Model of cellulase enzyme, produced by T. fusca, based on PDB structure 1JS4
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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 ("simple sugars") 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.
An important example is the cellulase produced mainly by symbiotic bacteria in the ruminating chambers of herbivores, that allows them to digest the cellulose from their vegetable 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-1,4-glucanase, beta-1,4-endoglucan hydrolase, endoglucanase D, 1,4-(1,3,1,4)-beta-D-glucan 4-glucanohydrolase), carboxymethyl cellulase (CMCase), 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 (EC 188.8.131.52) randomly cleave internal bonds at amorphous sites that create new chain ends.
- Exocellulases or cellobiohydrolases (EC 184.108.40.206) 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 (EC 220.127.116.11) or beta-glucosidases hydrolyse the exocellulase product into individual monosaccharides.
- Oxidative cellulases depolymerize cellulose by radical reactions, as for instance cellobiose dehydrogenase (acceptor).
- 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 (also known as processive) 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 (including humans) 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.
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 (mostly endoglucanases) that lack cellulose binding domains. These enzymes might have a swelling function.
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 (57,000 daltons) that separates the chains of crystalline cellulose, an endogucanase (about 52,000 daltons), an exoglucanase (about 61,000 dalton), and a beta-glucosidade (76,000 daltons).
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
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.
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.
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 (exo-acting cellulase enzymes produce little or no change in viscosit, they are limited by the fact that it is hard to define activity in conventional enzyme units (micromoles of substrate hydrolyzed or product produced per minute).
The lower DP cello-oligosaccharides (DP2-6) 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. Howeverm 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.
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 (due to small, soluble, dyed fragments) is measured. With the aid of a standard curve, the enzyme activity can be determined.
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