In native cellulolytic organisms, enzymes needed for cellulose hydrolysis — xylanase, endoglucanase, exoglucanase, and ^-glucosidase — are expressed either separately or in complexes called cellulosomes (Fig 2). Noncomplexed cellulase systems are characteristic of cellulolytic aerobic bacteria (such as Bacillus spp.) and fungi (such as Trichoderma spp.) (Lynd et al. 2002). Endoglucanase hydrolyzes amorphous cellulose randomly, leading to the formation of cello-oligosaccharides of varying chain length. Exoglucanases are highly selective enzymes and act on either the reducing or the nonreducing end of cello — oligosaccharides to liberate glucose or cellobiose, respectively. ^-Glucosidase hydrolyzes cellobiose into its glucose monomers (Lynd et al. 2002). Cellobiose inhibits both exoglucanase and endoglucanase. Hence, ^-glucosidase plays an important role in the overall process, because it prevents the accumulation of cellobiose (Shewale 1982).

Anaerobic bacteria such as Clostridium spp. usually produce complexed cellulases called cellulosomes. In cellulosomes, individual enzymes attach to a scaffoldin with their dockerin domains, while exposing the cellulose-binding domain. This complex enables proper synergy among endoglucanase, exoglucanase, and ^-glucosidase. (Bayer et al. 1998). Several chimeric scaffoldins have been engineered to position enzymes of higher activity together, and thereby increase the overall hydrolysis efficiency (Fig 2B) (Wen et al. 2009). Even though the large size of the cellulosomes restricts them to only the most readily accessible regions of cellulose, cellulosomes can hydrolyze cellulose more efficiently than free cellulases can (Wilson 2009).

Engineering efforts to increase the efficiency of cellulases and to enhance their kinetic properties have focused mainly on improving the specific activity by improving the thermal or the pH stability of the enzymes (Wen et al. 2009). However, a more important parameter to consider is the efficiency of access to the cellulose interior. While the active — site plays an essential role in other hydrolytic enzymes, the cellulose-binding domain constitutes the key module for cellulases (Bayer et al. 1998). In fact, the cellulose-binding domain determines the type of cellulase. Several efforts to establish a kinetic model for cellulose hydrolysis have failed because of the heterogeneous nature of the cellulosic substrate and the need for multiple enzyme activities (Kadam et al. 2004). In addition to enzyme-substrate proximity, enzyme-enzyme synergy should be considered as a factor for the efficient hydrolysis of cellulose. Whether any relationship or correlation between the crystallinity of lignocellulose and the rate of enzymatic hydrolysis exists remains unclear (Zhang and Lynd 2004). Moreover, the mechanism of cellulose hydrolysis remains incompletely understood, because some groups of cellulases have both exoglucanase and endoglucanase activities.

The low processivity of cellulases demands that the enzymes be replenished several times during the saccharification process. The economic feasibility of enzymatic hydrolysis of lignocellulose to simple sugars is limited by the poor kinetic properties of the enzymes. The use of cellulase-secreting microbes could be an economical alternative to the enzymatic saccharification process. With microbes, the enzymes can be continuously produced, secreted, and used to hydrolyze cellulose into simple sugars that could be directly fermented to ethanol (Fig 3). Thus, microbial fermentation of lignocellulose offers greater promise for economical bioethanol production.