Environmental Engineering Reference
In-Depth Information
In natural systems, these enzymes work synergistically to rapidly break down cellulose into
glucose for metabolism. Endoglucanases cleave cellulosic bonds at random, mostly amorphous
regions in the cellulose chain, thereby producing more chain ends for cellobiohydrolase attachment.
Cellobiohydrolases produce cellobiose and some cellotriose from mostly crystalline regions of
cellulose, attaching to chain ends and working progressively along the cellulose chain. The product
of this action, cellobiose, is a strong inhibitor of cellobiohydrolase activity. β-glucosidases work
on the soluble cellobiose and cellotriose products, breaking them into glucose monomers. All
of these enzymes can be produced as complexed enzyme (attached to the surface of the cell) or
noncomplexed (released into the environment) according to species (Lynd et al. 2002). To function
effectively, cellulolytic enzymes must be able to associate with the insoluble substrate, disrupt the
structure, and guide a single polymer chain through the catalytic domain (Eijsink et al. 2008).
Cellulase activity can be determined on several different substrates, natural and artificial,
each with its own advantages and disadvantages. Table 8.7 provides a summary of many of
the potential substrates. Filter paper is readily available and has a well-established assay
method. Avicel can be used to effectively measure exoglucanase activity because it is largely
crystalline with a low degree of polymerization. Carboxymethylcellulose (CMC) can be used
to measure endoglucanase activity, but methylation blocks progressive action of exoglucanases.
Lignocellulosic biomass is a heterogeneous matrix of diverse linkages of polysaccharides and
aromatic compounds, and hydrolysis of such biomass is much more complex than for pure
cellulose. Activities determined for enzymes on pure cellulose do not necessarily correlate with
activities on biomass (King et al. 2009).
taBle 8.7
substrates containing
β
-1,4-Glucosidic Bonds hydrolyzed by cellulases
and their detection
substrate
detection
enzymes
Soluble
Short chain (low DP cellodextrins
radiolabeled cellodextrins
b-methylumbelliferyl-oligosaccharides
β-nitrophenol-oligosaccharides)
CMC, HEC
Dyed CMC
RS, HPLC, TLC
TLC plus liquid scintillation
Fluorophore liberation, TLC
Chromophore liberation, TLC
RS, viscosity
Dye liberation
Endo, Exo, BG
Endo, Exo, BG
Endo, Exo, BG
Endo, Exo, BG
Endo
Endo
Insoluble
Crystalline
Cotton, microcrystalline cellulose (Avicel,
Valonia cellulose, bacterial cellulose
Amorphous cellulose
PASC, alkali-swollen cellulose, RAC
Dyed cellulose
Fluorescent cellulose
Chromogenic and fluorephoric derivatives
TNP-CMC
Fluram-cellulose
Pretreated biomass
RS, TSS, HPLC
Total, Endo, Exo
RS, TSS, HPLC, TLC
Dye liberation
Fluorophore liberation
Total, Endo, Exo
Total, Endo
Total
Endo
Endo, Total
Total
Chromophore liberation
Fluorophore liberation
HPLC, RS
Source:
Adapted from Zhang, Y-HP., Himmel, ME., and Mielenz, JR.,
Biotechnol Adv
, 24, 452-481,
2006.
RS, reducing sugars; TSS, total soluble sugars; HPLC, high-performace liquid chromatography; TLC,
thin layer chromatography; HEC, hydroxyethylcellulose; PASC, phosphoric acid swollen cellulose;
RAC, regenerated amorphous cellulose.
