Phenol Removal from Wastewater Using Tyrosinase Enzyme Immobilized in Granular Activated Carbon and Activated Chitosan Beads

Round 1

Reviewer 1 Report

In this manuscript, granular activated carbon (GAC) and activated chitosan beads (ACB) were employed to immobilize the tyrosinase enzyme. Subsequently, the tyrosinase-immobilized GAC and ACB were utilized as catalysts for the removal of phenol. While the content is intriguing, the manuscript requires significant revision to address the following suggestions before it can be accepted:

 

1. First of all, The title appears to be confusing, as the removal of phenol in this study is achieved through catalytic degradation rather than adsorption. Furthermore, the study reveals that tyrosinase-immobilized ACB outperforms tyrosinase-immobilized GAC. Therefore, the authors should revise the title to align it with the main finding of the study.

 

2. The introduction section, relevant recent literature using other methods should be introduced to benefit the readership, such as Environ. Sci. Technol., 2022, 56, 4356-4366; Small 2023, 2301044; Bioresource Technol., 2022, 348, 126818; etc.

 

3. Figure 2, Improve the quality of Figure 2 and ensure that a scale bar is provided for clarity.

 

4. The authors claim that enzyme immobilization in ACB support occurs through covalent bonding. However, it is essential to provide experimental data or evidence to support this statement.

 

5. Investigate whether the loading dosage of the enzyme affects catalytic performance. High enzyme loading amounts could potentially block the pores of the resulting materials, and studying the relationship between enzyme loading and catalytic performance would be beneficial.

 

6. Streamline the conclusion to emphasize the main finding of the study. Avoid duplicating content from the abstract, and keep the conclusion concise and directly related to the study's outcomes.

 

In this manuscript, granular activated carbon (GAC) and activated chitosan beads (ACB) were employed to immobilize the tyrosinase enzyme. Subsequently, the tyrosinase-immobilized GAC and ACB were utilized as catalysts for the removal of phenol. While the content is intriguing, the manuscript requires significant revision to address the following suggestions before it can be accepted:

 

1. First of all, The title appears to be confusing, as the removal of phenol in this study is achieved through catalytic degradation rather than adsorption. Furthermore, the study reveals that tyrosinase-immobilized ACB outperforms tyrosinase-immobilized GAC. Therefore, the authors should revise the title to align it with the main finding of the study.

 

2. The introduction section, relevant recent literature using other methods should be introduced to benefit the readership, such as Environ. Sci. Technol., 2022, 56, 4356-4366; Small 2023, 2301044; Bioresource Technol., 2022, 348, 126818; etc.

 

3. Figure 2, Improve the quality of Figure 2 and ensure that a scale bar is provided for clarity.

 

4. The authors claim that enzyme immobilization in ACB support occurs through covalent bonding. However, it is essential to provide experimental data or evidence to support this statement.

 

5. Investigate whether the loading dosage of the enzyme affects catalytic performance. High enzyme loading amounts could potentially block the pores of the resulting materials, and studying the relationship between enzyme loading and catalytic performance would be beneficial.

 

6. Streamline the conclusion to emphasize the main finding of the study. Avoid duplicating content from the abstract, and keep the conclusion concise and directly related to the study's outcomes.

 

Quality of English language is fine.

Author Response

Responses to reviewer #1.

 

In this manuscript, granular activated carbon (GAC) and activated chitosan beads (ACB) were employed to immobilize the tyrosinase enzyme. Subsequently, the tyrosinase-immobilized GAC and ACB were utilized as catalysts for the removal of phenol. While the content is intriguing, the manuscript requires significant revision to address the following suggestions before it can be accepted:

The authors appreciate the time and effort to evaluate this manuscript. A point-by-point explanation of the revisions are listed below.

 

1. First of all, The title appears to be confusing, as the removal of phenol in this study is achieved through catalytic degradation rather than adsorption. Furthermore, the study reveals that tyrosinase-immobilized ACB outperforms tyrosinase-immobilized GAC. Therefore, the authors should revise the title to align it with the main finding of the study.

