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. Cellulose breakdown is of considerable economic importance, because it makes a major constituent of plants available for consumption and use in chemical reactions. Because cellulose molecules bind strongly to each other, cellulolysis is relatively difficult compared to the breakdown of other polysaccharides such as starch. Most mammals have only very limited ability to digest dietary fibres such as cellulose importance of tertiary structure of enzymes themselves. Several different kinds of cellulases are known, which differ structurally and mechanistically.
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. Avicelase has almost exclusively exo-cellulase activity, since avicel is a highly micro-crystalline substrate. 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.
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. Both binding of substrates and catalysis depend on the three-dimensional structure of the enzyme which arises as a consequence of the level of protein folding. The amino acid sequence and arrangement of their residues that occur within the active site, the position where the substrate binds, may influence factors like binding affinity of ligands, stabilization of substrates within the active site and catalysis. The substrate structure is complementary to the precise active site structure of enzyme. Changes in the position of residues may result in distortion of one or more of these interactions. Additional factors like temperature, pH and metal ions influence the non-covalent interactions between enzyme structure.
The enzyme is categorised as an endoglucanase, which internally cleaves β-1,4 -glycosydic bonds in cellulose chains facilitating further degradation of the polymer. Different species in the same family as T. Maritima make cellulases with different structures. These enzymes hydrolyse the substrate carboxymethyl cellulose. Binding of the substrate in the active site induces a change in conformation which allows degradation of the molecule. In many bacteria, cellulases in-vivo are complex enzyme structures organized in supramolecular complexes, the cellulosomes. The number of sub-units making up cellulosomes can also determine the rate of enzyme activity. The three types of reaction catalyzed by cellulases:1. Furthermore, cellulases are widely used in textile industry and in laundry detergents.
Analytical scientists have developed a number of alternative methods. The decrease in viscosity is directly proportional to the cellulase activity. 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. 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.
With the aid of a standard curve, the enzyme activity can be determined. 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 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. What is the rate limiting enzyme in gluconeogenesis cellulase activity of a given sample is directly proportional to the quantity of phenolate liberated which can be measured using a spectrophotometer.