Laccase-Oriented Immobilization Using Concanavalin A as an Approach for Efficient Glycoproteins Immobilization and Its Application to the Removal of Aqueous Phenolics

Round 1

Reviewer 1 Report

The work pertaining to the presented manuscript deals with the immobilization of the laccase enzymes onto Immobead 150P carrier via two mechanisms. The activity of the resultant complex was thoroughly probed at various temperature and pH conditions, and the removal potential of the complex was assessed using three target phenolic compounds. Upon reading the manuscript, the following issues were raised:

- The colloidal stability of the carrier (Immobead 150P), which is an important issue in the immobilization of enzymes, was not discussed. What is the aggregation status of used carrier since the colloidal stability of popular enzymes carriers such as latexes can be affected by the pH as well as the ionic strength (since multivalent salts were used). If such information is, the authors shall mention such information where appropriate.

- Enzyme carriers often reach a saturation point, how did the authors decide on the dose of Con-A as well as the laccase, such that the used doses won't leave free (non-bound) enzymes in the different preparations. Also, is random immobilization possible during oriented immobilization?

- Why do the free and random immobilized enzymes have better performance below 70°C (section 3.3). Also, since 70 °C was the optimal temperature, why did the enzyme preparations experience the most significant decrease with time at 70 °C in Figure 3, even within few minutes of pre-incubation (according to the fit). Similarly, in Figure 5, the maximum reduction in activity occurred around the optimal pH.

- What about the enzyme leaching after the formation of the enzyme-carrier complex during complex isolation, and how was the isolation performed during reusability check, such details has to be added to the manuscript.

- Generally, the authors made no literature comparison for the presented results such as temperature/pH profile and reusability for similar enzyme complex. Results of similar studies have to be mentioned where appropriate?   

- In the removal of the phenolic compounds, what is the fate and toxicity of the end products, compared to their initial counter part, such information was not mentioned?

- The description of the experimental procedure in Section (2.6) is quite ambiguous, the author shall provide more details on the experimental procedure and the setup especially how the ABTS is removed to terminate the enzymatic reaction and the order of addition of components.

- In a number of places, the author used the term "standard assay conditions", these conditions shall be unambiguously stated.

- More details on the experimental procedure have to be added in (section 2.7 and 2.8).

- In Section (2.9), the experimental procedure and conditions, at which the outstanding activity (A) and thermodynamic parameters are determined,  have to be detailed. Also, A ought to be unambiguously defined.

- There are a lot of experimental conditions such as pH, temperature, buffers, at which various measurements were performed, the authors are advised to generate a comprehensive table where the conditions for the various measurements are listed, that makes it easy to follow the various measurements .

- The manuscript contain a number of punctual typos such as superscripts/subscripts,  line 216, (lin) in equation (4),  line 538, line 550. The authors shall revise the manuscript and correct such occurrences.

Author Response

1. The colloidal stability of the carrier (Immobead 150P), which is an important issue in the immobilization of enzymes, was not discussed. What is the aggregation status of used carrier since the colloidal stability of popular enzymes carriers such as latexes can be affected by the pH as well as the ionic strength (since multivalent salts were used). If such information is, the authors shall mention such information where appropriate.

Response:

Immobead 150 is an epoxy support with a high surface area that is formed by cross-linked copolymers of methacrylate bearing oxirane groups. Given the enzyme carrier requirements of large surface area, narrow pore size distribution, well-defined pore geometry, and thermal/mechanical stability, this polymer is a promising candidate for enzyme immobilization, and has been employed to immobilize different enzymes by taking advantage of its capacity to adsorb or form covalent linkages.

Since Immobead 150P is a macroporous carrier, and due to its macro-size, it tends to precipitate, and for that all its reactions should be conducted under stirring.

