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Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability

Moraïs, Sarah and Stern, Johanna and Kahn, Amaranta and Galanopoulou, Anastasia P. and Yoav, Shahar and Shamshoum, Melina and Smith, Matthew A. and Hatzinikolaou, Dimitris G. and Arnold, Frances H. and Bayer, Edward A. (2016) Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability. Biotechnology for Biofuels, 9 . Art. No. 164. ISSN 1754-6834. PMCID PMC4973527. http://resolver.caltech.edu/CaltechAUTHORS:20160816-065945854

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Abstract

Background: The concerted action of three complementary cellulases from Clostridium thermocellum, engineered to be stable at elevated temperatures, was examined on a cellulosic substrate and compared to that of the wild-type enzymes. Exoglucanase Cel48S and endoglucanase Cel8A, both key elements of the natural cellulosome from this bacterium, were engineered previously for increased thermostability, either by SCHEMA, a structure-guided, site-directed protein recombination method, or by consensus-guided mutagenesis combined with random mutagenesis using error-prone PCR, respectively. A thermostable β-glucosidase BglA mutant was also selected from a library generated by error-prone PCR that will assist the two cellulases in their methodic deconstruction of crystalline cellulose. The effects of a thermostable scaffoldin versus those of a largely mesophilic scaffoldin were also examined. By improving the stability of the enzyme subunits and the structural component, we aimed to improve cellulosome-mediated deconstruction of cellulosic substrates. Results: The results demonstrate that the combination of thermostable enzymes as free enzymes and a thermostable scaffoldin was more active on the cellulosic substrate than the wild-type enzymes. Significantly, “thermostable” designer cellulosomes exhibited a 1.7-fold enhancement in cellulose degradation compared to the action of conventional designer cellulosomes that contain the respective wild-type enzymes. For designer cellulosome formats, the use of the thermostabilized scaffoldin proved critical for enhanced enzymatic performance under conditions of high temperatures. Conclusions: Simple improvement in the activity of a given enzyme does not guarantee its suitability for use in an enzyme cocktail or as a designer cellulosome component. The true merit of improvement resides in its ultimate contribution to synergistic action, which can only be determined experimentally. The relevance of the mutated thermostable enzymes employed in this study as components in multienzyme systems has thus been confirmed using designer cellulosome technology. Enzyme integration via a thermostable scaffoldin is critical to the ultimate stability of the complex at higher temperatures. Engineering of thermostable cellulases and additional lignocellulosic enzymes may prove a determinant parameter for development of state-of-the-art designer cellulosomes for their employment in the conversion of cellulosic biomass to soluble sugars.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1186/s13068-016-0577-zDOIArticle
https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-016-0577-zPubMed CentralArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4973527/PubMed CentralArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Received: 12 June 2016. Accepted: 27 July 2016. Published online: 4 August 2016. The authors appreciate the technical assistance of Daniel Liapman. This research was supported by the F. Warren Hellman Grant for Alternative Energy Research in Israel in support of alternative energy research in Israel to E.A.B. administered by the Israel Strategic Alternative Energy Foundation (I-SAEF). Additional support was obtained by a Grant (No. 1349) to E.A.B. from the ISF Israel Science Foundation (ISF), a European Union Contract, Area NMP.2013.1.1-2: Self-assembly of naturally occurring nanosystems: CellulosomePlus Project number: 604530 and an ERA-IB Consortium (EIB.12.022), acronym FiberFuel. This research was also supported by the Establishment of an Israeli Center of Research Excellence I-CORE Center No. 152/11) managed by the Israel Science Foundation, by the Weizmann Institute of Science Alternative Energy Research Initiative (AERI) and the Helmsley Foundation (the Leona M. and Harry B. Helmsley Charitable Trust), and Grants from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel. E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry. Authors’ contributions: SM designed the research, performed the experiments and wrote the manuscript. JS and MAS designed and performed the experiments for the selection of the best-performing exoglucanase mutant. SY designed and performed the experiments for the selection of the β-glucosidase mutant. AK and APG designed the methodology for thermostability assay of the cellulosomal components. MS produced and purified the proteins. SM, JS, SY, AK, APG, DGH, FHA and EAB analyzed the results. SM, DGH, FHA and EAB wrote the manuscript. All authors read and approved the manuscript. The authors declare that they have no competing interests.
Funders:
Funding AgencyGrant Number
Israel Strategic Alternative Energy FoundationUNSPECIFIED
Israel Science Foundation1349
European UnionNMP.2013.1.1-2
European Research Area Industrial Biotechnology (ERA-IB Consortium)EIB.12.022
I-CORE Program of the Planning and Budgeting Committee152/11
Weizmann InstituteUNSPECIFIED
Leona M. and Harry B. Helmsley Charitable TrustUNSPECIFIED
Binational Science Foundation (USA-Israel)UNSPECIFIED
Maynard I. and Elaine Wishner Chair of Bio-organic ChemistryUNSPECIFIED
Subject Keywords:Thermostable cellulases Multi-enzyme complex Designer cellulosomes Clostridium thermocellum
PubMed Central ID:PMC4973527
Record Number:CaltechAUTHORS:20160816-065945854
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160816-065945854
Official Citation:Enhancement of cellulosome-mediated deconstruction of cellulose by improving enzyme thermostability Sarah Moraïs, Johanna Stern, Amaranta Kahn, Anastasia P. Galanopoulou, Shahar Yoav, Melina Shamshoum, Matthew A. Smith, Dimitris G. Hatzinikolaou, Frances H. Arnold and Edward A. Bayer Biotechnology for Biofuels 2016 9:164 DOI: 10.1186/s13068-016-0577-z
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:69636
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:16 Aug 2016 20:37
Last Modified:16 Aug 2016 20:37

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