Poster Presentation 1B-18

 

The Mechanism of Cellulose Hydrolysis by Thermobifida fusca Cellulases

 

Tina Jeoh,1* David B. Wilson,2 A. Brad Anton3 and Larry P. Walker4

1Work was done as Ph.D. Researcher at Cornell University

  Current Address: National Bioenergy Center, National Renewable Energy Laboratory

1617 Cole Boulevard

Golden, CO 80401

Phone:  (303)384-7777

Fax:  (303)384-7752

Email:  tina_jeoh@nrel.gov

 

2Department of Molecular Biology and Genetics, Cornell University

 

3School of Chemical and Biomolecular Engineering, Cornell University

 

4Department of Biological and Environmental Engineering Department, Cornell University

 

 

 

In this presentation, we highlight two aspects of the mechanism of cellulose hydrolysis by Thermobifida fusca cellulases.  We first present data to explain the synergistic mechanism by binary mixtures of Cel5A, Cel6B and Cel9A.  Following, we present a mechanistic model developed from classical heterogeneous enzyme kinetic mechanisms describing cellulose hydrolysis by individual cellulases. 

 

Previously, we reported cooperative binding by the cellulase components in synergistic binary mixtures.  We hypothesized that enhanced concentrations of cellulase bound to the substrate surface increases access to the substrate, thus resulting in the synergistic effect.  We have since collected more data on the binary mixtures to reveal a second mechanism for synergism.  In the endocellulase-exocellulase mixtures studied (primarily the Cel5A+Cel6B mixture), it appears that the endocellulase also increases access to reactive sites for the exocellulase.  This effect can be measured at low total cellulase loading (0.25 mmoles/g cellulose) and was verified by experiments using CDCel5A pretreated bacterial microcrystalline cellulose (BMCC).

 

The cellulase-cellulose system is heterogeneous, with soluble cellulases acting on the insoluble substrate, cellulose.  The classical heterogeneous enzyme kinetics model is comprised of two coupled equations - a reversible adsorption-desorption reaction and an irreversible hydrolysis reaction.

 

                                                                                (1)

 

                                                                                            (2)

 

Where Ef, Eb, s and P are the free enzyme, bound enzyme, free binding sites on the substrate and product, respectively, and ka, kd and kr are kinetic constants for the reaction.  The cellulase-cellulose system is a classic example of a heterogeneous enzyme reaction and can be modeled by the mechanisms shown in Equations 1 and 2.  Although the closed system can be described by only two ordinary differential equations, these are nonlinearly coupled with no analytical solution.  We have solved the ODEs using singular perturbation method, thus obtaining asymptotic solutions that simultaneously describe time dependent bound cellulase and product concentrations.  The model was simultaneously fit to time course binding concentration and reducing sugar measurements collected for reactions with fluorescence-labeled T. fusca Cel5A, Cel6B or Cel9A on BMCC at various cellulase/substrate loadings.  The model provides good estimates of the kinetic parameters, ka, kd and kr, as well as provides key information on substrate characteristics affecting cellulase-cellulose interactions.