Cellulaza ферменттері және олардың маңызы

Class of enzymes

Cellulase (; systematic name 4 β D glucan 4 glucanohydrolase) is any of several enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharides:

Endohydrolysis of (1→4) β D glucosidic linkages in cellulose, lichenin and cereal β D glucan

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. Cellulases break down the cellulose molecule into monosaccharides ("simple sugars") such as β glucose, or shorter polysaccharides and oligosaccharides. Cellulose breakdown is of considerable economic importance, because it makes a major constituent of plants available for consumption and use in chemical reactions. The specific reaction involved is the hydrolysis of the 1,4 β D glycosidic linkages in cellulose, hemicellulose, lichenin, and cereal β D glucans. 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 like cellulose by themselves. In many herbivorous animals such as ruminants like cattle and sheep and hindgut fermenters like horses, cellulases are produced by symbiotic bacteria. Endogenous cellulases are produced by a few types of animals, such as some termites, snails, and earthworms. Recently, cellulases have also been found in green microalgae (Chlamydomonas reinhardtii, Gonium pectorale and Volvox carteri) and their catalytic domains (CD) belonging to GH9 Family show highest sequence homology to metazoan endogenous cellulases. Algal cellulases are modular, consisting of putative novel cysteine rich
carbohydrate binding modules (CBMs), proline/serine (PS) rich linkers in addition to putative Ig like and unknown domains in some members. Cellulase from Gonium pectorale consisted of two CDs separated by linkers and with a C terminal CBM. 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 β D glucanase (β 1,4 glucanase, β 1,4 endoglucan hydrolase, endoglucanase D, 1,4 (1,3;1,4) β D glucan 4 glucanohydrolase), carboxymethyl cellulase (CMCase), avicelase, celludextrinase, cellulase A, cellulosin AP, alkali cellulase, cellulase A 3, 9.5 cellulase, celloxylanase and pancellase SS. Enzymes that cleave lignin have occasionally been called cellulases, but this old usage is deprecated; they are lignin modifying enzymes.

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. 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 Thermotoga maritima species make cellulases consisting of 2 β sheets (protein structures) surrounding a central catalytic region which is the active site. 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. They can contain, but are not limited to, five different enzymatic subunits representing namely endocellulases, exocellulases, cellobiases, oxidative cellulases and cellulose phosphorylases wherein only exocellulases and cellobiases participate in the actual hydrolysis of the β(1→4) linkage. The number of sub units making up cellulosomes can also determine the rate of enzyme activity. Multidomain cellulases are widespread among many taxonomic groups, however, cellulases from anaerobic bacteria, found in cellulosomes, have the most complex architecture consisting of different types of modules. For example, Clostridium cellulolyticum produces 13 GH9 modular cellulases containing a different number and arrangement of catalytic domain (CD), carbohydrate binding module (CBM), dockerin, linker and Ig like domain. The cellulase complex from Trichoderma reesei, for example, comprises a component labeled C1 (57,000 daltons) that separates the chains of crystalline cellulose, an endoglucanase (about 52,000 daltons), an exoglucanase (about 61,000 dalton), and a β glucosidase (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. Multimodular cellulases are more efficient than free enzyme (with only CD) due to synergism because of the close proximity between the enzyme and the cellulosic substrate. CBM are involved in binding of cellulose whereas glycosylated linkers provide flexibility to the CD for higher activity and protease protection, as well as increased binding to the cellulose surface. details of beta glucosidase activity of cellulase]]

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 in medicine as a treatment for phytobezoars, a form of cellulose bezoar found in the human stomach, and it has exhibited efficacy in degrading polymicrobial bacterial biofilms by hydrolyzing the β(1 4) glycosidic linkages within the structural, matrix exopolysaccharides of the extracellular polymeric substance (EPS).

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. DNSA Method Cellulase activity was determined by incubating 0.5 ml of supernatant with 0.5 ml of 1% carboxymethylcellulose (CMC) in 0.05M citrate buffer (pH 4.8) at 50 °C for 30 minutes. The reaction was terminated by the addition of 3 ml dinitrosalicylic acid reagent. Absorbance was read at 540 nm. 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 viscosity), 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. 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.

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.

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 (β glucosidase) 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.