Economy Assessment for the Chiral Amine Production with Comparison of Reductive Amination and Transamination Routes by Multi-Enzyme System

Round 1

Reviewer 1 Report

Article “Economy Assessment for the Chiral Amine Production with Comparison of Reductive Amination and Transamination Routes by Multi-enzyme system”.

Huo Heyu, Yao Guangxiao and Wang Shizhen

In general, the answers to the comments of the reviewer are satisfactory. This applies to the first version of the article.

During the re-revision of the article, the following shortcomings were noted.

The Abstract is still unsatisfactory.

1. The second phrase has no verb and therefore requires correction.
2. Further, the authors claim that the work is about the synthesis of the S-enantiomer. But in the scheme (Fig. 3) the R-enantiomer is shown.
3. It is unclear what makes it possible to increase the activity of amine dehydrogenase so strongly, by 4-5 times.
4. In Table 1, the last line, 150 $ must be indicated, otherwise the amount is incorrect.
5. In the Table 2 and Table 6, column 2 should be labeled “Volume” instead of “Type”.

Author Response

Response to Reviewer’s comments

During the re-revision of the article, the following shortcomings were noted.

The Abstract is still unsatisfactory.

1. The second phrase has no verb and therefore requires correction.

Response: It has been corrected.

2. Further, the authors claim that the work is about the synthesis of the S-enantiomer. But in the scheme (Fig. 3) the R-enantiomer is shown.

Response: Fig. 3 has been corrected.

3. It is unclear what makes it possible to increase the activity of amine dehydrogenase so strongly, by 4-5 times.

Response: It has been rephrased as follow:

Improved amine dehydrogenase by 4-5 folds, the conversion rate can be increased from 31% to 80%-90%. Therefore, the unit price will drop to 0.5-0.6$/g. Therefore, reductive amination will has a great advantage than transamination in the case of same bioprocess cost [11].

4. In Table 1, the last line, 150 $ must be indicated, otherwise the amount is incorrect.

Response: It has been corrected.

5. In the Table 2 and Table 6, column 2 should be labeled “Volume” instead of “Type”.

Response: It has been corrected.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

no big mistakes in the manuscript. please double double check some minor grammar mistakes. 

Author Response

Response to Reviewer’s comments

no big mistakes in the manuscript. please double double check some minor grammar mistakes.

Response: The language has been polished.

 

Reviewer 3 Report

Authors have made an interesting approach to the biocatalytic preparation of a chiral amine, trying to study the economic assesment of this process by two different approaches. The starting point sounds good, but the description of the processes and the discussion must be improved. In some cases, the results are very schematic, without explanation in the text about the data and the results. There are no conclusions apart to establish some obvious statements, so the manuscript does not apport any novely apart for some tables. Some aspects that have to be improved are:

• Introduction should cover the use of amine dehydrogenases for the preparation of amines, as this system is the one emplyed to compare with the use of transaminases.
• Author should explain why a blender is used in the process.
• Lines 89-92. These data are presented as a table caption, giving only the amounts without a clear explanation. Authors must explain in the text how they have established these values, not only pushing numbers.
• There is no description of table 3 neither table 4 on the text. Authors must comment them, it is really unclear to read a manuscript in which table are placed one after another without a text that can help to clarify what is expressed.
• In view of the results achieved, it seems that transaminase-catalyzed process is more efficient than the one catalyzed by amine dehydrogenases, but it is completely unnecessary to establish "if we can enhance the activity of amine dehydrogenase....". This is obvious. It will be the same for transaminase. It is not very logic that after this work, the only thing that authors can establish is that if the enzyme is improved, the process will be improved. This is something really known.
• Method section: Enzymatic processes must be described in deep in this section, clearly indicating amounts, times, temperatures....
• Only ten references for a research like this is a very low number of references in which support the results obtained.

Author Response

Response to Reviewer’s comments

Authors have made an interesting approach to the biocatalytic preparation of a chiral amine, trying to study the economic assesment of this process by two different approaches. The starting point sounds good, but the description of the processes and the discussion must be improved. In some cases, the results are very schematic, without explanation in the text about the data and the results. There are no conclusions apart to establish some obvious statements, so the manuscript does not apport any novely apart for some tables. Some aspects that have to be improved are:

1. Introduction should cover the use of amine dehydrogenases for the preparation of amines, as this system is the one emplyed to compare with the use of transaminases.

