Enzymatic Synthesis of O-Methylated Phenophospholipids by Lipase-Catalyzed Acidolysis of Egg-Yolk Phosphatidylcholine with Anisic and Veratric Acids
Round 1
Reviewer 1 Report
This paper reports lipase-catalyzed acidolysis reactions of egg-yolk phosphatidylcholine (PC) with anisic (ANISA) and veratric (VERA) acids. The most effective commercially available immobilized lipases and the effects of different solvent, substrate molar ratio, temperature, enzyme loading and time of the reaction on the process of incorporation of ANISA into the phospholipids were well evaluated. This study provides valuable information for environmentally friendly one-step enzymatic synthetic method of biologically active compounds. However, there are a few points which should be clarified. The comments to this paper are described below:
- Page 6; line 243-244
Please describe information, if any, on the reason why no further increase in the incorporation of ANISA when the substrate molar ratio was beyond 1:15, such as aggregation of ANISA or inhibition of lipase activity by ANISA.
- Page 8; line 278-281
A large difference was observed for the incorporation of ANISA into PLs fraction between enzyme loads of 20% and 30%. If possible, please provide explanation for the reason.
Author Response
Anna Gliszczyńska
Department of Chemistry,
Wroclaw University of Environmental and Life Sciences,
Norwida 25, PL-50-375 Wroclaw, Poland
E-mail: [email protected]
Editor
Catalysts
Dear Editor,
We would like to thank you very much for giving us the possibility to improve the manuscript of our paper “Studies on the enzymatic synthesis of O-methylated phenophospholipids by lipase-catalyzed acidolysis of egg-yolk phosphatidylcholine with anisic and veratric acids” (Catalysts-792540). Here we would like to present and comment on the changes we have made according to the Reviewers’ suggestions and give some explanations of some issues that had been raised by Reviewers.
General comments:
All changes were highlighted in yellow in revised version of the manuscript.
According to Reviewers comments the manuscript was corrected by a native speaker. All language changes are visible in red color.
According to the Reviewer 1 comments:
Please describe information, if any, on the reason why no further increase in the incorporation of ANISA when the substrate molar ratio was beyond 1:15, such as aggregation of ANISA or inhibition of lipase activity by ANISA.
Response:
According to the Reviewer suggestion we added appropriate paragraph in the section 2.4
“At the substrate molar ratio 1:20 we observed that incorporation of ANISA into the phospholipid fraction drastically decreased. This phenomenon may be explained by the inhibition effect of high concentration of the substrate in the reaction medium on the enzyme activity. As highlighted above, solubility limitation of the substrate might also be a key factor. The increase of substrate amounts higher than the solubility limitation may not increase the concentration of substrate available for reaction, instead increasing viscosity and decrease mass transfer, and thus might result in a decrease of incorporation”.
A large difference was observed for the incorporation of ANISA into PLs fraction between enzyme loads of 20% and 30%. If possible, please provide explanation for the reason.
Response:
We agree with the Reviewer that non-linear trend of incorporation of ANISA into phospholipid fraction with the increase amount of enzyme is not a common phenomenon. We added the possible explanation for this in the section 2.6.
“A large difference observed for incorporation of ANISA into phospholipid fraction between enzyme loads of 20% and 30% (w/w) could be the result of slow rate for acyl-enzyme complex formation. Jakovetić et al. reported four times higher reaction rate constant for ethyl cinnamate than for ethyl-p-coumarate [46]. The higher incorporation at the presence of the higher amount of lipase (30% (w/w)) can be then the result of increases the probability of enzyme-substrate collisions and subsequent reaction of esterification. Overall, optimum enzyme load was estimated as 30%”.
According to the Reviewer 2 comments:
GC was used to analyze both fatty acids and methoxy derivatives benzoic acid. However, no results are shown in the work. Authors must prepare a supporting file in which they come with all the chromatograms (also HPLC ones).
