CELLULASE

CELLULASE

beta-cellotriose = 61788-77-0 = D-(+)-Cellotriose

CAS: 9012-54-8
European Community (EC) Number: 232-734-4
Molecular Formula: C18H32O16
Molecular Weight: 504.4
IUPAC Name: (2S,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6S)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2R,3S,4R,5R,6R)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol

Cellulases are extremely important enzymes both industrially and in the natural world, because they play a major role in the global carbon cycle by degrading insoluble cellulose to soluble sugars. 
Cellulases are the most diverse class of enzymes that catalyze the hydrolysis of a single substrate, because there are seven different protein folds among the ten true cellulase families. There are three types of cellulases, endoglucanases, exocellulases, and processive endoglucanases, which have different modes of action and different structures. 
In common with other enzymes that hydrolyze insoluble substrates, most cellulases contain a substrate-binding domain and a catalytic domain (CD). 
Certain cellulases act synergistically on crystalline cellulose, with the specific activity and extent of hydrolysis of the mixture being much higher than that of the sum of those properties for the enzymes acting alone. 
There are three different strategies used by cellulolytic microorganisms to degrade the cellulose in plant cell walls: secretion of a synergistic set of free cellulases, production of multienzyme complexes called cellulosomes, and an unknown mechanism that does not require processive cellulases, which are major components in the other two mechanisms.

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. 
The name cellulase 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 beta-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-beta-D-glycosidic linkages in cellulose, hemicellulose, lichenin, 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 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 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-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.

Five general types of cellulases based on the type of reaction catalyzed:

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 energy in fibrous plant material.

Cellulases are a complex group of enzymes which are secreted by a broad range of microorganisms including fungi, bacteria, and actinomycetes. 
In the natural environment, synergistic interactions among cellulolytic microorganisms play an important role in the hydrolysis of lignocellulosic polymer materials.
In fact, it is the combined action of three major enzymes which determines the efficiency of this process. 
They are exoglucanases, endoglucanases, and β-glucosidase. Microorganisms produce these enzymes in a diverse nature which determines their efficiency in cellulose hydrolysis. 
During the cellulose degradation reaction, the enzyme targets the β-1,4-linkages in its polymeric structure. 
This is an essential ecological process as it recycles cellulose in the biosphere. 
The application of this same scenario for industrial purposes is identified as an emerging area of research. 
Biofuel production, textile polishing and finishing, paper and pulp industry, and lifestyle agriculture are among the key areas where cellulase enzyme shows a broader potential. 

Cellulase is not a single enzyme. 
Cellulase is a group of enzymes which is mainly composed of endoglucanase and exoglucanases including cellobiohydrolases and β-glucosidase. 
Fungi, bacteria, and actinomycetes are recorded to be efficient cellulase enzyme producers in the natural environment. 
These microorganisms must secrete cellulases that are either free or cell surface bound. 
Their enzyme production efficiency and the enzyme complex composition are always diverse from each other.
Although both aerobic and anaerobic microorganisms produce these enzymes, aerobic cellulolytic fungi, viz., Trichoderma viride and T. reesei, are excessively studied. 
The enzyme breaks β-1,4-linkages in cellulose polymer to release sugar subunits such as glucose. 
This notion is applied in industries either cellulose is utilized as a raw material or cellulose degradation is a must.

According to recent enzyme market reports, the key areas of the industry where cellulase enzyme is increasingly being applied are healthcare, textile, pulp and paper, detergent, food, and beverages. 
Cellulase's wide application in coffee processing, wine making, and fruit juice production is related to food and beverage segment. 
In other industrial applications, it is broadly used to produce laundry detergents and cleaning and washing agents. 
Cellulase is also being highly recognized as an effective alternative to available antibiotics for treatment of biofilms produced by Pseudomonas. 
Therefore, the potential of cellulases to fight against antibiotic-resistant bacteria is an amazing trend which will overcome problems in the healthcare sector.

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.

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 beta-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.

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.

Cellulase enzymes account for a significant shore of the world enzyme market. 
Trichoderma reesei is the major fungus for industrial cellulase production. 
Its genome encodes 10 cellulases and 16 hemi-cellulases.
It produces two exoglucanases (CBH I and CBH II), about eight endoglucanases (EGI–EGVIII), and seven β-glucosidases (BG I–BG VII). 
Cellulases are inducible enzymes and the regulation of the cellulase production is finely controlled by activation and repression mechanism. 
The production of cellulolytic enzyme is induced only in presence of the substrate, and is repressed when easily utilizable sugars are available. 
This happens with the help of fine-tuned cooperation of the transcription factors. 

Cellulases hydrolyze cellulose under mild conditions compared to inorganic or organic acid. 
Generally, cellulases like CBH II consist of core and cellulose-binding domains and a linker that binds the two domains. 
The core domain contains an active center to hydrolyze cellulose in a catalytic manner and the subsites that interact with cellulose chain close to the active center. 
The cellulose-binding domains consist of amino acids having aromatic rings such as tyrosine or tryptophan, and these aromatic rings of the cellulose-binding domains attach to hydrophobic plains of cellulose chains through van der Waals force. 
The active center of the core domains of cellulases can then attack the cellulose chain. 
The subsites of the core domains can give some mechanical stress to the cellulose chain, and one of the glucose residues of the cellulose chain is forced to have the unstable boat form. 
Thus, remarkable reduction of activation energy to hydrolyze cellulose can be achieved.

