Abstract
Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.
Document Type
Article
Publication Date
8-31-2017
Digital Object Identifier (DOI)
https://doi.org/10.1074/jbc.M117.803270
Funding Information
This work was supported, in part, by a subcontract from the Office of Biomass Program, within the Department of Energy Office of Energy Efficiency and Renewable Energy; the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (Formas; Grant 213-2013-1607; principal investigator: J. S.); and the National Science Foundation (NSF) under Grant 1552355 (principal investigator: C. M. P.).
Related Content
This article contains supplemental Figs. S1–S5.
The atomic coordinates and structure factors(code 5OA5) have been deposited in the Protein Data Bank (http://wwpdb.org/).
Repository Citation
Goedegebuur, Frits; Dankmeyer, Lydia; Gualfetti, Peter; Karkehabadi, Saeid; Hansson, Henrik; Jana, Suvamay; Huynh, Vicky; Kelemen, Bradley R.; Kruithof, Paulien; Larenas, Edmund A.; Teunissen, Pauline J. M.; Ståhlberg, Jerry; Payne, Christina M.; Mitchinson, Colin; and Sandgren, Mats, "Improving the Thermal Stability of Cellobiohydrolase Cel7A from Hypocrea jecorina by Directed Evolution" (2017). Chemical and Materials Engineering Faculty Publications. 41.
https://uknowledge.uky.edu/cme_facpub/41
Supplemental Figs. S1–S5
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Notes/Citation Information
Published in The Journal of Biological Chemistry, v. 292, no. 42, p. 17418-17430.
This research was originally published in The Journal of Biological Chemistry. Frits Goedegebuur, Lydia Dankmeyer, Peter Gualfetti, Saeid Karkehabadi, Henrik Hansson, Suvamay Jana, Vicky Huynh, Bradley R. Kelemen, Paulien Kruithof, Edmund A. Larenas, Pauline J. M. Teunissen, Jerry Ståhlberg, Christina M. Payne, Colin Mitchinson, and Mats Sandgren. Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution. J. Biol. Chem. 2017; 292:17418-17430. ©2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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