Continuous Flow Glycolipid Synthesis Using a Packed Bed Reactor
Round 1
Reviewer 1 Report
Dear authors, please find bellow comments and suggestion for you work entitle « continuous flow biocatalysis for glycolipid synthesis using a packed bed reactor : lipase-catalyzed transesterification in a biphasic solvent system. »
The present article is certainly within the scope of the journal and of interest to the reader. However : The main limitation in my opinion is the lack of further caracterization of the immobilized enzyme and support.
Abstract :
it is well written and presenting the challenges associated with glycolypids
1 Introduction :
line 73 2M2B must be defined as it is the first time you use this abbreviation
line 76 and figure 1 : it should be placed in the material and methods section not at the and of the introduction
2- results : are consistent with the experiments carried out.
3- discussion is consistent with the experiments carried out.
4- materials and methods is generally satisfactory and most of the part are easily understandable and carried out according to state of the art standards except for the part of the enzymatic immobilisation of the lipase to the support which is key to this article
line 175 : XK columns might be purchased empty. What is the resin used in the column ? What is the column volum? What is the functionnalisation used to immobilize the lipase ? Have the author tried to study the immobilisation parameters ?
Is it possible to reuse the immobilized column to carry out other reactions. How many column volums is it possible to maitain a proper yield?
It would also have been great to have a batch non immobilized reaction presented as a control in the experiments
5 – Conclusion
line 232 how much higher ?
Line 236 what would be the main advantages of trying these other reactor set-up ?
Author Response
Dear Reviewer 1,
thank you for your insightful comments that helped to improve the manuscript substantially.
2M2B as defined, we are sorry that we missed this in line 73.
Figure 1 was moved to the methods section.
We applied a commercially available immobilized lipase in our process. We are sorry that this was not clear, we specified that in the methods section, as well as in the abstract and results part and introduced the lipase formulation in the introduction. The commercially available lipase formulation Novozym 435® produced by the company Novozymes (Denmark) is a well-known and extensively characterized/studied biocatalyst. The carrier used for the immobilization is a macroporous polyacrylic resin (Lewatit VP OC 1600), the latter relies on adsorption mechanism. The Lipase B from Candida antarctica itself is produced industrially from recombinant Aspergillus oryzae. More details can be found in this highly extensive review: “Novozym 435: the “perfect” lipase immobilized biocatalyst?” from Ortiz et al. 2019. We can hardly provide real new information on the characterization of this biocatalyst, the possibilities for new developments in its use are in the opposite broader.
The process runs stable for at least 72h without any loss of enzyme activity. We added this data in the results section.
Unfortunatly, a batch process with a biphasic system was not possible. As soon as we have extensive amounts of water present in batch we see no product formation due to hydrolysis. However, we studied the reaction in batch in a hydrophilic DES which was published earlier (reference 25, Hollenbach et al 2020) and did a comparison to that in the discussion.
Numbers were implemented in the comparison in the discussion in line 232.
Other continuous flow systems do not realize on fix packed beds but on rotating beds for example. The supplier claim increased heat and mass transfer due to an efficient stirring and packing of the biocatalyst into cartridges. The idea would have a “competition” between reactors so to say.
Reviewer 2 Report
Dear Editor,
in the manuscript entitled: “Continuous flow biocatalysis for glycolipid synthesis using a packed bed reactor: Lipase-catalyzed transesterification in a biphasic solvent system”, Hollenbach and co-authors describes the use of Novozyme 435 in the production of glycolipids through a continuous flow approach.
Although the results seem interesting, some paragraphs appear scarce and benefit from a deeper description to help the reader understand the logic of the experiments, the results, and the conclusions of the work. For example, the use of N-435 as an immobilized lipase is not clear until the beginning of section 4.
Analytical comments:
Title: in my opinion title is too long and not very informative of the content of the work.
Pag. 1 Line 29. Novozyme 435 lipase should be better introduced. Please consider the following review: https://doi.org/10.1039/C9CY00415G.
