There are 14 cellulase families listed on the CAZy web site: (http://afmb. cnrsmrs. fr/CAZY/ fam/acc_GH. html). Several of these families, 10, 26, 51, 74, mainly contain other types of glycosyl hydrolases, with only a few members having cellulase activity but, even if these families are excluded, there are still ten cellulase families. The enzymes in any given family show significant sequence homology with some or all of the other family members. All members of a glycosyl hydrolase family have the same basic protein fold and utilize the same catalytic mechanism but their substrate specificities can be quite different. Because there is a wide range of amino acid sequences that can give the same protein fold, several families share the same fold, even though the sets of sequences in each family show little similarity between the families.

There are nearly twice as many cellulase families, as are present in the next largest group of hydrolases, the seven xylanase families. Furthermore, there are seven different protein folds among the known cellulase structures, and a structure is not yet known for one family. There are two possible reasons why there is so much cellulase diversity. One is that the actual substrate of most cellulases is not pure cellulose but rather plant cell walls, which are extremely diverse and complex, containing many other components, some of which are bound to the cellulose fibrils (21). The other reason is that cellulose itself is quite complex with both crystalline and amorphous regions. It appears that cellulases are under positive selection, as when the DNA and protein sequences of two related cellulase genes were compared there were nearly as many DNA changes that caused an amino acid change (nonsynonymous) as there were DNA changes that did not change the amino acid (synonymous) (22).

There are three functionally different types of cellulases: endocellulases, also called en — doglucanases, exocellulases, also called cellobiohydrolases, and processive endocellulases, which were discovered later (23). To completely hydrolyze cellulose to glucose, a fourth enzyme, p glucosidase, is required, which hydrolyzes the soluble oligosaccharides produced by the cellulases to glucose. Many aerobic fungi secrete a p glucosidase as part of their crude cellulase, while most cellulolytic aerobic bacteria do not, and their p glucosidases are usually cytoplasmic. Some organisms, mainly anaerobic bacteria, contain cellobiose phosphorylase, also called dextrin phosphorylase, which converts cellobiose and soluble dextrins to glucose and glucose-1-phosphate, conserving the energy in the cellobiose linkage (24). All endo — cellulase CDs, whose structures have been determined, have an open active site, as would be expected, since they are able to bind to the interior of long cellulose molecules (25). In contrast, all exocellulases have their active sites in a tunnel, consistent with their processive activity (26). In the case of glycosyl hydrolase family GH-48 enzymes, only part of the active site is in the tunnel, but these enzymes are just as processive as family GH-7 enzymes, where the entire active site is in the tunnel (27). There are two classes of exocellulases (28); one class attacks the nonreducing end of a cellulose molecule and all known members of this class are

Glc(+1)

Thermobifida fusca Cel6AD117A

Figure 11.1 Model of the three-dimensional structures of the catalytic domains of the endocellulase, T fusca Cel6A and the exocellulase, T. reeseiCel6A.

in family GH-6. Members of the other class attack the reducing end of a cellulose chain and all aerobic fungal members of this class are in family GH-7, while the bacterial members are in family GH-48 (see Figure 11.1). It is interesting that the anaerobic fungal members of this class are in family GH-48, rather than in family GH-7 (29). All exocellulases act processively, sequentially cleaving cellobiose residues from a cellulose molecule, so that they are also called cellobiohydrolases. It has been claimed that the T. reesei exocellulase, Cel6A, can act as an endocellulase and that is the reason it can synergize with T. reesei exocellulase, Cel7A (30); however, it has been shown that all of the hydrolysis in a synergistic mixture of these two enzymes results from exocellulolytic activity (20).

There are a number of claims in the literature that specific enzymes are exocellulases, when they are actually endocellulases. In particular, Clostridium thermocellum, CBHA (31) is clearly an endocellulase, as shown by the open active site seen in its X-ray structure (32) and this was confirmed by a set of assays, which showed that it behaved like an endocel — lulase in three different assays: higher activity on CMC then other substrates, reducing the viscosity of CMC and producing 40% insoluble reducing sugars from filter paper, while exocellulases produce from 5 to 8% insoluble reducing sugars from filter paper (33). An­other example is Cel6A from the anaerobic rumen fungus, Neocallimastix patriciarum, which has very high activity relative to other family GH-6 exocellulases but not relative to family GH-6 endocellulases (34). By all the above tests, this enzyme turned out to be a true endocellulase (Wilson, D. B., unpublished). It is often stated that an enzyme is an exocellulase because it produces cellobiose as its major soluble product, but this is true of many endocellulases. Some workers have claimed that only exocellulases have activity on para-nitrophenyl-^-cellobioside but that is not true, as many endocellulases hydrolyze this substrate.

All well-documented processive endocellulases are in family G-9, which is the largest cel­lulase family and includes most plant cellulases, animal cellulases, many bacterial cellulases

and surprisingly, very few fungal cellulases (see Figure 11.2). Processive endoglucanases have an open active site cleft like all endocellulases but in addition they contain a family 3 CBM, which is rigidly attached to the C-terminus of the CD (35). The two domains are oriented so that a cellulose chain can bind simultaneously to both domains. The family 3c CBMs, that are present in processive endocellulases, differ from families 3a and 3b CBMs, in that they lack the conserved aromatic residues, which cause the high affinity for cellulose. Although the 3c CBMs bind very weakly to cellulose, it has been shown that they are necessary for the processive activity of these enzymes (36).

There do not appear to be major differences between the CD families of the cellulases present in cellulosomes and the families of cellulases secreted by aerobic microorganisms, as most cellulase families contain cellulases from both types of microorganisms. However, all known GH-7 cellulases are from aerobic fungi or termites, and there are no known GH-6 cellulases produced by anaerobic bacteria. Furthermore, all GH-12 cellulases appear to be produced by aerobic microorganisms, but this is currently a small family that only contains endocellulases. At this time, all known GH-48 cellulases are exocellulases and this is the only cellulase family that does not contain endocellulases.

There have been a number of studies that analyzed the properties of the cellulose that remained after significant hydrolysis had occurred by a pure cellulase, to try to identify the preferred sites of attack for that cellulase; i. e., amorphous or crystalline regions, as well as how the enzyme has changed the average chain length of the cellulose. A study of T reesei Cel7A, an exocellulase, and T. reesei Cel7B, an endocellulase, found as expected that Cel7A did not cause large changes in the cellulose chain length while Cel7B did (37). A study of four Cellulomonasfimi cellulases acting on Sigma cellulose found that Cel5A rapidly reduced the chain length but Cel6A had a lesser effect on chain length, even though it also is an en — docellulase. Both enzymes increased the crystallinity of the residual cellulose, suggesting that they preferentially degrade amorphous regions in the cellulose. The two exocellulases tested, Cel6B and Cel48A, had no effect on chain length and Cel6B increased crystallinity of the residual cellulose while Cel48A decreased its crystallinity (38). Four synergistic mixtures were tested and none of them caused significant differences in crystallinity. Another study of comparable enzymes from T. fusca showed that both the endocellulase Cel5A and the ex — ocellulase Cel6B primarily digested amorphous cellulose, while the processive endocellulase Cel9A digested both types of cellulose (39).