For that, we presented a two-step procedure to immobilize laccase enzyme on Immobead 150P with the application of Con-A. By this, the method offered the stability of Immobead 150P and the affinity of Con-A to immobilize glycoproteins via molecular recognition of their glycosidic residues and, in the meantime, shields the unfavorable interactions between enzymes and the support surface and thus prevents enzyme deactivation.

For that, we added a short paragraph about Immobead nature in the Introduction section (lines: 66-72)

2. Enzyme carriers often reach a saturation point, how did the authors decide on the dose of Con-A as well as the laccase, such that the used doses won't leave free (non-bound) enzymes in the different preparations. Also, is random immobilization possible during oriented immobilization?

Response:

The process of Con A-glycoprotein self-assembly was achieved on the basis of the method described by Li et al., 2013 (doi:10.1002/jctb.4091). Regarding the saturation points, we did different trials to attain the suitable doses of Con-A and laccase (data not shown), on the basis of our published article (https://doi.org/10.1016/j.enzmictec.2021.109865) to define the exact doses required for the highest immobilization yield. Additionally, we followed the decrease in protein and activity during oriented and random laccase immobilization using Concavalin A on the modified Immobead 150 to define the proper time course of each tested support (Supplementary file).

To be sure that there is no free (non-bound) enzyme in the different preparations, as we mentioned in the Methods section (2.5), the Immobead 150P/Con-A complex was washed with an excess of distilled water to remove the un-bound Con-A. Additionally, after enzyme immobilization, the whole support washed with excess of distilled water and finally with phosphate buffer (100 mM, pH 7.0) to eliminate the unbound laccase.

Regarding the possibility of random immobilization occurring during oriented immobilization? Well, this could be possible to happen, but it will be in a little amount, due to the occupation of Immobead 150P reactive arms (glyoxyl groups) by the bound Con-A.

3. Why do the free and random immobilized enzymes have better performance below 70°C (section 3.3). Also, since 70 °C was the optimal temperature, why did the enzyme preparations experience the most significant decrease with time at 70 °C in Figure 3, even within few minutes of pre-incubation (according to the fit). Similarly, in Figure 5, the maximum reduction in activity occurred around the optimal pH.

Response:

The reaction rate of the immobilized enzymes is usually much lower than that of free enzymes because of the lower activity of immobilized systems and also steric hindrance. This is usually due to the required higher activation energy in case of immobilized form. Lettera et al., [https://doi.org/10.1016/j.foodchem.2015.10.074.] found that the higher response of the free enzyme at a more extensive temperature range is attributable to the reduction in flexibility of the immobilized enzyme.

On the same way, due to the difference between the construction of both random and oriented forms, it is supposed that they have different affects by different reaction factors.

Regarding the significant decrease with time at 70 °C, in the optimum temperature experiment, the activities of free and immobilized laccases were estimated only for 1.0 and 4 min in their reaction mixtures. On the other hand, thermal stability was conducted by heating the enzyme preparations (up to 6 h) before using it in the reaction. So, more or less, they are not correlated to each other as they are different effects and also due to the broad scale (in h) of stability experiment.

Similarly, regarding the significant decrease with time at pH 3.0, in the optimum pH experiment, the activities of free and immobilized laccases were estimated only for 1.0 and 4 min respectively, in their reaction mixtures. On the other hand, pH stability was conducted by incubating the enzyme preparations (up to 6 h) before using it in the reaction. So, more or less, they are not correlated to each other as they are different effects and also due to the broad scale (in h) of stability experiment.

Additionally, as mentioned in discussion part, the optimum pH value is depending on the substrate and its redox potential, so laccases have variable optimal pH levels regarding to substrate. Here, laccase has shown the greatest action on ABTS in acidic conditions at pH 3.0. On the other hand, stability experiment depending on the stability of the protein structure and shape in specific pH values for large time courses.  

4. What about the enzyme leaching after the formation of the enzyme-carrier complex during complex isolation, and how was the isolation performed during reusability check, such details has to be added to the manuscript.

