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PLOS One

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Enhanced Fibrinolytic Enzyme Production by Oidiodendron maius through Green Bioprocessing of Agro-Industrial residue

Reviewer’s Comments:

The manuscript presents an interesting study on improving fibrinolytic enzyme production using mutagenized strains of Oidiodendron maius, combined with low-cost agro-industrial substrates. The topic is relevant to biotechnology, enzyme engineering, and the development of eco-friendly bioprocesses for thrombolytic agents. The work includes extensive experimental data covering isolation, mutagenesis, fermentation optimization, purification, biochemical characterization, and kinetic/thermodynamic analysis.

While the study has clear novelty as particularly the use of O. maius mutants for enhanced fibrinolytic enzyme production. However, it requires improvement in scientific clarity, methodological detail, statistical rigor, language quality, and structural organization. The manuscript is far too lengthy and repetitive, and several data presentations need careful revision.

Overall, the manuscript shows potential but requires revision before publication.

1. Abstract section is too long and should be condensed.

2. Please include more recent citations in Introduction section as several citations are outdated.

3. The section on thrombotic diseases is disproportionately long relative to the focus of the study.

4. Provide details of instrumentation in materials and method section and also clarify buffer compositions.

5. Please report all biochemical tests briefly which were used for the confirmation of Oidiodendron maius.

6. What is the purpose of using wheat-bran? Why it has been used specifically? Elaborate its importance.

7. Why did the authors test the fungal capability to grow at 60 oC? Is it a thermophilic fungus?

8. What is the purpose of 40% and 60% sediments mentioned but not explained in the purification section?

9. Avoid repeating numerical values extensively in text and only mention in the tables or supplementary data.

10. In Discussion section, excessive citation of unrelated bacterial studies were observed, focus more on fungal systems.

11. The manuscript is excessively long and contains large sections of repeated explanations that could be condensed. The Discussion section repeats many Results with minimal interpretation.

12. Some references are duplicated.

13. What limitations exist in current fibrinolytic enzyme sources.

14. The mutagenesis procedures require clearer explanation of exposure conditions (UV wavelength inconsistently stated as 240 nm; standard UV mutagenesis uses 254 nm).

15. Authors should explicitly state the number of replicates per experiment.

16. All the figures should have proper labels, axis titles, units.

17. The purification tables contain inconsistencies between U/mL and U/mg.

18. The paper mentions multiple mutants were tested, but only the ethidium bromide mutant's data are highlighted in the abstract. A table of comparison of all the mutants, highlighting the best data of ethidium bromide mutant would strengthen the argument.

19. The manuscript contains numerous grammatical errors and unclear sentences.

Reviewer #2: The manuscript presents an extensive investigation into the enhancement of fibrinolytic enzyme production in Oidiodendron maius through mutagenesis, followed by purification and characterization of enzyme activity. The work is scientifically relevant, methodologically rich, and demonstrates a clear link between experimental findings and potential application in thrombolytic therapy. The novelty — particularly the first reported hyperproduction and kinetic profiling from mutated O. maius strains — is of significance.

However, the manuscript requires some editorial and structural refinement to improve clarity, cohesion, and scientific readability. The volume of information is high, with several sections appearing overly descriptive and repetitive. With revision, this work could become impactful within enzyme biotechnology and biomedical application domains.

The results contain large blocks of information without sectional breaks. This makes it difficult for readers to locate important findings. I suggest authors to make subsections such as:

• Mutagenesis optimization

• Fermentation parameter influence

• Purification profile of wild vs mutants

• Kinetic & thermodynamic analysis

• Clot lysis and biomedical relevance

There is strong literature support, but results and citations are sometimes interwoven in a way that disrupts flow. Recommended structure:

Result → Interpretation → Supporting literature, rather than alternating statements.

Language, clarity, and grammar

Several sentences are long, repetitive, or grammatically weak. Conciseness is needed. For example:

“Our results are revealed…” → “Our results revealed…”

“These findings correlate well…” appears repeatedly and could be merged.

A focused language edit would substantially improve readability.

The manuscript mentions novelty, but it would be strengthened by explicitly stating:

• How much improvement was achieved versus wild type

• What makes BBTI-EB superior mechanistically

• Next step towards scale-up / drug development

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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: Urmil Bansal

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Enhanced Fibrinolytic Enzyme Production by Oidiodendron maius through Green Bioprocessing of Agro-Industrial residue

PONE-D-25-56032R1

Dear Dr. Gulle-e-Faran,

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.

