A Multicomponent Butyrylcholinesterase Preparation for Enzyme Inhibition-Based Assay of Organophosphorus Pesticides
Round 1
Reviewer 1 Report
In this paper, two materials for producing butyrylcholinesterase preparations stable in storage and use has been studied. The results are good. But a few need attention:
(1) In introduction part, other materials should be mentioned.
(2) Less than 2 months as shown in Figure 2a.
(3) The storage conditions are not stated in the method.
(4) What is the MAC in abstract?
(5) Figure 6 is not detailed?
Author Response
Dear reviewer,
Thank you for your comments and suggestions. Here are our answers and corrections.
Remark 1
In introduction part, other materials should be mentioned.
Answer 1
Page 2, Lines 47-54. We have added information on materials suitable for enzyme immobilization in introduction part:
«Immobilized enzymes are more stable and more resistant to environmental changes [Home, 2013]. Various nanoparticles (copper nanoflowers [Lambhiya, 2021], magnetic MnO2 [Huang, 2021]) or functionalized multi-walled carbon nanotubes [Dwivedee, 2017] can be used as a carrier for enzyme immobilization. These methods make it possible to produce enzyme preparations that are stable when working in organic solvents and resistant to high temperatures, providing the possibility of numerous applications in enzymatic catalysis. However, polymers of various origins are more often used to immobilize cholinesterase [Doretti, 1999; Mehrani, 2004; Cho, 1999; White, 2002].»
Other materials suitable for cholinesterase immobilization are described in the Discussion section.
Remark 2
Less than 2 months as shown in Figure 2a.
Answer 2
Page 3, Line 92. We have revised the sentence: «The activity of the enzyme in the starch or gelatin gel stored less than two months changed insignificantly (Figure 3a), suggesting effective stabilization of the enzyme by these carriers».
Remark 3
The storage conditions are not stated in the method.
Answer 3
Page 7, Lines 248-249. We have added new information on storage conditions in «Materials and Methods»:
«The produced preparations were stored at 4°C and 45-50% relative humidity».
Remark 4
What is the MAC in abstract?
Answer 4
MAC means Maximum Allowable Concentration. It is a criterion of safe level of exposure to a hazardous or toxic process, chemical, etc., that can be contained in environmental objects (for example, in water). Page 5, Line 146. The abbreviation has been decoded.
Remark 5
Figure 6 is not detailed?
Answer 5
We have added the missing legend to Figure 6.
Reviewer 2 Report
Authors proposed a new method to a BChE-based preparation stable in storage and use. The topic is interesting, simple and straight forward. I would recommend publication with minor revision:
-Move the sentence (line 91-93 in page 3) to Page 2 line 78. Also, more discussion is needed in this part.
-The gelatin shows higher activity than starch. Is this because higher amount of starch is present and it effects the enzyme activity? Please discuss this in the manuscript.
-In Figure 6, chart legends are missing
Author Response
Dear reviewer,
Thank you for your comments and suggestions. Here are our answers and corrections.
Remark 1
Move the sentence (line 91-93 in page 3) to Page 2 line 78. Also, more discussion is needed in this part.
Answer 1
We have moved the sentence to the specified location and added a new sentence (Page 3, lines 88-89): «Keeping BChE active indicates the absence of restrictions arising from the diffusion of DTNB from the polymer matrix of the carrier».
Remark 2
The gelatin shows higher activity than starch. Is this because higher amount of starch is present and it effects the enzyme activity? Please discuss this in the manuscript.
Answer 2
It was previously shown that in both starch and gelatin gel, the activity of BChE varies slightly at different concentrations of gels [Lonshakova-Mukina, V.I.; Esimbekova, E.N.; Kratasyuk, V.A. Thermal inactivation of butyrylcholinesterase in starch and gelatin gels. Catalysts 2021, 492. 10 p. DOI: 10.3390/catal11040492]. The decrease in the activity yield of the enzyme included in the starch gel during the transition to the solid phase may be due to differences in the processes of rehydration of the dried gel. We discussed it on lines 208-214: «The stabilizing effects of starch and gelatin on BChE differ in strength. The decrease in the activity of the enzyme immobilized in starch gel may be associated with the greater ability of starch, compared to gelatin, to bind water, which is the substrate in the reaction catalyzed by BChE. Thus, the osmotic effect may occur inside the starch carrier, resulting in a reduction in the enzyme activity. In addition, the effects of starch and gelatin polymer networks on the rate of diffusion of the substrate and/or inhibitor to the enzyme may differ in strength».
Remark 3
In Figure 6, chart legends are missing
Answer 3
We have added the missing legend to Figure 6.
Reviewer 3 Report
Lonshakova-Mukina et al. have developed a new method of producing preparations of butyrylcholinesterase (BChE) co-immobilized in either a starch or gelatin gel. The authors demonstrated that both starch and gel were effective methods for storing BChE for at least 300 days. In addition, the sensitivity of the co-immobilized enzyme to several inhibitors was reported. This development would allow for rapid enzyme inhibition-based assay of organophosphorus compounds (OPC) in aqueous solutions.