Response:

From the results of the reusability of laccase immobilized using oriented and random immobilization, where ten operational cycles (each round is 4.0 min) were achieved. The rounds of the laccase reaction were stopped by the removal of the substrate from the reaction medium. Consequently, the immobilized enzyme was gently washed by distilled water and then it was returned to a fresh re-action medium, to start a new reactivity cycle.

For that, if there is an enzyme leaching after the formation of the enzyme-carrier complex, it was appeared as a notable gradual decrease in the activity after each cycle, and this is not happened as reported in the results (Fig 1).

Regarding the isolation performed during reusability check, the rounds of the laccase reaction were stopped by the removal of all reaction medium except immobilized laccase through filtration (line: 200-201). Consequently, the immobilized enzyme was gently washed by distilled water and then it was returned to a fresh reaction medium, to start a new reactivity cycle (Section 2.6).

5. Generally, the authors made no literature comparison for the presented results such as temperature/pH profile and reusability for similar enzyme complex. Results of similar studies have to be mentioned where appropriate?

Response:

More literature comparison for the presented results was added in section 4.

6. In the removal of the phenolic compounds, what is the fate and toxicity of the end products, compared to their initial counter part, such information was not mentioned?

Response:

Actually, in the current study we aimed to utilize an innovative approach for the oriented immobilization of laccase as a glycoprotein on the surface of Immobead 150P utilizing the unique properties of Con-A as a bio-affinity ligand. Then, we intend to offer available model of microbial enzymes might be used to remediate contaminated wastewater in an economical and environmentally benign manner. For that and due to the importance of this issue, we intend to do a further detailed study for the bioremediation process including degradation mechanisms and resulted products and their effects on the surrounding ecosystem.

7. The description of the experimental procedure in Section (2.6) is quite ambiguous; the author shall provide more details on the experimental procedure and the setup especially how the ABTS is removed to terminate the enzymatic reaction and the order of addition of components.

Response:

Section (2.6) was rephrased to be clearer and more details on the experimental procedure were amended.

8. In a number of places, the author used the term "standard assay conditions", these conditions shall be unambiguously stated.

Response:

The phrase "standard assay conditions" mean that the conditions are as mentioned in Section 2.3 (Enzyme activity estimation). This was considered to avoid unnecessary wording repetition. Otherwise, we went through the document to clear what was not described in the methods part.

9. More details on the experimental procedure have to be added in (section 2.7 and 2.8).

Response:

More details on the experimental procedure were added in (section 2.7 and 2.8).

10. In Section (2.9), the experimental procedure and conditions, at which the outstanding activity (A) and thermodynamic parameters are determined, have to be detailed. Also, A ought to be unambiguously defined.

Response:

The experimental procedure and conditions, at which the residual enzyme activity (A) and thermodynamic parameters are determined, were detailed previously in the section 2.8 (Thermal and pH stability), in addition to the details present in the section 2.9. Also, A was re-expressed as the residual enzyme activity.

11. There are a lot of experimental conditions such as pH, temperature, buffers, at which various measurements were performed, the authors are advised to generate a comprehensive table where the conditions for the various measurements are listed, that makes it easy to follow the various measurements .

Response:

Thank you for this suggestion, but as the reviewer mentioned there are a lot of experimental conditions such as pH, temperature, buffers, at which various measurements were performed. For that generate such a comprehensive table where the conditions for the various measurements are listed, will make the following of conditions for every experiment as a difficult and an exhausting mission. So, we think putting such information under each experiment will make it easy to follow.

12. The manuscript contain a number of punctual typos such as superscripts/subscripts,  line 216, (lin) in equation (4),  line 538, line 550. The authors shall revise the manuscript and correct such occurrences.

Response:

Thank you. The whole manuscript was revised to correct the punctual typos.

Reviewer 2 Report

I have some questions and concerns that should be addressed by the authors as they revise the manuscript:

Line 21 -Should be place a degree Centigrade sign after 50.