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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,

Faiz Ahmad Joyia, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

Reviewer #4: (No Response)

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

The PLOS Data policy

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #3: The manuscript presents extensive work with promising enhancement of fibrinolytic enzyme production. Purification results presented in the manuscript are potentially impactful for enzyme biotechnology. However, to make it readable and reliable for publication it requires some changes

Comment 1: In Methods it is mentioned that production flasks were incubated at 37°C/120 rpm for 24 days, but in same section and Results, fermentation time is written as 12–72 h with maxima at 24–36 h, creating a major inconsistency. Please confirm the actual fermentation duration used for enzyme production and revise the manuscript accordingly.

Comment 2: UV mutagenesis parameters are inconsistent and include an implausible wavelength “25440 nm” and exposure time is reported differently (e.g., 1 hour vs 50 minutes). There is confusion in text, either 240 or 254 nm. Please provide one definitive UV protocol (wavelength, lamp type, intensity/dose, distance, exposure time/intervals, shielding) and keep it consistent across sections of article.

Comment 3: The statistical analysis section seems too vague, its two-factor randomized design with ANOVA, but does not specify the factors tested, whether interactions were evaluated, what post-hoc multiple-comparison procedure was used, how ANOVA assumptions were checked, or whether “triplicates” are biological vs technical replicates. Please revise to report n for each experiment, the exact ANOVA model (strain × parameter level), interaction results, post-hoc method (e.g., Tukey), assumption checks, and make sure every figure clearly states mean ± SD/SE and what the error bars represent.

Comment 4: The study measures fibrinolysis using fibrin-plate/punch-hole clear zone diameters yet reports activity as U/mL without defining what 1 unit represents. Please standardize the activity reporting by explicitly defining 1 U (and the calculation for U/mL), including any standard curve/reference enzyme, or add a quantitative spectrophotometric/chromogenic assay to support diffusion-based measurements.

Comment 5: The study is framed as green bioprocessing, but the strain improvement relies on ethidium bromide mutagenesis. Its better to either restrict “green” to the agro-industrial residue substrate aspect or tone down the green claim and clearly acknowledge this limitation. Please also add a brief biosafety and waste-disposal compliance statement describing how ethidium bromide was handled, decontaminated and disposed of according to institutional regulations.

Comment 6: Please ensure all figures have complete axis titles and units.

Comment 7: It is mentioned that bands from SDS-PAGE were excised. After removal, these bands were used for downstream applications.

Comment 8: Ensure consistency in mutant naming (BBTI-EB-150 / BBTI-E / etc.) across text, tables and figures.

Reviewer #4: The manuscript presents an interesting and timely study focusing on sustainable enzyme production using agro-industrial waste substrates. The topic is scientifically relevant, particularly in the context of green biotechnology, value-added bioprocessing, and the growing demand for fibrinolytic enzymes with potential therapeutic applications. The approach of utilizing agro-industrial residues aligns well with current trends in sustainable biomanufacturing and circular bioeconomy principles.

Overall, the study addresses a meaningful research question and has the potential to contribute to the field of industrial microbiology and enzyme biotechnology. However, several aspects of the manuscript require clarification, methodological refinement, and language improvement to enhance its scientific rigor and presentation quality.

1. Please include at least two additional relevant keywords in the Abstract to improve indexing and search visibility.

2. The last two paragraphs of the Introduction contain several grammatical issues. Careful language editing is required to improve clarity and readability.

3. In the Materials and Methods section, kindly recheck the names of the chemicals and their respective compositions for accuracy and consistency.

4. The rationale for selecting wheat bran as the preferred agro-industrial residue should be clearly justified. Please elaborate on its selection criteria and relevance to this study.

5. Please specify the type and molecular weight cut-off of the dialysis membrane used for desalting.

6. The subsection titled “Ammonium sulfate precipitation for partial purification” in the Results section requires revision to minimize grammatical and scientific inaccuracies. The description should be made more precise and technically sound.

7. The Discussion section would benefit from improvement in scientific language and deeper critical interpretation of the results in relation to existing literature.

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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 #3: No

Reviewer #4: Yes: Sher Muhammad

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