Enantioselective Transesterification of Allyl Alcohols with (E)-4-Arylbut-3-en-2-ol Motif by Immobilized Lecitase™ Ultra

Round 1

Reviewer 1 Report

The authors continue a line of papers describing the utility of lecitase ultra as an enzyme for biotechnology and chemistry applications. Here, they describe immobilized enzyme that engages in transesterification. Although similar to other papers of theirs, it is noteworthy that this chemistry relies on anhydrous reaction conditions necessary to see transesterification rather than hydrolysis. Although many examples of this sort of process do exist, even with this enzyme, it remains a meaningful challenge to address with enzyme reactions that must be conducted under anhydrous conditions, far away from biological conditions.

 

The manuscript may represent a step forward and present new knowledge in this field. However, primary data is needed to convince the readers of the conclusions drawn. In particular, immobilization efficiency seems to be quite low, and that poor level of active enzyme brings significant concerns and questions about subsequent measurements. Furthermore, the loadings here are likely >50% immobilized enzyme by mass, compared to substrate. As such, the process demonstrates quite limited real-life utility.

 

Although a future manuscript might be suitable for publication, significantly improved and additional data is needed.

 

1. The broken y-axes are not appropriate presentation. Authors can provide two different y-axes (left and right) for ee and yield, but the current setup cannot stay.
2. Please provide primary data for ee measurements, including racemic traces, and for product yield.
3. Products should be characterized by NMR, and those spectra provided.
4. Immobilization seems to be extremely inefficient. In a typical case, 40,000 U of enzyme (4 mL, 10,000 U/g) is immobilized onto 1 g of Supelite, to give product with 0.06 U/g * 1g = 0.06 U. Are we to assume that immobilization is ~0.0002% efficient?
5. Please provide primary data and more explanation of activity (U) measurements. The low concentration of active enzyme creates many possible sources of error, and I do not understand how the authors get quantitative kinetics from a single time point.
6. Given the clear acceleration of activity with added water in the original report (Ref 84), why do these authors use ethanol as a quench? Are we fully convinced that halts reaction?
7. What of background reaction in these studies, and/or complicating absorption by iron particles at 410 nm in this measurement?
8. Please have a professional editor or native English speaker edit the manuscript for language and clarity.

Author Response

Dear Reviewer,

we would like to thank you for your critical comments which helped us to improve  our paper. According to your suggestions we corrected the manuscript and made some changes in the text which are highlighted in yellow. We enclose the "point by point" list of our corrections and explanations in the attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this manuscript by Leśniarek, Gładkowski and co-workers an enantioselective transesterification of allyl alcohols is described. The kinetic resolution of racemic allyl alcohols was carried out  with (E)-4-phenylbut-3-en-3-ol motif by immobilized Lecitase^TM Ultra.

 

The research topic is interesting and essential, the manuscript is well-written and demanding, the methods applied are adequately described and the results are clearly presented.

 

I believe that this work might be of interest to the readers of Catalysts, and recommend for publication in the present form.

Author Response

Reviewer 2

We would like to thank the Reviewer for appreciating our work and positive review of the manuscript.

Reviewer 3 Report

The present manuscript describes a series of experiments for the use of an immobilized form of LecitaseTM Ultra for the transesterification reaction. Although overall novelty of the results are low as also stated and duly referenced in the introduction by the authors the manuscript is well written and could be accepted providing some important lacks or integration or improvements of description. Authors should check the following

1. past applications of the enzyme in degumming must be clarified, line 44
2. applied instead of used? line 92
3. Importance of the products of the reaction must be briefly cited in the Introduction also shortening the long description about uses of enzyme in immobilized form
4. Pay attention to plural accordance, line 106. Moreover I found not useful here the mention of alchohols listed in table 2 if the latter is not anticipated before table 1, it creates confusion
5. A better description shared with experimental part of the form of immobilized material should be furnished at this stage in the paper
6. line 156 A better description of how authors assessed enantiomeric excesses must be furnished here and in Material (integration peak areas?). Please indicate retention time of substrate and products in the gas-chromatographic analysis and for TLC used in the preparative work. It could be much better if for chemical assessment of structures some NMR spectroscopy results were given, at least a 1H NMR with authentic material as comparison
7. The way to present results in Figures is of doubtful effect. While the lower part of the graph is generally clear the upper part could be replaced by numbers, also give plenty experimental details also in the legend of figures, some data are not present in the various legend (amount of enzyme? for figure 2 for example)
8. line 201 authors stated about toluene results indicating the data the worst obtained, it simply does not seem so to me (see hexane for example)
9. line 221-230 this part could be shortened also for the "no-result" picture here, the mention of alcohol dehydrogenase seems to be hardly correlated and a clear hypothesis seems not be mentioned in molecular terms about enzyme/template of immobilization structures.
10. line 235 "from" ?
11. line 279 "the preparation was separated from the reaction"?
12. line 281 "was used to resolution" ?
13. the concept here is not clear "Both activities are not closely correlated", as it is inserted in the operational stability of the enzyme preparation in toto, authors must be clear; is the same active site for hydrolysis of PNP substrate and transesterication reaction or not?
14. line 372 indication of stereochemistry for TLC mention is unappropriate
15. line 494 spectroscopic data is vague

Author Response

Dear Reviewer,

Thank you for detailed review of our paper, which helped us to improve our manuscript. The list of our corrections and explanations are in the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I am satisfied that the changes improve the manuscript. Publication now seems warranted.

Reviewer 3 Report

Authors answered fully to all my questions and improved the quality of presentation of their results, thus this piece of work can be published