Immobilization of a Bienzymatic System via Crosslinking to a Metal-Organic Framework

Round 1

Reviewer 1 Report

The present paper describes the covalent attachment of two relevant industrial enzymes (glucose oxidase and chloroperoxidase) on zirconium-based MOF supports. The paper shows some nice results. However, some revisions are required before it could be considered for publication, as follows:

- Title: I recommend change the current title. In this, immobilization of enzymes were performed under fixed conditions. Optimization approaches were not performed. This information should also be reported in Abstract section.

- Abstract: The better experimental results should be briefly described in this section such as maximum immobilized protein loading, catalytic activity and reusability. It is more interesting to the readers.

 

- Introduction – lines 50 and 58: Replace “solid substrates” by “solid matrices” or “solid supports”.

 

- Introduction – lines 67/81: How is this system different to other reports to merit publication? Please, report. A variety of articles has been recently reported in the literature to immobilize such enzymes.

 

- Results and discussion: All experimental data should be represented with error bars. Explain the number replications used in the study.

 

- Materials: The authors should better explain some properties of the biocatalyst used in this study – free (powder or liquid extract). The recommend report protein concentration, catalytic activity, and speficic activity.

 

- Illustrative schemes for support preparation, chemical modification via activation, and enzyme immobilization (attachment of nucleophilic groups on the enzyme surface with introduced reactive groups on the support surface) should be incorporated to this manuscript.

 

- Results: Give immobilization yield and expressed activity (see Boudrant et al., 2019).

https://doi.org/10.1016/j.procbio.2019.11.026

 

- The influence of activating agentes on the immobilization process and catalytic performance of the enzymes should be better discussed (enzyme orientation on the support surfaces, preferential immobilization on external or internal surfaces, mass transfer limitations,  distorting effects, etc.).

Author Response

Thank you for your careful review of our manuscript. We have taken your constructive comments and revised our manuscript. We have addressed your concerns, point-by-point.

- Title: I recommend change the current title. In this, immobilization of enzymes were performed under fixed conditions. Optimization approaches were not performed. This information should also be reported in Abstract section.

Title changed to “Immobilization of a bienzymatic system via cross-linking to a metal-organic framework”

- Abstract: The better experimental results should be briefly described in this section such as maximum immobilized protein loading, catalytic activity and reusability. It is more interesting to the readers.

 

Added text: “The DSG crosslinked composites displayed enhanced activity relative to free enzyme, and all crosslinked enzyme/MOF composites demonstrated recyclability with at least 30% of the activity being retained after four catalytic cycles.” 

 

- Introduction – lines 50 and 58: Replace “solid substrates” by “solid matrices” or “solid supports”.

 

The word “substrate” changed to support or matrices throughout the text.

 

- Introduction – lines 67/81: How is this system different to other reports to merit publication? Please, report. A variety of articles has been recently reported in the literature to immobilize such enzymes.

 

Text added: “The results of this report will aid researchers in utilizing CPO as a biocatalyst that is more active and has greater recyclability.” 

 

 

- Results and discussion: All experimental data should be represented with error bars. Explain the number replications used in the study.

 

Enzyme assays were run in triplicate and error bars were added to the bar graph. This information was also added to the text.

 

- Materials: The authors should better explain some properties of the biocatalyst used in this study – free (powder or liquid extract). The recommend report protein concentration, catalytic activity, and speficic activity. 

 

Catalytic activity has been defined in equation 1 in terms of Units as defined as M/min.

 

- Illustrative schemes for support preparation, chemical modification via activation, and enzyme immobilization (attachment of nucleophilic groups on the enzyme surface with introduced reactive groups on the support surface) should be incorporated to this manuscript. 

 

Illustrative scheme has been added showing the crosslinking of the MOF to the enzyme.

 

Figure 5 has moved to the top of the second page to become Figure 1.

 

- Results: Give immobilization yield and expressed activity (see Boudrant et al., 2019).

https://doi.org/10.1016/j.procbio.2019.11.026

 More information added to address this concern including how enzyme activity was calculated. Specifically, immobilization by electrostatics has been added for further comparison of activity and recyclabilities. This reference was also added into the text.

 

- The influence of activating agentes on the immobilization process and catalytic performance of the enzymes should be better discussed (enzyme orientation on the support surfaces, preferential immobilization on external or internal surfaces, mass transfer limitations,  distorting effects, etc.).

 

We have added text that discusses these concerns.

