Review Reports

Round 1

Reviewer 1 Report

Ln 52-53. "Plants can be used in the production but the evidence of extraction of plant’s glutaminases is not very easy due to less feasible approaches". Unclear, please, rewrite

Ln 130. The part should be named : Identification of the isolate.

Ln. 152 Table 3 is incorrect. Should be Identified as Achromobacter xylosoxidans strain RSHG1,Sequence homology with Achromobacter xylosoxidans

 According to the Fig. 11, The maximum velocity of purified L-glutaminase is 444 U/mg/ml. If this value is correct then the title of Y axes in figures 3-8 are incorrect. Because according to these titles the specific L-glutaminase activity of cell lysate is 3000-5000 U/mg/ml. It is impossible. The total U of activity is more appropriate in Figures 3-8. Please, correct.

According to the L-glutaminase assay (Ln 381-392) the 10 mM of substrate L-glutamine are added to the assay solution. At this substrate concentration, the specific activity should be >100 U/mg/ml according to Fig.11. However, the shown in part 2.4.2 specific activity values are 40-60 U/mg/ml in the standard assay.  These values are contrary to the values from Figure 11. Please, clarify and correct.

Author Response

Response to the Comments:

Reviewer 1

 

1. Ln 52-53. "Plants can be used in the production but the evidence of extraction of plant’s glutaminases is not very easy due to less feasible approaches". Unclear, please, rewrite

Response: Line 52-53 rewritten as “Plants are also involved in L-glutaminase production. The extraction from plants is not well studied..”

2. Ln 130. The part should be named: Identification of the isolate.

Response: Ln 130 changed  now written“. Identification of the Bacterial isolate”

3. Should be Identifiedas Achromobacter xylosoxidans strain RSHG1, Sequence homology with Achromobacter xylosoxidans

Response: we have changed in the table G1identified as Achromobacter xylosoxidans strain RSHG1, and in the next column Sequence homology with Achromobacter xylosoxidans.

4. According to Fig. 11, The maximum velocity of purified L-glutaminase is 444 U/mg/ml. If this value is correct then the title of Y axes in figures 3-8 are incorrect. Because according to these titles the specific L-glutaminase activity of cell lysate is 3000-5000 U/mg/ml. It is impossible. The total U of activity is more appropriate in Figures 3-8. Please, correct.

Response: To address this issue we have calculated all units again from raw data (from all absorbance values of enzyme assay and protein assay). And made all the graphs again.

5. According to the L-glutaminase assay (Ln 381-392), 10 mM of substrate L-glutamine is added to the assay solution. At this substrate concentration, the specific activity should be >100 U/mg/ml according to Fig.11. However, the shown in part 2.4.2 specific activity values are 40-60 U/mg/ml in the standard assay.  These values are contrary to the values from Figure 11. Please, clarify and correct.

Response: mistakenly IU/mL/min values were divided by total protein. We recalculated whole data before and after purification from raw data.

Reviewer 2 Report

The authors of the manuscript " Characterization of a New L-glutaminase produced by Achromobacter xylosoxidans G1, isolated from an Expired hydrolyzed L-glutamine sample" presents the study of new L-glutaminase and its characterization. The article is written in a clear and understandable way. The selection of literature is appropriate and logical, there are no unnecessary citations.

However, the article needs some improvements from current version.

1. The English need improvement, since there are some grammatical and syntax errors in the manuscript (For example, "on 30 °C", "sources" instead of source, "an" instead of "a" in line 109
2. There are a few typing mistakes as well, and authors are advised to carefully proof-read the text (For example space between number and unit, subscript for the chemical formula, L-glutaminases, lower case for the G or upper case, please be consistent throughout the manuscript, typo in figure 7, typo in line 218 and 229, double spaced between Achromobacter xylosoxidans and Table 3 in line 135, and many more)
3. Check the abbreviations throughout the manuscript and introduce the abbreviation when the full word appears the first time in the text and then use only the abbreviation (For example, Km and Vmax
4. I realized the author mentioned the number of replicates in the end. However, it’s good to have n=3 in the context.
5. Figure 9 and 11: consider labeling the curves.
6. Figure 10, 12, 13, 14, and 15: consider changing the color. It’s very difficult to read.

Author Response

Point by point reply to Comments of Reviewer 2:

1. The English need improvement, since there are some grammatical and syntax errors in the manuscript (For example, "on 30 °C", "sources" instead of source, "an" instead of "a" in line 109

Response: English corrections are done in consultation with English experts.

Response: we have tried to remove all grammatical and syntax errors

2. There are a few typing mistakes as well, and authors are advised to carefully proof-read the text (For example space between number and unit, subscript for the chemical formula, L-glutaminases, lower case for the G or upper case, please be consistent throughout the manuscript, typo in figure 7, typo in line 218 and 229, double spaced between Achromobacter xylosoxidansand Table 3 in line 135, and many more)

Response: We have changed L-glutaminases, lower case for the G or upper case, and corrected typo mistakes in figure 7, line, 218, 229, and double space of table 3.

3. Check the abbreviations throughout the manuscript and introduce the abbreviation when the full word appears the first time in the text and then use only the abbreviation (For example, Km and Vmax

Response: The data has been rewritten as suggested.

4. I realized the author mentioned the number of replicates in the end. However, it’s good to have n=3 in the context.

Response: Done accordingly

5. Figures 9 and 11: consider labeling the curves.

Response: Labelled the curves in Figure 9 and 11

6. Figures 10, 12, 13, 14, and 15: consider changing the color. It’s very difficult to read.

Response: corrected according to the suggestions. Colours of the graphs have been changed.

 

Round 2

Reviewer 1 Report

Now the article sounds much better, the authors improved the manuscript significantly. However, minor corrections should be done.

