Page 61 - Advances in bioenergy (2016)

buried in pockets or caves; therefore, these enzymes have little or no activity on polymeric
cellulose. 34

Cellulases belong to at least eight unrelated protein folds (Table 2.1), which are diffentiated
into even more protein families in the CAZy database. For instance, Cel44A endoglucanase
from Clostridium thermocellum (PDB: 2E0P) has an (β/α) -fold belonging to family GH44,
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sharing the same topology with other cellulases including cellobiohydrolases and β-
glucosidases, members of completely different GH families. Recently, an endoglucanase from
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C. thermocellum (CtCel124) that is distantly related to lytic transglycosylases initially
positioned in family GH23, introduced a novel GH family 124 that exhibits only members with
endoglucanase activity. As cellulases belong to many different GH families, although having
different structural characteristics, we can presume that these enzymes are representatives of a
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large class of nonhomologous isofunctional enzymes. This means that these enzymes catalyze
the same reaction, while having evolved independently and are unrelated in structure and
sequence. Therefore, cellulases from each GH family must be treated as independent cases in
any type of genomic analysis, resulting in a huge increase of the amount of data analysis. 36

In literature, two are the main classical mechanisms leading to the inversion or retention of the
anomeric carbon configuration. Transglycosylation events are restricted to retaining enzymes.
The stereochemistry of catalysis is dictated by the spatial arrangement of the catalytic groups
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in the active site of the enzyme. Enzymatic glycoside hydrolysis normally requires the
presence of two catalytic groups, which in most cases have been observed to be either
aspartate or glutamate residues. A novel cellulase fold was determined for a C. thermocellum
cellulosomal enzyme that showed low activity by itself but acted synergistically with other
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cellulases. It is surprising that there is no evidence for a catalytic base, although it has a
catalytic acid. It seems that the limiting step for the hydrolysis of crystalline cellulose is not the
bond cleavage but the way that cellulose molecule binds into the active site of the catalytic
domain of cellulase, leading to the complete understanding of the important residues that
participate in the binding step. 37

Synergistic Interaction

Multiple enzymatic activities are required in cellulose hydrolysis leading to soluble sugars that
can be metabolized by fermenting microorganisms. These activities include β-1,4-
endoglucanases, exoglucanases including D-cellodextranases and cellobiohydrolases and,
finally, β-glucosidases (Figure 2.2). Endoglucanases randomly cut internal sites on amorphous
cellulose surfaces, generating new chain ends. Cellobiohydrolases act in a processive manner
on the reducing or nonreducing ends of cellulose and liberate cellobiose as major product. β-
Glucosidases hydrolyze soluble cellodextrins and cellobiose to D-glucose and thus relieve the
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system from end product inhibition. Cellodextrinases hydrolyze soluble
cellooligosaccharides, producing cellobiose but exhibit little or no activity against insoluble
cellulose or carboxymethylcellulose (CMC). In comparison with many other GHs that act on
natural substrates, cellulases are known to interact on crystalline cellulose showing low
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catalytic activity. For example, several endoglucanases and cellobiohydrolases from H.