Response:
According to the Reviewer suggestion we added to the manuscript Supplementary Materials where we present the HPLC and GC chromatograms.
Figure 3: It is indeed strange that the incorporation of ANISA into the phospholipid decreases so drastically when a 1:20 ratio is used. I would have expected it to have the same trend as the 1:15 ratio. The authors should include considerations in this regard in the paragraph (line 244)
Response:
According to the Reviewer suggestion we added appropriate paragraph in the section 2.4
“At the substrate molar ratio 1:20 we observed that incorporation of ANISA into the phospholipid fraction drastically decreased. This phenomenon may be explained by the inhibition effect of high concentration of the substrate in the reaction medium on the enzyme activity. As highlighted above, solubility limitation of the substrate might also be a key factor. The increase of substrate amounts higher than the solubility limitation may not increase the concentration of substrate available for reaction, instead increasing viscosity and decrease mass transfer, and thus might result in a decrease of incorporation”.
lines 128-130: "Lack of lipase affinity towards VERA can be explained then by electron effect, where electron donating groups on the ring cause deactivation of electrophilic carbon from carboxylic group, which is responsible for nucleophilic attack on the hydroxy group of glycerol": a carboxylic group is not a nucleophile that attacks an alcohol, rather the opposite is true! In addition, VERA may be less reactive due to a steric effect - the authors should perhaps make a comparison with another acid with a similar steric hindrance but with an electron attractor group, such as vanillin (4-Hydroxy-3-methoxybenzaldehyde).
Response:
Thank you, Reviewer has right we removed incorrect part of the sentence.
line 151, scheme 1: The reaction mechanism they propose makes sense only if Novozym 435 is selective for primary alcohols. I believe this is the case (due to the steric hindrance), but they must cite an article that mentions this selectivity before making any mechanism hypotheses. To test this hypothesis, they could synthesize a PC with an ether group instead of the primary acetate, which cannot be hydrolyzed, and then see if the secondary ester hydrolyses by migration (which in that case cannot occur).
Response:
Reviewer has right that Novozym 435 is considered as a non-regiospecific lipase but in the majority of lipid modifications it showed high selectivity towards the sn-1 position of TAGs and PC. Hence, we proposed this pathway for the preformed reaction of acidolysis of PC with ANISA. According to the Reviewer suggestion we added in the manuscript appropriate references confirming this.
line 49: fib re?
Response:
Thank you we corrected it and all other language errors.
line 384: It should be High Performance Liquid Chromatography (formerly known as high-pressure liquid chromatography)
Response:
Thank you, we corrected it using indicated by the Reviewer terminology.
line 392: TEA abbreviation (Triethylamine)
Response:
We added the full name of the reagent.
Error bars are shown in all graphs. it is necessary to report in the captions what they refer to (standard deviation?) and the number experiments performed to calculate them
Response:
We fully agree with the Reviewer, we added the appropriate information in the captions.
We would like to express our thanks to Reviewers for very valuable comments, which help us to improve our manuscript. We hope that our explanations and corrections are sufficient, and our paper will be finally accepted for publication.
With best regards,
Anna Gliszczyńska
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This article from Marta Okulus and Anna Gliszczyńska describes the enzymatic synthesis of phospholipids containing anisic acid or veratric acid. The authors test four different commercial lipases in their immobilized form and show that just one of them displays a significant activity on the substrates tested. They also select the more efficient solvent system, substrate ratio and temperature to carry out the reaction.
The article itself does not present a high degree of novelty. The scientific literature on lipases is boundless. However, the results they obtain are interesting. Binding these methoxy derivatives of benzoic acid to a phospholipid could actually increase their bioavailability as is reported for other similar molecules.
From a methodological point of view, the work does not present very critical issues. The work is very simple without any kind of more detailed investigation of the reaction mechanisms. Nevertheless, the experiments are correctly described both from the methodological point of view and in the actual results.
Major issues:
The English used to write the manuscript must be reviewed extensively. There are many errors in the text which sometimes make it difficult to be understood.
GC was used to analyze both fatty acids and methoxy derivatives benzoic acid. However, no results are shown in the work. Authors must prepare a supporting file in which they come with all the chromatograms (also HPLC ones).
Figure 3: It is indeed strange that the incorporation of ANISA into the phospholipid decreases so drastically when a 1:20 ratio is used. I would have expected it to have the same trend as the 1:15 ratio. The authors should include considerations in this regard in the paragraph (line 244)
lines 128-130: "Lack of lipase affinity towards VERA can be explained then by electron effect, where electron donating groups on the ring cause deactivation of electrophilic carbon from carboxylic group, which is responsible for nucleophilic attack on the hydroxy group of glycerol": a carboxylic group is not a nucleophile that attacks an alcohol, rather the opposite is true! In addition, VERA may be less reactive due to a steric effect - the authors should perhaps make a comparison with another acid with a similar steric hindrance but with an electron attractor group, such as vanillin (4-Hydroxy-3-methoxybenzaldehyde).
line 151, scheme 1: The reaction mechanism they propose makes sense only if Novozym 435 is selective for primary alcohols. I believe this is the case (due to the steric hindrance), but they must cite an article that mentions this selectivity before making any mechanism hypotheses. To test this hypothesis, they could synthesize a PC with an ether group instead of the primary acetate, which cannot be hydrolyzed, and then see if the secondary ester hydrolyses by migration (which in that case cannot occur).
minor issues:
line 49: fib re?
line 384: It should be High Performance Liquid Chromatography (formerly known as high-pressure liquid chromatography),
line 392: TEA abbreviation (Triethylamine)
Error bars are shown in all graphs. it is necessary to report in the captions what they refer to (standard deviation?) and the number experiments performed to calculate them
Considering that the authors have already published a similar article on "catalysts" having the same theme, I suggest accepting also this work provided that English is thoroughly reviewed, and the suggested changes are made.
Author Response
Anna Gliszczyńska
Department of Chemistry,
Wroclaw University of Environmental and Life Sciences,
Norwida 25, PL-50-375 Wroclaw, Poland
E-mail: [email protected]
Editor
Catalysts
Dear Editor,
We would like to thank you very much for giving us the possibility to improve the manuscript of our paper “Studies on the enzymatic synthesis of O-methylated phenophospholipids by lipase-catalyzed acidolysis of egg-yolk phosphatidylcholine with anisic and veratric acids” (Catalysts-792540). Here we would like to present and comment on the changes we have made according to the Reviewers’ suggestions and give some explanations of some issues that had been raised by Reviewers.
General comments:
All changes were highlighted in yellow in revised version of the manuscript.
According to Reviewers comments the manuscript was corrected by a native speaker. All language changes are visible in red color.
According to the Reviewer 1 comments:
Please describe information, if any, on the reason why no further increase in the incorporation of ANISA when the substrate molar ratio was beyond 1:15, such as aggregation of ANISA or inhibition of lipase activity by ANISA.
Response:
According to the Reviewer suggestion we added appropriate paragraph in the section 2.4
“At the substrate molar ratio 1:20 we observed that incorporation of ANISA into the phospholipid fraction drastically decreased. This phenomenon may be explained by the inhibition effect of high concentration of the substrate in the reaction medium on the enzyme activity. As highlighted above, solubility limitation of the substrate might also be a key factor. The increase of substrate amounts higher than the solubility limitation may not increase the concentration of substrate available for reaction, instead increasing viscosity and decrease mass transfer, and thus might result in a decrease of incorporation”.
A large difference was observed for the incorporation of ANISA into PLs fraction between enzyme loads of 20% and 30%. If possible, please provide explanation for the reason.
Response:
We agree with the Reviewer that non-linear trend of incorporation of ANISA into phospholipid fraction with the increase amount of enzyme is not a common phenomenon. We added the possible explanation for this in the section 2.6.
“A large difference observed for incorporation of ANISA into phospholipid fraction between enzyme loads of 20% and 30% (w/w) could be the result of slow rate for acyl-enzyme complex formation. Jakovetić et al. reported four times higher reaction rate constant for ethyl cinnamate than for ethyl-p-coumarate [46]. The higher incorporation at the presence of the higher amount of lipase (30% (w/w)) can be then the result of increases the probability of enzyme-substrate collisions and subsequent reaction of esterification. Overall, optimum enzyme load was estimated as 30%”.
According to the Reviewer 2 comments:
GC was used to analyze both fatty acids and methoxy derivatives benzoic acid. However, no results are shown in the work. Authors must prepare a supporting file in which they come with all the chromatograms (also HPLC ones).
Response:
According to the Reviewer suggestion we added to the manuscript Supplementary Materials where we present the HPLC and GC chromatograms.
Figure 3: It is indeed strange that the incorporation of ANISA into the phospholipid decreases so drastically when a 1:20 ratio is used. I would have expected it to have the same trend as the 1:15 ratio. The authors should include considerations in this regard in the paragraph (line 244)
Response:
According to the Reviewer suggestion we added appropriate paragraph in the section 2.4
“At the substrate molar ratio 1:20 we observed that incorporation of ANISA into the phospholipid fraction drastically decreased. This phenomenon may be explained by the inhibition effect of high concentration of the substrate in the reaction medium on the enzyme activity. As highlighted above, solubility limitation of the substrate might also be a key factor. The increase of substrate amounts higher than the solubility limitation may not increase the concentration of substrate available for reaction, instead increasing viscosity and decrease mass transfer, and thus might result in a decrease of incorporation”.
lines 128-130: "Lack of lipase affinity towards VERA can be explained then by electron effect, where electron donating groups on the ring cause deactivation of electrophilic carbon from carboxylic group, which is responsible for nucleophilic attack on the hydroxy group of glycerol": a carboxylic group is not a nucleophile that attacks an alcohol, rather the opposite is true! In addition, VERA may be less reactive due to a steric effect - the authors should perhaps make a comparison with another acid with a similar steric hindrance but with an electron attractor group, such as vanillin (4-Hydroxy-3-methoxybenzaldehyde).
Response:
Thank you, Reviewer has right we removed incorrect part of the sentence.
line 151, scheme 1: The reaction mechanism they propose makes sense only if Novozym 435 is selective for primary alcohols. I believe this is the case (due to the steric hindrance), but they must cite an article that mentions this selectivity before making any mechanism hypotheses. To test this hypothesis, they could synthesize a PC with an ether group instead of the primary acetate, which cannot be hydrolyzed, and then see if the secondary ester hydrolyses by migration (which in that case cannot occur).
Response:
Reviewer has right that Novozym 435 is considered as a non-regiospecific lipase but in the majority of lipid modifications it showed high selectivity towards the sn-1 position of TAGs and PC. Hence, we proposed this pathway for the preformed reaction of acidolysis of PC with ANISA. According to the Reviewer suggestion we added in the manuscript appropriate references confirming this.
line 49: fib re?
Response:
Thank you we corrected it and all other language errors.
line 384: It should be High Performance Liquid Chromatography (formerly known as high-pressure liquid chromatography)
Response:
Thank you, we corrected it using indicated by the Reviewer terminology.
line 392: TEA abbreviation (Triethylamine)
Response:
We added the full name of the reagent.
Error bars are shown in all graphs. it is necessary to report in the captions what they refer to (standard deviation?) and the number experiments performed to calculate them
Response:
We fully agree with the Reviewer, we added the appropriate information in the captions.
We would like to express our thanks to Reviewers for very valuable comments, which help us to improve our manuscript. We hope that our explanations and corrections are sufficient, and our paper will be finally accepted for publication.
With best regards,
Anna Gliszczyńska
Author Response File: Author Response.pdf
