Binding, cellulolytic enzymes

Enzymatic action can be defined on three levels operational kinetics, molecular architecture, and chemical mechanism. Operational kinetic data have given indirect information about cellulolytic enzyme mode of action along with important information useful for modeling cellulose hydrolysis by specific cellulolytic enzyme systems. These data are based on measurement of initial rates of enzyme hydrolysis with respect to purified celluloses and their water soluble derivatives over a range of concentrations of both substrate and products. The resulting kinetic patterns facilitate definition of the enzyme s mode of action, kinetic equations, and concentration based binding constants. Since these enable the enzymes action to be defined with little direct knowledge of its mechanistic basis, the rate equations obtained are referred to as operational kinetics. The rate patterns have enabled mechanisms to be inferred, and these have often coincided with more direct observations of the enzyme s action on a molecular level [2-4]. 

Work needs to be done to find the specific activity of purified enzymes on the cellulose structure, especially the synergism between different cellulolytic enzymes. With this understood, one can then undertake the task of studying the endoglucanase-cellobiohydrolase system. This requires the identification of binding sites, reaction sites, and changes in cellulose structure as a result of the enzyme action. 

This is a preliminary report of our work. Our results to date indicate that cellulolytic enzymes may be found extracellularly, both in the culture medium and attached to the surface of hyphae, and, perhaps, intracellu-larly. In addition, they may be found in active and inactive forms in all places. The binding of the enzymes to the mycelium and the natural release mechanism is unknown. In order to investigate induction and repression mechanisms and the influence of chemical and environmental factors on the synthesis of cellulolytic enzymes, total enzyme activity must be measured. Further work along these lines is in progress. 

Lemos, M. A., Teixeira, J. A., Domingues, M. R. M., Mota, M., and Gama, F. M., The enhancement of the cellulolytic activity of cellobiohydrolase I and endoglucanase by the addition of cellulose binding domains derived from Trichoderma reesei. Enzyme Microbial Technol 2003, 32, (1), 35—40. 

Fig. 5. Modular nature of cellulolytic proteins. The enzyme illustrated (cellobiohydrolase) binds to the cellulose microfibril through the cellulose binding domain while the catalytic domain binds a single cellulose chain at a free end. Arrows representing the active site within the catalytic domain show the ability of cellobiohydrolase to remain bound to the substrate while the hydrolyzed product, cellobiose, is released. This allows the enzyme to progressively hydrolyze cellobiose from cellulose...

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