Advances in Recombinant Lipases: Production, Engineering, Immobilization and Application in the Pharmaceutical Industry

Round 1

Reviewer 1 Report

The review article entitled “Advances in recombinant lipases: Production, engineering, immobilization and application in the pharmaceutical industry” sent to Catalysts concerns a very current and constantly developing topic of the use of Lipases in the synthesis and kinetic resolution of chiral active substances.

The article is written clearly and understandably. The division of the collected material is logical and consistent. The section on the description of obtaining enzymes is comprehensive and is a source of knowledge for people who want to start this topic or already work in it. However, the lack of any exemplary structures of lipases taken, for example, from the PDB database, causes a slight dissatisfaction. This would allow a better understanding of the structural differences, in particular influencing catalytic activity, such as, for example, the presence of a lid in protein structure.

A very interesting fragment for the reader is the part describing the use of enzymes immobilized on carriers, in particular on those that allow their reuse. However, it’s a pity that the authors have omitted e.g. publications describing the immobilization of lipase from Candida Rugosa on magnetic nanoparticles coated with polysaccharides like chitosan and with collagen, in the case of which hyperactivation was observed.

The part concerning the application of catalytic systems in the pharmaceutical industry is the most interesting but the shortest part of the work. The authors focused mainly on aryl propionic acid derivatives. Unfortunately, references to other drugs, such as beta blockers (atenolol etc.) for which lipases were used, were omitted.

In addition, in the case of kinetic separations or syntheses, the enantiomeric excess (ee%) and yield should be given, and where possible, in the case of immobilized lipase, the number of catalytic cycles in which the recovered enzyme was used.

In summary, the work is well written and interesting, and the suggestions included in the review will only allow for a better and complete presentation of the subject.

Author Response

Dear Reviewer 1,

We have carefully revised the manuscript according to the suggestions. The itemized list of changes addressing the comments is found below and the changes are marked using the track changes in the manuscript. 

The review article entitled “Advances in recombinant lipases: Production, engineering, immobilization and application in the pharmaceutical industry” sent to Catalysts concerns a very current and constantly developing topic of the use of Lipases in the synthesis and kinetic resolution of chiral active substances.

Thank you for this comment.

The article is written clearly and understandably. The division of the collected material is logical and consistent. The section on the description of obtaining enzymes is comprehensive and is a source of knowledge for people who want to start this topic or already work in it. However, the lack of any exemplary structures of lipases taken, for example, from the PDB database, causes a slight dissatisfaction. This would allow a better understanding of the structural differences, in particular influencing catalytic activity, such as, for example, the presence of a lid in protein structure.

Thank you for the comment. We inserted exemplary structures of two lipases (Figure 2).

A very interesting fragment for the reader is the part describing the use of enzymes immobilized on carriers, in particular on those that allow their reuse. However, it’s a pity that the authors have omitted e.g. publications describing the immobilization of lipase from Candida Rugosa on magnetic nanoparticles coated with polysaccharides like chitosan and with collagen, in the case of which hyperactivation was observed.

A discussion of a report on the immobilization of lipase from Candida rugosa on magnetic nanoparticles coated with polysaccharides like chitosan and with collagen was added in the text.

The part concerning the application of catalytic systems in the pharmaceutical industry is the most interesting but the shortest part of the work. The authors focused mainly on aryl propionic acid derivatives. Unfortunately, references to other drugs, such as beta blockers (atenolol etc.) for which lipases were used, were omitted.

Thank you for your comment. We have considered your suggestion and expanded the topic mentioned with an example of a beta-blocker (atenolol). Moreover, we have included two paragraphs regarding granted patents using CALB for enzymatic synthesis of pharmaceuticals. We also added a table with more examples (Table 3).

In addition, in the case of kinetic separations or syntheses, the enantiomeric excess (ee%) and yield should be given, and where possible, in the case of immobilized lipase, the number of catalytic cycles in which the recovered enzyme was used.

Thank for your suggestion. The topic has been revised in order to include this information where it was possible.

In summary, the work is well written and interesting, and the suggestions included in the review will only allow for a better and complete presentation of the subject.

Thank you very much for the very constructive comments.

Reviewer 2 Report

This manuscript reviews work on lipases, their expression, and applications. It contains useful information and overall is in a decent shape and is enjoyable to read. However, it contains lack of diagrams/figures. Like recombination methods or applications shown in block diagrams for an overview would make a better review paper.

Please discuss the productivity of recombinant lipases. How do they compare with natural lipases? There’s little information in the manuscript about fermentation. Since the title indicates that this manuscript reviews ‘production,’ then production on large scale and issues, cost etc. need to be discussed more.

“nlike  esterases  that  act  on  soluble  substrates,  lipases  act  on  lipids.” The reactions are still carried out in solution form. Do you mean in this paragraph that lipases act on emulsified lipids in water at different pH?? Oil in water type liquid phase. “called  interfacial  activation” oil/water interface. Otherwise it can also mean air-water interface.

Are all lipids insoluble in water?

“Many tricks are available to optimize a production process.” “capacity may be pushed to its limit” “an infinity of modeling software has been created” You can use better scientific language. There are many such sentences. Check elsewhere.

 Not everybody knows what chaperons are. Please explain what they are and their role in the context of protein secretion.

