Polymer-Assisted Biocatalysis: Polyamide 4 Microparticles as Promising Carriers of Enzymatic Function

Round 1

Reviewer 1 Report

The article is an interesting piece of work. The authors mentioned that the work is the first study describing non-covalent PA4-laccase conjugates with all aspects of their preparation and characterization. The goal of the work was to investigate three new micron-sized particulate porous supports based on PA4, with and devoid of magnetic susceptibility. The authors compared two immobilization strategies (adsorption-immobilized (PA4@iL) and entrapped (PA4@eL)). Analysing the results of these two immobilization strategies they found morphological and the immobilization efficiency differences. The authors proved that PA4 microparticles could be used as possible enzyme supports for biotechnological applications. As was discussed in the article, the possibility of application of different immobilization strategies and using the magnetic field as external stimulus could open the way of using of PA4 in smart green catalytic systems. The article may be interesting to the broader scientific community. The authors comprehensively described  the metodology, results and discussion sections. The results were clearly presented also in supplementary filles. My recommendation is to accept the article for possible publication in “ Catalysts” in present form.

Author Response

We thank to the respected reviewer for the recommendation to accept the manuscript in its initial form.

Reviewer 2 Report

This paper describes the potential of magnetic-responsive polyamide 4 (PA4) microparticles obtained by low-temperature using activated anionic ring-opening polymerization as supports for immobilization of laccase. The authors used two immobilization strategies: (i) physical adsorption of laccase and (ii) in situ encapsulation (entrapment) of laccase during the activated anionic ring-opening polymerization of PA4 synthesis. The morphology and the crystalline structure of the PA4-laccase preparations were analyzed by microscopy, spectral and X-ray scattering techniques. The enzyme activities toward 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) were also compared. The reusability of the immobilized biocatalysts was evaluated and the activity of the immobilized laccase was tested in decolorization reactions of malachite green and bromophenol blue. This paper covers a field of great interest to Catalysts readers. However, some points need to be addressed. The questions raised are as follows:

Line 133. I suggest the authors describe the three microparticulate PA4 supports fabricated (PA4, PA4-Fe and PA4-Fe₃O₄) the first time they appear in the text.

Line 157. Define TGA.

Line 226. Define IE.

Table 3. What is the activity expressed in each preparation?

Table 5. Again, what is the expressed activity of the different preparations?

Figure 10. The legend of the figures must be self-explanatory. Add the experimental conditions.

Figure 11a. Correct y-axis legend.

It is necessary to add a more in-depth discussion comparing the results obtained here with other previous works. The laccase studied in this work has been immobilized by different strategies in numerous scientific articles. In this way, it would be easier to highlight the novelties and improvements of the immobilization strategy presented by the authors in this paper.

Author Response

We would like to thank for the reviewer´s notes and also for the criticism that helped improve this submission.

Point-by-point answers:

Q1 Line 133. I suggest the authors describe the three microparticulate PA4 supports fabricated (PA4, PA4-Fe and PA4-Fe₃O₄) the first time they appear in the text.

A1 The three microparticulate supports represent fine powders with white (PA4), grey (PA4-Fe) or brownish color (PA4-Fe₃O₄). This description is introduced in p. 3, lines 134-5.

Q2 Line 157. Define TGA.

A2 TGA = thermogravimetric analysis. This abbreviation appears first in the footer of Table 1 so it was defined there. TGA is also defined in Section 3.2 (line 808).

Q3 Line 226. Define IE.

A3 IE = immobilization efficiency. In the revised text it is was explained in line 220 where it first appears and in the footer of Table 3 (line 239). 

Q4 Table 3. What is the activity expressed in each preparation?

A4 Column 1 in Table 3 represents the activity of the laccase that remained in the supernatant after completing the immobilization process. The dimension is µkat.mL^-1. The data in this table help calculate the amount of the immobilized laccase. The activity of the immobilized laccase in each one of the preparations is shown in Table 7, section 2.4.

Q5 Table 5. Again, what is the expressed activity of the different preparations?

A5 Table 5 does not display activity data. As it was mentioned in the previous query, this table shows parameters necessary to calculate the amount of the entrapped laccase. The activity of the three different preparations (PA4@eL series) is given in Table 7, as compared to the other three samples (PA4@iL series).

Q6 Figure 10. The legend of the figures must be self-explanatory. Add the experimental conditions.

A6 The experimental conditions for each cycle in Figure 11 were included in the legends as required. For better understanding, more details about the leaching study were given in the Experimental part (Section 3.3.4, lines 889-893, highlighted in red)   

Q7 Figure 11a. Correct y-axis legend.

A7 Done as required.

Q8 It is necessary to add a more in-depth discussion comparing the results obtained here with other previous works. The laccase studied in this work has been immobilized by different strategies in numerous scientific articles. In this way, it would be easier to highlight the novelties and improvements of the immobilization strategy presented by the authors in this paper.

A8 The publications reporting the use of polyamides as enzyme supports are very scarce, in most of the cases the carrier investigated is PA6 as films, fibers or textile structures. Throughout the original manuscript and especially in the ‘Conclusions’ we highlighted the novelty of the present study. Summarizing, we describe for the first time the biodegradable PA4 porous microparticles, prepared by us by a low-temperature polymerization, without and with magnetic susceptibility, as supports for immobilization of enzymes. Moreover, we showed that this immobilization can be done in two different ways – by adsorption and entrapment. The results of our decolorization studies favorably compare to other authors data using laccase on alginate/chitosan supports [46] or free laccase + mediator [47]. Judging by the activity test measurements, we have attained higher amounts of immobilized laccase on our PA4 supports, as compared to PA6 film supports [21] or carbon nanotubes/polysulfone membranes [30]. These cited references offer adequate comparison to immobilization systems close to ours. We feel that a comparison to completely different supports that can be found in the literature i.e., inorganic metal oxides, silica, nanoclays, or organic biopolymers such as alginate, cellulose or chitosan, or other synthetic polymers (polyacrylonitrile, some ionomers, etc.) would not be very appropriate. We hope that the respected reviewer will accept this point of view.

Author Response File: Author Response.pdf