R: The authors agree with the reviewer's comment. The title of the manuscript was revised to "Phenol removal from wastewater using Tyrosinase enzyme immobilized in granular activated carbon (GAC) and activated chitosan beads (ACB)" in order to better align it with the content of the manuscript. 

1. The introduction section, relevant recent literature using other methods should be introduced to benefit the readership, such as Environ. Sci. Technol., 2022, 56, 4356-4366; Small 2023, 2301044; Bioresource Technol., 2022, 348, 126818; etc.

R: The references were updated in order to include more current work that aligns with the manuscript’s subject.

1. Figure 2, Improve the quality of Figure 2 and ensure that a scale bar is provided for clarity.

R: The authors apologize if the quality of Figure 2 compromises the manuscript's comprehension. We were not able to improve it since MEV analyses were performed in another Laboratory and these are the only images available. Nevertheless, the scale bars that were previously left out were reintroduced to such images. 

1. The authors claim that enzyme immobilization in ACB support occurs through covalent bonding. However, it is essential to provide experimental data or evidence to support this statement.

R: In the manuscript, a brief explanation was included about the covalent bonds between ACB and Tyrosinase, including a reference (G. Romero, L.M. Contreras, C. Aguirre Céspedes, J. Wilkesman, J.M. Clemente-Jiménez, F. Rodríguez-Vico, F.J. Las Heras-Vázquez, Efficiency Assessment between Entrapment and Covalent Bond Immobilization of Mutant β-Xylosidase onto Chitosan Support, Polymers (Basel). 15 (2023) 3170) that previously discussed the matter (lines 273-280):

 

“The immobilizations in GAC and chitosan beads occur mainly by physical adsorption, so enzyme desorption is more accessible in case of operational changes. The ACB Support was subjected to a chemical activation process with glutaraldehyde, a cross-linking agent, which introduces aldehyde functional groups into the chitosan spheres that form covalent bonds with the amine (NH2) and thiol (SH) functional groups of Tyrosinase [34]. Some authors have observed that during immobilization of enzymes in GAC, the percentage of immobilized enzymes usually stabilizes in less than 120 minutes of contact between the enzyme and the support [28–30].” 

1. Investigate whether the loading dosage of the enzyme affects catalytic performance. High enzyme loading amounts could potentially block the pores of the resulting materials, and studying the relationship between enzyme loading and catalytic performance would be beneficial.

R: The authors appreciate the suggestion and agree that such evaluation makes a lot of sense. However, the main focus of this work was to evaluate the capacity of the supports to produce an efficient material to be used in phenol removal, that could be easily separated, stored and reused. The evaluation of different enzyme loads is an excellent suggestion for a future study. 

1. Streamline the conclusion to emphasize the main finding of the study. Avoid duplicating content from the abstract, and keep the conclusion concise and directly related to the study's outcomes.

R: The authors appreciate the suggestion. Some information was removed from the Conclusion section in order to make it less repetitive and its first paragraph was altered to highlight the main achievements of the study.

Lines 515-520: “The tyrosinase enzyme from a crude extract was immobilized on granular activated carbon (T-GAC) and activated chitosan beads (T-ACB). It was possible to obtain enzymatic immobilizations of up to 70% on GAC and up to 100% on ACB in a contact time of 120 minutes, keeping the temperature at 298K and pH at 7.0. Overall, ACB seems to be a better support for the immobilization of Tyrosinase than GAC, presenting better results for phenol oxidation efficiencies, material’s reuse and stability over storage.”

Reviewer 2 Report

The aim of the manuscript entitled “Phenol adsorption from wastewater using Tyrosinase enzyme immobilized in granular activated carbon (GAC)” is to investigate the immobilization of the Tyrosinase enzyme within granular activated carbon (GAC) and activated chitosan beads (ACB) as cost-effective supports, and to evaluate their effectiveness in removing phenol from wastewater. The study focuses on optimizing the immobilization process and assessing the phenol removal efficiency of the enzyme-immobilized materials, with the goal of providing environmentally friendly and economically viable solutions for mitigating phenol's impact on industrial wastewater in compliance with Brazilian legislation. The manuscript fits with the scope of the journal but has many issues.

