Fabrication and Characterization of Dextranase Nano-Entrapped Enzymes in Polymeric Particles Using a Novel Ultrasonication–Microwave Approach

Round 1

Reviewer 1 Report

Lot of abbreviations are used in this paper , it would be better if authors creating a separate table for this

There is no ethical approval statement

There is no recommendations

The most used methodology without references

There is no reference for the statistical analysis

Some journal names were written abbreviated , while others were not ---why ??? same style should be ---apply for all

Some cited references need to be more update

The most used references contained more than 6 authors ---why ???? should be 6 at the maximum plus etal with the last ones (example :Ref(7,8,22,25----etc) apply for all

Huge number of references (36) ???

There is no highlights for the manuscript

The abstract---needs to be more modified and contained background, methods , results and conclusion

LN/26----biopolymer gel---detailed that

The author (s) did not mention anything about the applications of nanotechnology in the different fields of life

LN/39-40---add purification , molecular mass , degradation and clinical applications to the keywords

LN/279---add reference

LN/287--- why at PH 5 and T 40 ????

LN/296---Ionotropic gelation -----give more details

LN/344----there is no reference for SEM and why not TEM ???

Write like Table (1):---- apply for all also for the figures

Discussion should be based upon discussing the obtained results with those of the previous investigators results

LN/274 ----Materials and methods should be written after introduction and before the results and discussion (RESONABLE)

LN/192-193----what are the magnification power used ????

LN/175-----write the magnifying power (X)

Why all figures not colored for better resolution ????

LN/177---results should be dissociated from the discussion

Discussion should be more concise and based upon discussing the obtained results with those of the previous investigators results

What are the applications of enzymes immobilization in our life ???

What about the drawbacks of using microwaves irradiations ???

Are there any possible chance to occur mutations to the enzyme protein and so cancer probabilities increased ???

Author Response

Reviewer 1:

Lot of abbreviations are used in this paper, it would be better if authors creating a separate table for this.

A: The number of abbreviations was reduced in the manuscript according to the reviewer’s advice.

There is no ethical approval statement.

A: Ethical approval statement was provided.

There is no recommendations.

A: The recommendation was included in the conclusions part.

The most used methodology without references

A: References were added to methods as per the reviewer’s advice.

Some journal names were written abbreviated , while others were not ---why ??? same style should be ---apply for all

A: Modified according to the reviewer’s advice.

Some cited references need to be more update

A: Cited references were updated according to the reviewer’s advice.

The most used references contained more than 6 authors ---why ???? should be 6 at the maximum plus etal with the last ones (example: Ref (7,8,22,25-etc) apply for all

A: Modified according to the reviewer’s advice.

Huge number of references (36)???

A: This paper discussed the synergetic effects of two approaches (ultrasonication and microwave) which needs more citations to enhance the paper and improve the discussion through the manuscript.

There is no highlights for the manuscript

A: highlights was provided in the separated file. Please see the attached.

The abstract---needs to be more modified and contained background, methods, results and conclusion

A: Modified according to the reviewer’s advice.

LN/26----biopolymer gel---detailed that

A: More detail was provided according to the reviewer’s advice.

The author (s) did not mention anything about the applications of nanotechnology in the different fields of life

A: this point was added to introduction according to the reviewer’s advice.

LN/39-40---add purification , molecular mass , degradation and clinical applications to the keywords

A: Modified according to the reviewer’s advice.

LN/279---add reference

A: Reference was added.

LN/287--- why at PH 5 and T 40?

A: We are following the previous method described by [14–16]. The optimum temperate was 40 C and pH was 5 which give the maximum enzyme activity and reaction rate.

LN/296---Ionotropic gelation -----give more details

A: More details was provided according to the reviewer’s advice.

LN/344----there is no reference for SEM and why not TEM ???

A: Reference was added. TEM is not available in our lab only have SEM which could give same results.

Write like Table (1):---- apply for all also for the figures

A: Modified according to the reviewer’s advice.

