Improved Catalytic Performance of Lipase Eversa^® Transform 2.0 via Immobilization for the Sustainable Production of Flavor Esters—Adsorption Process and Environmental Assessment Studies

Round 1

Reviewer 1 Report

The authors evaluated the production of various flavor esters through esterification utilizing an immobilized lipase in a solvent-free system.

This study used Eversa® Transform 2.0 as a biocatalyst, which had not been used in the production of food-grade flavor esters, and kinetic and mass transfer studies of the adsorption process were performed to elucidate the interaction of the enzyme with the PSty-DVB surface.

The purpose and methods of the study are carefully presented and the results support the purpose of the study in part.

However, the following major questions should be answered.

1.     It should be understood to what extent economic or commercialization levels have been achieved compared to commercially available immobilized lipases such as the well-known Novozym 435.

2.     The loading (usage) of lipase used in the reaction system should be provided on a unit basis. (Please also provide a definition of the activity of the unit, e.g., the ability to esterify a unit molar substrate per unit time).

 

3.     Strategies for improving enzyme reusability should be presented more specifically by comparison with other enzyme-reusing studies.

Author Response

1. It should be understood to what extent economic or commercialization levels have been achieved compared to commercially available immobilized lipases such as the well-known Novozym 435.

Response

The reviewer is correct. The final cost of the heterogeneous biocatalyst prepared in this study was incorporated to the revised manuscript and compared to commercially available immobilized lipases such as the well-known Novozym 435, as suggested (see comments in Section 2.4 – Lines 403-408):

From an economic standpoint, the application of the heterogeneous biocatalyst prepared in this study is highly interesting on an industrial scale, since its average final cost may be estimated of around $300 per Kg, and this is cheaper than a well-known commercially available immobilized biocatalyst Novozym^® 435 (the cost is $700 per Kg [59]), and this is the most used heterogeneous biocatalyst to produce flavor esters via esterification [60].

 

2. The loading (usage) of lipase used in the reaction system should be provided on a unit basis. (Please also provide a definition of the activity of the unit, e.g., the ability to esterify a unit molar substrate per unit time).

 

Response

The reviewer is correct. However, the loading (usage) of lipase used in the reaction system were provided on a protein amount basis due to its high purity. Our results confirm very similar results between immobilization yield (based on disappeared units of activity during the adsorption process) and immobilized protein amount values. As suggested, we provided the definition of the activity of the unit. This information can be observed in Section 3.3. – Lines 453-457:

The catalytic activity of the enzyme was assayed by olive oil emulsion hydrolysis method [61]. One international unit (U) of hydrolytic activity was defined as being the mass of lipase required to release 1 µmol of free fatty acids per minute at pH 8.0 (100 mmol.L^-1 sodium phosphate), and 37 °C and 200 rpm [61].

 

3. Strategies for improving enzyme reusability should be presented more specifically by comparison with other enzyme-reusing studies.

 

Response

The reviewer is correct. This information was reported in the revised version – see Conclusion Section (Lines 587-591):

Further studies should be conducted using fusel oil as alcohol source from the bioethanol distilleries, evaluating the influence of relevant factors, including water molecules removal strategies that improves the catalytic performance and operational stability of immobilized lipases on esters production in batch or continuous processes.

Thanks for your contributions to greatly the quality of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

The research area is very timely. Solvent-free conversion of naturally occurring raw materials using biocatalysts is one of the important objectives of the modern chemical industry. The authors successfully recorded and used one of the modified enzyme preparations of the Novozym company during the production of octanoic acid esters. During the tests, the immobilized preparation was compared with the free enzyme, and better results were achieved with the immobilized version in three cases. The authors demonstrated that the immobilized biocatalyst they produced can be successfully reused in 6 consecutive steps. At the same time, all of the examined processes are considered environmentally friendly methods. The article is clear and well-edited.
The theme is opportune and usefull.
I have just one comment.
In line 360 and 368 the remaining acid conversion (for fusel oil) is listed with two different values (45% and 43%). Which is the true? Please correct it! The authors implemented a well-structured experimental design and correctly evaluated the obtained results. The train of thought is easy to follow, and the figures and tables are easy to interpret. After correcting the objectionable errors, the announcement is worth publishing.

Author Response

In line 360 and 368 the remaining acid conversion (for fusel oil) is listed with two different values (45% and 43%). Which is the true? Please correct it! The authors implemented a well-structured experimental design and correctly evaluated the obtained results. The train of thought is easy to follow, and the figures and tables are easy to interpret. After correcting the objectionable errors, the announcement is worth publishing.

 

Response

The reviewer is correct. Sorry for this mistake. These values were carefully revised and corrected, as suggested (see Lines 367-370).

These values correspond to a loss of the original activity of the biocatalyst of around 28% (from 62.5% in the first batch to 45% in the sixth batch) and 53% (from 82.5% in the first batch to 39% in the sixth batch) using fusel oil and commercial isoamyl alcohol, respectively.

 

We also corrected these values in Abstract section:

Reusability tests revealed high retention of the original activity of the immobilized lipase after six successive batch reactions using isoamyl alcohol (47%) and fusel oil (72%).

Thanks for your contributions to greatly the quality of the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The revised manuscript was significantly improved by the authors.

The paper is now can be accepted for publication in Catalysts.