The main problem with this manuscript is its novelty. I do not see an essential difference compared to some already published works (https://doi.org/10.1021/bp00015a002). Motivation and novelty should be thoroughly explained. That problem aside, the manuscript has a few weak points. First, it is not clear what happens to phenol after oxidation. Do the authors have evidence that degradation occurs or that a quinone is formed? What is adsorbed, and on which substrate? All this should be explained in detail, and a removal mechanism should be suggested. Why would oxidation using immobilized tyrosinase be more successful compared to the free enzyme? It does not make sense. Also, SEM cannot be evidence for enzyme immobilization. Is the same spot on the material photographed?

Minor changes required. 

Author Response

Responses to reviewer #2.

 

The aim of the manuscript entitled “Phenol adsorption from wastewater using Tyrosinase enzyme immobilized in granular activated carbon (GAC)” is to investigate the immobilization of the Tyrosinase enzyme within granular activated carbon (GAC) and activated chitosan beads (ACB) as cost-effective supports, and to evaluate their effectiveness in removing phenol from wastewater. The study focuses on optimizing the immobilization process and assessing the phenol removal efficiency of the enzyme-immobilized materials, with the goal of providing environmentally friendly and economically viable solutions for mitigating phenol's impact on industrial wastewater in compliance with Brazilian legislation. The manuscript fits with the scope of the journal but has many issues.

The main problem with this manuscript is its novelty. I do not see an essential difference compared to some already published works (https://doi.org/10.1021/bp00015a002). Motivation and novelty should be thoroughly explained. That problem aside, the manuscript has a few weak points. First, it is not clear what happens to phenol after oxidation. Do the authors have evidence that degradation occurs or that a quinone is formed? What is adsorbed, and on which substrate? All this should be explained in detail, and a removal mechanism should be suggested. Why would oxidation using immobilized tyrosinase be more successful compared to the free enzyme? It does not make sense. Also, SEM cannot be evidence for enzyme immobilization. Is the same spot on the material photographed?

 

 

R: The authors appreciate the time and effort to evaluate the manuscript. As for the novelty of the study, the authors agree that it should be further highlighted. A paragraph was included at the end of Introduction section in order to highlight the novelty of the study.

Lines 78-86: “To the best of our knowledge, this study is the first one to assess the immobilization of Tyrosinase directly from a crude extract and not from a purified enzyme sample, which enables the use of agricultural waste as a source of such enzyme and might reduce production costs associated with purification steps. Additionally, the immobilization was performed on  low-cost supports, which tends to make the technology more accessible and economically viable for application in different scenarios, including the treatment of industrial wastewater and contaminated effluents [24,25]. Finally, the study also presents stability results in relation to storage, which has been little studied for these supports, thus, presenting valuable results for the literature.”

Furthermore, the study focuses on developing a cost-effective material for the removal of phenol from wastewater and also aims to fit such wastewater to the Brazilian legislation for discharge. Although the evaluation of the mechanisms of phenol (or o-quinone) is an excellent suggestion for future studies, it was not that main focus of this work.

Finally, the clarify the benefits of using immobilized enzymes instead of free ones, a paragraph was included in the manuscript.

Lines 334-345: “The higher activity of the free enzyme can be explained by some factors such as conformation and accessibility, since when the enzyme is immobilized on a solid support its active conformation can be altered due to the bonds that hold it to the support; Restriction of Movement, since immobilizing the enzyme on a solid support can restrict its movement and its ability to access substrates; Limited Diffusion, because when the enzyme is immobilized, the diffusion of substrates and products to and from the enzyme's active site can be limited; and Steric Inhibition, where immobilization of the enzyme on a solid support can result in steric inhibition, where the solid support directly interferes with the conformation and activity of the enzyme. However, the main focus of enzyme immobilization is not to enhance Tyrosinase enzymatic oxidation when compared to free enzymes, but to allow its reuse, enhance its long-term stability and the ability to easily recover the enzyme from the reaction medium.”

