length decreases. Therefore, the higher productivity of lauric acid under microwave irradiation (50 W) compared to myristic acid and palmitic acid possibly refers to the selective heating of more polar molecules under the influence of microwave irradiation. However, the temperature of a microwave assisted reaction is controlled by power. The reaction temperature increases with increasing potency. As microwave power increases, the rate of reorientation of polar molecules under microwave irradiation increases. In microwave irradiation-assisted esterification, the amount of heat generated depends on the concentration of polar molecules that are induced. The ability of a substance to be polarized is measured by the dielectric constant. The dielectric constant decreased with increasing temperature and power. Therefore, at higher powers the dipolar polarization of substances is affected. Therefore, with the increase in power from 50 W to 100 W or 200 W, there may have been a change in the dielectric constant of the fatty acids. Thus, the bioprinted BCL showed higher productivity with increasing fatty acid carbon length.

 

Suggestion 7:

- Table 1~3: please provide the intensity of MW irradiation for the secondary structure by FTIR of lipases.

Answer 7: We agree with the suggestions, and the necessary modification is described in P7, line 190: “MW (50 W)”, P7, line 194: “MW (50 W)” and P8, line 198: “MW (100W) and, P11, line 317 and 318: “after the reaction times (lauric acid and myristic acid – 50 W and palmitic acid – 100 W)”.

 

Suggestion 8:

- Table 3: As seen in table 3, the change of α-helix of lipases is irregular. Please clarify the interrelation ship between productivity and secondary structure of BCL printed with palmitic acid at different time.

Answer 8: The reduction of the α-helix content indicates the displacement of the BCL lid, which facilitates the access of the substrate to the active site, favoring the increase of enzymatic activity. Thus, the more the α-helix content decreases, the greater the displacement of the lipase lid and, consequently, the more exposed the active site of the enzyme, making it easier for the substrate to access. Another point to be observed associated with the α-helix is the β sheet. The increase in the β sheet content makes the BCL structure more rigid due to the loss of intermolecular hydrogen bonding between water molecules and the enzyme surface, making the enzyme more active. The greater the decrease in α-helix content and the greater the increase in β-sheet content, the greater the bBCL productivity. Therefore, the best productivity result obtained in 25 min with palmitic acid corroborates the secondary structure data, as it has the lowest α-helix content associated with the highest β-sheet content compared to the other analyzed times.

 

Suggestion 9:

- Figures 5 and 6: How about the stability of unprinted BCL?

Answer 9: We understand your question, but with unprinted BCL it is not possible to evaluate the stability. As it is a free enzyme, it is not possible to recover it for analysis of operational stability/reuse and storage time.

Author Response File: Author Response.pdf

Reviewer 2 Report

de Melo et al. studied the combined effect of bioprinted lipases with acids fatty acids in microwave-assisted esterification reactions. The manuscript reported some interesting results. The paper can be accepted after minor revision:

1. Introduction section does not show the comprehensive research progress of the esterification reactions. The background knowledge on this area needs to be further reviewed, by consulting the recently published articles, for example: Journal of Industrial and Engineering Chemistry, 2023, 117, 85-102; Journal of the Taiwan Institute of Chemical Engineers, 2022, 133, 104277; Journal of Industrial and Engineering Chemistry, 2022, 114, 1-18; Fuel Processing Technology Volume 239, January 2023, 107558.

2. Did the author compare the esterification properties of other catalysts with the catalyst reported in this paper. Better to make a table to compare the performances.

3. Did the microwave irradiation have many attractive advantages compared to conventional heating in this paper？

4. Figure 5. What is the reason of the decrease of catalytic performance？

5. What are the steps of catalyst regeneration？

 

Author Response

Reviewer #2

 

Suggestion 1:

- Introduction section does not show the comprehensive research progress of the esterification reactions. The background knowledge on this area needs to be further reviewed, by consulting the recently published articles, for example: Journal of Industrial and Engineering Chemistry, 2023, 117, 85-102; Journal of the Taiwan Institute of Chemical Engineers, 2022, 133, 104277; Journal of Industrial and Engineering Chemistry, 2022, 114, 1-18; Fuel Processing Technology Volume 239, January 2023, 107558.

