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Highly thermostable fungal cellobiohydrolase I (Cel7A) engineered using predictive methods

Komor, Russell S. and Romero, Philip A. and Xie, Catherine B. and Arnold, Frances H. (2012) Highly thermostable fungal cellobiohydrolase I (Cel7A) engineered using predictive methods. Protein Engineering, Design and Selection, 25 (12). pp. 827-833. ISSN 1741-0126. https://resolver.caltech.edu/CaltechAUTHORS:20130107-104235257

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Abstract

Building on our previous efforts to generate thermostable chimeric fungal cellobiohydrolase I (CBH I, also known as Cel7A) cellulases by structure-guided recombination, we used FoldX and a ‘consensus’ sequence approach to identify individual mutations present in the five homologous parent CBH I enzymes which further stabilize the chimeras. Using the FoldX force field, we calculated the effect on ΔG_Folding of each candidate mutation in a number of CBH I structures and chose those predicted to be stabilizing in multiple structures. With an alignment of 41 CBH I sequences, we also used amino acid frequencies at each candidate position to calculate predicted effects on ΔG_Folding. A combination of mutations chosen using these methods increased the T_50 of the most thermostable chimera by an additional 4.7°C, to yield a CBH I with T_50 of 72.1°C, which is 9.2°C higher than that of the most stable native CBH I, from Talaromyces emersonii. This increased stability resulted in a 10°C increase in the optimal temperature for activity, to 65°C, and a 50% increase in total sugar production from crystalline cellulose at the optimal temperature, compared with native T.emersonii CBH I.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1093/protein/gzs058DOIArticle
http://peds.oxfordjournals.org/content/25/12/827PublisherArticle
ORCID:
AuthorORCID
Romero, Philip A.0000-0002-2586-7263
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© The Author 2012. Published by Oxford University Press. Received July 13, 2012; Revision received July 13, 2012; Accepted August 13, 2012. First published online: September 7, 2012. The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. This work was supported by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office.
Funders:
Funding AgencyGrant Number
Army Research Office (ARO)W911NF-09-0001
Subject Keywords:CBH I/Cel7A/cellulase/protein engineering
Issue or Number:12
Record Number:CaltechAUTHORS:20130107-104235257
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20130107-104235257
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:36199
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:07 Jan 2013 22:41
Last Modified:26 Nov 2019 20:14

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