Magnetically Responsive PA6 Microparticles with Immobilized Laccase Show High Catalytic Efficiency in the Enzymatic Treatment of Catechol

Round 1

Reviewer 1 Report

The manuscript submitted by Nadya et al. investigates the properties of laccase immobilized on polyamide supports in the context of its applications to the enzymatic treatment of catechol. The results presented by the authors are interesting both for cognitive reasons and for their potential application in the remediation of toxic diphenol pollutants. The manuscript is properly constructed but very carelessly, the objectives are stated clearly but from my point of view, the discussion of results needs to be refined. The part of the results was not discussed at all. More references to literature would enrich the manuscript. The figures, tables and signatures need to be corrected. There are also numerous editorial errors in the text. Detailed comments for consideration are provided below.

Comment 1#

Page 1, 2, 3

# in text is "Versicolor" instead of "versicolor"

Comment 2#

Page 3

# wt% - No spaces

# ca. 6 wt.% - spelling

Comment 3#

Table 1 is shifted in relation to the text. Incorrect format. No standard deviations in the table.

Comment 4#

Figure 1 is shifted in relation to the text. Part of the text is on the figure. Indications are illegible. Please correct figure 1

Comment 4#

Page 4

# in text is " Morphilogy" instead of "Morphology"

Comment 5#

Page 5

# in text is “Z-potential measurements” instead of “zeta potential measurements”.

# Figure 2 without caption, shifted in relation to the text.

Comment 6#

# The use of the UV method (280 nm) to measure protein (laccase) is not appropriate due to its limitations, the authors should use the Bradford method as the most optimal for enzymatic research.

Comment 7#

# Figure 3 is shifted in relation to the text.

# Figure 4, 5 and 6 - correct the figure caption.

# Table 3 - correct the caption and notes, no standard deviations in the table.

Comment 8#

#Table 4 is incorrectly placed in the text. Change the values in the table to two decimal places.

Comment 9#

# Figure 7, 8, 10, 11 and 12 – correct the figures and captions

# Figure 7b shows interesting differences between pH PA6@L conjugates which are not discussed in the text.

Comment 10#

#Table 5 and 6 is shifted in relation to the text. Correct the table and caption.

Comment 11#

# Page 16, correct the heading 2.6 (table is on the heading)

Comment 12#

# Page 20 - in the text Katchem (Check Republic) instead of Katchem (Czech Republic)

# Page 22 – in the text pH 5 instead of pH 4; signature to the kinetic parameters formula shifted in the text

Comment 13#

Correct the references according to the journal's guidelines.

Author Response

On behalf of all coauthors I thank for the general opinion and the detailed comments provided by the respected Referee 1. We apologize for the bad formatting of some parts of the original submission. It must have resulted in the conversion of the doc file to pdf during the submission. All critical notes related to this issue were taken care of by applying the new template of the journal. The references were also formatted accordingly. This time we checked both doc and pdf before submission and hope to have resolved all formatting problems. 

Language corrections

Correct "…Versicolor" to "…versicolor" throughout the text.  Done.

“Wt%” should be without spaces; remove the “ca.” abbreviation. Done.

On page 4 correct "Morphilogy" to "Morphology". Done.

In page 5 and throughout the text: correct “Z-potential…” to “zeta potential …”. Done.

Page 20 – change “Check Republic” to “Czech Republic”. Done.

Queries related to the content

Q. The use of the UV method (280 nm) to measure protein (laccase) is not appropriate due to its limitations, the authors should use the Bradford method as the most optimal for enzymatic research.

