β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

Round 1

Reviewer 1 Report

The authors give a comprehensive overview of applying ß-N-acetylhexosaminidases for the the synthesis of carbohydrates by trans-glycosylation for actual and future commercial purposes and thus is of high interest to the biotech community. The manuscript is very well structured and easy to read. The detailed tables perfectly complement the text.

However, I suggest to add some more reaction shemes, which mix up the text and visualize like the general reaction described in lines 40-43 and in some parts of section "3. Increased trans-glcosylation activity by reaction engineering"

In lines 99 and 102 Figure 1 is written instead of Figure 2. Please correct it.

Although the tables and written draft is very comprehensive, it become not clear if the enzymes were used in all studies in purified, isolated form or maybe also as crude extract or even wildtype or recombinant whole-cell catalyst. Due to sidereactions one may assume that only purified enzymes can be applied!? Please, introduce a few words to your manuscript giving an answer to this questions. Furthermore, I would be great if you add some information on the production of the enzymes: are the wildtype enzymes produced recombinantly? are produced exclusively intracellular or are they studies using secretion to the culture medium to enable cheap and easy purification?

All in all it was a great pleasure reading this fantastic and interesting review! The authors can be proud on their work!

Author Response

The authors give a comprehensive overview of applying ß-N-acetylhexosaminidases for the the synthesis of carbohydrates by trans-glycosylation for actual and future commercial purposes and thus is of high interest to the biotech community. The manuscript is very well structured and easy to read. The detailed tables perfectly complement the text.

However, I suggest to add some more reaction shemes, which mix up the text and visualize like the general reaction described in lines 40-43 and in some parts of section "3. Increased trans-glcosylation activity by reaction engineering"

Re: We added a new figure 3 on p. 9 showing structures of activated donor substrates, the new figure 4 on p. 23 showing the LNT II synthesis using pNP-GlcNAc and the new figure 6 on p. 26 showing the LNTII/LNT synthesis using Glc-oxa.

In lines 99 and 102 Figure 1 is written instead of Figure 2. Please correct it.

Re: We changed the text accordingly. See p. 4, lines 105+108

Although the tables and written draft is very comprehensive, it become not clear if the enzymes were used in all studies in purified, isolated form or maybe also as crude extract or even wildtype or recombinant whole-cell catalyst. Due to sidereactions one may assume that only purified enzymes can be applied!? Please, introduce a few words to your manuscript giving an answer to this questions. Furthermore, I would be great if you add some information on the production of the enzymes: are the wildtype enzymes produced recombinantly? are produced exclusively intracellular or are they studies using secretion to the culture medium to enable cheap and easy purification?

Re: We added the following paragraph to the text in section 2, p. 3, lines 88-92: “Furthermore, GH20 β-N-acetylhexosaminidases are usually well-expressed in a recombinant host (either cytosolic or secreted) or can be easily purified from wild-type cultivations since these enzymes are often secreted by their natural host organism. This facilitates simple purification of the enzymes for biocatalytic purposes, which was applied in >95% of the literature studied for this article.”

All in all it was a great pleasure reading this fantastic and interesting review! The authors can be proud on their work!

Reviewer 2 Report

The review article submitted by Anne Meyer and co-workers summarises several decades of research directed at the use of N-acetylhexosaminidases in the biocatalytic synthesis of glycosides containing a GlcNAc moiety. The article is very well researched, structured and written, and it presents a large body of biotransformation data in tabular format, which I'm convinced will become a valuable resource for researchers in the field. Since the manuscript is of excellent quality, my comments and suggestions mainly pertain to a few specific aspects of wording and data presentation, and I recommend acceptance after minor revision.

(1) Abstract: Uppercase N's representing nitrogen in compound names (e.g., N-acetylglucosamine) should be typeset in italic, as they are in the other parts of the manuscript.

(2) line 76: The term "biosynthesis" typically refers to the production of a compound in natural metabolism, while in this context it refers to non-natural biocatalytic transformations. It should therfore be replaced with "biocatalytic synthesis".

(3) Figure 1: I suggest to redraw the left-hand GlcNAc moiety in the bivalent lectin ligand to a canonical chair conformation (i.e., one in which the lower half of the chair protrudes out of the drawing plane and is hence drawn with bold/wedged bonds).