Discussion should be based upon discussing the obtained results with those of the previous investigators results

A: Modified according to the reviewer’s advice. More discussion and citation form the previous investigators results were added in this part.

LN/274 ----Materials and methods should be written after introduction and before the results and discussion (RESONABLE)

A: Thank you for your comment, we are following the authors guidelines of the Journal.

LN/192-193----what are the magnification power used ????

A: 160X was used in this study.

LN/175-----write the magnifying power (X)

A: Done according to the reviewer’s advice.

Why all figures not colored for better resolution?

A: We modify some figures to be colored.

LN/177---results should be dissociated from the discussion. Discussion should be more concise and based upon discussing the obtained results with those of the previous investigators results

A: Modified according to the reviewer’s advice. More discussion and citation form the previous investigators results were added in this part.

What are the applications of enzymes immobilization in our life ???

During the past several years, the immobilized enzymes have a wide range of applications including:

1. Applications in food industry such as sugar manufacturing, fish, beverage industries, and dairy products.
2. Biosensors for metabolite control and in situ measurements of environmental pollutants.
3. Immobilized enzymes also find significant application in drug metabolism, biodiesel and antibiotic production, and bioremediation.
4. Wastewater management.
5.  

The widespread usage of immobilized enzymes is largely due to the fact that they are cheaper, environment friendly, and much easier to use when compared to equivalent technologies.

Are there any possible chance to occur mutations to the enzyme protein and so cancer probabilities increased ???

A: Protein structures have a certain flexibility which allows them to slightly modulate their conformations to maximize the transition state stabilization in response to the steric perturbations induced by mutations. The synergetic effects of ultrasonication and microwave may lead to mutations of  the enzyme protein which will be investigated in separated study.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript proposed in the title of ‘Fabrication and characterization of dextranase nano-entrapped enzyme onto polymeric particles using a novel ultrasonication- microwave approach’. The author provided well organized scientific article. However, there are several questions, which authors should make the explanations.

 

1.              Introduction: please provide an explanation, what is the application of dextrose immobilization or why select dextrose to immobilize on alginate bead.

2.              Please clarify the procedure, how to use UMS for enzyme immobilization. Did the author use simultaneous US and MS or the sequence of US and then MS to immobilize enzymes as explained in lines 83 to 84 and lines 114 to 115.

3.              The author mentioned that when the time length was more than 30 min, the LE and IY were decreased because of the sonicating prolonged period of time. However, the author only used 15 min for US as indicated in line 114. Please provide more explanation.

4.              The control immobilization enzyme should be present in FTIR and SEM results and provided the comparison.

5.              How can the author confirm that it is nano-entrapped enzyme?

6.              Table 2 presents the t1/2, therefore, the Equation to calculate t1/2 should be given.

7.              Figure 6 should provide the R2 for all linear plots.

8.              What is the temperature range for investigation the Arrhenius plot?

9.              Please provide the value of t1/2 at 60C to support this statement “Additionally, compared to IE prepared without ultrasound treatment, IE prepared by UMS displayed a lower t1/2 value at 60°C.

10.           Why was the stability of this immobilized enzyme less than that of dextranase immobilized on chitosan?

 

11.           There was no Figure 8 as indicated in line 300.

Author Response

Reviewer 2:

This manuscript proposed in the title of ‘Fabrication and characterization of dextranase nano-entrapped enzyme onto polymeric particles using a novel ultrasonication- microwave approach’. The author provided well organized scientific article. However, there are several questions, which authors should make the explanations.

1. Introduction: please provide an explanation, what is the application of dextrose immobilization or why select dextrose to immobilize on alginate bead.

A: More information about provided in the introduction, according to the reviewer’s advice.

2. Please clarify the procedure, how to use UMS for enzyme immobilization. Did the author use simultaneous US and MS or the sequence of US and then MS to immobilize enzymes as explained in lines 83 to 84 and lines 114 to 115.

A: The combination of MS and US was used sequentially. More information was added in the manuscript according to the reviewer’s advice.

3. The author mentioned that when the time length was more than 30 min, the LE and IY were decreased because of the sonicating prolonged period of time. However, the author only used 15 min for US as indicated in line 114. Please provide more explanation.

