Studies Of Thermobifida Fusca Cellulases Cel9A And Cel48A Using A Novel Kinetic Modeling Approach For The Enzymatic Digestion Of Cellulose.

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Abstract

Lignocellulosic biomass is a potential source of sustainable transportation fuels, but efficient enzymatic saccharification of cellulose is a key challenge in its utilization. Enzymatic digestion of cellulose is a complex, heterogeneous process. An enzyme must repeatedly take multiple steps to hydrolyze the substrate to product. In addition, most bulk cellulose contains crystalline, semi-crystalline, and amorphous fractions, whose ratio likely changes during digestion. Access to substrate, rather than hydrolysis, is widely accepted to be the rate limiting step, as we showed experimentally using Cel9A, and the rate of digestion rapidly and continuously drops off as digestion proceeds. Predictive kinetic models typically incorporate different parameters and constants to account for the possible factors responsible for such behavior. Cellulose hydrolysis by individual cellulases and their mixtures can be modeled with a simple two-parameter model based on a modified classical kinetics scheme. Analogous to a fractal kinetics approach, the specific activity constant is replaced with a time-dependent activity coefficient in order to account for the continuous decrease in the digestion rate. The parameter that quantifies the time dependence of the digestion rate is an intrinsic constant for a given cellulase or mixture on a given substrate. The developed kinetic model was utilized in studies aimed to understand the function of aromatic residues located near the active site tunnel entrance of Cel48A and to develop a distinct model of synergistic cooperation between Cel48A and Cel9A. Additional experiments were carried out to establish experimentally the catalytic base in family 48 glycoside hydrolases.

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2013-08-19
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Cellulases; Thermobifida fusca; Cel9A; Cel48A; cellulose; biomass; synergism
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Wilson, David B
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Nicholson, Linda K
Chen, Peng
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Biochemistry
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Ph. D., Biochemistry
Degree Level
Doctor of Philosophy
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Government Document
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dissertation or thesis
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