PONE-D-24-53783Gama Rays Mediated Improvement of Catalytic Efficiency and Thermostability of Glucoamylase by Replacing Active Site Leucine to Isoleucene from Super Koji (Aspergillus oryzae)PLOS ONE

Dear Dr. Ali,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 30 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Habibullah Nadeem

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript.

3. We note that your Data Availability Statement is currently as follows: “All relevant data are within the manuscript and in Supporting Information files.”

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.   

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

7. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitled "Gama Rays Mediated Improvement of Catalytic Efficiency and Thermostability of Glucoamylase by Replacing Active Site Leucine to Isoleucine from Super Koji (Aspergillus oryzae)” presents results of (random) gamma-ray induced mutation of the A. oryzae glucoamylase, which results in one positive point mutation on the enzyme that shows that the single point mutation L103I provides improved thermostability of the enzyme and improved kcat. The manuscript is well fitted to the journal scope.

So, I recommend that the manuscript to be accepted for publication in PLOS ONE. The data could be of high interest for scientific community working in the field. However, before publication the author should addressed the following minor changes / points;

INTRODUCTION

The introduction part clearly summarizes the current state of the topic and addresses the limitations of current knowledge in this field with clear and appropriate research question. However, the following minor changes need to addressed by authors;

Line 44: Comma missing after ‘However’.

Line 44-45: Replace the sentence ‘However their major short coming of low catalytic activity lead to consume high energy and restrictions in starch processing and fermentation’ with ‘However, their lower catalytic activity leads to consume high energy and restrictions in starch processing and fermentation’.

Line 61-63: Add reference (s) for the sentence ‘Random mutagenesis induced by γ-rays may contribute in enhancement of industrial enzymes production; hence γ-ray mediated mutagenesis of A. niger made the mutant GA highly efficient in substrate hydrolysis’.

Line 65: Comma missing after ‘Previously’.

Line 71: Remove the ‘data not shown’.

MATERIALS AND METHODS

The study design and methods are appropriate for the research question with enough detail for each experiment. However, the following minor changes need to addressed by authors;

Line 110: Add the paragraph under ‘In silico sequence analysis’ to heading ‘Sequencing and in-silico point mutation identification in GA gene’.

Line 113-112: Add full name for first time to ‘EMBOSS, EMBL-EBI and NCBI’.

Line 139: Remove ‘s’ from GA.

RESULTS

The authors presented results clearly and accurately and all the relevant data been included as well as the data described in the text consistent with the data in the figures and tables. However, the following minor changes need to addressed by authors;

Line 205-206: Replace the sentence ‘DNA Extraction and PCR amplification of A. oryzae Mutant M-60(5) and parent strain indicated the size of genomic DNA and cDNA of GA genes as 2,039 bp and 2,241 bp, respectively’ with ‘The sequenced data analysis revealed the size of genomic DNA and cDNA of GA genes as 2,039 bp and 2,241 bp, respectively’.

Line 213: Incorrect abbreviations for A. Awamori (AA), A. nigar (AN), A. oryzae (AO). Write the correct abbreviations.

Line 255: Replace the word ‘consider’ with ‘considered’.

Line 267: Replace the word ‘consider’ with ‘considered’.

DISCUSSION

The authors logically explained the findings and compared the findings with current findings in the research field. However, the following minor changes need to addressed by authors;

Line 351: Replace the word ‘may’ with ‘might’.

Line 352-354: Replace the sentence ‘In the current study, it is supposed that the improvement in catalytic efficiency of GAs in mutant was linked with the change in a Leu203 to Ile203 at active site of GA Mutant M-60(5)’ with ‘we believe that improvement in catalytic efficiency of GAs in mutant was linked with the change in a Leu203 to Ile203 at active site of GA Mutant M-60(5)’.

Line 391-392: The sentence ‘that shows decline in optimal activity above 50 °C, pH 5.5’ should be written as ‘which showed a decline in optimal activity above 50 °C, pH 5.5’.

Line 419-421: Replace the sentence ‘Similarly, previously we had reported about the improvement in kinetic properties due to γ-rays mutagenesis on thermodynamics of starch hydrolysis’ with ‘Similarly, we already reported γ-rays mutagenesis-based improvement in kinetic properties on thermodynamics of starch hydrolysis’.

