CBMs play an important role in the ability of cellulases to degrade crystalline cellulose. They have little or no effect on the activity of most cellulases on a soluble cellulose derivative, CMC, amorphous cellulose or oligosaccharides (40). One role of a CBM is to anchor the cellulase to the insoluble cellulose, so that the CD remains close to the substrate. The flexible linker that separates the CBM from the CD allows the CD to access regions of the cellulose, adjacent to the bound CBM. Some workers have proposed that a CBM can also disrupt the structure of cellulose, making it more accessible to the CD (41, 42) but this is still controversial (43). This activity would be equivalent to the Cx activity proposed by Reese, in his early discussion of the nature of cellulases (44). CBMs have also been reported to target cellulases to specific regions of cellulose, presumably to the regions where they will be most active (45). It has been shown that family 2 CBMs can diffuse on the surface of cellulose without dissociation; giving them the ability to readily access new regions of a cellulose particle after the CD has hydrolyzed cellulose near the original site of binding (46). Other CBMs that bind to crystalline cellulose may also have this ability, since they appear to bind in a similar way.

There are many CBM families with 45 listed on the CAZy web site (http://afmb. cnrs- mrs. fr/CAZY/fam/acc_CBM. html). Not all CBMs bind cellulose, as many families con­tain chitin-binding domains, xylan-binding domains, or mannose-binding domains. Some CBMs can bind to several polymers, while others are specific for only one. Labeled CBMs are being used to stain plant materials and different members of a given family can give very different staining, showing that there is even greater binding specificity than is seen with pure substrates (47). Almost all known enzymes that have high activity on insoluble substrates contain a substrate-binding domain, in addition to a CD, so that the presence of such a domain is a general property of this type of enzyme.

All fungal cellulase CBMs are in family I and they are small, containing about 30 AA. Most aerobic bacterial cellulase CBMs are in family 2 and they are larger, containing about 120 AA. The CBMs on cellulosomal scaffoldins are in family 3. Most of the CBMs in these three families bind to crystalline cellulose and have a relatively flat-binding surface that usually contains three aromatic residues spaced, so they can bind to three adjacent glucose residues in a cellulose molecule. They also contain a number of residues, which can hydrogen bond to the cellulose chain, but site-directed mutagenesis has shown that the aromatic residues are essential for high affinity binding, while the other residues play a secondary role (48). The CBMs in families 4 and 6 bind to single cellulose molecules and their binding sites are in a groove (49). A number of cellulases contain multiple CBMs and in some cases they are from the same family and in other cases they are from different families. It has been shown that the affinity of a protein containing two CBMs can be significantly higher than one with only one CBM (50). Atomic force microscopy of the binding of a family I CBM to cellulose found that the bound CBM was present in aggregates, not as single domains (49).

Binding of a cellulase to cellulose is an important step in hydrolysis and there have been many studies of cellulase binding (50-54). Cellulases that contain a CBM bind tightly to cellulose, but cellulase CDs bind cellulose weakly. Mutations that dramatically reduce activity can lead to enhanced CD binding, showing that the cleavage products bind more weakly than the intact substrate. The extent of binding is directly related to the accessible surface area of the substrate, and accessible surface area also determines the rate of hydrolysis of a substrate. Much of the surface area of cellulose is in the interstitial region between microfibrils of cellulose (irregularly shaped pores), so that the size of a cellulase will affect how much of the cellulose surface area is available for its binding. There is good evidence that binding to cellulose does not fit the Langmuir isotherm, and in many cases binding appears to be irreversible (53). At low protein concentrations, several family 2 CBMs were shown to bind reversibly and in this region binding might be to the external surface, but at higher extents of binding, where binding might be occurring in pores, it was irreversible (53). In a study where the binding of a mixture of two cellulases was studied, synergism in binding was observed in several mixtures (54).