Cellulase is any of several enzymes produced chiefly by fungi, bacteria, and
protozoans that catalyze cellulolysis, the decomposition of cellulose and of
some related polysaccharides; specifically, the hydrolysis of the
1,4-beta-D-glycosidic linkages in cellulose, hemicellulose, lichenin, and
cereal beta-D-glucans. Cellulases break down the cellulose molecule into
monosaccharides such as beta-glucose, or shorter polysaccharides and
oligosaccharides. The name is also used for any naturally occurring mixture or
complex of various such enzymes, that act serially or synergistically to
decompose cellulosic material. Most mammals have only very limited
ability to digest dietary fibres such as cellulose, by themselves. Important
example are, the cellulase produced mainly by symbiotic bacteria in
ruminants like cattle and sheep and in hindgut fermenters like horses that
allows them to digest the cellulose from their grass diet. Cellulases are also
produced by a few other types of organisms, such as some termites.
Several different kinds of cellulases are known, which differ structurally and
mechanistically. Synonyms, derivatives, and specific enzymes associated with the
name "cellulase" include endo-1,4-beta-D-glucanase-beta-D-glucan
4-glucanohydrolase), carboxymethyl cellulase, avicelase, celludextrinase,
cellulase A, cellulosin AP, alkali cellulase, cellulase A 3, 9.5 cellulase,
and pancellase SS. Enzymes that cleave lignin are occasionally called
cellulases, but this is usually considered erroneous.
Types and action Five general types of cellulases based
on the type of reaction catalyzed: Endocellulases randomly cleave internal
bonds at amorphous sites that create new chain ends.
Exocellulases or cellobiohydrolases cleave two to four units from the ends
of the exposed chains produced by endocellulase, resulting in
tetrasaccharides or disaccharides, such as cellobiose. Exocellulases are further
classified into type I, that work processively from the reducing end of
the cellulose chain, and type II, that work processively from the nonreducing
end. Cellobiases or beta-glucosidases
hydrolyse the exocellulase product into individual monosaccharides.
Oxidative cellulases depolymerize cellulose by radical reactions, as for
instance cellobiose dehydrogenase. Cellulose phosphorylases depolymerize
cellulose using phosphates instead of water.
Avicelase has almost exclusively exo-cellulase activity, since avicel is
a highly micro-crystalline substrate. Within the above types there are also
progressive and nonprogressive types. Progressive cellulase will continue to
interact with a single polysaccharide strand, nonprogressive cellulase will
interact once then disengage and engage another polysaccharide strand.
Cellulase action is considered to be synergistic as all three classes of
cellulase can yield much more sugar than the addition of all three separately.
Aside from ruminants, most animals do not produce cellulase in their bodies
and can only partially break down cellulose through fermentation, limiting
their ability to use energy in fibrous plant material.
Structure Most fungal cellulases have a two-domain
structure, with one catalytic domain and one cellulose binding domain, that are
connected by a flexible linker. This structure is adapted for working on an
insoluble substrate, and it allows the enzyme to diffuse two-dimensionally on a
surface in a caterpillar-like fashion. However, there are also cellulases that
lack cellulose binding domains. These enzymes might have a swelling function.
= Cellulase complexes = In many bacteria, cellulases in-vivo are
complex enzyme structures organized in supramolecular complexes, the
cellulosomes. They contain roughly five different enzymatic subunits
representing namely endocellulases, exocellulases, cellobiases, oxidative
cellulases and cellulose phosphorylases wherein only endocellulases and
cellobiases participate in the actual hydrolysis of the β(1→ 4) linkage.
The cellulase complex from Trichoderma reesei, for example, comprises a
component labeled C1 that separates the chains of crystalline cellulose, an
endoglucanase, an exoglucanase, and a beta-glucosidase.
Numerous "signature" sequences known as dockerins and cohesins have been
identified in the genomes of bacteria that produce cellulosomes. Depending on
their amino acid sequence and tertiary structures, cellulases are divided into
clans and families. Mechanism of cellulolysis
Uses Cellulase is used for commercial food
processing in coffee. It performs hydrolysis of cellulose during drying of
beans. Furthermore, cellulases are widely used in textile industry and in
laundry detergents. They have also been used in the pulp and paper industry for
various purposes, and they are even used for pharmaceutical applications.
