Cellulolytic Enzyme Production and Enzymatic Hydrolysis for Second-Generation Bioethanol Production - Biotechnology in China: Biofuels and Bioenergy - page 8

after treatment with purified CBD of CBH I from Penicillium janthinellum; this
suggests the dissociation of small fibrils from cellulose assisted by the CBD [ 32 ],
and agrees with the hypothesis that the CBD disrupts the regional hydrogen net-
work of crystalline cellulose. The ''free'' polysaccharide chain released from this
CBD-assisted disruption is then recognized by the CD and subsequently enters its
catalytic tunnel. Four cellobiose units enter the tunnel, where cleavage of the
glycosidic bond takes place [ 35 , 36 ]. The energy released from this reaction is used
in changing the enzymatic structure, causing the enzyme molecule to move for-
ward along the polysaccharide chain, and compressing the linker [ 37 ]. Further
cleavage results in further compression of the linker and eventually results in the
dissociation of the CBD region from the polysaccharide chain despite the tight
bond between the three Tyr residues at its active site and the pyranoid rings on the
polysaccharide chain [ 38 , 39 ]. The CBD then moves forward by four cellobiose
units (the length of the linker), rebinds with the polysaccharide chain, and
again disrupts the regional hydrogen-bond network for further digestion by the CD
[ 31 , 40 ]. This clear intramolecular synergism between the CBD, the CD, and the
linker helps the CBH I molecule crawl along the polysaccharide chain for its
complete digestion. A dual function is proposed for the CBD in this model:
binding and, consequently, increasing the regional concentration of CBH I on the
cellulose surface, and disrupting the hydrogen-bond network and, subsequently,
the crystalline surface [ 29 ]. Indeed, real-time observations of the movement of
CBH I from T. reesei and its CD along cellulose were made using a high-speed
atomic force microscope; the similar movement rates and therefore the hydrolysis
rates of the holoenzyme and the CD suggest that the CBD does not participate in
catalysis but rather helps increase the regional enzyme concentration on the cel-
lulose surface [ 41 ]. The role of the CBD in the disruption of the hydrogen-bond
network could not be identified in these experiments, but a role cannot be ruled out
either because unlike the highly crystalline and highly pure cellulose from green
algae used in this investigation, the ''regular'' substrates for CBH are cellulose
from plants in a more complicated environment. It was suggested that the product
of cellulose degradation by CBH I, cellobiose, is an inhibitor of CBH I. This
inhibition mechanism was investigated, and it was found that the binding of cel-
lobiose to the active site of CBH I prevents further reactions, and that cellobiose
induces conformational change in CBH I, thereby decreasing its activity [ 42 ].
This model explains a phenomenon long observed by scientists: the rate of
cellulose hydrolysis by cellulases drops drastically during extended digestion.
CBH I only hydrolyzes the one polysaccharide chain residing on the surface of
the crystalline cellulose because binding of the CBD is required for disruption
of the hydrogen-bond network. The changes in the packing and arrangement of
microfibrils during later stages of hydrolysis lead to inhibition of CBH activity, as
observed by Wang et al. [ 28 ].
This model for cellulose biodegradation is complicated in itself, but further evi-
dence suggests that this may not be the full story. Ma et al. [ 43 ] showed that the CBH I
from T. reesei is not uniformly adsorbed onto cellulose; some enzyme molecules are
reversibly bound to cellulose, but the binding is irreversible for others. It was further