Response: Introduction about amine dehydrogenase for biosynthesis of chiral amine has been added into manuscript as follow:

Amine dehydrogenases dependent on NADH can catalyze the direct reductive amination of ketones with high enantioselectivity. Multi-enzyme biosynthesis of chiral amine based on amine dehydrogenases is with the advantages of simple reaction system and easy co-enzyme regeneration.

 

2. Author should explain why a blender is used in the process.

Response: The explanation of why blender was used has been added into manuscript as follow: Stirred bioreactors are widely applied for biosynthesis due to the advantages such as easy scale-up, good fluid mixing and alternative impellers.

 

3. Lines 89-92. These data are presented as a table caption, giving only the amounts without a clear explanation. Authors must explain in the text how they have established these values, not only pushing numbers.

Response: Details of reaction condition have been added into manuscript.

 

4. There is no description of table 3 neither table 4 on the text. Authors must comment them, it is really unclear to read a manuscript in which table are placed one after another without a text that can help to clarify what is expressed.

Response: Description has been added into manuscript for Table 3 and 4 as follow:

Energy consumption, including stirring, heating, and also waste treatment during the process of transamination is calculated (Table 3). The labor costs are shown in Table 4.

 

5. In view of the results achieved, it seems that transaminase-catalyzed process is more efficient than the one catalyzed by amine dehydrogenases, but it is completely unnecessary to establish "if we can enhance the activity of amine dehydrogenase....". This is obvious. It will be the same for transaminase. It is not very logic that after this work, the only thing that authors can establish is that if the enzyme is improved, the process will be improved. This is something really known.

Response: The description has been rephrased. We agree with reviewer that enhanced activity both suitable for amine dehydrogenases and transaminases. The activity of amine dehydrogenase is much lower than that of transaminase. Compared with long study history of transaminase, research about amine dehydrogenases were carried out in recent 10 years. It is promising that great enhancement of amine dehydrogenases can be achieved by modern biotechnology.

 

6. Method section: Enzymatic processes must be described in deep in this section, clearly indicating amounts, times, temperatures....

Response: Details of reaction condition have been added into manuscript after Figure 1 and Figure 3.

 

7. Only ten references for a research like this is a very low number of references in which support the results obtained.

Response: More references have been added into manuscript as follow:

5. Gruber, P.; Carvalho, F.; Marques, M.P.C.; O'Sullivan, B.; Subrizi, F.; Dobrijevic, D.; Ward, J.; Hailes, H.C.; Fernandes, P.; Wohlgemuth, R., et al. Enzymatic synthesis of chiral amino-alcohols by coupling transketolase and transaminase-catalyzed reactions in a cascading continuous-flow microreactor system. Biotechnology and Bioengineering 2018, 115, 586-596
6. Caparco, A.A.; Pelletier, E.; Petit, J.L.; Jouenne, A.; Bommarius, B.R.; Berardinis, V.; Zaparucha, A.; Champion, J.A.; Bommarius, A.S.; Vergne‐Vaxelaire, C. Metagenomic mining for amine dehydrogenase discovery. Advanced Synthesis & Catalysis 2020, 362, 2427-2436.
7. Liu, J.; Li, Z. Enhancing cofactor recycling in the bioconversion of racemic alcohols to chiral amines with alcohol dehydrogenase and amine dehydrogenase by coupling cells and cell‐free system. Biotechnology and Bioengineering 2019, 116, 536-542.
8. Tseliou, V.; Knaus, T.; Masman, M.F.; Corrado, M.L.; Mutti, F.G. Generation of amine dehydrogenases with increased catalytic performance and substrate scope from ε-deaminating L-Lysine dehydrogenase. Nature Communications 2019, 10.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Authors have addressed some of the comments made in the first revision, but the manuscript still has to be improved in order to be published at Catalysts.