Reviewer 3 Report

Manuscript deals with the Tyrosinanse enzyme immobilized in GAC in order to obtain phenol from wastewater. The subject is important for environmental application. However, there is one comment that the authors should consider before publication.

11.      The introduced novelties in this paper should be clearly presented; the knowledge gap needs to be clearly addressed in the Introduction.

Author Response

Responses to reviewer #3.

 

Manuscript deals with the Tyrosinanse enzyme immobilized in GAC in order to obtain phenol from wastewater. The subject is important for environmental application. However, there is one comment that the authors should consider before publication.

The authors appreciate the time and effort to evaluate this manuscript.

 

11. The introduced novelties in this paper should be clearly presented; the knowledge gap needs to be clearly addressed in the Introduction.

R: A paragraph was included in the manuscript at the end of Introduction section in order to highlight the novelty of the study.

Lines 78-86: “To the best of our knowledge, this study is the first one to assess the immobilization of Tyrosinase directly from a crude extract and not from a purified enzyme sample, which enables the use of agricultural waste as a source of such enzyme and might reduce production costs associated with purification steps. Additionally, the immobilization was performed on  low-cost supports, which tends to make the technology more accessible and economically viable for application in different scenarios, including the treatment of industrial wastewater and contaminated effluents [24,25]. Finally, the study also presents stability results in relation to storage, which has been little studied for these supports, thus, presenting valuable results for the literature.”

Reviewer 4 Report

This manuscript described the phenol removal from wastewater using Tyrosinase enzyme immobilized in granular activated carbon (GAC) and in activated chitosan beads (ACB). This work is acceptable for publication in the journal "Water" after major revision. In my opinion, some issues should be addressed to improve this manuscript. The reasons for this are as follows:

1.             I think the title does not fully capture the manuscript content. First: Not only phenol adsorption was investigated, but also enzymatic oxidation as a method of phenol removal. Second: Two supports (carbon and chitosan) were investigated Why is there only one in the title?

2.             The novelty of the work must be clearly addressed and discussed, compare your research with existing research findings and highlight novelty.

3.             Reference citation numbers should be placed in square brackets, i.e. [ ], and placed inside the punctuation, for example [4]. or [1–3], and all the references should be listed separately and as the last section at the end of the manuscript. Do not use superscript text to indicate citations. https://www.mdpi.com/authors/references

4.             The reference list contains only 3 references for the last five years, none for 2022 and 2023. Is the problem presented in the manuscript not current? I recommend updating the reference list.

5.             Section Materials and Methods: Lines 137 – 139. Why were the reactions performed when different amounts of T-GAC and T-ACB were added?

6.             How Enzymatic activity, Phenol Removal Rate, removal efficiency was calculated? Why is the phenol removal rate in percent (fig. 6, 7, 8)?

7.             «Fig. 3 shows the comparison between the FTIR spectra of GAC before immobilization and after immobilization of tyrosinase enzyme… The spectrum of support after immobilization presents peaks indicating the presence of histidine… The appearance of two bands on 3700 - 3600 cm^-1 region in FTIR spectrum of GAC after immobilization (b), corresponding to the stretching frequency of the N-H binding, is likely due to the interaction of tyrosinase with the activated carbon matrix» Do these FTIR data not indicate a chemical immobilization rather than a physical one?

8.             Lines 242-244. What is the difference between chitosan beads and activated chitosan beads (ACB)? And why is enzyme immobilization on ACB covalent? Specify which covalent bonds occur.

9.             Lines 242-244. What is the difference between chitosan granules and activated chitosan granules? And why is the enzyme immobilization on ACB covalent? Specify which covalent bonds occur.

10.          Lines 250 – 260. Immobilization rate as a percentage? It's probably a degree. Which figures or tables represent the immobilization degree? How was it calculated?

