Heinzelman, Pete and Snow, Christopher D. and Smith, Matthew A. and Yu, Xinlin and Kannan, Arvind and Boulware, Kevin and Villalobos, Alan and Govindarajan, Sridhar and Minshull, Jeremy and Arnold, Frances H. (2009) SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation That Contributes Markedly to Stability. Journal of Biological Chemistry, 284 (39). pp. 26229-26233. ISSN 0021-9258 http://resolver.caltech.edu/CaltechAUTHORS:20091013-093450880
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A quantitative linear model accurately (R^2 = 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5 °C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5 °C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55–56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations.
|Additional Information:||Copyright © 2009 by American Society for Biochemistry and Molecular Biology. Received June 16, 2009. First Published on July 22, 2009, doi: 10.1074/jbc.C109.034058. This work was supported by grants from the Army-Industry Institute for Collaborative Biotechnologies and the Caltech Innovation Institute. The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7 and Tables S1–S5.|
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|Deposited By:||George Porter|
|Deposited On:||22 Oct 2009 19:00|
|Last Modified:||26 Dec 2012 11:28|
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