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Cellulase is any of several
enzymes Enzymes () are protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery Kendrew in 1958 ...
produced chiefly by
fungi A fungus (plural The plural (sometimes abbreviated An abbreviation (from Latin ''brevis'', meaning ''short'') is a shortened form of a word or phrase, by any method. It may consist of a group of letters, or words taken from the full ...

fungi
,
bacteria Bacteria (; common noun bacteria, singular bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of prokaryotic microorganisms. Typ ...

bacteria
, and
protozoan Protozoa (singular protozoon or protozoan, plural protozoa or protozoans) is an informal term for a group of single-celled eukaryote Eukaryotes () are organism In biology, an organism () is any organic, life, living system that ...
s that
catalyze that utilizes a low-temperature oxidation catalyst to convert carbon monoxide to less toxic carbon dioxide Carbon dioxide (chemical formula ) is a colorless gas with a density about 53% higher than that of dry air. Carbon dioxide molecules ...

catalyze
cellulolysis Cellulose is an organic compound with the chemical formula, formula , a polysaccharide consisting of a linear chain of several hundred to many thousands of glycosidic bond, β(1→4) linked glucose, D-glucose units. Cellulose is an important struc ...
, the decomposition of
cellulose Cellulose is an organic compound In chemistry Chemistry is the study of the properties and behavior of . It is a that covers the that make up matter to the composed of s, s and s: their composition, structure, properties, behavior ...

cellulose
and of some related
polysaccharide Polysaccharides (), or polycarbohydrates, are the most abundant carbohydrate A carbohydrate () is a biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallograp ...
s. 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
monosaccharide Monosaccharides (from Greek#REDIRECT Greek Greek may refer to: Greece Anything of, from, or related to Greece Greece ( el, Ελλάδα, , ), officially the Hellenic Republic, is a country located in Southeast Europe. Its population is ap ...
s ("simple sugars") such as beta-
glucose Glucose is a simple with the . Glucose is the most abundant , a subcategory of s. Glucose is mainly made by and most during from water and carbon dioxide, using energy from sunlight, where it is used to make in s, the most abundant carbohydr ...

glucose
, or shorter polysaccharides and
oligosaccharide An oligosaccharide (/ˌɑlɪgoʊˈsækəˌɹaɪd/; from the Greek ὀλίγος ''olígos'', "a few", and σάκχαρ ''sácchar'', "sugar") is a saccharide is a disaccharide A disaccharide (also called a double sugar or ''biose'') is the sug ...
s. 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 Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution Substitution may refer to: Arts and media *Chord substitution, in music, swapping one chord fo ...

hydrolysis
of the 1,4-beta-D-
glycosidic linkage A glycosidic bond or glycosidic linkage is a type of covalent bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate. A glycosidic bond is formed between the hemiacetal or hemiketal group ...
s in cellulose,
hemicellulose A hemicellulose (also known as polyose) is one of a number of heteropolymer, heteropolymers (matrix polysaccharides), such as arabinoxylans, present along with cellulose in almost all embryophyte, terrestrial plant cell walls.Scheller HV, Ulvskov H ...

hemicellulose
,
lichenin Lichenin, also known as lichenan or moss starch, is a complex glucan A glucan is a polysaccharide derived from D-glucose, linked by glycosidic bonds. Many beta-glucans are medically important. They represent a drug target for antifungal medicatio ...
, and cereal beta-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 fermentation, hindgut fermenters like horses, cellulases are produced by symbiotic bacteria. Endogenous cellulases are produced by a few types of metazoan 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 Glycoside hydrolase family 9, 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.Guerriero G, Sergeant K, Legay S. Hausman J-F, Cauchie H-M, Ahmad I, Siddiqui KS. 2018 Novel insights from comparative in silico analysis of green microalgae cellulases. Int. J. Mol. Sci. 19 (6), 1782. 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 have occasionally been called cellulases, but this old usage is deprecated; they are lignin-modifying enzymes.


Types and action

Five general types of cellulases based on the type of reaction catalyzed: *Endoglucanase SS, Endocellulases (EC 3.2.1.4) randomly cleave internal bonds at amorphous sites that create new chain ends. * Exocellulases or cellobiohydrolases (EC 3.2.1.91) 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 3.2.1.21) 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 food energy, 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 (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 beta-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. Cellulases produced by the species Coprinopsis Cinerea consists of seven protein strands in the shape of an enclosed tunnel called a beta/alpha barrel. 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.


Cellulase complexes

In many bacteria, cellulases in-vivo are complex enzyme structures organized in Supramolecular assembly, 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 dalton (unit), daltons) that separates the chains of crystalline cellulose, an endoglucanase (about 52,000 daltons), an exoglucanase (about 61,000 dalton), and a beta-glucosidase (76,000 daltons).Worthington Biochemical Corporation (2014)
Cellulase
Accessed on 2014-07-03
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.


Mechanism of cellulolysis


Uses

Cellulase is used for commercial food processing in coffee. It performs
hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution Substitution may refer to: Arts and media *Chord substitution, in music, swapping one chord fo ...

hydrolysis
of cellulose during drying of coffee bean, 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. Medically, Cellulase is used 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).


Measurement

As the native substrate,
cellulose Cellulose is an organic compound In chemistry Chemistry is the study of the properties and behavior of . It is a that covers the that make up matter to the composed of s, s and s: their composition, structure, properties, behavior ...

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).


Cellooligosaccharide substrates

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 Ion chromatography, 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 (due to small, soluble, dyed fragments) 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 (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.


See also

* Cellulose 1,4-beta-cellobiosidase, an efficient cellulase * Cellulase unit, a unit for quantifying cellulase activity


References


Further reading

* * The Merck Manual of Diagnosis and Therapy, Chapter 24 * * {{Glycoside hydrolases Enzymes Cellulose