 

Reviewer 2 Report

The work submitted for review deals with a new way to immobilize two enzyme proteins (glucose oxidase (GOx) and chloroperoxidase (CPO)) which, from a practical point of view, could be an interesting and important issue. However, while reading the paper, a number of remarks came to my mind that prevent the publication of the paper in its current form: 

1. Improvement in terms of appropriate vocabulary e.g. immobilization on carrier, matrix rather than substrate (line 46, 50 etc)

2. general language correction necessary

3. written haphazardly and messy-  line 88 carboxyl group rather than carboxylic acid

4. fig 4. how do you know that higher enzymatic activity? Bradford method gives information about the amount of protein and not about the activity itself

5. Production of CPO-Acetone precipitation without prior biomass separation?

6. methods are described in an unclarified way- e.g. The absorbance of the mixture was taken by centrifuging out the MOF for 30 seconds and taking out supernatant to avoid scattering at the following time intervals: 3 minutes, 8 minutes, and 15 minutes.

7. Authors' participation is omitted

8. In general, the work is written messily and lacks in-depth analysis of the results of the experiments carried out

Author Response

Thank you for your careful review of our manuscript. We have taken your constructive comments and revised our manuscript. We believe that our results will provide a framework of how to prepare a biocatalyst with CPO which is of interest to the biocatalysis community because of its versatility as an enzyme. We have addressed your concerns, point-by-point.

1. Improvement in terms of appropriate vocabulary e.g. immobilization on carrier, matrix rather than substrate (line 46, 50 etc)

Text fixed to exclude substrate.

2. general language correction necessary
3. written haphazardly and messy-  line 88 carboxyl group rather than carboxylic acid

Thank you for this find. We have changed carboxyl to carboxylic acid.

4. fig 4. how do you know that higher enzymatic activity? Bradford method gives information about the amount of protein and not about the activity itself

You are correct. Enzyme activity is measured by the chlorination assay and not by Bradford.

5. Production of CPO-Acetone precipitation without prior biomass separation?

 

Yes, the biomass is separated via centrifugation. Text added to clarify this.

6. methods are described in an unclarified way- e.g. The absorbance of the mixture was taken by centrifuging out the MOF for 30 seconds and taking out supernatant to avoid scattering at the following time intervals: 3 minutes, 8 minutes, and 15 minutes.

MOF in solution contributes to light scattering that is not associated with the absorbance spectrum of the monochlorodimedone. MOF must be taken out of solution to run the assay. Text added for clarification.

7. Authors' participation is omitted

Authors’ contribution will be added prior to publication since it was added to the submission form.

8. In general, the work is written messily and lacks in-depth analysis of the results of the experiments carried out

The revised text adds more insight into the data and more analysis has been added.

Reviewer 3 Report

The study reported the use of the UiO-66-NH₂, a zirconium-based metal-organic framework (MOF), to immobilize the enzymes, glucose oxidase (GOx,) and chloroperoxidase (CPO), for the investigation of the enzymatic activity and recyclability of single immobilization of CPO and the doubly immobilized CPO and GOx. The effects of using two different crosslinkers, disuccinimidyl glutarate (DSG) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinidimide (NHS) were compared. The results showed that both single enzymatic (CPO) and bienzymatic (GOx/CPO) systems crosslinked with DSG exhibited enhanced enzyme activity compared to free enzymes. The study provides the novelty of using CPO and GOx onto UiO-66-NH₂ by two different crosslinkers and the comparison of the biocatalytic properties of single enzymatic and bienzymatic systems. The manuscript is suggested to be accepted after modifications. 

 

1.     The figures were usually inserted in the main text after the paragraph of its first citation. The positions of Figures 1, 2, and 4 have to be replaced.

2.     The results of monochlorodimedone (MCD) assay to determine the chlorination activity of CPO was shown in Figure 2. However, only GOx/CPO-linker@UiO-66-NH₂ was labeled without the information of the cross-linker used. It is suggested to show the results of four composites, including CPO-EDC@UiO-66-NH₂, CPO-DSG@UiO-66-NH₂, GOx/CPO-EDC@UiO-66-NH₂, and GOx/CPO-DSG@UiO-66-NH₂.   

3.     The description of the figure legend of the inset plot in Figure 4 was ambiguous.

4.     The authors described that the single system CPO-DSG@UiO-66-NH₂ retained the highest activity after four catalytic cycles. However, according to Figure 4, it was CPO-EDC@UiO-66-NH₂ retained the highest activity. The CPO-DSG@UiO-66-NH₂ retained the highest percentage of activity after four catalytic cycles among four composites. The authors have to clarify the results and the descriptions.

