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Engineering Thermostable Fungal Cellobiohydrolases


Wu, Indira (2013) Engineering Thermostable Fungal Cellobiohydrolases. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/V80F-X625.


Meeting the world's growing energy demands while protecting our fragile environment is a challenging issue. Second generation biofuels are liquid fuels like long-chain alcohols produced from lignocellulosic biomass. To reduce the cost of biofuel production, we engineered fungal family 6 cellobiohydrolases (Cel6A) for enhanced thermostability using random mutagenesis and recombination of beneficial mutations. During long-time hydrolysis, engineered thermostable cellulases hydrolyze more sugars than wild-type Cel6A as single enzymes and binary mixtures at their respective optimum temperatures. Engineered thermostable cellulases exhibit synergy in binary mixtures similar to wild-type cellulases, demonstrating the utility of engineering individual cellulases to produce novel thermostable mixtures. Crystal structures of the engineered thermostable cellulases indicate that the stabilization comes from improved hydrophobic interactions and restricted loop conformations by proline substitutions. At high temperature, free cysteines contribute to irreversible thermal inactivation in engineered thermostable Cel6A and wild-type Cel6A. The mechanism of thermal inactivation in this cellulase family is consistent with disulfide bond degradation and thiol-disulfide exchange. Enhancing the thermostability of Cel6A also increases tolerance to pretreatment chemicals, demonstrated by the strong correlation between thermostability and tolerance to 1-ethyl-3-methylimidazolium acetate. Several semi-rational protein engineering approaches – on the basis of consensus sequence analysis, proline stabilization, FoldX energy calculation, and high B-factors – were evaluated to further enhance the thermostability of Cel6A.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Protein engineering; directed evolution; fungal cellulases; cellobiohydrolases; thermostability;
Degree Grantor:California Institute of Technology
Division:Engineering and Applied Science
Major Option:Bioengineering
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances Hamilton
Thesis Committee:
  • Rees, Douglas C. (chair)
  • Arnold, Frances Hamilton
  • Tirrell, David A.
  • Mayo, Stephen L.
Defense Date:26 March 2013
Funding AgencyGrant Number
Army Research Office--Institute for Collaborative BiotechnologiesW911NF-09-D-0001
National Science FoundationMCB-0903817
Record Number:CaltechTHESIS:05022013-164749371
Persistent URL:
Related URLs:
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Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7657
Deposited By: Indira Wu
Deposited On:21 May 2013 18:17
Last Modified:08 Nov 2023 00:11

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