9
highly crystalline regions of cellulose where EGs are unable to bind productively.
Thus they are able to synergize with glycosyl hydrolases, likely as endo-acting
enzymes that act directly on the surface of crystalline cellulose. It is now known that
LPMOs require a reducing agent and molecular oxygen and a copper ion in the
active site (Payne et al. 2015). The electron donor can also be a co-secreted enzyme
like cellobiose dehydrogenase (CDH), the only known example of a secreted flavocytochrome (Dimarogona et al. 2012).
1.2.1.6 Mechanism of Cellulose Hydrolysis by Cellulases
With more and more studies on cellulase action being undertaken, it is now becoming clearer that our understanding of cellulose hydrolysis is probably not complete,
and there are paradigms not yet characterized. However, decades of research in this
field have given insights into a generally appreciated mechanism of action, and
recent discoveries like that of the LPMOs have improved that understanding. The
following mode of hydrolysis is a summary of what is currently accepted as the
mechanism of cellulose breakdown by cellulases. Cellulose structure is complex
with crystalline array of cellulose microfibrils with glucan chains interlinked
through hydrogen bonds. There are regions of disorder in the arrangement of glucan
chains along the cellulose microfibrils, which are called the amorphous regions. The
biomass-degrading enzymes work at the solid liquid interfaces, which implies that
a high concentration of catalytic units is required at the surface for efficient hydrolysis of the polymer. This is achieved by the unique adaptation of several of the
endo- and exoglucanases in having a three-domain structure with a carbohydrate
binding module (CBM) which attaches to the cellulose surface, a catalytic module
which does the actual hydrolysis, and a linker which serves mobility and also aids
the enhanced binding of the enzyme to the cellulose surface. A detailed description
of the structural features of cellulose-hydrolyzing enzymes is beyond the scope of
this chapter and the readers are directed to Payne et al. (2015) for a comprehensive
discussion on this topic. The typical three-domain structure aids in the processivity
of the exoglucanases and endoglucanases that possess this structure. Processive
enzymes are those which catalyze consecutive reactions without release of their
substrate, and in the cases of cellulases with this modular structure, they help to
keep the catalytic domain near the substrate (Teeri et al. 1998).
On recognition of a free chain end, the cellulase threads the chain into the tunnel
(exoglucanase) in the catalytic domain of the enzyme to form a catalytically active
complex (CAC). Hydrolysis occurs following a retaining or an inverting mechanism
(Davies and Henrissat 1995) depending on the type of enzyme and the product is
expelled. The processive cycle is continued with multiple events of hydrolysis
before finally dissociating from the chain and reinitiating the processive cycle at a
new site (Payne et al. 2015). The processive mechanism for exocellulase-mediated
cellulose hydrolysis is represented in Fig. 1.2.
In the case of endoglucanases, the processive cycle is different in that the chain
threading and product expulsion are omitted. The binding site of endoglucanase has
a cleft instead of a tunnel, which allows chain acquisition without threading. It is
now known that cellulose hydrolysis by the exoglucanases proceeds by movement
1 Enzymes for Bioenergy
highly crystalline regions of cellulose where EGs are unable to bind productively.
Thus they are able to synergize with glycosyl hydrolases, likely as endo-acting
enzymes that act directly on the surface of crystalline cellulose. It is now known that
LPMOs require a reducing agent and molecular oxygen and a copper ion in the
active site (Payne et al. 2015). The electron donor can also be a co-secreted enzyme
like cellobiose dehydrogenase (CDH), the only known example of a secreted flavocytochrome (Dimarogona et al. 2012).
1.2.1.6 Mechanism of Cellulose Hydrolysis by Cellulases
With more and more studies on cellulase action being undertaken, it is now becoming clearer that our understanding of cellulose hydrolysis is probably not complete,
and there are paradigms not yet characterized. However, decades of research in this
field have given insights into a generally appreciated mechanism of action, and
recent discoveries like that of the LPMOs have improved that understanding. The
following mode of hydrolysis is a summary of what is currently accepted as the
mechanism of cellulose breakdown by cellulases. Cellulose structure is complex
with crystalline array of cellulose microfibrils with glucan chains interlinked
through hydrogen bonds. There are regions of disorder in the arrangement of glucan
chains along the cellulose microfibrils, which are called the amorphous regions. The
biomass-degrading enzymes work at the solid liquid interfaces, which implies that
a high concentration of catalytic units is required at the surface for efficient hydrolysis of the polymer. This is achieved by the unique adaptation of several of the
endo- and exoglucanases in having a three-domain structure with a carbohydrate
binding module (CBM) which attaches to the cellulose surface, a catalytic module
which does the actual hydrolysis, and a linker which serves mobility and also aids
the enhanced binding of the enzyme to the cellulose surface. A detailed description
of the structural features of cellulose-hydrolyzing enzymes is beyond the scope of
this chapter and the readers are directed to Payne et al. (2015) for a comprehensive
discussion on this topic. The typical three-domain structure aids in the processivity
of the exoglucanases and endoglucanases that possess this structure. Processive
enzymes are those which catalyze consecutive reactions without release of their
substrate, and in the cases of cellulases with this modular structure, they help to
keep the catalytic domain near the substrate (Teeri et al. 1998).
On recognition of a free chain end, the cellulase threads the chain into the tunnel
(exoglucanase) in the catalytic domain of the enzyme to form a catalytically active
complex (CAC). Hydrolysis occurs following a retaining or an inverting mechanism
(Davies and Henrissat 1995) depending on the type of enzyme and the product is
expelled. The processive cycle is continued with multiple events of hydrolysis
before finally dissociating from the chain and reinitiating the processive cycle at a
new site (Payne et al. 2015). The processive mechanism for exocellulase-mediated
cellulose hydrolysis is represented in Fig. 1.2.
In the case of endoglucanases, the processive cycle is different in that the chain
threading and product expulsion are omitted. The binding site of endoglucanase has
a cleft instead of a tunnel, which allows chain acquisition without threading. It is
now known that cellulose hydrolysis by the exoglucanases proceeds by movement
1 Enzymes for Bioenergy