Ethanol treatment for sterilization, concentration, and stabilization of a biodegradable plastic–degrading enzyme from Pseudozyma antarctica culture supernatant

Takumi Tanaka; Ken Suzuki; Hirokazu Ueda; Yuka Sameshima-Yamashita; Hiroko Kitamoto

doi:10.1371/journal.pone.0252811

Peer Review History

Original SubmissionDecember 10, 2020
18 Mar 2021 Decision Letter - Olga Cristina Pastor Nunes, Editor PONE-D-20-38928 Ethanol treatment for sterilization, concentration, and stabilization of biodegradable plastic–degrading enzyme from Pseudozyma antarctica culture supernatant PLOS ONE Dear Dr. Hiroko Kitamoto, 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. Manuscript PONE-D-20-38928 reports a new method for sterilization, concentration and stabilization of a plastic–degrading enzyme. In general the manuscript is well organized and clear. However, as recommended by both reviewers, the discussion section should be improved, with a comparative analysis of the data obtained with that already reported in the literature. In addition, English must be improved before acceptance. Please submit your revised manuscript by 16/04/2021. 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Olga C. Nunes 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 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. 3. 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. 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. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [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: Yes 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: No 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 overall study seems interesting, however, few queries needs to be addressed. 1. The grammar is poor. The manuscript needs extensively revised. 2. Reframe the abstract. 3. Correct line no. 50 4. Edit line no. 130 5. Edit line no. 269 6. Edit line no. 282 7. Cite some more references Reviewer #2: It is preferable that the discussion section should include the comparative analysis of the specific activity and yield of the enzyme precipitated by the usual method and this method, in order to highlight the advantage / importance of this research work. The entire manuscript talks about this research study but it is good to compare the work with that of previous reports, so that it would give a more clear idea for the readers. ****** 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: Yes:** Subathra Devi C [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: PLos one review.docx https://doi.org/10.1371/journal.pone.0252811.r001
Revision 1
19 Apr 2021 Author Response [Response to Reviewer #1] The overall study seems interesting, however, few queries needs to be addressed. → We thank the reviewers for their helpful comments and suggestions. Below are our point-by-point responses to the reviewer’s comments. 1. The grammar is poor. The manuscript needs extensively revised. → We have modified the entire manuscript. The manuscript has been edited carefully by two native English-speaking professional editors from ELSS, Inc. (elss@elss.co.jp, http://www.elss.co.jp). All changes are highlighted in yellow. 2. Reframe the abstract. → We have edited the abstract to reflect the content of the manuscript more accurately as follows: Line nos. 24–42 (revised manuscript): Biodegradable plastics must be sufficiently stable to maintain functionality during use but need to be able to degrade rapidly after use. We previously reported that treatment with an enzyme named PaE, secreted by the basidiomycete yeast Pseudozyma antarctica can speed up this degradation. To facilitate the production of large quantities of PaE, here, we aimed to elucidate the optimal conditions of ethanol treatment for sterilization of the culture supernatant and for concentration and stabilization of PaE. The results showed that Pseudozyma antarctica completely lost its proliferating ability when incubated in ≥20% (v/v) ethanol. When the ethanol concentration was raised to 90% (v/v), PaE formed a precipitate; however, its activity was restored completely when the precipitate was dissolved in water. To reduce ethanol use, PaE was successfully concentrated and recovered by sequential ammonium sulfate precipitation and ethanol precipitation steps. Over 90% of the activity in the original culture supernatant was recovered and the specific activity was increased 3.4-fold. By preparing the enzyme solution at a final concentration of 20% (v/v) ethanol, about 60% of the initial activity was maintained at ambient temperature for over 6 months without growth of microbes. We conclude that ethanol treatment is effective for sterilization, concentration, and stabilization of PaE, and that concentrating PaE by sequential ammonium sulfate precipitation and ethanol. 