Visualizing Hydrophobic and Hydrophilic Enzyme Interactions during Immobilization by Means of Infrared Microscopy

Round 1

Reviewer 1 Report

Dear Editor,

in this manuscript, Pauli and co-Authors described the immobilization kinetics and distribution of CALB on two different resins, by infrared microscopy. This work highlighted that CALB was successfully immobilized in the first hours of the process and that the CALB activity is due to the enzyme localized on the surface.

Despite the scientific soundness of this work and the broad interest that the manuscript arouses, the description of the results appears to be very technical, suitable only for FTIR experts. This manuscript could be improved by better describing the method and interpretation of micro-FTIR data in language suitable even for non-expert readers such as enzymologists or biotechnologists.

Analytical comments:

Line 14. What do the Authors mean with “very hydrophobic” and “very hydrophilic” resin?

Line 46. An article was recently published in which the authors used micro-FTIR to study the secondary structure of immobilized CALB in the absence and presence of short-chain alcohols (https://doi.org/10.1002/biot.202100712). This work should be mentioned in the introduction or in the discussion.

Line 85. Reference is missing.

Line 87. The immobilization procedure should be better described. Some information is missing such as resin functionalization, immobilization yields etc. The SDS-PAGE analyzes reported in line 187 should be moved here. Furthermore, a quantification of the unbound CALB is necessary, the authors could quantify the CALB band by densitometry. I was wondering why the authors analyze unbound CALB with SDS-PAGE, which has low sensitivity, and do not measure the hydrolytic activity of CALB in the liquid fraction.

Line 94. It is not clear the relationship between CALB size and porosity of the resin. Please clarify.

Line 110. The description of the micro-FTIR spectra shown in Figures 1A and B is cryptic for non-expert readers. Please add information on the interpretation of the micro-FTIR spectra and how the authors obtained the spectra in Figure 1A and B.

Line 127. Again, the description of the 3D-FTIR-spectra and heatmaps is very cryptic. Please make this paragraph understandable by non-expert readers. I suggest adding arrows or box in figure 2 to highlight the spectral regions of interest.

Line 144. See the previous comments.

Line 172. Why did the authors choose these two catalytic reactions? Please clarify.

Line 179. It is not clear why the Authors showed both specific activity and relative activity in Figure 5. In my opinion, panels A and C and B and D in Figure 5 contain the same information; differences in the immobilization rate between the two resins are observed even without normalization. Moreover, it is not clear the number of replicates and why the error bars are not shown in all experimental points.

Line 213. The authors attributed the different behavior of CALB to the polarity of the resin. however, the immobilization procedures are also different, in one case adsorption and in the other covalent immobilization. In my opinion, the difference in the immobilization procedure should be considered when interpreting the data.

Line 315. The number of replicates is never mentioned in the text. I suppose there is a high heterogeneity in the immobilized samples, the spectra shown in the "Results" section are the average of how many measurements?

Line 329. Please specify the TBU abbreviation.

Line 369. Some procedures such as paraffin embedding, microtome slicing and 3D-printed are reported here for the first time. it is not clear with which procedure the samples shown in the "results" section were prepared.

Line 405. The supporting information contains some information that will help the reader understand the results. However, the presence of supporting information was mentioned here for the first time. Some figures should be moved to the main text to help non-expert readers.

Author Response

Dear editor, Dear reviewer,

We thank you for considering the possibility to publish our paper in the special edition of Catalysts.

We here declare that authors are aware of the submission and agree to its publication. The submission is original and is not under consideration for publication elsewhere.

We are here submitting a revised version of the paper according to last comments from referees and we hope is now acceptable for publication.

Please find attached a point-by-point response (in green) to reviewer 1. 

With warm regards,

Simona Serban, PhD

Purolite

Unit D, Llantrisant Business Park,
Llantrisant

South Wales, CF72 8LF, UK
Phone +44 1443 229334

Email: [email protected]

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors, please see the attached document.

Comments for author File: Comments.pdf

Author Response

Dear editor, Dear reviewer,

We thank you for considering the possibility to publish our paper in the special edition of Catalysts.

We here declare that authors are aware of the submission and agree to its publication. The submission is original and is not under consideration for publication elsewhere.

We are here submitting a revised version of the paper according to last comments from referees and we hope is now acceptable for publication.

Please find attached a point-by-point response (in green) to reviewer 2. 

With warm regards,

Simona Serban, PhD

Purolite

Unit D, Llantrisant Business Park,
Llantrisant

South Wales, CF72 8LF, UK
Phone +44 1443 229334

Email: [email protected]

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Dear Editor,

the Authors responded properly to all comments and the manuscript was improved.

My main concern is that some parts concerning the description of the micro-FTIR method, and the interpretation of the data have a language that is too technical and unsuitable for a broad audience such as that of Catalysts. The manuscript would be strengthened if the authors clarify these points better even to non-expert readers.

Analytical comments:

Line 108. Supplementary figures S3, S4 and S5 are very clear also for non-expert readers. Based on these simple schemes, how were the spectra in figure 1A and 3A obtained? It is very difficult to understand the relationship between the panel A and B in figure 1 and 3.

Line 197. It is not clear whether the sentence "Interestingly, the activity was always higher…" refers to experimental results or to literature data. Please clarify and rephrase. How do the Authors explain this behavior? Could it be that the covalent modifications inactivate the enzyme?

Figure 6 and 7. I suggest showing the SDS-PAGE in the region between 50 and 25 kDa. Moreover, the Authors should better comment on the results obtained with the two techniques.

Author Response

Dear editor, Dear reviewer,

 

We thank you for considering the possibility to publish our paper in the special edition of Catalysts.

We here declare that authors are aware of the submission and agree to its publication. The submission is original and is not under consideration for publication elsewhere.

We are here submitting a revised version of the paper according to last comments from the referee and we hope is now acceptable for publication.

Please find attached a point-by-point response to the reviewer. I also attached the manuscript with the tracked changes active to help track the modifications. 

Best regards,

Simona Serban

Author Response File: Author Response.pdf