Cellulase is used in the fermentation of biomass into biofuels, although this
process is relatively experimental at present. Cellulase is used as a
treatment for phytobezoars, a form of cellulose bezoar found in the human
stomach. Measurement of cellulase
As the native substrate, cellulose, is a water-insoluble polymer, traditional
reducing sugar assays using this substrate can not be employed for the
measurement of cellulase activity. Analytical scientists have developed a
number of alternative methods. = Viscometric substrates =
A viscometer can be used to measure the decrease in viscosity of a solution
containing a water-soluble cellulose derivative such as carboxymethyl
cellulose upon incubation with a cellulase sample. The decrease in
viscosity is directly proportional to the cellulase activity. While such
assays are very sensitive and specific for endo-cellulase, they are limited by
the fact that it is hard to define activity in conventional enzyme units.
= Cellooligosaccharide substrates = The lower DP cello-oligosaccharides are
sufficiently soluble in water to act as viable substrates for cellulase enzymes.
However, as these substrates are themselves 'reducing sugars', they are
not suitable for use in traditional reducing sugar assays because they
generate a high 'blank' value. However their cellulase mediated hydrolysis can
be monitored by HPLC or IC methods to gain valuable information on the
substrate requirements of a particular cellulase enzyme.
= Reduced cellooligosaccharide substrates =
Cello-oligosaccharides can be chemically reduced through the action of sodium
borohydride to produce their corresponding sugar alcohols. These
compounds do not react in reducing sugar assays but their hydrolysis products do.
This makes borohydride reduced cello-oligosaccharides valuable
substrates for the assay of cellulase using traditional reducing sugar assays
such as the Nelson-Symogyi method. = Dyed polysaccharide substrates =
These substrates can be subdivided into two classes-
Insoluble chromogenic substrates: An insoluble cellulase substrate such as
AZCL-HE-cellulose absorbs water to create gelatinous particles when placed
in solution. This substrate is gradually depolymerised and solubilised by the
action of cellulase. The reaction is terminated by adding an alkaline
solution to stop enzyme activity and the reaction slurry is filtered or
centrifuged. The colour in the filtrate or supernatant is measured and can be
related to enzyme activity. Soluble chromogenic substrates: A
cellulase sample is incubated with a water-soluble substrate such as
azo-CM-cellulose, the reaction is terminated and high molecular weight,
partially hydrolysed fragments are precipitated from solution with an
organic solvent such as ethanol or methoxyethanol. The suspension is mixed
thoroughly, centrifuged, and the colour in the supernatant solution is measured.
With the aid of a standard curve, the enzyme activity can be determined.
= Enzyme coupled reagents = Recently, new reagents have been
developed that allow for the specific measurement of endo-cellulase. These
methods involve the use of functionalised oligosaccharide
substrates in the presence of an ancillary enzyme. In the example shown,
a cellulase enzyme is able to recognise the trisaccharide fragment of cellulose
and cleave this unit. The ancillary enzyme present in the reagent mixture
then acts to hydrolyse the fragment containing the chromophore or
fluorophore. The assay is terminated by the addition of a basic solution that
stops the enzymatic reaction and deprotonates the liberated phenolic
compound to produce the phenolate species. The cellulase activity of a
given sample is directly proportional to the quantity of phenolate liberated
which can be measured using a spectrophotometer. The acetal
functionalisation on the non-reducing end of the trisaccharide substrate
prevents the action of the ancillary β-glucosidase on the parent substrate.
References Further reading
See also Cellulose 1,4-beta-cellobiosidase, an
efficient cellulase ^ Zafar M, Ahmed S, Khan MI, Jamil A.
"Recombinant expression and characterization of a novel
endoglucanase from Bacillus subtilis in Escherichia coli.". Mol Biol Rep. 41:
3292–302. doi:10.1007/s11033-014-3192-8. PMID 24493451.