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Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures

Wu, Indira and Arnold, Frances H. (2013) Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures. Biotechnology and Bioengineering, 110 (7). pp. 1874-1883. ISSN 0006-3592. doi:10.1002/bit.24864.

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Thermostability is an important feature in industrial enzymes: it increases biocatalyst lifetime and enables reactions at higher temperatures, where faster rates and other advantages ultimately reduce the cost of biocatalysis. Here we report the thermostabilization of a chimeric fungal family 6 cellobiohydrolase (HJPlus) by directed evolution using random mutagenesis and recombination of beneficial mutations. Thermostable variant 3C6P has a half-life of 280 min at 75°C and a T_50 of 80.1°C, a ∼15°C increase over the thermostable Cel6A from Humicola insolens (HiCel6A) and a ∼20°C increase over that from Hypocrea jecorina (HjCel6A). Most of the mutations also stabilize the less-stable HjCel6A, the wild-type Cel6A closest in sequence to 3C6P. During a 60-h Avicel hydrolysis, 3C6P released 2.4 times more cellobiose equivalents at its optimum temperature (T_opt) of 75°C than HiCel6A at its T_opt of 60°C. The total cellobiose equivalents released by HiCel6A at 60°C after 60 h is equivalent to the total released by 3C6P at 75°C after ∼6 h, a 10-fold reduction in hydrolysis time. A binary mixture of thermostable Cel6A and Cel7A hydrolyzes Avicel synergistically and released 1.8 times more cellobiose equivalents than the wild-type mixture, both mixtures assessed at their respective T_opt. Crystal structures of HJPlus and 3C6P, determined at 1.5 and 1.2 Å resolution, indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by introduced proline residues.

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Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2013 Wiley Periodicals, Inc. Received 2 December 2012; Revision received 21 January 2013; Accepted 28 January 2013; Accepted manuscript online 12 February 2013; Article first published online 1 March 2013 in Wiley Online Library. The authors thank E. M. Brustad, C. D. Snow, and the Molecular Observatory at the California Institute of Technology for assistance with high-throughput protein crystallography, X-ray data collection, and analysis. The Molecular Observatory is supported by the Gordon and Betty Moore Foundation, the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program at Caltech. The authors acknowledge the Caltech Innovation Initiative and the U.S. Army Research Office, Institute for Collaborative Biotechnologies (grant W911NF-09-D-0001) for funding the Cel6A work. A non-provisional patent application has been filed on some of the stabilizing mutations.
Funding AgencyGrant Number
Caltech Innovation Initiative (CI2)UNSPECIFIED
Army Research Office (ARO)W911NF-09-D-0001
Subject Keywords:biofuel; fungal cellulase; CBHII; enzyme thermostability; cellulase synergy; proline substitutions
Issue or Number:7
Record Number:CaltechAUTHORS:20130703-095728488
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Official Citation:Wu, I. and Arnold, F. H. (2013), Engineered thermostable fungal Cel6A and Cel7A cellobiohydrolases hydrolyze cellulose efficiently at elevated temperatures. Biotechnol. Bioeng., 110: 1874–1883. doi: 10.1002/bit.24864
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:39201
Deposited By: Jason Perez
Deposited On:03 Jul 2013 21:00
Last Modified:09 Nov 2021 23:43

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