Microbial cellulases have a wide range of potential applications in biotechnology. 
In several applications, they are used with supplement of hemicellulases, pectinases, ligninases and associated enzymes. 
In addition to lignocellulosic bioenergy, some most important applications of cellulases are in food, brewery and wine, animal feed, textile and laundry, pulp and paper industries, as well as in agriculture and many more. 
Humans lack the ability to digest cellulose fiber hence a digestive enzyme “Digestin” that contains cellulase has been commercialized. 
Cellulases have been applied successfully in textile wet processing and finishing of cellulose based textile to improve final quality of the products. 
Microbial cellulases and polysaccharides play important roles in fermentation processes to produce alcoholic beverages including beer and wine.
Uses of cellulases improve both quality and yield of the fermented products, hence, cellulases are supplemented during mashing or preliminary fermentation to hydrolyze glucan that help to reduce viscosity of wort and improve the filterability. 
In wine industry, cellulases, hemicellulases and pectinases have been adopted since their use improves color extraction, skin maceration, clarification and filterability and finally the quality of wine.
Malting of barley, preparation of grape juice for wine production and several other processes use cellulases derived from T. reesei. 
Cellulases and hemicellulases are immensely useful in animal feed to improve feed value by pretreatment of agricultural silage and grains by cellulases and hemicellulases. 
Application of these enzymes eliminates antinutritional factors of feed grains, enhance feed quality and also provide supplementary digestive enzymes such as proteases, amylases, glucanases and many more. 
In agricultural field, to control various crop diseases and pest, to enhance crop growth, mixture of cellulases, hemicellulases and pectinases has been used. 
The cellulolytic fungi T. reesei play a major role in agricultural industry by facilitating enhanced seed germination, plant growth and ultimately enhance crop yield. 
Trichoderma reesei cellulases are also widely used in detergent industry and waste management.

Cellobiohydrolases and endocellulases consist of a signal peptide that mediates secretion, a hinge region which is rich in Pro, Thr and Ser residues, a cellulose binding domain, and a catalytic domain. 
N- and O-glycosylated proteins are present in catalytic domain and hinge region, respectively. 
The bacterial and fungal cellulases usually consist of two or more functional and structural domains which are connected by a peptide linker. 
In aerobic organisms, cellulose binding domain binds to a catalytic domain whereas, dockerin domain joins to the catalytic domain in anaerobic organisms. 
Fungal cellulases consist of cellulose binding module (CBM) and a catalytic domain (CD); CBM is connected through a short polylinker region. 
In fungal cellulases, catalytic binding domain is comprised of less than 40 amino acid residues which also includes three conserved aromatic residues. 
According to the International Union of Biochemistry and Molecular Biology Enzyme Nomenclature, bacterial cellulases are grouped into EC 3.2.1.4 and are included in fourteen Glycosil Hydrolase (GH) families. 
Higher growth rates and genetic versatility of bacteria, emphasize the advantages and suitability of bacterial cellulases over fungal sources, although many fungal cellulases are commercially available.

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. 
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).

Interest in the application of cellulases in the pulp and paper industry has increased considerably during the last decade. 
The mechanical pulping processes such as refining and grinding of the woody raw material lead to pulps with high content of fines, bulk, and stiffness. 
While in contrast, biomechanical pulping using cellulases resulted in substantial energy savings (20–40%) during refining and improvements in hand-sheet strength properties.

Mixtures of cellulases (endoglucanases I and II) and hemicellulases have also been used for biomodification of fiber properties with the aim of improving drainage and beatability in the paper mills before or after beating of pulp. 
While endoglucanases have the ability to decrease the pulp viscosity with a lower degree of hydrolysis, cellulases have also been reported to enhance the bleachability of softwood kraft pulp producing a final brightness score comparable to that of xylanase treatment.

Cellulases alone, or used in combination with xylanases, are beneficial for deinking of different types of paper wastes. 
Most applications proposed so far use cellulases and hemicellulases for the release of ink from the fiber surface by partial hydrolysis of carbohydrate molecules.
It has been postulated that improvements in dewatering and deinking of various pulps result in the peeling of the individual fibrils and bundles, which have high affinity for the surrounding water and ink particles. 
The main advantages of enzymatic deinking are reduced or eliminated alkali usage, improved fiber brightness, enhanced strength properties, higher pulp freeness and cleanliness, and reduced fine particles in the pulp.
Moreover, deinking using enzymes at acidic pH also prevents the alkaline yellowing, simplifies the deinking process, changes the ink particle size distribution, and reduces the environmental pollution. 
Although enzymatic deinking can lower the need for deinking chemicals and reduce the adverse environmental impacts of the paper industry, the excessive use of enzymes must be avoided, because significant hydrolysis of the fines could reduce the bondability of the fibers.

Interestingly, the use of cellulases in improving the drainage has also been pursued by several mills with the objective to increase the production rate. 
Enzyme treatments remove some of the fines or peel off fibrils on the fiber surface and dissolved and colloidal substances, which often cause severe drainage problems in paper mills.
In this aspect, cellulases have shown considerable improvement in the overall performance of paper mills. 
Enzymatic treatment also destabilizes the lipophilic extractives in the filtrates and facilitates their attachment to thermomechanical pulping fibers. 
These enzymes are also used in preparation of easily biodegradable cardboard, manufacturing of soft paper including paper towels and sanitary paper, and removal of adhered paper.

SYNONYMS:

Cellulase

beta-cellotriose

(2S,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6S)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2R,3S,4R,5R,6R)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol

61788-77-0

D-(+)-Cellotriose

Beta-D-Glucopyranosyl-(1->4)-Beta-D-Glucopyranosyl-(1->4)-Beta-D-Glucopyranose

CT3

33404-34-1

CHEBI:41753

ZINC8220386

DB01697

W-110244

Q26840894

WURCS=2.0/1,3,2/[a2122h-1b_1-5]/1-1-1/a4-b1_b4-c1