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Computationally-Guided Thermostabilization of the Primary Endoglucanase from Hypocrea jerorina for Cellulosic Biofuel Production


Lee, Toni Marie (2014) Computationally-Guided Thermostabilization of the Primary Endoglucanase from Hypocrea jerorina for Cellulosic Biofuel Production. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9S180G0.


The creation of thermostable enzymes has wide-ranging applications in industrial, scientific, and pharmaceutical settings. As various stabilization techniques exist, it is often unclear how to best proceed. To this end, we have redesigned Cel5A (HjCel5A) from Hypocrea jecorina (anamorph Trichoderma reesei) to comparatively evaluate several significantly divergent stabilization methods: 1) consensus design, 2) core repacking, 3) helix dipole stabilization, 4) FoldX ΔΔG approximations, 5) Triad ΔΔG approximations, and 6) entropy reduction through backbone stabilization. As several of these techniques require structural data, we initially solved the first crystal structure of HjCel5A to 2.05 Å. Results from the stabilization experiments demonstrate that consensus design works best at accurately predicting highly stabilizing and active mutations. FoldX and helix dipole stabilization, however, also performed well. Both methods rely on structural data and can reveal non-conserved, structure-dependent mutations with high fidelity. HjCel5A is a prime target for stabilization. Capable of cleaving cellulose strands from agricultural waste into fermentable sugars, this protein functions as the primary endoglucanase in an organism commonly used in the sustainable biofuels industry. Creating a long-lived, highly active thermostable HjCel5A would allow cellulose hydrolysis to proceed more efficiently, lowering production expenses. We employed information gleaned during the survey of stabilization techniques to generate HjCel5A variants demonstrating a 12-15 °C increase in the temperature at which 50% of the total activity persists, an 11-14 °C increase in optimal operating temperature, and a 60% increase over the maximal amount of hydrolysis achievable using the wild type enzyme. We anticipate that our comparative analysis of stabilization methods will prove useful in future thermostabilization experiments.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:cellulase, thermostability, endoglucanase, computational protein design, biofuels
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Mayo, Stephen L.
Thesis Committee:
  • Rees, Douglas C. (chair)
  • Arnold, Frances H.
  • Tirrell, David A.
  • Shan, Shu-ou
  • Mayo, Stephen L.
Defense Date:28 October 2013
Record Number:CaltechTHESIS:11172013-001549340
Persistent URL:
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URLURL TypeDescription adapted for ch.2 CentralArticle adapted for ch.2
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8027
Deposited By: Toni Lee
Deposited On:20 Sep 2016 16:30
Last Modified:04 Oct 2019 00:03

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