A: For UMS treatment, a constant US of 40 W at 25 kHz for 15 min was used combined with MS of 60 W at various incubation (0-40 min). The results were compared with the immobilized enzymes prepared by MS, US and without UMS treatments, separately,  at same incubation (0-40 min),  as be seen in figure (2 A & B).

4. The control immobilization enzyme should be present in FTIR and SEM results and provided the comparison.

A: We have used pure Ca-alginate as a control to confirm the immobilization process and provided the comparison.

5. How can the author confirm that it is nano-entrapped enzyme?

A: The nano-entrapped of the enzyme into Ca-alginate gel was confirmed by comparing the FT-IR spectrum of the pure alginate with that for IE. The spectrum of the IEs produced by the UMS, MS, and US revealed a strong peak at a wavelength of 1650 cm^-1 that corresponded to the C=O bond of the amino acid, which is a characteristic peak of the second helix of the dextranase enzyme and correlates to the amide I and amide II groups.

6. Table 2 presents the t1/2, therefore, the Equation to calculate t1/2 should be given.

A: Equation was provided according to the reviewer’s advice.

7. Figure 6 should provide the R2 for all linear plots.

A: R2 was provided in table according to the reviewer’s advice.

8. What is the temperature range for investigation the Arrhenius plot?

A: The temperature range for investigation the Arrhenius plot was 327.8-307.5K as shown in figure (6C). It was calculated as 3.05-3.25 k^-1 from 1/T_x 10³ (k^-1).

9. Please provide the value of t1/2 at 60C to support this statement “Additionally, compared to IE prepared without ultrasound treatment, IE prepared by UMS displayed a lower t1/2 value at 60°C.

A: t1/2 was calculated only at 50°C. This point was corrected in the manuscript.

10. Why was the stability of this immobilized enzyme less than that of dextranase immobilized on chitosan?

A: Both are biopolymer carbohydrate commonly used in immobilization process and drug release Alginate is composed of alternating blocks of 1–4 linked α-L-guluronic and β-D-mannuronic acid residues, and chitosan is consisting of β (1–4)-linked 2-amino-2-deoxy-D-glucose (D-glucosamine) and 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine) units. the molecular weight of chitosan also evidently influenced the immobilization efficiency. 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

This paper entitled ` Fabrication and characterization of dextranase nano-entrapped enzyme onto polymeric particles using a novel ultrasonication-microwave approach` investigates the improved method for immobilization of dextranase produced by Chaetomium erraticum onto Ca-alginate nanoparticles for further utilization for dextran hydrolysis. Even though presented results indicate the importance of investigation in this field, presentation of obtained results is quite unclear and thorough revision of the paper should be undertaken before accepting the manuscript. Therefore, I suggest a major revision of the paper.

Specific comments:

- Firstly, obtained Ca-particles should be more thoroughly characterized (primarily, when stating that nanoparticles were used, their nano-scale size should be demonstrated). Also, entrapment implies that enzyme is trapped inside the particles, and in this paper `entrapment onto` phrase is frequently used – authors should explain this. Regarding this, what was the authors` reasoning for using nano-entrapped enzyme for hydrolysis of macromolecules, such as dextran?

- Material and method section should be completely re-written. It should be more informative, and also it seems that large parts of methods are omitted. For example: Section `3.10. Kinetics and thermodynamic studies of IE` (page 11) it states `Using a constant enzyme concentration and varying amounts of soluble dextran (0.1– 10%) at 50°C, the enzymatic kinetic constants for free and IE produced via UMS treatment were determined. The relationship between substrate concentration and the rate of enzyme reaction was investigated using first-order kinetics.`

This is not true!!! The relationship between substrate concentration and the rate of enzyme reaction follows Michaelis-Menten kinetics, and the linearization of this equation was used for determination of Km, Vm, and kcat (in results section). The mentioned first first-order kinetics was used for thermodynamics calculations.

