β-Mannosidase from Cellulomonas fimi: Immobilization Study and Application in the β-Mannoside Synthesis

Round 1

Reviewer 1 Report

In the present work, the authors focused on the immobilization of beta-mannosidase. They tried several carriers and different conditions. The functionality of immobilized mannosidase was demonstrated by a transglycosilation reaction leading to the synthesis of manopyranosyl (1-6) glucopyranoside. Immobilization of mannosidas is still very little studied, so I think that the publication of this article will be beneficial. The work is well and carefully written. I have just minor comments. In the introduction, I would delete the paragraph on lines 59-63. This is the well-known knowledge about enzyme immobilization and does not need to be mentioned. In the names of compounds, "D" must be written in small caps, and in "O-acetyl" O must be italicized. It is not uniform throughout the work.

I have no comments.

Author Response

Reviewer #1

In the present work, the authors focused on the immobilization of beta-mannosidase. They tried several carriers and different conditions. The functionality of immobilized mannosidase was demonstrated by a transglycosilation reaction leading to the synthesis of manopyranosyl (1-6) glucopyranoside. Immobilization of mannosidas is still very little studied, so I think that the publication of this article will be beneficial. The work is well and carefully written. I have just minor comments.

We wish to thank Reviewer 1 for his/her positive comment and his/her careful reading of the manuscript. A point-by-point response to the issues he/she raised is reported below.

In the introduction, I would delete the paragraph on lines 59-63. This is the well-known knowledge about enzyme immobilization and does not need to be mentioned.

We have followed the recommendations and deleted the sentence.

In the names of compounds, "D" must be written in small caps, and in "O-acetyl" O must be italicized. It is not uniform throughout the work.

We have followed the recommendations and tried to be consistent throughout the work.

Reviewer 2 Report

The authors describe the immobilization of beta-mannosidase and its application in the synthesis of beta-mannoside. The procedures are not original but the sceening reported on transglycosylation acceptors and carriers can be useful for those working in this field. In my opinion the manuscript must be revised expecially in the experimental part.

Below are my observations:

lines 107-108 and 115-118:  it is not clear why those solvents, that I would non define green, were chosen. In addition, in ref. 14 ionic liquids and solvents from biomass are mentioned in this regard. The authors should clarify this point.

line 426: the appropriate reference should be 23 not 22.

lines 435-433; 450-457467-474 and so on: it is the same immobilization procedure and it should be written only once as a general procedure citing ref. 17. The same applies to other identical experimental procedures, citing the appropriate single reference.

References 22 and 24 are not necessary and should be omitted and replaced by more appropriate ones already present in the text.

Author Response

Reviewer #2

The authors describe the immobilization of beta-mannosidase and its application in the synthesis of beta-mannoside. The procedures are not original but the sceening reported on transglycosylation acceptors and carriers can be useful for those working in this field. In my opinion the manuscript must be revised expecially in the experimental part.

We wish to thank Reviewer 2 for his/her suggestions. A point-by-point response to the issues raised is reported below.

lines 107-108 and 115-118:  it is not clear why those solvents, that I would non define green, were chosen. In addition, in ref. 14 ionic liquids and solvents from biomass are mentioned in this regard. The authors should clarify this point.

t-BuOH is a preferred solvent (see ref. Byrne et al., Sustain Chem Process, 2016, 4, 7), while glycerol formal is considered a green solvent (see ref. Jordan et al., Chem. Rev. 2022, 122, 6749−6794). ACN and THF are not green solvents but they are generally employed for sugar solubilization. Since we knew that not all tested solvents can be considered green in the text line 115 we have written green between brackets. We have also tested one IL in our reaction but we could not observe also in this case the disappearance of pNMan by TLC monitoring as for the other co-solvents. In the first draft of the manuscript we did not added this trial since with acceptor 2d we tested just one IL, now we have add also this trial (See line 108 and new Figure S3). However, in a preliminary screening with acceptor 2b we have also tried a second IL (1-butyl-3-methylimidazoliumhexafluorophosphate) but it was not completely miscible with reaction media and some precipitation (probably of the reagents) was observed even when we lowered it concentration to 20% v/v from 30% v/v. This second IL was not tested with acceptor 2d, so it has not been reported in the main manuscript. 

line 426: the appropriate reference should be 23 not 22.

As suggested, we have eliminated reference 22 and maintained just reference 23.

lines 435-433; 450-457 467-474 and so on: it is the same immobilization procedure and it should be written only once as a general procedure citing ref. 17. The same applies to other identical experimental procedures, citing the appropriate single reference.

We have modified the text following reviewer’s suggestions. However, lines 433-435 describe the immobilization on GLX-agarose while ref. 17 describes the derivatization of aminic Sepabeads with DFF and its application in the immobilization of two different enzymes. In ref. 17 agarose is not used as support. Thus, for DFF-EDA-AG we have cited ref. 23 for the preparation of EDA-AG starting from GLX-AG and subsequently we have followed ref. 17 for DFF derivatization.

References 22 and 24 are not necessary and should be omitted and replaced by more appropriate ones already present in the text.

As suggested, we have eliminated references 22 and 24 and maintained just reference 23.