A. We understand the point of view of the respected reviewer. However, let us have in mind the following. During this study altogether 4 different immobilizations of laccase on all four PA6 MP supports were performed. All immobilization processes were carried out at absolutely the same conditions – pH, temperature, laccase solution concentration, quantity of PA6 MP, time duration of immobilization, and rate of shaking during the incubation. After the first immobilization, we studied the use of three methods for quantification of the laccase content in the supernatant, namely direct measurement of the UV absorption at 286 nm, Bradford method and also bicinchoninic acid assay (BCA). Various measurements were made in one point and the largest differences of laccase content (± 30% dispersion) were observed by the BCA method. Most probably this is due to the fact that the BCA method relies on reduction of protein-Cu²⁺ complex, which is apparently affected by the laccase Cu active centers. The most reliable data with higher reproducibility and negligible differences between the individual measurements were obtained by the direct UV method. As explained in the footer of Table 2, the standard deviation of the laccase amounts in this case was in the range of 3-5%. Quantification of the laccase content by the Bradford method led to standard deviations of 10-15%. This can be explained with the fact that all colorimetric methods (including the Bradford) are indirect and as such they are very sensitive to temperature, time duration of complex formation, presence of contaminations in the supernatant, etc. For all these reasons we chose the UV direct method for this study. In our case the laccase peak to be measured is shifted from 280 (as is in many other proteins) to 286 nm, which is an additional advantage. A short text in this sense was placed in p. 22 of the manuscript.

Q. Figure 7b shows interesting differences between pH PA6@L conjugates which are not discussed in the text.

A. We agree with this comment of the respected reviewer and introduced the following text as additional explanation of Fig. 7b.

“As seen from Fig. 7, laccase activity is strongly dependent on the pH value and exhibits the highest activity at pH 4-5, i.e. around its isoelectric point. The PA6Fe-L and PA6FeP-L samples even showed slightly increased activity as compared to free laccase. This can be due to some synergism between Fe0 and Cu active sites of the laccase already noted in the comments to Table 4. This effect was less pronounced with the PA6F_e3O₄-L sample, that contains Fe²⁺/Fe³⁺ ions. It is noteworthy that in the wider pH range of 3-6, the PA6@laccase conjugates retain more than 80% of their activity”.

Q. More references to literature would enrich the manuscript.

A. In this study we quoted 65 literature references. The studies on polyamides as enzyme supports are very scarce and, to the best of our knowledge, we mentioned all of them. We feel that mentioning completely different supports found in the literature (e.g., nanoclays, alginates, quitosan, PAN, etc.) would not be very appropriate here. We hope the respected reviewer will accept this point of view.

Reviewer 2 Report

Dencheva et al. present in their manuscript a detailed study on the immobilization of Trametes Versicolor laccase on neat and iron-doped porous polyamide 6 microparticles as well as their use for the oxidation of catechol. Since diphenols and especially catechol are toxic pollutants of soil and water, such systems might be used for the removal of such compounds form the environment,
The authors describe in detail the extensive characterization of the morphology and the crystalline structure of the particles. The enzyme activity of the immobilized laccase, the kinetics of the oxidation of the 2,2'-azino-bis-(3- ethylbenzothiazoline-6-sulfonic acid) and the reusability of the immobilized laccase-conjugates were tested as well.
However, the manuscript's title claims that "Magnetically Responsive PA6 Microparticles… "are prepared and studied, but the magnetic properties of the microparticles are neither characterized nor studied in the manuscript. The authors just determined the iron content and indirect effects of the incorporated iron species on other particles' properties. A proper characterization of the particles' magnetic properties and the incorporated magnetic fillers' properties and distribution should be added. Besides, the authors should show how these magnetic properties are needed for the intended applications.
In addition, the authors published in 2020 in this journal a somewhat similar study on "Polymer-Assisted Biocatalysis: Polyamide 4 Microparticles as Promising Carriers of Enzymatic Function" (Catalysts 2020, 10, 767; doi:10.3390/catal10070767), where almost the same characterization experiments are carried out just with polyamide 4 instead of polyamide 6 particles prepared in a similar way and also modified with the same iron species. As mentioned in the present manuscript, the new polyamide 6 systems show slightly different properties, such as fewer layers and a higher activity of the adsorbed laccase. Still, I wonder if these differences justify such an extended scientific article on a very similar system. The authors should point out and explain the importance of their present manuscript compared to their work from last year.
In most tables (Table 1, 2,3,4, and 6), data are given with many digits, but the measurement uncertainties are missing. This information has to be added, especially if small differences between the present samples or relative to the data from an article from 2020 are compared.