Minor comments

1. Please, change U/mg/min for U/mg everywhere, because  1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per minute under the specified conditions of the assay method.
2. In part 4.7 "phosphate buffer" should be added after 0.5M
3. Please, add the amino acid sequence of the new L-glutaminase or its number in the manuscript.

Author Response

All the corrections are also highlighted. We will highly appreciate and request to please consider the submission for publication in your journal Catalyst.

 

Response to the Comments:

Reviewer 1

 

1. Please, change U/mg/min for U/mg everywhere, because  1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of one micromole of substrate per minute under the specified conditions of the assay method.

Response: We have changed U/mg/min by U/mg in all figures and text.

2. In part 4.7 "phosphate buffer" should be added after 0.5M

Response: We have added phosphate buffer after 0.5M in 4.7.

1. Please, add the amino acid sequence of the new L-glutaminase or its number in the manuscript.

 Response: We have not done protein sequencing or MALDI-TOF. So at this stage, we cannot add a sequence or number of amino acids.

We look forward to hearing about the outcomes of the review process.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript in question is a report on the partial purification of a glutaminase from a bacterium that was isolated from old glutamine samples. Given the wealth of recent literature on glutaminases from various microorganisms, this work does not quite meet the required criteria regarding novelty of approach and importance of results. The discussion consists merely of a list of data on other, already known glutaminases without making clear what the role or the advantages or disadvantages of the new enzyme might be. There is no example for an application that could illustrate what the new enzyme may be good for, or better than others that are already established.

Reviewer 2 Report

The article is devoted to the preliminary characterization of a new L-glutaminase from the novel strain of Achromobacter xylosoxidans. A new enzyme is always interesting, it is like a discovery.  However, the peculiar features of a new L-glutaminase (in comparison with the already described in the literature L- glutaminases) remained unclear. Maybe, this is because of bad English.  And definitely, numerous points should be clarified:

Major comments:

1. Please, clarify, what is “an old glutamine sample”? Describe the sample in details….
2. First, The new L-glutaminase should be identified. Give it the name and use it in the text. The used through the text ” L-glutaminase by Achromobacter xylosoxidans G1”, “G1 L-glutaminase” are awkward for understanding. Second, The name of the organism should be presented fully only once – the first time, then only in short form: xylosoxidans G1. Please, correct the whole text.
3. It is incorrect to calculate the Michaelis-Menten kinetic parameters using the partially purified enzyme. These parameters characterize the reaction, catalyzed by the purified enzyme. And these parameters should be calculated from concentration dependence using nonlinear regression analysis. This experiment should be repeated correctly with a purified enzyme.
4. 1 unit of Km (Michaelis constant) is 1 M (or mM etc.). The authors refer to [51], where the correct units are used.
5. To satisfy the audience of the Catalyst the biochemical units of the enzyme activity should be recalculated for U/mg/min. Unit IU/ml is preferable for pharmaceutical analysis and is not used in enzymology.
6. 2.1.1. primary screening and 2.1.2. secondary screening should be combined into one section. The name of the organism should be removed from this section. The organism gets the name only after 16S rRNA analysis. In this section, it should be identified as isolate…
7. Ln 261. The homology with Achromobacter xylosoxidans strain GN050 is written to be 92,02%. However, in conclusions is written: “revealed G1 strain 99 % homology to Achromobacter xylosoxidans strain GN050”. Where is the true value? If it is 99%, can we consider G1 as a new organism? Was G1 deposited to the microorganism database?
8. I did not notice the difference between the earlier described L-glutaminases and a new one described in the article. Please, itemize them in the conclusions.
9. English needs serious correction.
10. In the Introduction the reasons for searching a new (one more) L-glutaminase should be described.
11. I suggest another title of the article: Characterization of a new L-glutaminase from the old glutamine sample.

 

Minor comments

1. In Material and Method section, the concentrations of phosphate buffers should be added everywhere.
2. Ln 354-355 . Did you check the pH after the addition of 0.5 ml of 40 mM L-glutamine solution to the 0.5 ml of phosphate buffer of unknown concentration? Please, clarify this point.
3. The information from the Tables 2 and 3 should be presented in text format.
4. Ln257-Ln266. The text of this section is unclear and should be rewritten.
5. Ln199-200. The incubation conditions should be added in the text and in the Figure 10 Caption.
6. The name of Table 5 is unclear. It is about enzyme purification. Should be rewritten.
7. Section 4.5 of Materials and methods. Optimization of culture conditions for L-Glutaminase production (Ln 392) or in section 2.3.1. Optimization of parameters for glutaminase production The experimental conditions should be added.

Effect of pH – Describe buffers and add the temperature of the experiment

Effect of Temperature – What is the рН value

Effect of Carbon source. Effect of Nitrogen source, Effect of Inducers – pH and temperature of the experiments should be added.

8. 2: Characterization for kinetic parameters…. Should be Characterization of kinetic parameters
9. Ln 12-14: After L-glutaminase screening, culture was purified and observed of morphological characters and biochemical testing of bacterial strains along with 16S rDNA sequence homology testing.. Would be better: the unknown organism was purified and described morphologically as well biochemically characterized, including 16S rDNA sequence homology testing
10. 33 «without being expressing themselves in the last product»–Please, rewrite the sentence..
11. 48 «Due to complex organization, animals are not well known in the field of enzymatic isolation from their tissues» Unclear, please, rewrite the sentence
12. Ln 55-56 “Requests for chemicals utilized in ventures are expanding step by step to improve the procedures” - unclear, please, rewrite…
13. Ln 60 – “as a biosensor for deciding glutamine and glutamate”- unclear, please, rewrite…
14. Ln155-156 The sentence is unclear and should be rewritten.
15. Ln 173 Unclear Figure 4 caption. Please, Rewrite it.