Review Reports - Immobilization and Catalytic Properties of Limonene-1,2-Epoxide Hydrolase

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The authors have addressed all the comments and suggestions raised in the previous review. The revised manuscript shows significant improvement in terms of clarity, data presentation, and discussion of results.

Author Response

Thank you very much for your effort and help.

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors have revised the manuscript.

Author Response

Thank you very much for your effort and help.

Reviewer 3 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The authors have amended the text of the article where possible based on my comments. They removed the idea of the commercial appeal of the immobilized limonene-1,2-epoxide hydrolase (LEH), which cleared up confusion about the feasibility of using immobilized LEH in industrial practice.

I recommend that the authors reiterate in their conclusion (as they so well noted in their responses to my comments) that they have achieved the first immobilization of limonene 1,2-epoxide hydrolase via covalent binding to solid commercial supports, and the results obtained represent a pilot study for future research aimed at studying the influence of the immobilization method, conditions, and support properties on the activity yield, and may be of interest and useful to other authors wishing to begin their own research in this area.

Author Response

Reviewer's comment:

Authors' response:

In line with your recommendation, we have revised the conclusion and in the last paragraph we have explicitly stated that this work represents the first immobilization of limonene-1,2-epoxide hydrolase by covalent binding to commercially available supports. We have also emphasized the pilot nature of the study and its importance for future research aimed at optimizing immobilization methods.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Kaniaková et al. regards the immobilization of limonene-1,2-epoxide hydrolase (LEH) on Immobead 150P and various Purolite® methacrylate-based carriers, and their application in the hydrolysis of cis-/trans-(+)-limonene-1,2-epoxide. The biocatalyst, immobilized on Purolite
LifetechTM ECR8309M modified with glutaraldehyde and 1,4-diaminobutane, was applied in 10 reaction cycles at 30 °C and retained 62% of the initial activity
in the presence of 10% (v/v) acetonitrile and 75% in its absence.

In general, the results of this work are interesting and the conclusions are supported by the experimental details, but some minor points should be taken into consideration prior to publication:

- The introduction is somewhat long and could be made more concise by reducing the general background on epoxide hydrolases and emphasizing more clearly the knowledge gap (study on LEH immobilization).

- In the general discussion of immobilised enzymes, the manuscript could benefit from referencing recent comprehensive reviews that summarise state-of-the-art immobilisation technologies and sustainable materials. For example: Robescu et al., Molecules 2025, 30(4), 939; Patti et al., ChemSusChem 2025, 18, e202402007.

- The section describing carrier modifications is overly detailed. Consider moving part of this content to the Supplementary Materials and keeping only the essential schemes in the main text.

- The overall activity recovery after immobilization is rather low (<10%). The authors acknowledge this, but the discussion could be expanded to address possible causes (e.g., enzyme orientation on the support, conformational changes, or pore diffusion effects).

- Minor typographical issues should be corrected.

- Ensure consistency in abbreviations

Reviewer 2 Report

Comments and Suggestions for Authors

The study immobilized limonene-1,2-epoxide hydrolase (LEH) on various carriers to enhance its stability and performance. The immobilized enzyme showed improved thermal resistance and pH stability, making covalent immobilization an effective method for its application. Here are some points that could be improved:

Line 37-39: “LEHs do not contain an α/β-fold, a core domain of eight β-strands connected by α-helices, which is characteristic of α/β-fold hydrolases.” Here the authors should give the structure figure of LEH in SI for clarity.
Figure 3: The mechanism of pH influencing relative activity should be analyzed. Why does the enzyme activity seem more stable after immobilization at different pH? Is there any exact activity (not relative activity) data at different pH?
Figure 4: Why does the optimum temperature increase after immobilization? Is there any evidence of the relativity of this phenomenon with the proposed multipoint covalent attachment or increased substrate solubility?
Current Eq (3) used for Figure 5, 6 and 7 is a reaction. Should it be Eq (5)? Meanwhile, the R2 value of each fitting should be provided (the data fitting in Figure 5 seems with large bias).
Line 97-99: “The reported advantages of immobilization include enhanced operational, thermal, and storage stability, as well as improved enantioselectivity [1].” To highlight the importance of enzyme immobilization work, this paper also needs to be cited: Bioresource Technology, 2020, 308, 123271

Reviewer 3 Report

Comments and Suggestions for Authors

The article "Improving the Properties of Limonene-1,2-epoxide Hydrolase by Immobilizing on Commercial Carriers" addresses a relevant topic and possesses elements of scientific novelty. However, the manuscript leaves a very ambiguous impression.

On the one hand, the authors use an enzyme with promising industrial applications and a commercially available carrier for its immobilization. They achieved a high immobilization yield (98.5-100%), but the values for retained activity are negligible and do not even reach 10%.

With such a low percentage of retained activity, further discussions on pH and temperature activity profiles, as well as thermal and operational stability, may lose their relevance, despite the fact that the authors conducted these experiments quite thoroughly.

First and foremost, the authors should compare the parameters they obtained for the immobilization of limonene-1,2-epoxide hydrolase with the results presented by other authors. It would be best to present this comparison in a table format.

Secondly, and even more importantly, the authors need to prove the commercial (or other) rationale for immobilizing limonene-1,2-epoxide hydrolase using their proposed method. That is, they must demonstrate that the increase in thermostability and operational stability after immobilization fully compensates for the low percentage of retained enzyme activity. Without this proof, I see no merit in publishing this article.

The Сonclusion should also be written in the vein of justifying the use of costly immobilization steps to enhance the catalytic efficiency and commercial appeal of limonene-1,2-epoxide hydrolase, despite its low retained activity post-immobilization.

If providing the requested proof of the commercial (or other) viability of their proposed costly immobilization steps proves impossible, I suggest to the authors consider other possible chemical modifications of the carriers they use or revise the immobilization methodology from the perspective of adding chemical agents that protect the enzyme's active site during the immobilization process.