3. Correct line no. 50 → We have corrected this sentence, and have added references. Line nos. 45–47 (revised manuscript): Since large-scale production of the plastics started in the 1950’s, more than 8 billion tons of plastic products have been used worldwide, and more than 6 billion tons have been disposed of as waste [1,2]. In addition, we have edited the Introduction (see changes highlighted in yellow in the manuscript file) 4. Edit line no. 130 → We have modified this sentence to describe in more detail how the concentration of purified PaE was calculated: Line nos. 131–139 (revised manuscript): The concentration of purified PaE was calculated as described previously [13]. In short, the purified PaE solution was appropriately diluted, and its absorbance was measured at 280 nm (A280). Then, the concentration of PaE was calculated according to the following equation: A280 (M−1 cm−1) = 5,500nW + 1,490nY + 125nC [17,18], where M (=20,362.41) is the average molecular weight calculated using GENETYX software ver. 9 (GENETYX, Tokyo, Japan), nW is the number of Trp residues (=1), nY is the number of Tyr residues (=7), and nC is the number of disulfide bonds (=2), per polypeptide chain based on the mature amino acid sequence of PaE [6]. 5. Edit line no. 269 → We have edited line no 269 and have added a new paragraph above it as follows: Line nos. 278–284 (revised manuscript): When PaE is used for BP product degradation, it is desirable to be able to sterilize the culture media after enzyme production, concentrate the enzyme from a large amount of the culture supernatant, and both stabilize and store the enzyme for a long period of time. Here, we explored the possibility of using ethanol treatment to achieve these objectives and investigated the optimal conditions. First, we examined the degree of inactivation of P. antarctica cells following treatment with various concentrations of ethanol. In addition, we have edited the Discussion (see changes highlighted in yellow in the manuscript file) 6. Edit line no. 282 → We have edited line no. 282 and its surrounding lines as follows: Line nos. 299–302 (revised manuscript): After storage at ambient temperature for 206 days, the PaE activity of the samples with ethanol concentrations of 10% or 25% remained at 67% or 60% of the initial value, respectively, which was higher than the activity remaining at higher ethanol concentrations (Fig 2a). 7. Cite some more references → We have cited eight new references. In addition, we have corrected the reference style to match the style and format of PLOS ONE. [Response to Reviewer #2] It is preferable that the discussion section should include the comparative analysis of the specific activity and yield of the enzyme precipitated by the usual method and this method, in order to highlight the advantage / importance of this research work. The entire manuscript talks about this research study but it is good to compare the work with that of previous reports, so that it would give a more clear idea for the readers. → We thank the reviewers for their helpful comment and the suggestion. We tried to compare this work with that of previous reports. We previously reported purification of PaE from the culture filtrate of Pseudozyma antarctica (Kitamoto et al, 2011; reference no. 12 in the revised manuscript). In this report, enzyme-producing microbe can be removed from the culture supernatant by passage through a cell impermeable membrane using a dedicated facility, however, the concentration and preservation steps are still necessary. Therefore, we have added a new paragraph and new a sentence in the Discussion as follows: Line nos. 278–282 (revised manuscript): When PaE is used for BP product degradation, it is desirable to be able to sterilize the culture media after enzyme production, concentrate the enzyme from a large amount of the culture supernatant, and both stabilize and store the enzyme for a long period of time. Here, we explored the possibility of using ethanol treatment to achieve these objectives and investigated the optimal conditions. Line nos. 334–335 (revised manuscript): The ethanol treatment serves all the requirements for the preparation of enzyme. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0252811.r002
24 May 2021 Decision Letter - Olga Cristina Pastor Nunes, Editor Ethanol treatment for sterilization, concentration, and stabilization of a biodegradable plastic–degrading enzyme from Pseudozyma antarctica culture supernatant PONE-D-20-38928R1 Dear Dr. Kitamoto, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Olga C. Nunes 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: Yes 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: Authors have addressed all the comments. The manuscript can be accepted now in the current form. No further comment Reviewer #2: (No Response) ****** 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:** Subathra Devi C https://doi.org/10.1371/journal.pone.0252811.r003
Formally Accepted
26 May 2021 Acceptance Letter - Olga Cristina Pastor Nunes, Editor PONE-D-20-38928R1 Ethanol treatment for sterilization, concentration, and stabilization of a biodegradable plastic–degrading enzyme from Pseudozyma antarctica culture supernatant Dear Dr. Kitamoto: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Olga Cristina Pastor Nunes Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252811.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .