Laccase Cross-Linked Ultraporous Aluminas for Sustainable Biodegradation of Remazol Brilliant Blue R

Round 1

Reviewer 1 Report

The authors describe immobilization processes of laccase on ultraporous aluminas. This reviewer recommends publication after major revisions. Here are my concerns that need to be addressed.

1. How are you measuring loading of enzyme? An activity assay is not sufficient since enzyme may behave differently when immobilized so quantitation must be quantitated by other methods.

2. Figure 1b, wouldn’t there also be a mixture of  laccase active site orientations?

3. Figure 2. What buffer was the enzyme dissolved in prior to FTIR (if phosphate) make sure the peaks you see aren’t associated with phosphate buffer. Figure 2 c, did you confirm the C=N peak you assign, it does not to seem to be much different than the material alone. Since this is the significant result, there needs to be more evidence of crosslinking.

Author Response

Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 2 Report

 

 

The manuscript is written in a  tangled way and it is difficult to follow.

There are some inaccuracies in the introduction:

The authors refer to conventional immobilization method of enzymes "notably oxidoreductases": these methods are general for all enzymes, not notably for any particular class of them

Further they claim that “the proper selection of supporting materials of different origins  … usually ensures enhanced enzyme stability ….  “I totally disagree: the only selection of support does not ensure enzyme stabilization

"abundant presence of many functional groups ….  may facilitate the versatility of inorganic 87 materials ": this is imprecise. Inorganic materials may present abundant groups but the functionality is added to the material, in other words, inorganic materials are functionalized, exactly the same as the organic ones.

"Trametes versicolor": the right way to write Linnaean nomenclature of living beings is with italic letters

 

 

Schiff bases are formed by nucleophilic attack of unprotonated amine groups on the carbonyl group of aldehyde. In section 2.1 the authors explain that the pH of the crosslinking process is between 7 and 9 because of the higher reactivity of GA in this range. pKa of the amine groups from Lys and Arg is higher than 9.  Similarly, many authors define pKa of APTES equal or higher than 10. So, within this pH 7-9 range, these amines must be protonated and therefore they are unreactive. Then, the authors should confirm the pH value at which this process is performed or explain how the reaction takes place at pH lower than 9 (at the beginning of the section “sequential immobilization” they mention “neutral pH”. Does it mean that the process is at pH 7.0?)

fig 1c : UPA powders-Laccase: Since pI of laccases is generally low, around 4-4.5, at the pH mentioned (between 7 and 9) the net charge of laccase is negative. So the interaction with support would take place via electrostatic interactions. Remaining amine groups of laccase would be available for reaction with GA if pH was over 9 or 10. This means a different orientation of enzyme immobilized this way, which is not considered even when the authors claim that orientation may affect AR% of the biocatalysts

Pore diameters of the UPA materials are not given, nor the amount of laccase available for immobilization (therefore enzyme loading is not known).

No explanations are provided for the drastic drop of activity of laccase T./UPA(γ) after a few reaction cycles. Considering that the enzyme is just linked through non covalent electrostatic attraction, the pH at which these catalysts are washed after each cycle may be responsible for enzyme release.

The authors present kinetic studies on RBBR depletion and enzymatic degradation. But they do not present the chemical reaction nor the corresponding explanations on how the whole process takes place. I do not agree with the sequence of adsorption/catalytic process. Do authors suggest saturation of the powder, so no RBBR can access inside porous particles? Why do catalyst can only act on soluble RBBR?

Only UPA(γ) is used, maybe this can be related to a higher pore size than the other UPA materials, but pore diameters are not given.

 

a and b from figure 4 are not described in the legend

In my opinion there is a great deal of work in this manuscript that is not focused and developed in a precise and systematic way. It would have to be refocused and rewritten adding many explanations. In other words, in my opinion it cannot be published as is

 

 

Author Response

Please see the attachment."

Author Response File: Author Response.pdf

Reviewer 3 Report

 

Authors reported immobilization of laccase onto alumina via crosslinking approach. Then the immobilized enzyme was used for biodegradation of Remazol Brilliant Blue R under optimal reaction conditions.

The following comments should be considered before publication.

1. English should be improved. Some sentences are very long and confusing.

2. The scientific names of species and genus should be italicized.

3. SEM and TEM images and XRD spectra of the alumina should be added in the characterization section. In addition, surface area and pore size should be analyzed by N₂ adsorption-desorption isotherms.

4. In case of “laccase T. cross-linked UPA(γ) biocatalysts”, It is essential to determine what portion of dye is removed by adsorption and what portion is biodegraded. Using suitable blanks would be useful.

5. Kinetic experiments are missed in the “Materials and methods” section.

Author Response

Please see the attachment."

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have made a great effort to answer and explain the requested points of the manuscript and improve its the general presentation. In my opinion they have done a good job and it can now be published.

Reviewer 3 Report

I believe the manuscript has been sufficiently improved to warrant publication in Catalysts.