Author Response

On behalf of all coauthors I thank for the general opinion, the important queries and suggestions provided by the respected Referee 2. We tried to respond in detail and changed the submission where necessary. We hope to have thereby improved the manuscript.

Q1: …the manuscript's title claims that "Magnetically Responsive PA6 Microparticles… "are prepared and studied, but the magnetic properties of the microparticles are neither characterized nor studied in the manuscript. The authors just determined the iron content and indirect effects of the incorporated iron species on other particles' properties. A proper characterization of the particles' magnetic properties and the incorporated magnetic fillers' properties and distribution should be added. Besides, the authors should show how these magnetic properties are needed for the intended applications.

A1: We thank to the respected reviewer for this important comment. We obtained the magnetization curves of the Fe-containing samples in a VSM equipment. The results were compared in the new Figure S4 in the Supplementary Materials. In page 12 of the revision we added a new text explaining the VSM studies:

“The two Fe-containing PA6FeP and PA6Fe supports were studied in a vibrating sample magnetometer (VSM) at 23ºC. Notably, their magnetization curves were similar to that of the pure Fe (Figure S4 in the Supplementary Materials). The saturation values of the two PA6 supports were 13.44 emu/g and 12.70 emu/g respectively. With a saturation value of the neat Fe powder of 196 emu/g, the content of the ferromagnetic material in the two supports was found to be in the 6.5-6.8%, almost coinciding with that found by TGA (Table 1). Evidently, the VSM data of the PA6-based supports are strictly proportional to their Fe content without other changes in the hysteresis loop.”

In page 16 the following text on the magnetic properties was added:

“Fig. 10d visualizes also the attraction of the Fe-containing conjugates by constant magnets, which is an advantage enhancing their easy and complete removal from the reaction mixture and subsequent repeated reuse.”

Q2: In addition, the authors published in 2020 in this journal a somewhat similar study on "Polymer-Assisted Biocatalysis: Polyamide 4 Microparticles as Promising Carriers of Enzymatic Function" (Catalysts 2020, 10, 767; doi:10.3390/catal10070767), where almost the same characterization experiments are carried out just with polyamide 4 instead of polyamide 6 particles prepared in a similar way and also modified with the same iron species. As mentioned in the present manuscript, the new polyamide 6 systems show slightly different properties, such as fewer layers and a higher activity of the adsorbed laccase. Still, I wonder if these differences justify such an extended scientific article on a very similar system. The authors should point out and explain the importance of their present manuscript compared to their work from last year.

A2: The present study on PA6-laccase conjugates and the previous one on PA4-laccase systems differ in several crucial aspects. The PA6 microparticulate supports were synthesized by AAROP at 120ºC and atmospheric pressure, while for the PA4 supports temperatures as low as 40ºC and vacuum were needed for the polymerization to produce microparticles. These differences in the reaction conditions resulted in different topography, morphology and crystallization behavior of the particulate supports. Thus, the much porous PA6-based supports led to a laccase immobilization efficiency of 77-92%, while with the PA4@L conjugates these values were lower, varying in the 53-81% range. Moreover, the relative laccase activity of the PA6@L conjugates reached 106%, i.e., some of them were more active than the native laccase itself at the same reaction conditions. Meanwhile, the maximum relative activity of the PA4-laccase conjugates did not exceed 60%. Another important difference is that in the paper about the PA4-laccase conjugates two different immobilization techniques were applied – physical adsorption and entrapment during the AAROP. The latter technique is impossible with the PA6 supports. All these findings suggest different but possibly complementary applications of the two types of laccase conjugates, which, in our opinion, justifies their detailed investigation.

This text was placed in the Conclusions. We hope it gives an appropriate answer of the referee’s query. 

Q3: In most tables (Table 1, 2,3,4, and 6), data are given with many digits, but the measurement uncertainties are missing. This information has to be added, especially if small differences between the present samples or relative to the data from an article from 2020 are compared.

A3: We thank for this quite relevant comment. Wherever possible, we rounded off the values to the second meaningful digit. As seen for the comment above, the differences in the most important parameters between the PA6- and PA4-based samples are quite significant to be affected by this procedure