(4) line 106: the l in "longum" should be italic

(5) Figure 2: All electron pairs involved in the mechanism should be drawn. Currently, the N lone pair involved in the first step and the O lone pair involved in the second step are not shown. Moreover, in mechanistic drawings localised bonds are preferable to delocalised bonds, but if the authors want to stick with the delocalised drawing of carboxylates, they should at least draw the Glu carboxylate symmetrically (like they have done for the Asp carboxylate).

(6) line 115: replace "is super high" with "are exceptionally high"

(7) line 130: replace "from" with "of"

(8) line 147: replace "hardly" with "rarely"

(9) line 188: replace "side product" with "byproduct" (see W. Watson, Org. Process Res. Dev. 2012, 16, 1877-1877, doi: 10.1021/op300317g)

(10) line 232: correct double "a" ("a a thioglycoligase")

(11) line 246: replace "on" with "of"

(12) line 250: "Schmölzer et al. [71] recently reported a significantly lower yield (12% internal standard yield)." Where in this publication did the authors find this value? Table 1 of the cited article reports only activities, while Figure S6 in the SI shows an analytical yield of approx. 35% after 3 h (and >40% after 1-2 h) for wild-type BbhI. Besides, "internal standard yield" is not a generally excepted (and, likely, not generally understood) term. I suggest to use "yield based un HPLC analysis using an internal standard". I know it's a lengthy term, but it's also much less ambiguous.

(13) line 370: "but the obtained yield was much lower (18%; Table 5) [71]." Again, I didn't find this value in the original article. Figure S6 shows >40% yield after 3 h and >70% yield after 1 h.

(14) line 386: change "catalyze transfer a GlcNAc moiety" to "catalyze transfer of a GlcNAc moiety"

(15) lines 398, 399: change "Two mutants exhibited a doubled (R577K, Figure 4A) respectively quadrupled 398 (W882H, Figure 4A) yield of LNT II from reactions..." to "The two mutants R577K and W288H (Figure 4A) led to a doubled and quadrupled yield of LNT II, respectively, in reactions..." (see, for example: https://www.springer.com/gp/authors-editors/authorandreviewertutorials/writinginenglish/use-of-respectively/10252704)

(16) line 435: "backwards catalysis" is not a generally accepted term and should be omitted altogether

(17) line 439: "fostered" sounds awkward to me in a chemical context. Replace with "catalyzed", or (if you're looking for an alternative for the latter) with "facilitated" or "promoted".

(18) line 446: replace "varied" with "altered"

(19) lines 450, 451: replace "obvious substrates" with "an obvious substrate"

(20) The font use in Table 1 is inconsistent (some compound names are typeset in a mixture of Palatino Linotype and Times New Roman). In Table 3, p. 8, one "beta" is typeset in Calibri.

Author Response

The review article submitted by Anne Meyer and co-workers summarises several decades of research directed at the use of N-acetylhexosaminidases in the biocatalytic synthesis of glycosides containing a GlcNAc moiety. The article is very well researched, structured and written, and it presents a large body of biotransformation data in tabular format, which I'm convinced will become a valuable resource for researchers in the field. Since the manuscript is of excellent quality, my comments and suggestions mainly pertain to a few specific aspects of wording and data presentation, and I recommend acceptance after minor revision.

(1) Abstract: Uppercase N's representing nitrogen in compound names (e.g., N-acetylglucosamine) should be typeset in italic, as they are in the other parts of the manuscript.

Re: We changed the text accordingly. See p. 1, lines 9-27.

(2) line 76: The term "biosynthesis" typically refers to the production of a compound in natural metabolism, while in this context it refers to non-natural biocatalytic transformations. It should therfore be replaced with "biocatalytic synthesis".

Re: We changed the text accordingly. See p. 2, l. 76.

(3) Figure 1: I suggest to redraw the left-hand GlcNAc moiety in the bivalent lectin ligand to a canonical chair conformation (i.e., one in which the lower half of the chair protrudes out of the drawing plane and is hence drawn with bold/wedged bonds).

Re: The left-hand GlcNAc moiety was already drawn as the reviewer suggested – but we have redrawn the bivalent lectin to show the right-hand GlcNAc moiety in the way the reviewer suggested. See Figure 1 on p. 3.