Line 41- The reference 6 is a review paper. It will be helpful if the author cites reference/references where it talks about the electron transfer mechanism.

Line 123- replace “provided” with purchased from

Table 2- R2 value for free form at 50C is low. Do you have any explanation for this?

Table 3- delta G and H have space in their unit should be kJ/mol

Line 433- pyrimethanil is not a phenolic compound. It’s a commonly used fungicide. It will be better to rewrite the sentence.

Overall, the authors methodology has a fair way to go before it can be considered as being broadly applicable for Laccase-oriented efficient glycoproteins immobilization and its application to the removal of aqueous phenolics- as there is extremely stiff competition in this area. However, this work is a good step in the right direction towards understanding Laccase-oriented immobilization using Concanavalin A and this mechanistic study fits in nicely to the remit of Sustainability and is of an appropriate impact and novelty for this journal.

Author Response

1. Line 21 -Should be place a degree Centigrade sign after 50.

Response:

Thank you. A degree Centigrade sign after 50 was added.

2. Line 41- The reference 6 is a review paper. It will be helpful if the author cites reference/references where it talks about the electron transfer mechanism.

Response:

More references talks about the electron transfer mechanism were added.

3. Line 123- replace “provided” with purchased from

Response:

“Provided” was changed by “purchased from”

4. Table 2- R2 value for free form at 50C is low. Do you have any explanation for this?

Response:

For R-squared, higher R-squared values indicate that the data points are closer to the fitted values. While higher R-squared values are good, they don’t tell you how far the data points are from the regression line. Additionally, R-squared is valid for only linear models. So, the relatively lower R-squared value (0.765) for free form at 50 °C may be due to the deviation between fitting results and the stability results trend.

5. Table 3- delta G and H have space in their unit should be kJ/mol

Response:

Thank you. They were corrected.

6. Line 433- pyrimethanil is not a phenolic compound. It’s a commonly used fungicide. It will be better to rewrite the sentence.

Response:

The sentence was re-phrased to deal with the comment.

7. Overall, the authors methodology has a fair way to go before it can be considered as being broadly applicable for Laccase-oriented efficient glycoproteins immobilization and its application to the removal of aqueous phenolics- as there is extremely stiff competition in this area. However, this work is a good step in the right direction towards understanding Laccase-oriented immobilization using Concanavalin A and this mechanistic study fits in nicely to the remit of Sustainability and is of an appropriate impact and novelty for this journal.

Response:

Thank you so much for such positive evaluation.

Reviewer 3 Report

The article describes the immobilization of a laccase enzyme to a resin support using a bioaffinity protein to orient the laccase. The modified material is to be used for water purification. While the approach is not entirely new, as the Con-A system is already well known, the article nevertheless describes an interesting method for resin supports.

The paper is well written, but some sentences are worded rather bulky, so a linguistic simplification could help the readers. The main issue with the article is the lack of control values in the biodegradation experiments. Some points need revision, which are mentioned below or highlighted in the attached file.

Line 64: Before this, briefly explain what Immobead 150P is

Line 80: write the full name of Con-A (since abbreviation appearing for the first time)

Lines 98-102: the information in this section duplicates with previous section

Line 129: the phenolic substances are missing in Materials & Methods

Line 163: Throughout the whole paper, the phrase "purified or aminated laccase" occurs, but it is not clear what exactly the difference is. Isn't the aminated form also purified? Is the purified form more of a "non-aminated" form?

Line 167: What does "pre-activated" mean here?

Line 254: HBT is not listed or explained

Line 257: The term "abiotic action" is unclear. What is it and wouldn't "adsorption effects" be more appropriate?

Line 259: Details of the HPLC measurement are missing

Scheme 1: The graph is too inaccurate, the aldehyde groups should be converted after immobilization. Currently it looks like an adsorption.