Also, methods should be informative enough so that anyone can reproduce the same experiments and the term ` a constant enzyme concentration` does not say anything (same loaded protein concentration, same immobilized activity concentration?) and it is rather important for comparison of obtained results.

This is just illustration on one example, the authors are advised to completely rewrite this section and give information about quantities, volumes, concentrations…

- Results and discussion

Discussion is lacking throughout the whole manuscript, literature review should be introduced within the manuscript.

Results should be more carefully and more clearly written. Most of the figures and their captions are unclear and misleading. Within the Figure 1. caption implies that both LE and IY of the EI (it should be IE) onto Ca-alginate beads produced with and without UMS treatments are presented, and there are two lines red (MS) and black (UMS). What are they showing??? LE or IY??? It is impossible that they can show both!!! The same comment is applicable for Figure 2, and Figure 6 caption is inaccurate.

Additionally, when calculating LE, obtained particles should be rinsed for suspended enzyme, and then LE should be calculated.

Author Response

Reviewer 3:

This paper entitled ` Fabrication and characterization of dextranase nano-entrapped enzyme onto polymeric particles using a novel ultrasonication-microwave approach` investigates the improved method for immobilization of dextranase produced by Chaetomium erraticum onto Ca-alginate nanoparticles for further utilization for dextran hydrolysis. Even though presented results indicate the importance of investigation in this field, presentation of obtained results is quite unclear and thorough revision of the paper should be undertaken before accepting the manuscript. Therefore, I suggest a major revision of the paper.

Specific comments:

- Firstly, obtained Ca-particles should be more thoroughly characterized (primarily, when stating that nanoparticles were used, their nano-scale size should be demonstrated). Also, entrapment implies that enzyme is trapped inside the particles, and in this paper `entrapment onto` phrase is frequently used – authors should explain this. Regarding this, what was the authors` reasoning for using nano-entrapped enzyme for hydrolysis of macromolecules, such as dextran?

Entrapment is the physical enclosure of enzymes in a small space. Matrix entrapment and membrane entrapment, are the two major methods of entrapment. In this study we used Matrices entrapment which was used for enzyme immobilization in biopolymeric materials such as Ca-alginate. In addition, we have used nano-entrapped enzyme for hydrolysis of macromolecules for  many reasons:

1. To improve the efficiency of enzyme utilization in biotechnological processes.
2. Immobilization makes it simple to separate the enzyme from the product, which reduces or completely eliminates protein contamination of the product
3. To increase the recyclability.
4. To reduce the cost of the enzymatic hydrolysis of dextran in several industrial area including sugar industry and synthesis of isomaltooligosaccharides, which have been shown to exhibit prebiotic effects.

- Material and method section should be completely re-written. It should be more informative, and also it seems that large parts of methods are omitted. For example: Section `3.10. Kinetics and thermodynamic studies of IE` (page 11) it states `Using a constant enzyme concentration and varying amounts of soluble dextran (0.1– 10%) at 50°C, the enzymatic kinetic constants for free and IE produced via UMS treatment were determined. The relationship between substrate concentration and the rate of enzyme reaction was investigated using first-order kinetics.`This is not true!!! The relationship between substrate concentration and the rate of enzyme reaction follows Michaelis-Menten kinetics, and the linearization of this equation was used for determination of Km, Vm, and kcat (in results section). The mentioned first first-order kinetics was used for thermodynamics calculations.

A: This point was modified in the manuscript according to the reviewer’s advice.

 

Also, methods should be informative enough so that anyone can reproduce the same experiments and the term ` a constant enzyme concentration` does not say anything (same loaded protein concentration, same immobilized activity concentration?) and it is rather important for comparison of obtained results.This is just illustration on one example, the authors are advised to completely rewrite this section and give information about quantities, volumes, concentrations…

A: Material and methods section was rewritten and more information was provided according to the reviewer’s advice.

