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Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination

Chang, Chia-Jung and Lee, Cheng-Chung and Chan, Yueh-Te and Trudeau, Devin L. and Wu, Mei-Huey and Tsai, Chih-Hsuan and Yu, Su-May and Ho, Tuan-Hua David and Wang, Andrew H.-J. and Hsiao, Chwan-Deng and Arnold, Frances H. and Chao, Yu-Chan (2016) Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination. PLoS ONE, 11 (3). Art. No. e0147485. ISSN 1932-6203. PMCID PMC4795783. http://resolver.caltech.edu/CaltechAUTHORS:20160328-142111017

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Abstract

Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 3_(10) helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1371/journal.pone.0147485DOIArticle
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147485PublisherArticle
http://dx.doi.org/10.1371/journal.pone.0147485.s001DOISupporting Information
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795783/PubMed CentralArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2016 Chang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: December 1, 2015; Accepted: January 5, 2016; Published: March 17, 2016. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. This work was supported by grants MOST105-2321-B-001-024 and MOST104-2811-B-001-112 from Ministry of Science and Technology, and 5202402020-4006 from Academia Sinica, Taiwan, ROC. The authors have declared that no competing interests exist. Author Contributions: Conceived and designed the experiments: CJC CCL SMY THH AHW CDH FHA YCC. Performed the experiments: CJC CCL YTC DLT MHW. Analyzed the data: CJC CCL CHT SMY THH AHW CDH FHA YCC. Contributed reagents/materials/analysis tools: THH AHW CDH FHA YCC. Wrote the paper: CJC CCL CHT SMY THH AHW CDH FHA YCC.
Funders:
Funding AgencyGrant Number
Ministry of Science and Technology (Taipei)MOST105-2321-B-001-024
Ministry of Science and Technology (Taipei)MOST104-2811-B-001-112
Academia Sinica of Taiwan5202402020-4006
PubMed Central ID:PMC4795783
Record Number:CaltechAUTHORS:20160328-142111017
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160328-142111017
Official Citation:Chang C-J, Lee C-C, Chan Y-T, Trudeau DL, Wu M-H, Tsai C-H, et al. (2016) Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination. PLoS ONE 11(3): e0147485. doi:10.1371/journal.pone.0147485
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:65711
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:28 Mar 2016 23:41
Last Modified:28 Mar 2016 23:41

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