Immobilization of Phospholipase D for Production of Phosphatidylserine by a Pickering Emulsion Strategy
, Shujing Zhang †, Dianqing Liu, Zhiqi Huang, Yuxin Ge, Jiayi Hou, Fuping Lu and Yihan Liu *
Round 1
Reviewer 1 Report
The article «Immobilization of Phospholipase D for Production of Phosphatidylserine by A Pickering Emulsion Strategy» is attracted to a relevant topic and has a high applied value.
Nowadays, considerable attention was focused on the phospholipase D - mediated synthesis of phosphatidylserine. The application of free phospholipase is usually limited by high cost, poor reusability, and low stability. In this study, phospholipase from Streptomyces antibiotics was immobilized on SiO2 through physical adsorption to develop saPLD@SiO2. The stability and reusability of saPLD@SiO2 were higher than those of free phospholipase.
The article is well structured, written in sufficient detail and logically.
Major Comments:
When discussing the results, the authors should present a comparative analysis of the activity, stability and reusability of their phospholipase with the data of other authors.
It would be good to supplement the article with data on kinetics, namely Km, Vmax, Kcat.
Minor Comments:
Line 15: “stability … were” should be replaced with “stability … was”.
Line 81: Candida rugosa should be italicized.
Figures 1, 5, 6, 7, 10: the bottom of error bars is not visible.
Figure 9f: the top part of error bars is not visible.
Figure 1 caption and Figure 1 should be placed on the same page. Similar for Figure 5.
Line 246: may be “generally”?
If units of measurement are repeated, they should be specified once:
Lines 357-358: instead of “0.25 U, 0.5 U, 0.75 357 U, and 1.0 U” it will be better “0.25, 0.5, 0.75, and 1.0 U”
Line 359: instead of “10 °C, 20 °C, 30 °C, and 40 °C” it will be better “10, 20, 30, and 40 °C”
Similar for Lines 361, 386, 389,
The heading of Section 3.7 and the section itself should be placed on the same page.
Minor editing of English language required
Author Response
Reviewer #1
The article «Immobilization of Phospholipase D for Production of Phosphatidylserine by A Pickering Emulsion Strategy» is attracted to a relevant topic and has a high applied value.
Nowadays, considerable attention was focused on the phospholipase D - mediated synthesis of phosphatidylserine. The application of free phospholipase is usually limited by high cost, poor reusability, and low stability. In this study, phospholipase from Streptomyces antibiotics was immobilized on SiO2 through physical adsorption to develop saPLD@SiO2. The stability and reusability of saPLD@SiO2 were higher than those of free phospholipase.
The article is well structured, written in sufficient detail and logically.
- When discussing the results, the authors should present a comparative analysis of the activity, stability and reusability of their phospholipase with the data of other authors.
Response: Thanks for your insightful advice.
“which is similar to the PLD (from Streptomyces sp.) immobilized on ZnO nan-owires/macroporous SiO2 or the PLD (from Streptomyces sp. LD0501) immobilized on SiO2.” has been added in line 183-185 of page 5 in the Revised Manuscript.
“In addition, PLD (from S. halstedii) was immobilized on the cell surface of P. pastoris, and the relative PS yield was still above 40% after 4 repeated cycles” has been added in line 220-222 of page 7 in the Revised Manuscript.
“The results are similar to that of the PLD (from Streptomyces sp.) immobilized on epoxy resin-based hierarchical porous polymers.” has been added in line 230-232 of page 7 in the Revised Manuscript.
- It would be good to supplement the article with data on kinetics, namely Km, Vmax, Kcat.
Response: Thanks for your advice on this detail.
First, the activity of PLD to catalyze the transphosphatidylation was evaluated by analyzing the yield of PS from PC and l-serine. The reaction mixture was incubated for 20 min with rapid shaking at 200 rpm and 40 °C, then the reaction was terminated by adding chloroform/methanol (2:1, v/v) solution (4 mL), followed by extracting the phospholipids. After centrifugation for 10 min at 8500 g, the lower layer was harvested and detected using high-performance liquid chromatography (HPLC). Hence, too many intermediate steps make it difficult to accurately measure Km and Vmax, resulting in inaccurate Kcat values for the transesterification reaction. So far, none of the current articles on PLD, with more than 100 articles, have provided Km, Vmax, and Kcat values for the PLD transesterification reaction.
- Line 15: “stability … were” should be replaced with “stability … was”.
Response: Thanks for your helpful advice.
“stability … were” has been changed to “stability … was” in line 15 of page 1 in the Revised Manuscript.
- Line 81: Candida rugosa should be italicized.
Response: Thanks for your advice on this detail.
“Candida rugosa” been changed to “Candida rugosa” in line 77 of page 2 in the Revised Manuscript.
- Figures 1, 5, 6, 7, 10: the bottom of error bars is not visible.
Response: Thanks for your advice on this detail.
The bottom of error bars has been added in Figures 1, 5, 6, 7, 10 in the Revised Manuscript.
- Figure 9f: the top part of error bars is not visible.
Response: Thanks for your advice on this detail.
The top part of error bars has been added in Figure 9f in the Revised Manuscript.
- Figure 1 caption and Figure 1 should be placed on the same page. Similar for Figure 5.
Response: Thanks for your advice on this detail.
The Figure 1 caption and Figure 1 has been placed on the same page in line 117-120 of page 3 in the Revised Manuscript. The Figure 5 caption and Figure 5 has been placed on the same page in line 189-191 of page 6 in the Revised Manuscript.
- Line 246: may be “generally”?
Response: Thanks for your advice on this detail.
“genrally” been changed to “generally” in line 251 of page 8 in the Revised Manuscript.
- If units of measurement are repeated, they should be specified once:
Lines 357-358: instead of “0.25 U, 0.5 U, 0.75 357 U, and 1.0 U” it will be better “0.25, 0.5, 0.75, and 1.0 U”
Line 359: instead of “10 °C, 20 °C, 30 °C, and 40 °C” it will be better “10, 20, 30, and 40 °C”
Similar for Lines 361, 386, 389,
The heading of Section 3.7 and the section itself should be placed on the same page.
Response: Thanks for your insightful advice.
“0.25 U, 0.5 U, 0.75 357 U, and 1.0 U” has been changed to “0.25, 0.5, 0.75, and 1.0 U” in line 363-364 of page 11 in the Revised Manuscript.
“10 °C, 20 °C, 30 °C, and 40 °C” has been changed to “10, 20, 30, and 40 °C” in line 365 of page 11 in the Revised Manuscript.
“1 h, 20 h, 30 h, and 40 h” has been changed to “1, 2, 3, and 4 h” in line 366-367 of page 11 in the Revised Manuscript.
“40 °C, 50 °C, 60 °C 70 °C, and 80 °C” has been changed to “40, 50, 60, 70, and 80 °C” in line 391 of page 11 in the Revised Manuscript.
“40 °C, 50 °C, and 60 °C” has been changed to “40, 50, and 60 °C” in line 394 of page 11 in the Revised Manuscript.
The heading of Section 3.7 and the section itself have been placed on the same page in line 414 of page 12.
I greatly appreciate your help for improving this manuscript. I hope that the revised manuscript is now suitable for publication.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The paper is quite interesting and well-written. However, some issues have to be addressed:
Line 42: What is PC?
Fig. 2: I can see particle aggregates on fig. 2b. Can the authors comment on this? Maybe aggregation is due to the presence of the enzyme? The bar is barely visible.
Fig. 4: Why there's C-H stretching in the silica spectrum? Maybe an incomplete removal of the surfactant? 1i don't agree whith the enzyme-SiO2 spectrum interpretation. No mention is done to amide I and II, which indicate the presence of the enzyme (see for example Califano et al. Pure App. Chem. 91, 2019, 1583-1592; A. Barth. Biochim. Biophys. Acta (BBA) Bioenerg. 1767, 1073, 2007; Z. Zhou, A. Inayat, W. Schwieger, M. Hartmann. Microporous Mesoporous Mater. 154, 133, 2012.) I don't think that the band at 1865 is C=O stretching.
Fig. 9: (a) is without and with sonication (left and right)? I don't understand fig. 9 b and c: is there a solid on the vial's walls? Size bar is missing from fig. 9 d.
Line 302: a reaction scheme would be useful
Line 333: can the authors give more infrmation on SiO2? Porous or nonporous? Surface area, pore size and particle size.
Line 448: Covalent attachment? It seems that they performed only adsorption.
English is fine.
Line 15: was higher
Line 257: ethyl propionate?
Author Response
Reviewer #2
The paper is quite interesting and well-written. However, some issues have to be addressed.
- Line 42: What is PC?
Response: Thanks for your question.
“PC” has been changed to “Phosphatidylcholine (PC)” in line 42-43 of page 1 in the Revised Manuscript.
- Fig. 2: I can see particle aggregates on fig. 2b. Can the authors comment on this? Maybe aggregation is due to the presence of the enzyme? The bar is barely visible.
Response: Thanks for your advice on this detail.
We have removed the free enzyme by washing it many times before performing the test, so we are sure that fig. 2b is the enzyme immobilized on the surface of the carrier. In addition, the overall layout of fig. 2 has been adjusted, so that the bar is visible in line 127-129 of page 4 in the Revised Manuscript.
- Fig. 4: Why there's C-H stretching in the silica spectrum? Maybe an incomplete removal of the surfactant? 1i don't agree whith the enzyme-SiO2 spectrum interpretation. No mention is done to amide I and II, which indicate the presence of the enzyme (see for example Califano et al. Pure App. Chem. 91, 2019, 1583-1592; A. Barth. Biochim. Biophys. Acta (BBA) Bioenerg. 1767, 1073, 2007; Z. Zhou, A. Inayat, W. Schwieger, M. Hartmann. Microporous Mesoporous Mater. 154, 133, 2012.) I don't think that the band at 1865 is C=O stretching.
Response: Thanks for your advice on this detail.
“The C-H stretching…”“1865 cm-1 (carbonyl (C=O) stretching vibration),” has been deleted in the Revised Manuscript.
“The amide 1 band and amide 2 band are specific wavenumber ranges in the infrared spectrum used to represent vibration patterns in compounds containing an amide group[40]. The infrared amide 1 band is usually in the range of 1600-1800 cm-1, and the amide 2 band is typically in the range of 1470-1570 cm-1. An increase in vibration between the amide 1 and amide 2 bands can be seen after fixation of the saPLD, indicating a change in protein structure which could be due to the formation of new interactions between saPLD and SiO2.” has been added in line 159-165 of page 5 in the Revised Manuscript.
“1865 cm-1 (carbonyl (C=O) stretching vibration),” has been deleted in the Revised Manuscript.
- Fig. 9: (a) is without and with sonication (left and right)? I don't understand fig. 9 b and c: is there a solid on the vial's walls? Size bar is missing from fig. 9 d.
Response: Thanks for your insightful advice.
“Images of the aqueous and organic phases without sonicating” has been changed to “Images of the aqueous and organic phases without sonicating(left) and with soni-cating(right)” in line 299-300 of page 9 in the Revised Manuscript.
Figure 9b and Figure 9c have solid on the vial's walls.
Size bar has been added in figure 9 d in the Revised Manuscript.
- Line 302: a reaction scheme would be useful
Response: Thanks for your helpful advice.
The reaction scheme of the transphosphatidylation has been added in line 318-319 of page 9 in the Revised Manuscript.
- Line 333: can the authors give more information on SiO2? Porous or nonporous? Surface area, pore size and particle size.
Response: Thanks for your question.
SiO2 is a non-porous material. The manufacturer does not provide surface area data, but the particle size is specified to be 25-30 nm.
- Line 448: Covalent attachment? It seems that they performed only adsorption.
Response: Thanks for your advice on this detail.
“Covalent attachment” has been deleted in line454 of page 12 in the Revised Manuscript.
- Line 15: was higher
Response: Thanks for your advice on this detail.
“stability … were” has been changed to “stability … was” in line 15 of page 1 in the Revised Manuscript.
- Line 257: ethyl propionate?
Response: Thanks for your helpful advice.
“ethyl propiona” has been changed to “ethyl propionate” in line 262 of page 8 in the Revised Manuscript.
I greatly appreciate your help for improving this manuscript. I hope that the revised manuscript is now suitable for publication.
Author Response File: Author Response.pdf