Answer 1: We appreciate and agree your suggestions, and the modification can be seen in P2, lines 57-60: “Liow et al. [12] reviewed technologies for intensifying the synthesis of biodiesel through enzymatic esterification reactions and demonstrated improvement in terms of yield and reaction rate, with reduced enzymatic load and energy consumption in reactions under MW.” The other articles we studied and found very interesting, but the Liow et al. [12]  article is much more adherent to the proposal of our work.

 

Suggestion 2:

- Did the author compare the esterification properties of other catalysts with the catalyst reported in this paper. Better to make a table to compare the performances.

Answer 2:

We appreciate your suggestion. No, the catalysts reported in this paper were selected from previous studies and articles by our research group [2].

2. Brandão, L.M. de S.; Barbosa, M.S.; Souza, R.L.; Pereira, M.M.; Lima, Á.S.; Soares, C.M.F. Lipase Activation by Molecular Bioimprinting: The Role of Interactions between Fatty Acids and Enzyme Active Site. Biotechnol Prog 2021, 37, doi:10.1002/btpr.3064.

Suggestion 3:

- Did the microwave irradiation have many attractive advantages compared to conventional heating in this paper?

Answer 3: Yes, the use of microwave irradiation in the esterification reactions of BCL bioprinted with lauric acid increased productivity by approximately 33 X compared to reactions under conventional heating. Furthermore, there was a reduction in reaction time from 24 h under conventional heating to 25 min in reactions assisted by microwave irradiation. Thus, the results demonstrate the perspective of a new enzymatic route to obtain hyperactive catalysts, with the use of bioprinted lipases in esterification reactions under microwave irradiation.

.

Suggestion 4:

- Figure 5. What is the reason of the decrease of catalytic performance?

Answer 4: The literature reports that the heat produced by molecular friction induced in microwave-assisted reactions improves the performance of the synthesis in terms of increasing the reaction rate and reducing reaction times, resulting in greater obtaining of the products of interest in less time. Lipases have an intense dipole moment due to the functional groups, which is attributed to their polar nature. Microwaves influence polar molecules, increasing friction and collisions between them, which can induce conformation changes in the lipase structure that facilitate access of substrate molecules to the active site, making the enzyme more active. This occurs because microwave irradiation produces efficient internal heating through direct coupling of energy with solvent, substrate, and catalyst molecules in the reaction mixture. However, high reaction times cause a loss of enzymatic activity, resulting in conformational changes or inactivation, as can be seen in secondary structure analysis by FTIR.

 

Suggestion 5:

- What are the steps of catalyst regeneration?

Answer 5:  We appreciate your question and the explanation about the steps of catalyst regeneration is in P11-12, line 325-335, section 3.5: “3.5. Operational Stability. The operational stability of bBCL in the microwave-assisted esterification reactions was performed in consecutive 25 min-batches in the reaction conditions described in item ‘Productivity calculation’. After each cycle, the lipase was washed with octane to remove any remaining reagents and products. Subsequently, the bBCL was reused in a new cycle under the same reaction conditions. The residual fatty acid level was monitored at the end of each cycle by titration with 150 mM of the NaOH solution and the productivity calculation was performed. The first lipase activity was taken as 100%, and the relative activity is the percentage ratio of lipase activity to the initial activity. Enzyme deactivation models were fitted to experimental data using the “solver” function from Excel for Windows and used to estimate the half-life time of bBCL.”

 

The English correction was performed by https://www.proof-reading-service.com/en/. The correction certificate is attached.

 

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have addressed all the issues raised from the reviewers. The revised version is more readable than the original form. Therefore, I recommend it for acceptance in this present form.