Page 63 - Advances in bioenergy (2016)

cellulolytic enzymes during the breakdown of crystalline cellulose, but convincing
explanations at the molecular level have yet to be found, especially for the cooperation
between cellobiohydrolases. For example, the cooperation between endo- and exo-acting
cellulases is extensively described in literature, however, the degree of synergy observed is
usually less than that expected for a system in which the cellobiohydrolases depend entirely on
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β-1,4-endoglucanases for the provision of free chain ends. Cellobiohydrolases are critical to
the function of fungal systems, as, for example, Cel7A from Chrysosporium lucknowense
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comprises in excess of 50% of the cellulases secreted by this organism. However, the poor
understanding of the role of cellobiohydrolases is indicated by a recent investigation showing
that the deletion of two major cellobiohydrolases of C. thermocellum does not affect
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substantially cellulose breakdown. Moreover, cellulolytic systems may lack the pair of
cellobiohydrolases that act from the reducing and nonreducing ends of cellulose chains. 44,45
For example, the cellulose-degrading bacterium Cytophaga hutchinsonii does not contain
GH6, GH48, and GH7 cellobiohydrolases, although it is possible that C. hutchinsonii contains
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novel-type cellobiohydrolases. Recently, a complete cellulolytic system was reported to
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occur in the marine bacterium Saccharophagus degradans 2–40. The genome sequence of
this bacterium predicts that this organism does not contain any known processive
cellobiohydrolase genes and has 13 endocellulase genes of which 9 belong to a new class of
processive enzymes members of GH5 family. These processive endo-acting cellulases do not
require an additional domain for processivity, such as CBMs, suggesting that their processivity
may result from unusual subsite affinities that need to be determined by structural

crystallography. 47

Most of the cellulases, discovered to date, are modular proteins that in addition to their
catalytic domain, exhibit accessory domains such as CBMs, which are connected to the
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catalytic part with a flexible linker. The main role of these accessory modules is helping the
binding of cellulolytic enzymes on the polymer of cellulose, although CBMs might also
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participate in the initial disruption of cellulose fibers. It is considered that β-1,4-
endocellulases which exhibit CBMs, are keen on binding on the amorphous regions of
cellulose fibers creating accessible ends, whereas cellobiohydrolases continue hydrolysis by
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removing oligosaccharides from the ends of the disrupted polymer. Moreover, the
processivity of most endoglucanases is dependent upon their associated CBM modules, for
example, several bacterial GH9 endoglucanases depend upon the resident CBM3 module. 51

Anaerobic bacteria, such as Clostridium and Ruminococcus spp., or fungi such as
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Chytridomycetes have developed complexed cellulase systems called ‘cellulosomes’. The
assembly and spatial organization of enzymes in naturally occurring cellulosomes, which are
regarded as nanomachines for the efficient cellulose degradation, constitute the base of their
synergistic activity. In most cases, these complexes are bound to the cell surface; however, this
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does not seem to be true for all cellulosome biosynthesizing microorganisms. The
cellulosome architecture consists of multiple enzyme subunits, with different substrate
specificities and catalytic mechanisms, organized by scaffolding proteins. 52,53 Cellulosome
hydrolases, apart from the catalytic module, always contain at least one supplementary domain,