• The sentence included in the introduction about amine dehydrogenases is very short and does not give any relevant information. Please describe what reaction do these enzymes catalyze and include one review in the references.
• The reaction conditions of the transamination should be placed in the materials and methods section. The same thing for the reaction with AmDH. Experimental procedures should be included there and a brief description of the processes can be commented in the results and discussion section.
• The optical purity of the amine is not given in both processes? Has it been detemined? In case by which method? All the information about this should be provided.
• There is no yield given in the amine dehydrogenase reaction. Please provide it.
• It should be ok to give the purity of the amine obtained.
• In addition, which AA-ADH and Ch1AmDH concentrations are employed. Which is the concentration of 1-phenylethanol (is placed in Figure 3, but should be also in the text). NADH oxidase and its concentration is neither included in the text in lines 120-128 (should be moved to materials and methods).
• Table captions are described by a: and b:, but the information at line 90, lines 95 to 99, line 104, line 130, lines 135 to 139 and lines 142 and 146 should be included as text in the Results and discussion. They cannot be explained in that way, a proper paragraph commenting the results showed in the tables is mandatory. As I stated before, the manuscript is very schematic.
• Please include date of access of the websites (alibaba.com).
• I do not understand line 148 "consumables and waste disposal". What is this referred to?

 These papers sections must be rewritten in a better way in order to make the manuscript acceptable for a publication as Catalysts.

Author Response

The sentence included in the introduction about amine dehydrogenases is very short and does not give any relevant information. Please describe what reaction do these enzymes catalyze and include one review in the references.

R: Description of amine dehydrogenase has been added into manuscript. . Review of amine dehydrogenase has been added as: Amine dehydrogenases occur in nature. Nature Catalysis volume 2, pages288–289(2019)  

 

The reaction conditions of the transamination should be placed in the materials and methods section. The same thing for the reaction with AmDH. Experimental procedures should be included there and a brief description of the processes can be commented in the results and discussion section.

R：The reaction conditions of the transamination reaction have been placed in the materials and methods section.

 

The optical purity of the amine is not given in both processes? Has it been determined? In case by which method? All the information about this should be provided. It should be ok to give the purity of the amine obtained.

R：The ee of both systems was >99% (R). The optical purity was determined by GC-FID and NMR (1H-NMR, 13C-NMR).

 

There is no yield given in the amine dehydrogenase reaction. Please provide it.

R: This part of the experimental data is taken from the literature, and the references have been attached.

 

In addition, which AA-ADH and Ch1AmDH concentrations are employed. Which is the concentration of 1-phenylethanol (is placed in Figure 3, but should be also in the text). NADH oxidase and its concentration is neither included in the text in lines 120-128 (should be moved to materials and methods).

R：The concentration and reaction conditions of various enzymes have been introduced in the "Materials and Methods" section. The experimental data is taken from the supplementary materials in the literature, and the concentration of 1-phenylethanol is not involved, so the concentration cannot be labeled.

 

Table captions are described by a: and b:, but the information at line 90, lines 95 to 99, line 104, line 130, lines 135 to 139 and lines 142 and 146 should be included as text in the Results and discussion. They cannot be explained in that way, a proper paragraph commenting the results showed in the tables is mandatory. As I stated before, the manuscript is very schematic.

R：This part of the article has been re-edited.

 

Please include date of access of the websites (alibaba.com).

R: The date has been attached.

 

I do not understand line 148 "consumables and waste disposal". What is this referred to?

R：The line 148 'Consumables and Waste Disposals' have been moved to the top of table 7. The above refers to table 7, 8.

 These papers sections must be rewritten in a better way in order to make the manuscript acceptable for a publication as Catalysts

 

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Authors have slightly improved the manuscript and I will be willing to accept it after some minor corrections.

• Line 49. Why is reference 15 before references 6 and 7? Please renumber the manuscript references.
• Please write subscripts and superscripts when appropiate: NH₄Cl, NAD⁺, Na₃PO₄, m³,....
• Decide where to indicate the $ sign, if after of before the number.
• How you detemine the optical purity by 1H-NMR? I do not see this. Maybe you are referring to the compound chemical purity?

Author Response

Line 49. Why is reference 15 before references 6 and 7? Please renumber the manuscript references.

R：The document order issue has been corrected.