5.     It is suggested to show the compound names of all the compounds in Figure 5.

6.     The format of the references was different. In addition, references 61 and 62 were the same citations.

7.     The authors did not make critical conclusions, in the Abstract and the Conclusions Section, for this study. Instead, two studies were introduced to produce modafinil which can be described in the Introduction Section. It is suggested to discuss the advantages and disadvantages of four composites based on their enzymatic activity and recyclability, also improvements and future investigations. For example, the enzymatic activity of bienzymatic system GOx/CPO-DSG@UiO-66-NH₂ was high, however, the recyclability of which was low.    

 

Author Response

Thank you for your careful review of our manuscript. We have taken your constructive comments and revised our manuscript. We believe that our results will provide a framework of how to prepare a biocatalyst with CPO which is of interest to the biocatalysis community because of its versatility as an enzyme. We have addressed your concerns, point-by-point.

1.     The figures were usually inserted in the main text after the paragraph of its first citation. The positions of Figures 1, 2, and 4 have to be replaced.

Figures 1, 2, and 4 have been moved to be positioned after the paragraph in which they are first mentioned.

2.     The results of monochlorodimedone (MCD) assay to determine the chlorination activity of CPO was shown in Figure 2. However, only GOx/CPO-linker@UiO-66-NH2 was labeled without the information of the cross-linker used. It is suggested to show the results of four composites, including CPO-EDC@UiO-66-NH2, CPO-DSG@UiO-66-NH2, GOx/CPO-EDC@UiO-66-NH2, and GOx/CPO-DSG@UiO-66-NH2.   

Figure 2 was included to explain the MCD assay rather than present data for each of the individual materials. The text and the figure caption have been modified to explain  that this is representative data. 

Page 3, Lines 121-122 have been changed from “This assay is shown in Figure 2.” to ““Representative data from this assay is shown in Figure 2.” 

The caption for Figure 2 has been changed from “Chlorination of monochlorodimedone assay to assess CPO’s chlorination ability. Representative data of the UV/vis of the assay (A) showing formation of dichlorodimedone (B) over time as indicated by the decreasing absorption at 295 nm.” to “Figure 2. Chlorination of monochlorodimedone assay to assess CPO’s chlorination ability. Representative UV/vis data from the assay (A) showing formation of dichlorodimedone (B) over time as indicated by the decreasing absorption at 285 nm.”

3.     The description of the figure legend of the inset plot in Figure 4 was ambiguous.

“Inset plot of the percent retained activity of each composite per.” has been changed to “Inset plot shows the percent retained activity of each composite per milligram of enzyme/MOF composite.”

4.     The authors described that the single system CPO-DSG@UiO-66-NH2 retained the highest activity after four catalytic cycles. However, according to Figure 4, it was CPO-EDC@UiO-66-NH2 retained the highest activity. The CPO-DSG@UiO-66-NH2 retained the highest percentage of activity after four catalytic cycles among four composites. The authors have to clarify the results and the descriptions.

New figures have been generated and the results have been clarified.

5.     It is suggested to show the compound names of all the compounds in Figure 5. 

To make the chemical reactions in Figure 5 more clear, the figure caption has been updated from “Figure 5. The bienzymatic biocatalytic system of GOx/CPO cascade reaction of MCD assay.” to “Figure 5. The bienzymatic biocatalytic system of GOx/CPO cascade reaction of the MCD assay in which GOx utilizes glucose to produce H2O2, and CPO uses this H2O2 to chlorinate MCD. The decrease in MCD concentration is monitored by measuring the absorbance at 295 nm.”

6.     The format of the references was different. In addition, references 61 and 62 were the same citations.

This was corrected.

7.     The authors did not make critical conclusions, in the Abstract and the Conclusions Section, for this study. Instead, two studies were introduced to produce modafinil which can be described in the Introduction Section. It is suggested to discuss the advantages and disadvantages of four composites based on their enzymatic activity and recyclability, also improvements and future investigations. For example, the enzymatic activity of bienzymatic system GOx/CPO-DSG@UiO-66-NH2 was high, however, the recyclability of which was low. 

The following text was added to the Abstract (Page 1, Line 30) to summarize the key results and conclusions of the study:

“The DSG crosslinked composites displayed enhanced activity relative to free enzyme, and all crosslinked enzyme/MOF composites demonstrated recyclability with at least 30% of the activity being retained after four catalytic cycles.”