(4) line 106: the l in "longum" should be italic

Re: We changed the text accordingly. See p. 4, l. 112.

(5) Figure 2: All electron pairs involved in the mechanism should be drawn. Currently, the N lone pair involved in the first step and the O lone pair involved in the second step are not shown. Moreover, in mechanistic drawings localised bonds are preferable to delocalised bonds, but if the authors want to stick with the delocalised drawing of carboxylates, they should at least draw the Glu carboxylate symmetrically (like they have done for the Asp carboxylate).

Re: We changed the figure to clearly show the relevant lone pairs of the transition state stabilizing glutamate residue and the 2-acetoamidogroup to better illustrate the formation of the oxazoline intermediate in the substrate-assisted mechanism. See revised figure 2 on p. 4.

(6) line 115: replace "is super high" with "are exceptionally high"

Re: We changed the text accordingly. See p. 4, l. 121.

(7) line 130: replace "from" with "of"

Re: We changed the text accordingly. See p.5, l. 136.

(8) line 147: replace "hardly" with "rarely"

Re: We changed the text accordingly. See p. 5, l. 153.

(9) line 188: replace "side product" with "byproduct" (see W. Watson, Org. Process Res. Dev. 2012, 16, 1877-1877, doi: 10.1021/op300317g)

Re: We changed the text accordingly. See p. 8, l. 197 and p. 18, lines 224+247

(10) line 232: correct double "a" ("a a thioglycoligase")

Re: We changed the text accordingly. See p. 23, l. 281.

(11) line 246: replace "on" with "of"

Re: We changed the text accordingly. See p. 23, l. 295.

(12) line 250: "Schmölzer et al. [71] recently reported a significantly lower yield (12% internal standard yield)." Where in this publication did the authors find this value? Table 1 of the cited article reports only activities, while Figure S6 in the SI shows an analytical yield of approx. 35% after 3 h (and >40% after 1-2 h) for wild-type BbhI. Besides, "internal standard yield" is not a generally excepted (and, likely, not generally understood) term. I suggest to use "yield based un HPLC analysis using an internal standard". I know it's a lengthy term, but it's also much less ambiguous.

Re (12): see below

 (13) line 370: "but the obtained yield was much lower (18%; Table 5) [71]." Again, I didn't find this value in the original article. Figure S6 shows >40% yield after 3 h and >70% yield after 1 h.

Re (12) and (13): We sincerely apologize for this mistake. During extraction of the yield from Figure S6 in the original publication, the wrong y-axis was accidently used, which also led to misinterpretation of the data. We corrected the values in Table 5 and changed the text accordingly. See p. 23, lines 296-307 and table 5 on pages 14+15.

(14) line 386: change "catalyze transfer a GlcNAc moiety" to "catalyze transfer of a GlcNAc moiety"

Re: We changed the text accordingly. See p. 27, l. 442.

(15) lines 398, 399: change "Two mutants exhibited a doubled (R577K, Figure 4A) respectively quadrupled 398 (W882H, Figure 4A) yield of LNT II from reactions..." to "The two mutants R577K and W288H (Figure 4A) led to a doubled and quadrupled yield of LNT II, respectively, in reactions..." (see, for example: https://www.springer.com/gp/authors-editors/authorandreviewertutorials/writinginenglish/use-of-respectively/10252704)

Re: We changed the text accordingly. See p. 27, lines 454-455.

(16) line 435: "backwards catalysis" is not a generally accepted term and should be omitted altogether

Re: We changed the text accordingly. See p. 28, lines 489-492.

(17) line 439: "fostered" sounds awkward to me in a chemical context. Replace with "catalyzed", or (if you're looking for an alternative for the latter) with "facilitated" or "promoted".

Re: We changed the text accordingly. See p. 28, l. 495.

(18) line 446: replace "varied" with "altered"

Re: We changed the text accordingly. See p. 28, l. 502.

(19) lines 450, 451: replace "obvious substrates" with "an obvious substrate"

Re: We agreed and changed the text accordingly. See p. 28, lines 506-507.

(20) The font use in Table 1 is inconsistent (some compound names are typeset in a mixture of Palatino Linotype and Times New Roman). In Table 3, p. 8, one "beta" is typeset in Calibri.

Re: We adjusted the font in all tables again. See tables 1-10.