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A family of thermostable fungal cellulases created by structure-guided recombination

Heinzelman, Pete and Snow, Christopher D. and Wu, Indira and Nguyen, Catherine and Villalobos, Alan and Govindarajan, Sridhar and Minshull, Jeremy and Arnold, Frances H. (2009) A family of thermostable fungal cellulases created by structure-guided recombination. Proceedings of the National Academy of Sciences of the United States of America, 106 (14). pp. 5610-5615. ISSN 0027-8424. PMCID PMC2667002. doi:10.1073/pnas.0901417106. https://resolver.caltech.edu/CaltechAUTHORS:20090629-142611326

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Abstract

SCHEMA structure-guided recombination of 3 fungal class II cellobiohydrolases (CBH II cellulases) has yielded a collection of highly thermostable CBH II chimeras. Twenty-three of 48 genes sampled from the 6,561 possible chimeric sequences were secreted by the Saccharomyces cerevisiae heterologous host in catalytically active form. Five of these chimeras have half-lives of thermal inactivation at 63°C that are greater than the most stable parent, CBH II enzyme from the thermophilic fungus Humicola insolens, which suggests that this chimera collection contains hundreds of highly stable cellulases. Twenty-five new sequences were designed based on mathematical modeling of the thermostabilities for the first set of chimeras. Ten of these sequences were expressed in active form; all 10 retained more activity than H. insolens CBH II after incubation at 63°C. The total of 15 validated thermostable CBH II enzymes have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions. Selected purified thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures 7 to 15°C higher than the parent enzymes. These chimeras also hydrolyzed as much or more cellulose than the parent CBH II enzymes in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes. Generating this group of diverse, thermostable fungal CBH II chimeras is the first step in building an inventory of stable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1073/pnas.0901417106DOIArticle
http://www.pnas.org/content/106/14/5610.abstractPublisherArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667002/PubMed CentralArticle
ORCID:
AuthorORCID
Snow, Christopher D.0000-0002-7690-3519
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2009 by the National Academy of Sciences. Contributed by Frances H. Arnold, February 9, 2009 (sent for review January 19, 2009). We thank Iogen, Inc. (Ottawa, ON) for providing the YEp352/PGK91–1-ss vector and recommending use of the Δkre2 S. cerevisiae strain. This work was supported by the Army-Industry Institute for Collaborative Biotechnologies and the Caltech Innovation Institute. Author contributions: P.H., C.D.S., J.M., and F.H.A. designed research; P.H., I.W., and C.N. performed research; P.H., C.D.S., I.W., A.V., S.G., J.M., and F.H.A. analyzed data; and P.H., C.D.S., J.M., and F.H.A. wrote the paper. The authors declare no conflict of interest. This article contains supporting information online at www.pnas.org/cgi/content/full/0901417106/DCSupplemental.
Funders:
Funding AgencyGrant Number
Army Research Office (ARO)UNSPECIFIED
Caltech Innovation Initiative (CI2)UNSPECIFIED
Subject Keywords:biofuels; cellobiohydrolase; cellulose hydrolysis; Trichoderma reesei; CBH II
Issue or Number:14
PubMed Central ID:PMC2667002
DOI:10.1073/pnas.0901417106
Record Number:CaltechAUTHORS:20090629-142611326
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20090629-142611326
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:14455
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:12 Aug 2009 23:06
Last Modified:08 Nov 2021 23:11

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