11.          Why stirring conditions for enzyme immobilization have only been studied for GAC, and for ACB?

12.          Figure 5. Curves for free enzymes and T-GAC, T-ACB are comparable. What explains the rather high activity of the free enzymes? Maybe it does not need to be immobilized on the support?

13.          Was FTIR analysis of T-GAC and c T-ACB performed after phenol oxidation?

Comments for author File: Comments.pdf

Author Response

Responses to reviewer #4.

 

This manuscript described the phenol removal from wastewater using Tyrosinase enzyme immobilized in granular activated carbon (GAC) and in activated chitosan beads (ACB). This work is acceptable for publication in the journal "Water" after major revision. In my opinion, some issues should be addressed to improve this manuscript. The reasons for this are as follows:

The authors appreciate the time and effort to evaluate this manuscript. A point-by-point explanation of the revisions are listed below.

 

1. I think the title does not fully capture the manuscript content. First: Not only phenol adsorption was investigated, but also enzymatic oxidation as a method of phenol removal. Second: Two supports (carbon and chitosan) were investigated Why is there only one in the title?

R: The authors agree with the reviewer's comment. The title of the manuscript was revised to "Phenol removal from wastewater using Tyrosinase enzyme immobilized in granular activated carbon (GAC) and activated chitosan beads (ACB)" in order to better align it with the content of the manuscript.

1. The novelty of the work must be clearly addressed and discussed, compare your research with existing research findings and highlight novelty.

R: A paragraph was included in the manuscript at the end of Introduction section in order to highlight the novelty of the study.

Lines 78-86: “To the best of our knowledge, this study is the first one to assess the immobilization of Tyrosinase directly from a crude extract and not from a purified enzyme sample, which enables the use of agricultural waste as a source of such enzyme and might reduce production costs associated with purification steps. Additionally, the immobilization was performed on  low-cost supports, which tends to make the technology more accessible and economically viable for application in different scenarios, including the treatment of industrial wastewater and contaminated effluents [24,25]. Finally, the study also presents stability results in relation to storage, which has been little studied for these supports, thus, presenting valuable results for the literature.”

 

2. Reference citation numbers should be placed in square brackets, i.e. [ ], and placed inside the punctuation, for example [4]. or [1–3], and all the references should be listed separately and as the last section at the end of the manuscript. Do not use superscript text to indicate citations. https://www.mdpi.com/authors/references

R: The reviewer is right. The reference citations were corrected throughout the manuscript.

3. The reference list contains only 3 references for the last five years, none for 2022 and 2023. Is the problem presented in the manuscriptnot current? I recommend updating the reference list.

R: The references were updated in order to include more current work that aligns with the manuscript’s subject.

4. Section Materials and Methods: Lines 137 – 139. Why were the reactions performed when different amounts of T-GAC and T-ACB were added?

R: The amount of T-GAC and T-ACB were different since the materials presented different enzymatic activities. The distinct amounts of materials were used in order to unify the enzymatic activities available in both cases. A brief explanation was added to the manuscript to make it clear.

Lines 155-158: “As the supports had different immobilization degrees, in order to compare their efficiencies, it was established the same initial enzymatic activity (1500 U) for both supports and, for that reason, the reactions were carried out with different amounts of T-GAC and T-ACB.”

 

5. How Enzymatic activity, Phenol Removal Rate, removal efficiency was calculated? Why is the phenol removal rate in percent (fig. 6, 7, 8)?

R: Equations 1, 2 and 3 were added to the revised manuscript in order to clarify how such properties were calculated. Furthermore, we would like to clarify that phenol removal was expressed in terms of percentage since it denoted the removal efficiencies in each moment. The terms “phenol removal rate” were replaced by “phenol removal efficiency” throughout the manuscript to avoid this misconception.

 

6. «Fig. 3 shows the comparison between the FTIR spectra of GAC before immobilization and after immobilization of tyrosinase enzyme… The spectrum of support after immobilization presents peaks indicating the presence of histidine… The appearance of two bands on 3700 - 3600 cm-1 region in FTIR spectrum of GAC after immobilization (b), corresponding to the stretching frequency of the N-H binding, is likely due to the interaction of tyrosinase with the activated carbon matrix» Do these FTIR data not indicate a chemical immobilization rather than a physical one?

R: The authors understand that these results may lead to such conclusion. However, it was clear during the essays that the bond between GAC and the enzyme was not as strong as the one in T-ACB, mainly due the enzymatic activity shown by solutions after the contact time with T-GAC, which can be explained by enzyme desorption. This even led to the essays of reuse of support presented in Figure 7, which were performed only for GAC supports and were only possible due to this desorption. Either way, a brief explanation was introduced in the manuscript to clarify this issue.

Lines 245-250: “Although these results might suggest that chemical adsorption occurred during the immobilization of the enzyme on granular activated carbon, later tests showed a large desorption of the enzyme, which was proven by the determination of the enzymatic activity in the aqueous solution used in the phenol removal tests, which points that, although there may have been some chemical adsorption, the immobilization took place mainly by means of physical adsorption.”

 

7. Lines 242-244. What is the difference between chitosan beads and activated chitosan beads (ACB)? And why is enzyme immobilization on ACB covalent? Specify which covalent bonds occur.

R: Chitosan beads are the beads produced prior to the activation step. ACB are the chitosan beads after activation with glutaraldehyde, as described in lines 134-139:

“The chitosan beads (CB) were kept in a watch glass for 24 hours to be dehydrated and, consequently, have their mechanical strength increased. For more efficient immobilization, the dry CB were activated with glutaraldehyde (GA). Activation was performed by keeping the CB in contact with a solution containing up to 3.0% GA (m/V) under gentle stirring for 90 minutes. After this time, beads were vacuum filtered and stored under refrigeration.”

 

In the manuscript, a brief explanation was included about the covalent bonds between ACB and Tyrosinase, including a reference (G. Romero, L.M. Contreras, C. Aguirre Céspedes, J. Wilkesman, J.M. Clemente-Jiménez, F. Rodríguez-Vico, F.J. Las Heras-Vázquez, Efficiency Assessment between Entrapment and Covalent Bond Immobilization of Mutant β-Xylosidase onto Chitosan Support, Polymers (Basel). 15 (2023) 3170) that previously discussed the matter (lines 273-280).

 

“The immobilizations in GAC and chitosan beads occur mainly by physical adsorption, so enzyme desorption is more accessible in case of operational changes. The ACB Support was subjected to a chemical activation process with glutaraldehyde, a cross-linking agent, which introduces aldehyde functional groups into the chitosan spheres that form covalent bonds with the amine (NH2) and thiol (SH) functional groups of Tyrosinase [34]. Some authors have observed that during immobilization of enzymes in GAC, the percentage of immobilized enzymes usually stabilizes in less than 120 minutes of contact between the enzyme and the support [28–30].”

 

8. Lines 242-244. What is the difference between chitosan granules and activated chitosan granules? And why is the enzyme immobilization on ACB covalent? Specify which covalent bonds occur.

R: the response to comment 8 also apply to comment 9.

9. Lines 250 – 260. Immobilization rate as a percentage? It's probably a degree. Which figures or tables represent the immobilization degree? How was it calculated?

R: Equation 2 was added to the manuscript to clarify how immobilization degree was calculated. Additionally, we replaced the terms “immobilization rate” by “immobilization degree” throughout the manuscript, including in Tables 1 and 3, in order to align it with the understanding of the reviewer.

10. Why stirring conditions for enzyme immobilization have only been studied for GAC, and for ACB?

R: As explained in the manuscript (lines 304-308): “The low immobilization at low stirring speed is due to insufficient tyrosinase contact with the support. For higher stirring values, the mechanical inactivation of the enzyme occurs due to the increase of the contact area of the enzyme with air and with the Erlenmeyer walls. This phenomenon was reported by Colombiè et al. [37], Gikanga et al. [38], Menoncin et al. [39], and Wiesbauer et al. [40].”

The authors took the decision to not evaluate the stirring speed for chitosan beads since, as explained previously, it is a parameter that would affect immobilization regardless of the type of support. A sentence was included at the end of this paragraph to clarify this issue (lines 308-310): “Since both phenomena are expected to take place regardless of the type of support, all immobilizations in ACB were performed in the same optimum stirring speed as for GAC (15.7 rad/s).”

 

11. Figure 5. Curves for free enzymes and T-GAC, T-ACB are comparable. What explains the rather high activity of the free enzymes? Maybe it does not need to be immobilized on the support?

R: A paragraph was included in the manuscript to clarify this issue.

Lines 334-345: “The higher activity of the free enzyme can be explained by some factors such as conformation and accessibility, since when the enzyme is immobilized on a solid support its active conformation can be altered due to the bonds that hold it to the support; Restriction of Movement, since immobilizing the enzyme on a solid support can restrict its movement and its ability to access substrates; Limited Diffusion, because when the enzyme is immobilized, the diffusion of substrates and products to and from the enzyme's active site can be limited; and Steric Inhibition, where immobilization of the enzyme on a solid support can result in steric inhibition, where the solid support directly interferes with the conformation and activity of the enzyme. However, the main focus of enzyme immobilization is not to enhance Tyrosinase enzymatic oxidation when compared to free enzymes, but to allow its reuse, enhance its long-term stability and the ability to easily recover the enzyme from the reaction medium.”

 

12. Was FTIR analysis of T-GAC and c T-ACB performed after phenol oxidation?

R: Unfortunately, such analyses were not performed in time to be included in this study, but the authors agree that it would help further explain the oxidation process.

Round 2

Reviewer 1 Report

The quality of the manuscript has been improved accordingly.

Minor editing of English language required

Author Response

Thank you for your time and effort to improve our manuscript.

Reviewer 2 Report

The novelty elaboration is okay, but the rest of the comments are not addressed properly. The authors should revise carefully. 

Minor changes are required. 

Author Response

The authors apologize for not adressing your comments properly the first time. An extra effort was made from our part to clarify the issues. A point-by-point explanaition is given ahead.

"First, it is not clear what happens to phenol after oxidation. Do the authors have evidence that degradation occurs or that a quinone is formed? What is adsorbed, and on which substrate? All this should be explained in detail, and a removal mechanism should be suggested."

A new segment was added at the end of results and discussion section, which is entitled "O-quinone production and adsorption by the supports" (lines 527-558) in order to explain the destination of phenol oxidation products. We hope this clarifies the issue.

 

"Why would oxidation using immobilized tyrosinase be more successful compared to the free enzyme?"

A new paragraph was added to the manuscript (lines 351-357) to clarify this issue.

"Oxidation using immobilized enzymes are preferable for several reasons: immobilization offers greater stability, preserving the enzyme activity and enabling its use in many oxidation cycles. The facility of separation after oxidation provided by the supports simplifies the oxidation process and the product purification. Additionally, immobilization provides higher enzyme tolerance to high substrate concentrations, reduction in the enzymatic activity inhibition by scavenger compounds, and reduction of final product contamination."

 

"Also, SEM cannot be evidence for enzyme immobilization. Is the same spot on the material photographed?"

A new paragraph was added to the manuscript (lines 224-228) to clarify this issue.

"Although SEM micrographs are not direct evidence of Tyrosinase immobilization, it is a valuable analysis of the materials’ surfaces in micro or nanoscopic scales, highlighting the differences presented by the materials after immobilization. Also, when associated to FTIR analysis, SEM micrographs may provide a wide comprehension of the immobilization process and its effects on the enzyme."

 

 

Reviewer 4 Report

I am satisfied with the authors' responses. They have addressed all the comments. The revised manuscript may be accept in present form.

Author Response

Thank you for your time and effort to improve our manuscript.