Line 303: Enzyme immobilization is described extensively, but Con-A immobilization is not characterized enough, e.g., what was the yield?

Line 325: In enzyme biochemistry, the substrate would be ABTS (but that was converted). Clarification is needed for "removal of substrate", probably the Immobead particles are meant (i.e., support).

Figure 2 and next: It is unclear whether it is the aminated or purified form, and why one was chosen.

Table 3: As a note, the "better" enzyme constant is V_max/K_m (=V/K), see doi.org/10.1021/ed075p1153

Table 4 and 3.7: The main issue with the article is the lack of control values in the biodegradation of the phenolic substances. In principle, evidence that there was an enzymatic conversion is missing. The adsorption of the Immobeads seems to be very high, which could also explain the "removal". HPLC data should show that transformation products were formed. In addition, the values for the controls (Immobead without enzymes; and free enzyme) should be added in Table 4 for all phenolic substances. This would be the only way to decide whether the systems are suitable for enzymatic water treatment.

Line 478: Why should the amino groups in the aminated laccase have an effect on Con-A binding when it binds via the glycosyl residue? The amination could also affect the active center, or?

Line 481: The stability should depend on the immobilization method, since the support (Immobeads) was the same?

Line 492: Another reason for the temperature effect is that the Con-A-laccase complex should not have a covalent bond, but is supramolecular, which leads to weakening by thermal motion at higher temperatures.

Line 494: The increased temperature stability is not a proof of the superiority of laccase immobilization over fungal laccases (50-60°C), since here even the free laccase was already highest at 70°C.

Line 545: Steric hindrance was used to explain the low K_m values, but shouldn't it be the other way around? Low K_m values stand for a high substrate affinity. Steric hindrance should actually make substrate binding more difficult and thus lead to higher K_m values?

Comments for author File: Comments.pdf

Author Response

1. Line 64: Before this, briefly explain what Immobead 150P is

Response:

Immobead 150 was briefly explained in the same paragraph (lines: 66-72).

2. Line 80: write the full name of Con-A (since abbreviation appearing for the first time)

Response:

The full name of Con-A was written at its first appearance.

3. Lines 98-102: the information in this section duplicates with previous section

Response:

Thank you. The sentence was deleted to avoid duplicates with previous section.

4. Line 129: the phenolic substances are missing in Materials & Methods

Response:

The phenolic substances were added to Materials & Methods.

5. Line 163: Throughout the whole paper, the phrase "purified or aminated laccase" occurs, but it is not clear what exactly the difference is. Isn't the aminated form also purified? Is the purified form more of a "non-aminated" form?

Response:

Thank you. The clarified laccase was then aminated using EDA and EDC solutions at pH 4.75 as described in section 2.2. So, amination is a further step done for the purified form to enrich its amino groups content.

6. Line 167: What does "pre-activated" mean here?

Response:

"pre-activated" means that Con-A was pre-activated in 0.1 M phosphate buffer at pH 7.0 containing 0.1 M KCl, 0.1 mM CaCl₂, and 0.1 168 mM MnCl₂ for 15 h) and shaken at 200 rpm at 30 °C for 3 h [doi:10.1002/jctb.4091].

7. Line 254: HBT is not listed or explained

Response:

HBT was listed in Materials and Methods and explained in Results.

8. Line 257: The term "abiotic action" is unclear. What is it and wouldn't "adsorption effects" be more appropriate?

Response:

The term "abiotic action" was changed to "adsorption effects".

9. Line 259: Details of the HPLC measurement are missing

Response:

Details of the HPLC measurement were added.

10. Scheme 1: The graph is too inaccurate, the aldehyde groups should be converted after immobilization. Currently it looks like an adsorption.

Response: Thank you so much for this valuable comment. The scheme was corrected.

11. Line 303: Enzyme immobilization is described extensively, but Con-A immobilization is not characterized enough, e.g., what was the yield?

