Cellulases

For the engineer seeking to improve upon the natural process of converting plant biomass to fermentable sugars, the key challenge is to make cell wall depolymerization a more rapid and less costly process. The cost of biomass ethanol production has been reduced dramatically over the past two decades, to the point where the fuel is now competitive for the blending market, but further processing cost-reduction opportunities have been identified that would make it competitive as a pure fuel without subsidies (Lynd et al. 1996). Because the cost of producing the enzymatic catalysts proposed in the SSF process is a critical issue, the available enzymatic activity must be maximized to effectively incorporate cellulases into these process schemes. This requirement can be met by ensur­ing that the enzymes used are obtainable at minimal cost and of the highest specific activity, the highest possible stability, and optimal in terms of pH and temperature tolerance.

While cellulosic biomass is produced at a rate of nearly 3 x 109 tons per year and represents 50% of all available biomaterial, the biologically mediated depolymerization of this resource has eluded clear, precise definition at the molecular level. Although the biological depoly­merization of native plant matter requires a suite of glycoside hydrolases aided by chemical or mechanical conditioning, in many ways this problem is primarily one that focuses on the enzymes that act on cellulose. Many workers in the field agree that cellulose decrystallization and depolymerization are indeed the rate-limiting steps in biomass conversion (Himmel et al. 2007).

Hemicellulose removal by dilute acid treatment is a classical means of rendering biomass more amenable to cellulase action (Grohmann et al. 1985). Kong and coworkers (Kong et al. 1993) also showed that biomass with reduced acetylation responded significantly more favor­ably than native biomass to cellulase action. Biomass with reduced lignin content, or perhaps altered chemistry, appears to be more readily hydrolyzed by cellulases (Vinzant et al. 1997; Kristensen et al. 2007). The structural and reactive chemical features of the substrate (primar­ily defined as acetyl and lignin contents) can be pictured as controlling the accessibility of enzyme to cellulose; the degree of cellulose crystallinity can be visualized as controlling the hydrolytic rate (Jeoh et al. 2007).

The definitive enzymatic degradation of cellulose to glucose in fungi and most bacteria is generally accomplished by the synergistic action of three distinct classes of enzymes:

• The “endo-1,4-P-glucanases” or 1,4-P-D-glucan 4-glucanohydrolases (EC 3.2.1.4), which act randomly on soluble and insoluble 1,4-P-glucan substrates and are commonly measured by detecting the reducing groups released from carboxymethylcellulose (CMC). A relatively new subset of this class of cellulase, called “processive endoglu — canases,” has recently been classified (Reverbel-Leroy et al. 1997; Wilson et al. 1998).

• The “exo-1,4-P-D-glucanases,” including both the 1,4-P-D-glucan glucohydrolases (EC 3.2.1.74), which liberate D-glucose from 1,4-P-D-glucans and hydrolyze D-cellobiose slowly, and 1,4-P-D-glucan cellobiohydrolase (EC 3.2.1.91), which liberates D-cellobiose from 1,4-P-glucans.

• The “P-D-glucosidases” or P-D-glucoside glucohydrolases (EC 3.2.1.21), which act to release D-glucose units from cellobiose and soluble cellodextrins, as well as an array of glycosides.

Cross-synergism between endo — and exo-acting enzymes isolated from the same or differ­ent species, genera, or microbial families has been demonstrated many times (Wood and McCrae 1979; Coughlan et al. 1987; Eveleigh 1987). Exo-exo synergism was first reported in 1980 (Fagerstam and Pettersson 1980). It is currently believed that exo-endo synergism is explained best in terms of providing new sites of attack for the exoglucanases. The latter enzymes normally find available cellodextrin “ends” at the reducing and nonreducing termini of cellulose microfibrils. Random internal cleavage of surface cellulose chains by endoglu — canases provides numerous additional sites for attack by cellobiohydrolases. Therefore, each hydrolytic event by an endoglucanase yields both a new reducing and a new nonreducing site. Thus, logical consideration of catalyst efficiency dictates the presence of exoglucanases specific for reducing termini and nonreducing termini. The principle of interspecies inter­changeability of cellulase components is now the cornerstone of recombinant cellulase system design and construction. If indeed cellulase component enzymes are truly generalized in both structure and function, components can be selected and combined from a wide array of source organisms to form novel enzyme cocktails. For example, Trichoderma reesei cellobiohydro — lase I (CBH I) has been shown to be a powerful element in multi-enzyme mixtures using either fungal or bacterial endoglucanases (Baker et al. 1998).