Lipase B from Candida antarctica in Highly Saline AOT-Water-Isooctane Reverse Micelle Systems for Enhanced Esterification Reaction

Round 1

Reviewer 1 Report

This paper deals with the development of lipase reaction system using reverse micelle under high-saline environment.  The paper contains interesting results, however, there are some difficult points to understand.  So, the manuscript can be published after the following points are revised:

—     The merit of this research is not clear.  The authors should describe it, and also describe the advantage points.

—     In title, the authors wrote “Media engineering”.  However, the authors mainly discussed the saline concentration effects.  The authors should explain relationship media engineering and saline concentration.

Author Response

This paper deals with the development of lipase reaction system using reverse micelle under high-saline environment.  The paper contains interesting results, however, there are some difficult points to understand.  So, the manuscript can be published after the following points are revised:

—     The merit of this research is not clear.  The authors should describe it, and also describe the advantage points.

We consider your comments to be of great importance, so we are grateful. We agree with the reviewer in the need to clarify the merit of the research. Our goal is to study enzymes that require salt for biocatalysis, like halophilic archaeal lipases. Reverse micelles allow an halophilic environment inside. Our manuscript is the first approach to halophilic enzymes studies in this system, we use CALB as model because we know their halotolerance. The reviewer comment has been addressed by attaching the excerpts:

• Line 19-20. “Besides reverse micelles are promising systems for extreme halophilic lipases research.”
• Line 21. “halophilic enzymes”
• Lines 61-63. “Reverse micelles act as a biocompatible armor to host biocatalysts in an aqueous medium from the aggressive organic solvent, where this nanoreactor delimits an inner core of water and an outer organic solvent [34–36].”
• Lines 67-69 “Besides, these systems might offer significant support to halophilic biocatalysts, since this class of enzymes has exhibited rapid and irreversible inactivation when exposed to conditions below 1 M NaCl/KCl [33].”
• Lines 70-72. “It has been described in previous work [37] that lipase B from Candida antarctica (CalB) is suitable for ester synthesis reactions; however, its catalytic activity in the presence of high NaCl concentrations has been scarcely explored.”
• Lines 76-77. “emerging as an effective study model for the screening robust biocatalysts such CalB, and other halophilic lipases,”
• Line 454. “Therefore, reverse micelles present an advantage for halophilic lipases”
• Line 468-469. “we believe that reverse micellar systems are a breakthrough for biotechnological innovation on halophilic enzymes research.”

—     In title, the authors wrote “Media engineering”.  However, the authors mainly discussed the saline concentration effects.  The authors should explain relationship media engineering and saline concentration.

We decided to remove "media engineering" from the title. We are currently working on using this reverse micellar system on halophilic enzymes and we expect to publish these results in the near future. Therefore, this work represents the first approach. Thanks in advance for your suggestions, reviewer 1.

Reviewer 2 Report

Why was butyl oleate synthesis used as model system?

Please check the number of significant digits throughout the text, e.g.., 4.6160 nm±0.2271, there might be some to many. See, e.g., doi:10.1136/archdischild-2014-307149   

The legend of figure 1, more specifically the meaning associated to (A) and (B), should be made clearer.

How many replicate runs were performed to establish enzyme activity (under “Characterization of synthesis reactions in reverse micellar AOT/ Water/ Isooctane systems”)?

Minor issues

Some paragraphs are too long, e.g., in the introduction lines 23 to 28, please consider dividing in two. Other example is from lines 63 to 69 (in the latter, please clarify cause effect). Another is from lines 188 to 192 (in the latter, maybe attributed is not the most adequate verb, maybe suggested would be more adequate, please consider).  Check other cases throughout the text.

Line 58, “where, the microemulsion “, the comma should not be used here.

Line 217, “This consistent” should be “This is consistent”

Author Response

Why was butyl oleate synthesis used as model system?

We appreciate the reviewer comments that were addressed and are described below.

Our goal is to study enzymes that require salt for biocatalysis, like halophilic archaeal lipases. Reverse micelles allow an halophilic environment inside. Our manuscript is the first approach to halophilic enzymes studies in reverse micelles. We use CALB and butyl oleate synthesis as model because we know the CALB halotolerance and their capacity to perform this reaction. We are currently working on using this reverse micellar system on halophilic enzymes and we expect to publish these results in the near future. In order to clarify we rewrite some paragraphs:

• Line 9. “The butyl oleate synthesis by the lipase B from Candida antarctica (CalB) under extreme halophilic conditions”
• Lines 17-20. “This increase in the butyl oleate synthesis demonstrated the potential of reverse micelles as systems that enhance mass transport phenomena in heterogeneous biocatalysis. Besides reverse micelles are promising systems for extreme halophilic lipases research”
• Lines 70-72. “It has been described in previous work [37] that lipase B from Candida antarctica (CalB) is suitable for ester synthesis reactions; however, its catalytic activity in the presence of high NaCl concentrations has been scarcely explored.”

Please check the number of significant digits throughout the text, e.g.., 4.6160 nm±0.2271, there might be some to many. See, e.g., doi:10.1136/archdischild-2014-307149 

• Significant digits have been updated throughout the text. Lines 113-114, 144, 307

The legend of figure 1, more specifically the meaning associated to (A) and (B), should be made clearer.

• The legend was rewritten
• Lines 124-126. “Figure 1. Average size distribution in AOT/ Water/ Isooctane reverse micelle systems. (A) Effect of aqueous content and NaCl concentration on the size of reverse micelles, (B) Size of reverse micelles as a function of NaCl concentration at Wo=20.”

How many replicate runs were performed to establish enzyme activity (under “Characterization of synthesis reactions in reverse micellar AOT/ Water/ Isooctane systems”)?

• Lines 441. “All tests were run by triplicate.”

Minor issues

Some paragraphs are too long, e.g., in the introduction lines 23 to 28, please consider dividing in two. Other example is from lines 63 to 69 (in the latter, please clarify cause effect).

• Paragraphs were divided into lines 25-26, 67-72, 106-107, 156-157, 258-259, 287-288, 294-295.

Another is from lines 188 to 192 (in the latter, maybe attributed is not the most adequate verb, maybe suggested would be more adequate, please consider).  Check other cases throughout the text.

• The term “attributed” was replaced by “suggested” in line 203.

Line 58, “where, the microemulsion “, the comma should not be used here.

• The comma in line 60 was removed.

Line 217, “This consistent” should be “This is consistent”.

• "This is consistent" was corrected in the line 232.