Response:

Con-A immobilization is described and characterized also in the 3.1 section’s text, where we referred to it as the oriented immobilization process with a yield of 93.57% (Table 1).

12. Line 325: In enzyme biochemistry, the substrate would be ABTS (but that was converted). Clarification is needed for "removal of substrate", probably the Immobead particles are meant (i.e., support).

Response: Thank you. Clarification was done to make it more understandable.

13. Figure 2 and next: It is unclear whether it is the aminated or purified form, and why one was chosen.

Response:

At the end of section 3.2 (Reusability of the immobilized laccase preparations), we stated that “For that, oriented and random immobilization of the aminated laccase will be further studied through next experiments”. This decision is due to the higher immobilization efficiency (Table 1) and reusability (Figure 1) of the aminated laccase.

14. Table 3: As a note, the "better" enzyme constant is V_max/K_m(=V/K), see doi.org/10.1021/ed075p1153

Response:

Enzyme constant V_max/K_m was applied instead of K_m/V_max.

15. Table 4 and 3.7: The main issue with the article is the lack of control values in the biodegradation of the phenolic substances. In principle, evidence that there was an enzymatic conversion is missing. The adsorption of the Immobeads seems to be very high, which could also explain the "removal". HPLC data should show that transformation products were formed. In addition, the values for the controls (Immobead without enzymes; and free enzyme) should be added in Table 4 for all phenolic substances. This would be the only way to decide whether the systems are suitable for enzymatic water treatment.

Response:

Thank you. That’s right, as we mentioned in the results (section 3.7) and discussion, it is evidence already presented in previous literature that Immobeads have strong adsorptive activity. However, it is difficult in this case to determine the difference between adsorption-degradation. The only way is to operate during long time or successive cycles because the usual is to achieve the adsorption saturation, however, the enzyme activity follows the degradation of the pollutants. Thanks for your recommendation, these experiments will be performed in future studies.

16. Line 478: Why should the amino groups in the aminated laccase have an effect on Con-A binding when it binds via the glycosyl residue? The amination could also affect the active center, or?

Response:

Thank you. The sentence was reduced to avoid confusion.

17. Line 481: The stability should depend on the immobilization method, since the support (Immobeads) was the same?

Response:

We mean here that the immobilization complex specifications and steps could affect the stability due to the more sensitivity of some immobilization matrices such as Con-A (oriented) than only Immobead itself (random), which leads to the malfunction of different supports in different conditions.

18. Line 492: Another reason for the temperature effect is that the Con-A-laccase complex should not have a covalent bond, but is supramolecular, which leads to weakening by thermal motion at higher temperatures.

Response:

Thank you. The sentence was modified to fit that.

19. Line 494: The increased temperature stability is not a proof of the superiority of laccase immobilization over fungal laccases (50-60°C), since here even the free laccase was already highest at 70°C.

Response:

Yes, that’s true, but we mean the superiority of laccase forms under study (including the free form) over other some fungal laccases which aren’t stable at this range of temperature.

20. Line 545: Steric hindrance was used to explain the low K_mvalues, but shouldn't it be the other way around? Low K_m values stand for a high substrate affinity. Steric hindrance should actually make substrate binding more difficult and thus lead to higher K_m values?

Response:

Yes, we agree with that, but what we meant is that higher km values in the case of oriented immobilization could be due to steric hindrance.

 

Reviewer 4 Report

The abbreviation needs to write Concanavalin A (Con-A) for the fist time.

 English language and style are fine/minor spell check required

Author Response

The abbreviation needs to write Concanavalin A (Con-A) for the fist time.

Response:

Thank you for your comment. This error has been modified

 English language and style are fine/minor spell check required

Response:

The manuscript has been revised

Round 2

Reviewer 1 Report

The authors have implemented the recommended changes. Therefore, I recommend the publication of the manuscript.

Reviewer 3 Report

The comments and remarks were addressed by the authors, and the quality of the manuscript was improved.