"lipases also possess a lid or flap-like structure” you have written many times about the lipase 3D structure and how it changes in the vicinity of the substrate. Please include a diagram of a common lipase with proper labeling of the flap, helix sheets etc. and how it changes during catalysis.

 In the immobilization section, there is nothing written about recycling performance, loss of activity on immobilization and there is no figure/diagram.

In Fig. 1 – the enantiomeric structure of the desired active structure of ibuprofen, naproxen, ketoprofen is missing. Where is the S-enantiomer?

Author Response

Dear Reviewer 2,

We have carefully revised the manuscript according to the suggestions. The itemized list of changes addressing the comments is found below and the changes are marked using the track changes in the manuscript.

This manuscript reviews work on lipases, their expression, and applications. It contains useful information and overall is in a decent shape and is enjoyable to read. However, it contains lack of diagrams/figures. Like recombination methods or applications shown in block diagrams for an overview would make a better review paper.

We would like to thank you for this comment. A diagram representing recombination strategies for optimal lipase production was added to the text (Figure 1).

Please discuss the productivity of recombinant lipases. How do they compare with natural lipases? There’s little information in the manuscript about fermentation. Since the title indicates that this manuscript reviews ‘production,’ then production on large scale and issues, cost etc. need to be discussed more.

Indeed recombinant lipases can be achieved with higher yields compared to native systems. Discussion on productivity and bioprocess studies were added to the text.

“nlike  esterases  that  act  on  soluble  substrates,  lipases  act  on  lipids.” The reactions are still carried out in solution form. Do you mean in this paragraph that lipases act on emulsified lipids in water at different pH?? Oil in water type liquid phase. “called  interfacial  activation” oil/water interface. Otherwise it can also mean air-water interface.

We would like to thank you for this comment. In this paragraph, we just meant to distinguish lipases from esterases. We altered the text to have a more appropriate description.

Are all lipids insoluble in water?

The classic definition of lipids implies that lipids are water insoluble. However, we agree that there are derived lipids that has partial water solubility, including glycolipids and saponified lipids. For these reason we altered the text as explained in the previous comment.

“Many tricks are available to optimize a production process.” “capacity may be pushed to its limit” “an infinity of modeling software has been created” You can use better scientific language. There are many such sentences. Check elsewhere.

The sentences were altered.

Not everybody knows what chaperons are. Please explain what they are and their role in the context of protein secretion.

Molecular chaperones are proteins that assist the conformational folding or unfolding of other proteins. Their role in protein secretion is very important since they assist the target enzyme to be properly folded and modified in order to reach the ideal conformational structure and act efficiently on the substrate after secretion. A correct definition of chaperons were added in the  text.

"lipases also possess a lid or flap-like structure” you have written many times about the lipase 3D structure and how it changes in the vicinity of the substrate. Please include a diagram of a common lipase with proper labeling of the flap, helix sheets etc. and how it changes during catalysis.

Thank you for the comment. We added two lipase structures (Figure 2) with the lid in its open and closed conformation that will help readers to understand this topic.

In the immobilization section, there is nothing written about recycling performance, loss of activity on immobilization and there is no figure/diagram.

This information was added in the text.

In Fig. 1 – the enantiomeric structure of the desired active structure of ibuprofen, naproxen, ketoprofen is missing. Where is the S-enantiomer?

Thank you for the comments. We would like to clarify that the “R letter” in the figure represents the radicals that can be the ethyl-ibuprofen (1), ethyl-naproxen (2) and ethyl-ketoprofen (3), and not the R-enantiomer. The enantiomers R and S are represented in parenthesis in the figure. We explained this information in the figure caption.

Round 2

Reviewer 1 Report

I would like to thank the Authors for taking into account the Reviewer's suggestions. I believe that after editorial corrections the article is ready for publication.

Author Response

Reviewer 1,

I would like to thank the Authors for taking into account the Reviewer's suggestions. I believe that after editorial corrections the article is ready for publication.

We would like to thank you for the comment.

Reviewer 2 Report

Authors have addressed all the comments and the manuscript looks better than before. However, in the new Figure 2 caption, citation is missing. The rights and permissions from the publisher need to be taken for reusing this figure and should include a statement in the caption -adapted with permission from.. or reprinted with permission...

Figure 3 has not been pasted properly and not clear if it is an original image or taken from another article.

Author Response

Dear Reviewer 2,

The itemized list of changes addressing the comments is found below and the changes are marked using the track changes in the manuscript.

Authors have addressed all the comments and the manuscript looks better than before. However, in the new Figure 2 caption, citation is missing. The rights and permissions from the publisher need to be taken for reusing this figure and should include a statement in the caption -adapted with permission from.. or reprinted with permission...

Thank you for the comment. The Figure 2 was built using the molecular visualization system PYMOL based on information from PDB (Protein Data Bank), which is a public database. We also specified the access numbers of each structure. Therefore, we believe that there is no need for a statement in the caption adapted with any type of permission. We also specified that all figures were prepared by using molecular visualization software PYMOL.

Figure 3 has not been pasted properly and not clear if it is an original image or taken from another article.

Thank you for the comment. We pasted Fig 3 correctly now. The Figure 3 was designed by us, which means it is an original image. It was not taken from any other article. Therefore, there is no need for any permission.