Please write subscripts and superscripts when appropiate: NH4Cl, NAD+, Na3PO4, m3,....

R: Problem has been corrected.

Decide where to indicate the $ sign, if after of before the number.

R: Problem has been corrected.

How you detemine the optical purity by 1H-NMR? I do not see this. Maybe you are referring to the compound chemical purity?

R: The ees of amines were measured by GC using an Agilent 7890 A GC system, equipped with a FID detector and using a Varian Chrompack Chiracel Dex-CB column (25 m, 320 μm, 0.25 μm)

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Reviewer’s comments

 

The manuscript of “Economy Assessment for the Production of Chiral Amine by Comparison of Amine Dehydrogenase and Transaminase Multi-enzyme system” presented a work that calculation on the capitalized cost of Chiral Amine production comparison with the amine dehydrogenase route to the transamination route. This work gave a capital outlay of Chiral Amine production equipment and production processes. However, this work is less meaningful to the readers. We suggested the authors to make a major revise to the paper. There were some problems need to be revised below.

 

1. The authors listed a detailed cost of major equipment, labor cost, energy consumption and waste disposal during the manufacture of Chiral Amine in the manuscript. However, this calculation is meaningfulness to the readers due to the reason that the cost would have serious variances with the alteration of production place and the construction choice of time.

 

2. This work did not give the cost of determination instruments and the total construction cost of the production line.

 

3. The authors claimed “If we can enhance the activity of amine dehydrogenase by 4-5 folds to increase the conversion rate from 31% to 80%-90%, the unit price will drop to 0.5-0.6$/g.” at Line 58, Page 9. What is the judgement basis that the conversion rate promoted from 31% to 80%-90%? Please list the references.

 

4. The authors claimed “Considering the rising cost caused by immobilization, the last cost will be 12-20% of the initial cost.” at Line 165, Page 9. What is the basis of the judgement? Please list the references.

 

5. The authors claimed “Due to high enzyme activity, the conversion rate was 90% for transamination, while the conversion rate was 31% for reductive amination.” Please list the references.

 

6. The authors listed the cost distribution for chiral amine production by two conversion method in Table 9. The raw materials of amine dehydrogenase route cost lower than transaminase route. However, the conversion rate is the key variable that affect the cost of chiral amine production. If you can not give a valid conversion rate of the two routes, the raw materials cost is meaningfulness.

 

 

7. The conversion rate of the two routes is very critical for the final production cost. Please discuss the conversion rate difference of the two routes based on the reports and list the reference.

 

8. The authors should remove the content at Line 106-109, Page 6. This is description of format.

Reviewer 2 Report

The manuscript untitled "Economy Assessment for the Production of Chiral Amine by Comparison of Amine Dehydrogenase and Transaminase Multi-enzyme system" by H. Heyu, Y. Guangxiao, and W. Shizhen.

The manuscript is an interesting paper devoted to the comparative economic assessment of two bio-processes. The idea was good.

These materials could be useful for biological chemistry and medicinal chemistry specialists. 

The authors intended to prepare a very serious and thorough study, wich seems important for an economic evaluation of Chiral complexes production. Chiral compounds studies are a trend in modern bio-researchs. 

However, the manuscript raises any objections, and cannot be published in Catalysts in the present form.

Several details and inaccuracies should be noted. 

1. The title of the article should be revized. Since the calculations relate to one chiral amine, Its chemical name should be indicated.
2. Abstract need to better organized. 
3. Results and Discussion. Fig. 1. This scheme is plagiarism. It is presented without any indication of the authors of. Ref. [8]. Moreover, some of the names in the scheme are presented with errors and therefore difficalt to understand. For example, lacetate instead lactate.
4. Line 65. "... from the reaction mixture..."
5. Line 70. Figure 2.
6. Line 82. The cost of the total raw material is incorrected.
7. Table 2. Should be "Volume (m3) instead" Type". 
8. Table 3. Column "Volume (m3)". Why are all volumes equal to 5 m3? 
9. Cited literature should be ordered.
10. Ref. [7] is incorrected. Therefore the sheme in Fig. 3 is not possible to check.
11. Therefore, the manuscript needs serious editing