The binding of the cellulosome to microcrystalline cellulose has been suggested in the past to be mediated directly via the scaffoldin-based, cellulose-binding CBM (37, 38). In the original description of the cellulosome from C. thermocellum, a mutant was isolated that lacked cell-surface cellulosomes and consequently failed to bind to the substrate (27, 108). Since the scaffoldin is anchored to the cell surface via the various anchoring scaffoldins, the same CBM appears to be responsible for binding the entire bacterial cell to cellulosic substrates, as will be described below (109). Nevertheless, the C. thermocellum cellulosome also includes numerous enzymes that also bear family-3 CBMs (93, 95) that can potentially supplement the cellulose-binding role of the scaffoldin-based CBM.

The recombinant, scaffoldin-derived, cellulose-binding family-3 CBM was also shown to bind chitin but not xylan (110). The CBM binds to celluloses of very high crystallinity — even to cellulose of the highest known crystallinity, namely, from the cell walls of the al­gae, Valonia ventricosa. It also binds strongly to amorphous forms of cellulose, including phosphoric acid swollen cellulose of minimal crystallinity. The very high capacity to bind to the amorphous cellulose (111) likely reflects increased accessibility to the binding sites, rather than a preference for amorphous regions. In fact, the comparative crystal structures of different families of cellulose-binding CBMs suggest that, in each case, an aromatic strip of amino acids on the flat surface of the CBM molecule mediates strong binding with the glucose chain of the hydrophobic face of the cellulose surface (112). Additional hydrogen­bonding interactions between hydrophilic amino acids and polar groups on neighboring glucose chains are thought to provide additional protein-cellulose contacts that stabilize the strong interaction. Docking analyses thus suggest that a patch along three adjacent cellulose chains interacts with the flat surface of the CBM molecule, which indicates why cellobiose and other cellodextrins fail to inhibit CBM binding to the insoluble substrate (110).

The C. thermocellum genome includes several other, enzyme-borne family-3 CBMs that exhibit most or all of the latter proposed binding residues (113-115), all of which would presumably bind strongly to microcrystalline cellulose. However, the latter five or six en­zymes, lack dockerins and are thus free, non-cellulosomal enzymes. In addition, another sub-class of family-3 CBMs, termed CBM3c, is fused to a GH9 catalytic module. This type of family-3 CBM is modified in the same surface residues that are considered to bind crystalline cellulose, and the CBM3c is believed to assist the adjacent catalytic module in binding to structural intermediates of the substrate or to alter the mode of activity (113,116,117). The CBM3c fails to bind strongly to crystalline cellulose substrates, and thus plays an ancillary role in breakdown of the cellulose chain by the catalytic module. The balance of the other dockerin-containing, enzyme-borne CBMs lack several but not all of the cellulose-binding residues. In some cases (118,119), cellulose-binding properties have been reported, but their contribution to the primary strong recognition of crystalline cellulose by the cellulosome, as observed for the scaffoldin-based family-3 CBM, remains unclear.

The binding of the cellulosome to cellulose is inhibited by low ionic strength, and water can be used to release at least part of a cellulosome or CBM preparation from the cellu­lose matrix (111). Increasing the salt content of the medium increases the binding of the cellulosome to the substrate. Interestingly, maximal enzymatic activity of the cellulosome complex was observed at suboptimal conditions of adherence to the substrate. At low salt concentrations, the low enzymatic activity most likely reflects the lack of sufficient adsorp­tion, whereas above-optimum concentrations of salt, the reduced activities may be due to restricted mobility of the cellulosome under such conditions. Once attached, the cellulosome seems to remain static, as shown by laser bleaching experiments (unpublished results). A micrograph of cellulosomes bound to the surface of the super-crystalline Valonia cellulose is shown in Figure 13.3. Note the apparent cellulosome-induced stripping of the microfibrils from the surface of the cellulose crystal.

The organization of enzymes into cellulosomes ensures their multiple, concerted at­tachment to the substrate surface. This arrangement affords a distinct advantage over the distribution of free cellulases, since it allows multiple hydrolysis and facilitates access of additional appropriate cellulases and other enzymes (e. g., hemicellulases, pectate lyases, carbohydrate esterases) at or near the cleavage sites for enhanced processing of the sub­strate. The proximity ofcomplementary enzymes at the cellulose surface provides a suitable remedy for counteracting substrate recalcitrance, as will be further discussed below.