- Results and discussion

Discussion is lacking throughout the whole manuscript, literature review should be introduced within the manuscript.Results should be more carefully and more clearly written. Most of the figures and their captions are unclear and misleading. Within the Figure 1. caption implies that both LE and IY of the EI (it should be IE) onto Ca-alginate beads produced with and without UMS treatments are presented, and there are two lines red (MS) and black (UMS). What are they showing??? LE or IY??? It is impossible that they can show both!!! The same comment is applicable for Figure 2, and Figure 6 caption is inaccurate.

A: The effects of UMS treatment on LE and IY showed in Figures 1and 2, were provided in separated curves according to the reviewer’s advice. Figure 6 caption was modified.

 

Author Response File: Author Response.pdf

Reviewer 4 Report

The manuscript of Mohanad Bashari et al., entitled “Fabrication and characterization of dextranase nano-entrapped enzyme onto polymeric particles using a novel ultrasonication-microwave approach” is in general interesting, the results and discussions are presented in an appropriate manner, and the article is in general well written, but some studies are missing, and some aspects need to be improved /clarified. The issues that need to be addressed during the revision are listed below:

 

1. The introduction part offers too little information and should be improved.

 

2. The article has too many abbreviations and is difficult to read and follow. This should be considered and should be modified in the manuscript.

 

 

3. Figure 1 must be redone because it is not clear which data are those on the oY axis.

 

4. The quality of the IR spectra (Figure 3) and SEM images (Figure 5) must be improved.

 

 

5. A study of the effect of pH and temperature on the activity of immobilized dextranase is missing and must be done!

 

6. In the Materials and Methods section (page 9), more details on the HPLC analysis conditions, preparation of IE by ultrasonication treatment, and recycled hydrolysis of dextran must be given.

 

7. The calculation relationship of the specific activity must be indicated near Eq (2) that was used to calculate IY.

Author Response

Reviewer 4:

The manuscript of Mohanad Bashari et al., entitled “Fabrication and characterization of dextranase nano-entrapped enzyme onto polymeric particles using a novel ultrasonication-microwave approach” is in general interesting, the results and discussions are presented in an appropriate manner, and the article is in general well written, but some studies are missing, and some aspects need to be improved /clarified. The issues that need to be addressed during the revision are listed below:

 

1. The introduction part offers too little information and should be improved.

A: The introduction was improved according to the reviewer’s advice.

2. The article has too many abbreviations and is difficult to read and follow. This should be considered and should be modified in the manuscript.

A: Some abbreviations was deleted according to the reviewer’s advice.

3. Figure 1 must be redone because it is not clear which data are those on the oY axis.

A: Figure 1. was modified according to the reviewer’s advice.

4. The quality of the IR spectra (Figure 3) and SEM images (Figure 5) must be improved.

 A: Figure 3. was modified according to the reviewer’s advice.

5. A study of the effect of pH and temperature on the activity of immobilized dextranase is missing and must be done!

The industrial application and optimization of this immobilized enzyme in the reduction of dextran processing problems during sugar production processes and synthesis of isomaltooligosaccharides, are under investigation by our research group in two different projects. The two papers which will be publish very soon discuss the optimization of industrial parameters of the immobilized enzyme including the effect of pH and temperature on the activity of immobilized dextranase produced by UMS treatment.

6. In the Materials and Methods section (page 9), more details on the HPLC analysis conditions, preparation of IE by ultrasonication treatment, and recycled hydrolysis of dextran must be given.

A: More details were provided according to the reviewer’s advice.

 

 

7. The calculation relationship of the specific activity must be indicated near Eq (2) that was used to calculate IY.

A: Modified according to the reviewer’s advice.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

The issues that need to be addressed during the revision are listed below:

 

1. The article still has too many abbreviations and is difficult to read and follow.

 

 

2. On the oX axis of Figure 1 correct Pwoer (W) with Power (W).

 

4. The quality of the IR spectra (Figure 3) must be improved.

 

5. In page 13, lines 447-448 it is stated “In comparison to IE prepared without UMS, IE prepared with UMS demonstrated higher thermal stability and reusability.“ On what ground was reached that conclusion? because the study of temperature stability is missing.

 

6. The calculation relationship of the specific activity is not indicated near Eq (2).