In studying the action of cellulases on cellulose, different model cellulosic substrates are employed, which exhibit different levels of crystallinity and accessibility to enzyme, two of the major parameters that contribute to the overall recalcitrance of the substrate. It should also be remembered that the procedures used for the preparation of these model cellulose substrates result in a form, which is very different from that of the native cellulose microfibrils within the plant cell wall.

The least crystalline substrates used for such studies are the soluble derivatized forms, such as carboxymethyl cellulose or hydroxymethyl cellulose, which, due to the substituted groups, are essentially non-crystalline in nature. The soluble, derivatized celluloses are used as a substrate for endoglucanases that can cleave along the cellulose chain, providing that the derivatization is not too extensive. Exoglucanases begin at the chain ends, and, upon meeting the substituent group, immediately come to a halt. Thus, such soluble derivatized substrates can be used to differentiate between the endo — and exo-acting enzymes. It should be noted that cellulases tend to exhibit a spectrum between the endo — and exo-modes of

cellulolytic activity; and several enzymes are known to display an endo-processive action — i. e., the initial steps of cleavage may be endo-acting whereby the enzyme binds in some manner to the internal portion of the cellulose chain, whereupon the enzyme continues to degrade the substrate processively.

The next level of crystallinity is exemplified by phosphoric acid-treated celluloses, some­times referred to as amorphous cellulose, sometimes acid-swollen cellulose. The crystallinity of the latter substrate depends on the conditions used for treatment and is usually very low, although residual levels of crystallinity may be observed. In any case, some enzymes, whether endo — or exo-acting, degrade this type ofsubstrate readily, whereas others do not. In this case, the exact mechanistic features of substrate-versus-enzyme that generate extensive degradation are not understood.

Cotton cellulose is about 45% crystalline and is sometimes used as a model plant-derived substrate. Avicel, a commercial preparation of microcrystalline cellulose, is commonly used as a model substrate for examination of cellulolytic enzyme systems. Avicel is also about 45% crystalline (131, 132), but exhibits heightened recalcitrance owing to its large particle size (due to the drying of the material during its preparation) and consequent inaccessibility of the inner cellulose chains. Bacterial cellulose ribbons, produced by the bacterium Acetobacter xylinum, are about 65% crystalline, and BMCC (bacterial microcrystalline cellulose) is prepared by acid hydrolysis of amorphous regions of the bacterial cellulose ribbons, thus resulting in a crystallinity of about 70%. By far, however, the most crystalline and recalcitrant form of cellulose is derived from the Valonia ventricosa cell wall, which is close to 100% crystalline (131).

Despite the rather high crystallinity of bacterial cellulose ribbons, the C. thermocellum cellulosome completely dissolves this substrate relatively rapidly, within a 24-hour period under the conditions of the assay (Figure 13.5). Under the same conditions, a sluggish but relentless degradation of the highly recalcitrant Valonia cellulose is achieved, reaching near completion only after a 16-daytime interval (133).

The modified morphologies of the different substrates canbe followed ultrastructurally by transmission electron microscopy, and the images provide insight into the mechanism and extent of degradation of recalcitrant substrates (134-137). Cellulosome-induced degrada­tion ofbacterial cellulose ribbons was indicative of a digestion pattern suggesting a concerted assault of different types of cellulases (e. g., combined endo — and exo-acting) on the cellulose substrate (Figure 13.6), consistent with the spatial proximity of the two types of enzymes in the cellulosomes (133). In this context, the observed cleavage of the cellulose ribbons is the signature of an endo mode of action, and the observed defibrillation of the substrate (see Figure 13.6B) corresponds to processive action associated with exo-acting cellulases. The residual bacterial cellulose, following 85% degradation (Figure 13.6C), bears no resemblance to the fine ribbons that were originally subjected to cellulosome action.

The images of partially degraded, highly recalcitrant Valonia cellulose show a rather dif­ferent picture (Figure 13.7). Unlike the digestion pattern observed for the bacterial cellulose ribbons, the degradation of Valonia microcrystals is accompanied by distinctive digestive features, including crystal thinning and pointed tips, reminiscent of previously characterized features during the degradation of this substrate by individual and combined fungal cellu — lases. The thinning feature was previously characterized as a function of processive action by the exo-acting fungal cellobiohydrolase I (Cel7A), whereas pointed tips were associated with the action of the less processive “unidirectional” digestion (i. e., vis-a-vis non-reducing to reducing or vice versa) of cellobiohydrolase II (Cel6A) (135, 138).

Interestingly, the micrograph in Figure 13.7B shows both types of features. Even though over 95% of the cellulose have been digested, the individual residual cellulose microcrystals often show both features and some are apparently unchanged from the original images. It seems as if the individual cellulosomes from the same batch exhibit a wide diversity in their mode of action, probably related to the inherent heterogeneity in their enzyme content. The persistence of pointed tips, indicates unidirectional processivity (135, 138). Many of the properties of the intact cellulosome seem to be analogous to those of this particular processive enzyme. The direction of cleavage of the family-48 enzymes is from the reducing to non-reducing ends (139). The persistence of intact Valonia cellulose crystals, even after near-complete digestion may indicate that the rate-determining step in its degradation is the initial attack, once consummated, the crystal undergoes rapid degradation. Indeed, the rate-limiting step of cellulose degradation has been considered to be the separation of the individual cellulose chains from the crystal lattice. Once exposed, the battery of enzymes can deal both with the separated chain as well as the void left in the crystal.