J Polym Environ
In recent years there has been significant research into the
enzymes that degrade CA. Non-derivatized cellulose can
be hydrolyzed very efficiently by the cellulolytic enzyme
system consisting of several cellobiohydrolases (CBH),
acting from the reducing and non-reducing end of the
cellulose chain, and endoglucanases (EG) which acts randomly within the chain. This enzyme system is being used
commercially for the production of 2nd generation ethanol
from lignocellulosic material.
Cellobiohydrolases are able to attack amorphous and
crystalline cellulose regions, but are rather sensitive to all
types of cellulose substituents, including acetyl groups, due
to the fact that the active site of this enzyme class is in a
tunnel formed by stable surface loops [32]. The tunnelshaped active site restricts the hydrolysis to occurring at the
ends of the cellulose chain. Because hydrolysis continues
progressively, it may be stopped at the first substitution
encountered. Indeed cellobiohydrolases are believed to be
unable to attack cellulose derivatives, and the existence of
the active site inside a tunnel could explain that cellulose
derivatives would get stuck, depending on their size and
their charge (Fig. 3).
Cellobiohydrolase I (CBHI) cleaves ß-1,4-glycosidic
bonds with retention of configuration, yielding the ß-anomer as reaction product [33]. It starts its attack from the
reducing end, whereas cellobiohydrolase II (CBHII),
attacks the cellulose chain from the non-reducing end.
CBHII inverts the configuration at the glycosidic bond, so
that the first-formed cellobiose is the a-anomer [34]. Taking the structure of cellobiohydrolases into account it was
no wonder that a mixture of pure CBHI:CBHII:EGI:EGII = 6:2:1:1 was unable to degrade CA DS 2.5, since
acetyl cleaving enzymes were not present in this artificial
mixture [35]. However, 78% of cellophane and 43% of
unbleached kraft pulp were hydrolyzed within 6 h.
In contrast to exo-acting cellobiohydrolases, endoglucanases are only able to degrade disordered, amorphous
regions of cellulose, cutting at internal glycosidic bonds
[36]. However, advantageously their active site is more
open arranged and placed in a cleft, allowing a random
hydrolysis of the cellulose chain [37] (Fig. 4). Correspondingly endoglucanase-initiated hydrolysis of cellulose
derivatives is possible, the extent not only being dependent
on the degree of substitution, but also on the substituent
distribution along the cellulose chain [38] and the architecture of the enzyme protein [39]. For an effective
hydrolysis of CA, endoglucanase would be the most
favored especially if non-substituted or partly substituted
anhydroglucose units are present (Table 2).
In a careful study, the ability of TR Cel7B (endoglucanase I from Trichoderma reesei) to fragment CA of
increasing DS was described by Saake et al. [40]. The
degree of CA substitution ranged from 0.9 to 2.9, and the
degree of polymerization varied from 16,000 to 201,000.
Fig. 3 Water-soluble CA (DS 0.7) oligosaccharides (* DP12) after
incubation with cellobiohydrolases (CBH) of Trichoderma reesei, as
analyzed by anion exchange chromatography in NaOH medium
combined with pulsed amperometric detection. Upper left CBH I
alone. Upper right CBH I and CBH II acting together from reducing
and non-reducing end. Bottom left CBH II alone. Bottom right
Reference substrate. Retention time (RT) of substrates and products:
Glucose = 3 min; cellobiose = 5 min; cellotriose = 7 min; cellotetraose = 10 min; cellopentaose = 12 min; cellohexaose = 14 min;
celloheptaose = 15 min; cellooctaose = 16 min; cellononaose =
18 min; cellodecaose = 19 min; cello-undecaose = 20 min; cellododecaose (DP12) = 21 min
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