Pag. 2 Line 57. One of the main limitations of biocatalysis in non-conventional media is the enzymatic inactivation triggered by organic solvents. Please consider these publications (https://doi.org/10.1002/biot.202100712, https://doi.org/10.1016/j.ijbiomac.2020.02.145, https://doi.org/10.1021/acs.joc.1c01136, https://doi.org/10.1007/s11144-009-0103-4. and https://doi.org/10.1016/j.molcatb.2011.04.004) and discuss how continuous flow can overcome this limitation.
Pag. 2 Line 73. Please specify the abbreviation 2M2B
Pag. 3 Line 80. The “Results section” should be improved by a more in-depth description of the continuous flow system using in this work. Which is the concentration of organic solvent and reagents in the reaction? How did the Authors immobilize CALB?
Pag.3 Line 88. It is not clear how the authors chose the reaction temperatures. Why didn't the authors test 60°C and 80°C?
Pag. 3 Line 95. Also in this case the rationale of the choice of the organic solvent concentration is not clear.
Pag. 5 Line 108. It is not clear how the Authors calculated the water activity.
Pag. 5 Line 118. The discussion should be enhanced by a more detailed comparison between this process and others available in the literature both in terms of reaction conditions (reagents, co-solvents, etc.) as well as yield and productivity.
Pag. 5 Line 132. Please specify the abbreviation DES
Pag.6 Line 153. In figure 4 the main production of glycolipid is observed at 1.3 M of glucose and 0.97 Aw. The relationship between Aw and glucose concentration should be better discussed.
Pag.6 Line 169. The authors used N-435 as an immobilized lipase. Unfortunately up to this point it was not clear.
Figure. It is not clear how represent the letter “a” and “b”.
Author Response
Dear Reviewer 2,
thank you for your insightful comments that helped significantly to improve the manuscript substantially.
The title was modified.
We are sorry that we missed to introduce the biocatalyst properly. We fixed this issue.
Enzyme inactivation by organic solvents was implemented in the introduction and discussed that for our biphasic system we can avoid short chain alcohols as cosolvents.
The abbreviation 2M2B was specified.
We are sorry that we missed to mention the applied biocatalyst in the results section. We fixed this issue. We used the commercially available lipase formulation Novozym 435® which is produced by the company Novozymes (Denmark) and is a well-known and extensively characterized/studied biocatalyst. The carrier used for the immobilization is a macroporous polyacrylic resin (Lewatit VP OC 1600), the latter relies on adsorption mechanism. The Lipase B from Candida antarctica itself is produced industrially from recombinant Aspergillus oryzae.
We specified the relative flow rates in the results.
We additionally tested 60°C and 80°C as suggested and added this data.
Our starting point was the same flow rate for both feed solutions, meaning 0.25mL/min per solution with a total flow of 0.5mL/min. As we were interested in how the ratio of the two solutions affects productivity, we then changed the ratio to 25:75 (0.125 mL/min and 0.375 mL/min) and 75:25 (0.375 mL/min and 0.125 mL/min ) to test if this affects the reaction.
Water activity was measured using a water activity measuring device. We used the LabMaster-aw neo from Novasina.
Comparison between this process and others in literature was complemented by solvents and temperatures and pre-treatment conditions.
We specified the abbreviation DES, sorry that we missed this before.
Discussion concerning the relationship between water activity and sugar concentration has been elaborated.
Thank you for the comment, that we missed Novozym 435, precision has been from made earlier on (abstract, introduction, results)
Explanation on the letters in the figures was added in the figure captions. Letters represent statistically significant differences.
Round 2
Reviewer 1 Report
thank you for the revised version of the document.
Reviewer 2 Report
Dear Editor,
the Authors responded properly to all comments and the manuscript was improved.
I suggest adding a couple of sentences to line 87 to briefly describe the bioreactor system used by the authors.
