Characterization of two thermophilic cellulases from Talaromyces leycettanus JCM12802 and their synergistic action on cellulose hydrolysis

Yuan Gu; Fei Zheng; Yuan Wang; Xiaoyun Su; Yingguo Bai; Bin Yao; Huoqing Huang; Huiying Luo

doi:10.1371/journal.pone.0224803

Peer Review History

Original SubmissionJuly 30, 2019
9 Sep 2019 Decision Letter - Jean-Guy Berrin, Editor PONE-D-19-21437 Characterization of two thermophilic cellulases from Talaromyces leycettanus JCM12802 and their synergistic action on cellulose hydrolysis PLOS ONE Dear Mrs. Luo, 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. We would appreciate receiving your revised manuscript by Oct 24 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Revision 1
19 Oct 2019 Author Response Response to editor: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Answer: We have checked the manuscript carefully and changed the name of my files to ensure meet PLOS ONE's style requirements. 2. 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. Answer: I have provided the original uncropped and unadjusted gel image in the Supporting Information file. 3. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository. Answer: The gel image data has been provided in the Supporting Information file. 4. Figures are of poor quality and need to be replaced. Answer: The figures with high quality have been replaced and uploaded. 5. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Answer: I have finished the protocol and uploaded it, this is the link: dx.doi.org/10.17504/protocols.io.8g4htyw. Response to Reviewer 1: Reviewer #1: Several of the references are old and should be replaced or supplemented with more recent publications Answer: The latest references have been added and inserted into the corresponding position in the article and marked yellow. The followings are the six newly inserted references. 3. Touijer H, Benchemsi N, Ettayebi M, Janati A, Chaouni B, Bekkari H. Thermostable Cellulases from the Yeast Trichosporon sp. Enzyme research. 2019;1-6. (Line42) 6. Kamal S, Khan SU, Muhammad N, Shoaib M, Omar M, Kaneza P, et al. Insights on heterologous expression of fungal cellulases in Pichia pastoris. Biochemistry and Molecular Biology. 2018; 3(1): 15-35. (Line47) 15. Kamal S, Khan SU, Khan S, Shoaib M, Khan H, Man S, et al. Recent view on heterologous expression of thermostable fungal cellulases, focused on expression factory of Pichia Pastoris. International Journal of Basic Medical Sciences and Pharmacy. 2018; 7(2), 43-57. (Line66) 21. Li D C, Papageorgiou A C. Cellulases from thermophilic fungi: recent insights and biotechnological potential. Enzyme Research. 2019; 395-417. (Line72) 22. Xu X, Fan C, Song L, Li J, Chen Y, Zhang Y, et al. A novel CreA-mediated regulation mechanism of cellulase expression in the thermophilic fungus Humicola insolens. International Journal of Molecular Sciences. 2019; 20(15): 3693. (Line72) 30. Fang H, Zhao R, Li CF, Zhao C. Simultaneous enhancement of the beta–exo synergism and exo–exo synergism in Trichoderma reesei cellulase to increase the cellulose degrading capability. Microbial Cell Factories. 2019; 18(1): 9-23. (Line103) Response to Reviewer 2: Reviewer #2: I recommend this manuscript for publication in PLOS ONE. I suggest the following issue to address though. Speaking about the synergistic action, the Authors should make some thoughts and provide chemical scheme of hydrolysis reaction pathway for this scenario. It will simplify understanding the concept of enzymes action. Answer: We would like to take this opportunity to thank Reviewer 2 for his/her suggestions on my paper with kindly words. And the chemical scheme of hydrolysis reaction pathway of synergistic action was as follows: “Cooperation between many functionally related proteins is very important in biology. A prominent example is the apparent rate of substrate conversion increased by the addition of different combination cellulase enzymes to break down cellulose. A high level of coordination between the enzyme, e.g. exo/endo, exo/exo and endo/the BG synergistic effect, which is effective to hydrolyze crystalline cellulose required. [1] [2] [3] [4] There are three significant types of cellulases to be used basically for the efficient degradation of cellulose. And perhaps the most widely known mechanism remains the so-called endo/exo synergy model. In this form, cellobiohydrolases (CBHs), also called exo-glucanases, cleave cellobiose from the reducing and nonreducing ends producing crystalline ends of cellulose; Endo-glucanases (EGs) break recurrently the glycosidic bonds inside the amorphous part of the substrate creating new attack sites for the CBHs. The products of cellobiohydrolases and endoglucanases are able to inhibit the activities of their own enzymes, so finally, beta-glucosidases (BGLs) hydrolyze unconfined cellobiose and various soluble cellodextrins into glucose monomers. The efficient hydrolysis of cellulose requires the composition of a complete and balanced cellulase, with all components of the cellulase co-acting the enzymatic hydrolysis of cellulose. Synergy is used to describe the ability of collaboration between enzymes. [4] [5] [6] [7] [8] For my experiments, the enzymes of TlCel5A and TlCel6A were combined at different ratios, and the commercial β-glucosidase from Aspergillus fumigatu (Sigma-Aldrich) was added at the concentration of 10% (mol β-glucosidase/mol total enzyme) to hydrolyze cellobiose to glucose finally. When using Avicel, phosphoric acid swollen cellulose (PASC) or steam-exploded corn straw (SECS) as the substrate, combination of TlCel5A and TlCel6A showed significant synergistic action, releasing more reduced sugars than the individual enzymes. (Line270) 1. Shang B Z, Chu J W. Kinetic modeling at single-molecule resolution elucidates the mechanisms of cellulase synergy. ACS Catalysis. 2014; 4(7): 2216-2225. 2. Van Dyk J S, Pletschke B I. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes-factors affecting enzymes, conversion and synergy. Biotechnology Advances. 2012; 30(6):1458-1480. 3. Yang B, Dai Z, Shi-You Ding, et al. Enzymatic hydrolysis of cellulosic biomass. Biofuels. 2011; 2(4):421-450. 4. Kamal S, Khan S U, Muhammad N, et al. Insights on heterologous expression of fungal cellulases in Pichia pastoris. Biochemistry and Molecular Biology. 2018; 3(1): 1-15. 5. Olsen J P, Borch K, Westh P. Endo/exo-synergism of cellulases increases with substrate conversion. Biotechnology and bioengineering. 2017; 114(3): 696-700. 6. Badino S F, Christensen S J, Kari J, et al. Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes. Biotechnology and bioengineering. 2017; 114(8): 1639-1647. 7. Boisset C, Fraschini C, Schülein M, et al. Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase Cel6A from Humicola insolens and its mode of synergy with cellobiohydrolase Cel7A. Applied & Environmental Microbiology. 2000; 66(4): 1444-1452. 8. Fang H, Zhao R, Li C, et al. Simultaneous enhancement of the beta-exo synergism and exo-exo synergism in Trichoderma reesei cellulase to increase the cellulose degrading capability. Microbial cell factories. 2019; 18(1): 1-9. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0224803.r002
23 Oct 2019 Decision Letter - Jean-Guy Berrin, Editor Characterization of two thermophilic cellulases from Talaromyces leycettanus JCM12802 and their synergistic action on cellulose hydrolysis PONE-D-19-21437R1 Dear Dr. Luo, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Jean-Guy Berrin Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0224803.r003
Formally Accepted
1 Nov 2019 Acceptance Letter - Jean-Guy Berrin, Editor PONE-D-19-21437R1 Characterization of two thermophilic cellulases from Talaromyces leycettanus JCM12802 and their synergistic action on cellulose hydrolysis Dear Dr. Luo: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Jean-Guy Berrin Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0224803.r004

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