Specific Comments:
(1) Based on the text in lines 139-146, the reader should be able to understand the data presented in figure 6. However, the Figure legend is not at all clear. I am assuming that the orange columns are the control and the blue columns are the reactions that included a 5 minure incubation of the preparation with 0.055 mM pirimiphos-methyl solution.
Author Response
Dear reviewer,
Thank you for your comments and suggestions. Here are our answers and corrections.
Remark 1
Based on the text in lines 139-146, the reader should be able to understand the data presented in figure 6. However, the Figure legend is not at all clear. I am assuming that the orange columns are the control and the blue columns are the reactions that included a 5 minute incubation of the preparation with 0.055 mM pirimiphos-methyl solution.
Answer 1
We have added the missing legend to Figure 6. We have edited the caption of figure 6: «Residual activity of immobilized enzyme preparations based on starch gel vs. the time of incubation in the inhibitor solution ([pirimiphos-methyl] = 0.055 mM, [glyphosate] =10 mM). Each preparation disk contained 0.11 U of BChE and 0.2 mM DTNB. Green columns are the residual activity of soluble BChE after incubation in 0.055 mM pirimiphos-methyl solution».
Reviewer 4 Report
Here Authors reported a new method of producing butyrylcholinesterase (BChE) preparations stable. The BChE preparation is the enzyme co-immobilized with 0.2 M 5-5’- 9 dithiobis (2-nitrobenzoic acid) in starch or gelatin gel and the compare activity have been reported.
1. In figure 2 authors try to explore the activity of BChE in free and immobilize form, why authors took x axis maximum 110 mU only. could you used higher value also?? if not so why?
2. In figure 6 residual activity must be compare with free enzyme and also add the legend in this figure.
3. materials and methods part is very poor, less and there is no any references so authors need to improve it with suitable references. Some of the latest references are followings:
A. Development of nanobiocatalysts through the immobilization of Pseudomonas fluorescens lipase for applications in efficient kinetic resolution of racemic compounds
B. Xylanase from Acinetobacter pittii MASK 25 and developed magnetic cross-linked xylanase aggregate produce predominantly xylopentose and xylohexose from agro biomass
C. Production, immobilization and characterization of beta-glucosidase for application in cellulose degradation from a novel Aspergillus versicolor
D. Enzyme immobilization: an update
E. Immobilization of transaminase from Bacillus licheniformis on copper phosphate nanoflowers and its potential application in the kinetic resolution of RS-α-methyl benzyl amine
Author Response
Dear reviewer,
Thank you for your comments and suggestions. Here are our answers and corrections.
Remark 1
In figure 2 authors try to explore the activity of BChE in free and immobilize form, why authors took x axis maximum 110 mU only. could you used higher value also?? if not so why?
Answer 1
We did not check higher values of enzyme activity for a few reasons. First of all, when we use BChE with higher activity it results in lower sensitivity of enzyme preparation to the effect of toxic compounds. Moreover, the inclusion of more enzyme leads to an increase in the cost of the final product. The main idea of the study is to choose such enzyme activity in the preparation that lets us combine the high sensitivity of BChE to the effect of toxic compounds with the low cost of the preparation.
Remark 2
In figure 6 residual activity must be compare with free enzyme and also add the legend in this figure.
Answer 2
We have added the missing legend to Figure 6 and compared the residual activity of BChE preparation in the presence of pirimiphos-methyl and glyphosate with the soluble BChE.
Page 5, Lines 155-158. «For soluble BChE in the presence of 10 mM glyphosate solution, a complete loss of activity was observed regardless of the incubation time. The observed increase in the detection limit in this case is most likely due to the occurrence of serious diffusion and/or steric limitations in the polymer matrix of the starch gel.»
Remark 3
materials and methods part is very poor, less and there is no any references so authors need to improve it with suitable references. Some of the latest references are followings:
- Development of nanobiocatalysts through the immobilization of Pseudomonas fluorescens lipase for applications in efficient kinetic resolution of racemic compounds
- Xylanase from Acinetobacter pittii MASK 25 and developed magnetic cross-linked xylanase aggregate produce predominantly xylopentose and xylohexose from agro biomass
- Production, immobilization and characterization of beta-glucosidase for application in cellulose degradation from a novel Aspergillus versicolor
- Enzyme immobilization: an update
- Immobilization of transaminase from Bacillus licheniformis on copper phosphate nanoflowers and its potential application in the kinetic resolution of RS-α-methyl benzyl amine
Answer 3
Page 2, Lines 47-54. We have added information on materials suitable for enzyme immobilization to Introduction. We have added 8 new references:
«Immobilized enzymes are more stable and more resistant to environmental changes [11]. Various nanoparticles (copper nanoflowers [12], magnetic MnO2 [13]) or functionalized multi-walled carbon nanotubes [14] can be used as a carrier for enzyme immobilization. These methods make it possible to produce enzyme preparations that are stable when working in organic solvents and resistant to high temperatures, providing the possibility of numerous applications in enzymatic catalysis. However, polymers of various origins are more often used to immobilize cholinesterase [14-18].»
Other materials suitable for cholinesterase immobilization are described in Discussion.
Round 2
Reviewer 4 Report
All is okay now