CONCLUSION

The authors logically concluded the whole research work but needs to be addressed the following minor changes.

Line 441: Remove the word ‘drastic’.

Line 446: Replace the word ‘may’ with ‘might’.

REFERENCES

The references are correct and missing no key reference, as per my evaluation.

Reviewer #2: The authors mutated Aspergillus oryzae, also known as Super Koji, by gama rays for enhancing catalytic efficiency and thermostability of glucoamylase by replacing active site leucine to isoleucene. The point mutation altered the conformation of the glucoamylase active site that contributed to improve the functional energy, resulted the stabilization of transition state which made it thermostable and highly efficient in starch hydrolysis. I would like to recommend the research work to be published in PLOS ONE. However, the following minor suggestions should be addressed by authors before publication.

L 43. Use full name for the first time e.g. ‘R. delemar’.

L 47. Replace word ‘their’ with ‘its’.

L 49. The sentence ‘In recent era, strain improvement by site directed and random mutagenesis of fungi is an interesting research topic’ should be replaced with ‘In recent era, strain improvement by site directed and random mutagenesis of fungi is being used widely for industrial applications’.

L 59. Use short name for ‘Aspergillus oryzae’. Same consistency must be kept throughout the manuscript.

L 65-67. Support your claim by providing reference (if published) for ‘Previously we developed novel Aspergillus oryzae mutants by γ-rays’ mutagenesis for the enhanced production of thermostable α-amylases, which were also highly specific in the α-amylase production’.

L 72. Change the sentence ‘Novelty of current report is as it for the first time explains about effect of point mutation’ with ‘The current study reports for the first time effect of point mutation’.

L 78. The word ‘Maintenance’ should be started with small letter.

L 95. Ether use ‘hours’ or ‘hrs’. Correct the same in entire manuscript.

L 98. Mention the pore size of muslin cloth.

L 99. Which instrument was used for lyophilization? Provide model and manufacturer name etc.

L 135. Add brief procedure detail to Protein assay.

L 211. Add comma after ‘nucleotide position 703’.

L 289. Correct the ‘pointed out’ to ‘pointed’.

L 289. Correct the ‘does not’ to ‘did not’.

L 344. Add comma after ‘nucleotide position 703’.

L 360. Correct the ‘Similar’ to ‘similar’.

L 360. The word ‘Parent’ should be started with small letter.

L 380. Add comma after ‘Similar findings regarding proton donating residues were presented by [15]’.

L 386-388. This sentence is ambiguous and needs to be re-phrased ‘Dehareng and Dive [47] evaluated the ionization energies (IE) as a function of the conformation for α-L-amino acids and three to five conformations for the arginine, lysine, isoleucine, tyrosine and tryptophan were optimized’.

L 390. Add comma after ‘with optimum temp range of 45–55 °C (~80% of optimal activity)’.

L 396-400. This sentence is ambiguous, long and needs to be re-phrased ‘The Km of mutant M-60(5) GA for soluble starch hydrolysis at 50 °C, pH 5.0 was lower (0.06 mg ml-1) than the parental GA having 0.17 mg ml-1. Which is highly efficient than the already reported km values of naïve and modified enzyme i.e 0.34 and 0.29 respectively and 2.1mg/ml of glucoamylase GA-LZ2’.

L 431. Add comma after ‘strain was 274 kJ mol-1’.

L 432. Add comma after ‘mutant was as 237.02 kJ mol-1’.

L 434. Add comma after ‘mutant was 407.90 J mol-1K-1’.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Comments to Authors_PONE-D-24-53783.docx

Gama Rays Mediated Improvement of Catalytic Efficiency and Thermostability of Glucoamylase by Replacing Active Site Leucine to Isoleucene from Super Koji (Aspergillus oryzae)

PONE-D-24-53783R1

Dear Dr. Ali,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Habibullah Nadeem

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All the suggested changes are incorporated and I recommend the manuscript for publication in PLOS ONE.

Reviewer #2: Congratulations to all the authors for conducting outstanding research on the gamma-ray mediated improvement of catalytic efficiency and thermostability of glucoamylase by substituting the active site leucine with isoleucine from Super Koji (Aspergillus oryzae). You have thoroughly addressed all the comments, and I believe publishing your work in PLOS ONE will allow it to reach a wide audience, benefiting many in the field.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: Yes:  Muhammad Tayyab Akhtar

**********