The following text was added to the beginning of the conclusion:

“In this study, enzyme/MOF composites were prepared using two different crosslinkers, DSG and EDC, to immobilize CPO or GOx/CPO to the surface of UiO-66-NH2. Using this approach, four enzyme/MOF composite materials were prepared: bienzymatic composites GOx/CPO-DSG@UiO-66-NH2 and GOx/CPO-EDC@UiO-66-NH2, as well as single enzyme composites CPO-DSG@UiO-66-NH2, and CPO-EDC@UiO-66-NH2. The activity of these systems was measured using the MCD assay. While the EDC-coupled materials showed little activity, both DSG-coupled materials showed enhanced activity over the analogous free enzymes. All four crosslinked enzyme/MOF composites demonstrated enhanced recyclability, with ≥ 30% retained activity after four catalytic cycles. These results indicate that crosslinking enzymes to MOFs is an attractive biocatalysis strategy.”

Reviewer 4 Report

      This manuscript reported the preparation of an enzyme immobilization protocol via cross-linking to a metal-organic framework (MOF). This study investigates two different crosslinkers: disuccinimidyl glutarate (DSG) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinidimide (NHS) providing stable crosslinking of the MOF to the enzymes. The two crosslinkers are used to covalently bond chloroperoxidase (CPO) and glucose oxidase (GOx) onto UiO-66-NH2, and a comparison of the recyclability and enzymatic activity of the single immobilization of CPO and the doubly immobilized CPO and GOx was carried out.

     In general, the results of this work are interesting, and the conclusions are supported by the experimental details, but some major and minor points should be taken into consideration prior to publication:

     Major revisions:

-          To clarify better the focus of the manuscript, I suggest to insert in the Introduction a general scheme on the immobilization process.

-         -  In the Results and Discussion, recyclability experiments should be better explained.

-          To corroborate the immobilization process described in this paper, a protein leaching study should be considered.

      Minor revisions:

-      -    Page 2, line 58: the term substrate is misleading. Change substrate with support or a similar term.

-     -    Page 3, line 120: Remove double points.

-      -    Page 7, line 275: Change Chloroperoxidse with Chloroperoxidase.

-       -   Check the term crosslinking in whole text (or crosslinking or cross-linking).

-        -   Add the Author Contributions in the appropriate section.

       - Check the reference based on journal guidelines.

 

 

 

Author Response

Thank you for your careful review of our manuscript. We have taken your constructive comments and revised our manuscript. We believe that our results will provide a framework of how to prepare a biocatalyst with CPO which is of interest to the biocatalysis community because of its versatility as an enzyme. We have addressed your concerns, point-by-point.

 

     Major revisions:

-          To clarify better the focus of the manuscript, I suggest to insert in the Introduction a general scheme on the immobilization process.

 

Two new figures have been added to provide clarification of immobilization and crosslinking.

 

-         -  In the Results and Discussion, recyclability experiments should be better explained.

-          To corroborate the immobilization process described in this paper, a protein leaching study should be considered.

 

We plan to perform a leaching study in the future.

 

      Minor revisions:

-      -    Page 2, line 58: the term substrate is misleading. Change substrate with support or a similar term.

 

This has been done in all cases.

 

-     -    Page 3, line 120: Remove double points.

 

Thank you, this was corrected.

 

-      -    Page 7, line 275: Change Chloroperoxidse with Chloroperoxidase.

 

Thank you, this was corrected.

 

-       -   Check the term crosslinking in whole text (or crosslinking or cross-linking).

 

This was checked and corrected for consistency.

 

-        -   Add the Author Contributions in the appropriate section.

 

This will be done during copy editing.

 

       - Check the reference based on journal guidelines.

 

References have been corrected.

Round 2

Reviewer 2 Report

I would suggest moving some of the information from the conclusions to
the discussion. The summary should be short and informative, highlighting
the most important conclusions from the work. The text carboxylic acid group
appears several times in the text. In my opinion, there should be just a carboxylic
group. After making the above changes, it can be consider publishing this article
in the journal.

 

Author Response

Thank you for your careful review of our resubmission and we have responded to your feedback by editing the text that was suggested.

 

I would suggest moving some of the information from the conclusions to 

the discussion. The summary should be short and informative, highlighting 

the most important conclusions from the work. 

A whole paragraph was moved from the conclusion to the discussion along with text editing to conform to the discussion.

The text carboxylic acid group appears several times in the text. In my opinion, there should be just a carboxylic group. 

The text has been edited according to your advice in all cases.

After making the above changes, it can be consider publishing this article

in the journal.

Reviewer 4 Report

The Authors have satisfied all the requests suggested.

Author Response

Thank you for your thorough review of our resubmission.