Cellulose Hydrolysis Mechanisms
As described in the introductory section in Chapter 4, cellulose is a linear polysaccharide consisting of D-anhydroglucopyranose joined together by ^-1,4-glycosidic bonds with a degree of polymerization (DP) typically in the range of 100 to 14,000 [7-9]. Cellulose hydrolysis mechanism is a very widely studied subject, and the most accepted mechanism for enzymatic cellulose hydrolysis involves synergistic actions by all three major types of cellulases: endocellu — lase (endoglucanase) (EC 3.2.1.4), exoglucanase or cellobiohydrolase (EC 3.2.1.91), and в-glucosidase (EC 3.2.1.21) [9-13]. Endocellulases hydrolyze accessible intramolecular ^-1,4-glycosidic bonds of cellulose chains randomly to produce new chain ends; exoglucanases or cellobiohydrolase processively cleave cellulose chains at the ends to release soluble cellobiose, tetrasaccharides or glucose. The two cel — lobiohydrolases CBHI and CBHII work from opposite ends of the
cellulose chain. CBHI works processively from the reducing end, and CBHII works processively from the nonreducing end of cellulose. Then, P-glucosidases hydrolyze cellobiose to individual D-glucose molecules, and this will help to eliminate cellobiose inhibition.
This proposed mechanism is shown in Figure 6.3, where three hydrolysis processes occur simultaneously acting on cellulose in the solid and on soluble oligosaccharides in the liquid phase. After the initial adsorption of enzymes onto the surface of solid cellulose, the primary hydrolysis occurs on the surface of solid substrates releasing soluble sugars with a degree of polymerization (DP) up to 6 into the liquid phase upon hydrolysis by endocel — lulases and exoglucanases. The rate-limiting or slow steps for the whole process is the enzymatic depolymerization step performed by endocellulases and cellobiohydrolases types of cellulases. The liquid phase hydrolysis involves mainly the hydrolysis of the disaccharide cellobiose to D-glucose by P-glucosidases. Even though some P-glucosidases also hydrolyze longer cellodextrins [9], these reactions are known as secondary hydrolysis processes.
Cellulolytic systems can be associated into multienzymatic complexes (called cellulosomes) or unassociated as individual enzymes. In both cases, enzymes have a modular structure. The unassociated enzymes generally consist of a catalytic domain responsible for the hydrolysis reaction and of a cellulose-binding domain (CBD) mediating binding of the enzymes to the cellulose structure. The two domains are joined by a linker peptide, which must be sufficiently long and flexible to allow efficient orientation and operation of both domains.
The multienzymatic complexes or cellulosome are bound noncovalently to the cellulosome-integrating protein, and these modules are known as carbohydrate-binding module (CBM) to reflect the carbohydrate binding specificity of these complexes [14]. A number of carbohydrate-binding modules (CBM) have now been identified experimentally and several hundred presumed CBMs can be further identified on the basis of amino acid similarity.
Understanding the structural basis by which carbohydrate-binding module bind to their target ligands provides novel insights into the mechanisms of carbohydrate recognition and their mechanism of action. Currently 64 defined families of carbohydrate-binding modules (CBM) are known [15]. In general, carbohydrate-binding modules have three roles with respect to the function of their similar catalytic modules: (1) a proximity effect, (2) a targeting function, and (3) a disruptive function. In spite of the information available on these complex carbohydrate binding modules and on the structure of plant cell walls, application of this knowledge to cellulose degradation has met with limited success in understanding the complete hydrolysis process [16].