Define cellulose, Structure of cellulose, What are cellulases?

Define cellulose

Define Cellulose is an organic polysaccharide that consists of a straight chain of hundreds of β-linked D-glucose units.

Cellulose is the most abundant extracellular structural polysaccharide or organic polymer of all biomolecules in the biosphere.

Cellulose is present in all land plants, but without meat, eggs, fish, and milk. However, it is not metabolized by the human system.

It is the most common carbohydrate in the plant kingdom and comprises around 50% of all carbon in vegetation.

Cellulose occurs in the cell walls of plants, where it contributes significantly to the structure of the body.

All organisms that synthesize cellulose, including bacteria, algae, tunicates, and higher plants, have cellulose synthase proteins that catalyze the polymerization of glucan chains.

Even when the human body cannot digest cellulose, it serves as a source of fiber.

In nature, cellulose is a food source for a wide variety of organisms including bacteria, fungi, plants and protists, as well as a wide variety of invertebrates such as insects, crustaceans, annelids, molluscs and nematodes.

The mechanical resistance of the plant cell is attributed to the structural properties of the cellulose, as it can retain a partially crystalline state of aggregation even in an aqueous medium.

Cellulose is a homopolymer of a glucose derivative and therefore acts as an excellent source of fermentable sugars.

Cellulose is grown in the form of energy crops for the production of ethanol, ethers, acetic acid, etc.

The abundance of cellulose is due to the constant cycles of photosynthesis in higher plants, which synthesize about 1000 tons of cellulose.

Cellulose is a rigid, fibrous white solid that is insoluble in water but soluble in the ammonia solution of copper hydroxide.

Although it is insoluble in water, cellulose absorbs water and adds to most feces, and facilitates their elimination.

Structure of cellulose

Define cellulose, Structure of cellulose, What are cellulases?
  • The molecular weight of cellulose varies between 200,000 and 2,000,000, which corresponds to 1,250-12,500 glucose residues per molecule.
  • Cellulose consists of a D-glucose unit at one end with a C4-OH group as the non-reducing end and the terminating group as C1-OH as the reducing end.
  • The bond is formed by extracting a water molecule from the glycosidic OH group on carbon 1 of a β-D-glucose molecule and the alcoholic OH group on carbon 4 of the neighboring β-D-glucose molecule.
  • Anhydrocello-Biose is the repeating unit of cellulose.
  • The general structure of cellulose is the result of the joining of adjacent cellulose chains and layers through hydrogen bonds and van der Waals forces, resulting in parallel alignment.
  • This creates the crystalline structure of cellulose with straight and stable supramolecular fibers of high tensile strength and low accessibility.
  • The structure of cellulose is similar to the structure of amylose, except that the glucose units are linked by β-1,4-glucoside bonds.
  • The β-1,4-glycosidic bond in the structure creates a linear glucan chain in which all other glucose residues are rotated 180 ° against each other.
  • The cellulose molecule is very stable, with a half-life of 5-8 million years for the splitting of the β-glucosidic bond at 25 ° C.
  • Cellulose is of different types depending on its structure and accessibility; crystalline and non-crystalline, accessible and inaccessible.
  • Most of the cellulose found in wood is very crystalline with approx. 65% crystalline areas. The rest of the structure has a lower packing density, which leads to an amorphous or non-crystalline structure.
  • The accessibility of cellulose is used to define the availability of cellulose to water and microorganisms. Most of the crystalline cellulose surface is accessible while the rest of the structure is inaccessible.

What are cellulases?

Cellulases refer to a group of enzymes that catalyze the breakdown of cellulose into oligosaccharides, cellobiose, and glucose.

  • These enzymes are a class of enzymes produced by fungi and bacteria that aid in the hydrolysis of cellulose.
  • Cellulose is an important group of enzymes that play a fundamental role in both industry and nature.
  • In nature, cellulases are involved in the global carbon cycle by breaking down insoluble cellulose into soluble forms.
  • Cellulases are structurally distinct and diverse, and hydrolyze a single substrate, cellulose, although there are seven different protein folds within the family.
  • The complete cellulase enzyme system consists of three enzymes, exo-β-1,4-glucanases, endo-β-1,4-glucanases and β-1,4-glucosidases.
  • These enzymes work sequentially in a synergistic system to break down cellulose to produce a usable source of energy in the form of glucose.
  • Cellulases are generally divided into four main classes based on their mode of action; Exoglucanases, endoglucanases, β-glucosidases, and cellobiohydrolases.
  • These enzymes differ in structure and mode of action, but in some cases, the enzymes can act in sequence to produce the desired end product.
  • Cellulases also differ in different organisms such as fungi, and bacterial cellulases differ significantly in structure and function.
  • In contrast to bacterial cellulases, fungal cellulases consist of a carbohydrate-binding module (CBM) at the C-terminus, which is connected to the catalytic domain at the N-terminus by a short polylinker region.

Microorganisms involved in cellulose degradation (cellulolytic microorganisms)

A wide range of cellulolytic microorganisms, mainly fungi, and bacteria, have been identified over the years. The structure and mode of action of cellulases, which are produced by different microorganisms, are also different.

Cellulolytic mushrooms

  • Fungi are among the most active decomposing agents of organic material in general and of cellulose substrates in particular.
  • Cellulase-producing fungi are widespread among fungi and include species of Ascomycetes (Trichoderma reesei), Basidiomycetes (Fomitopsis palustris) with a few anaerobic species.
  • Among the fungi, soft rot is best known for producing cellulases, and among them Trichoderma is best studied.
  • Other known cellulase-producing soft rot are Aspergillus niger, Fusarium oxysporum, Neurospora crassa, etc.
  • In addition to soft rot, brown and white rot fungi are also actively involved in the breakdown of cellulose; however, the mechanism of action of these enzymes is significantly different.
  • Brown rot actively hydrolyzes cellulose during earlywood decomposition because it lacks exoglucanases. Some of the most common examples of these fungi are Poria placenta, Lenzites trabea, Coniophora puteana, and Tyromyces palustris.
  • White rot, on the other hand, is mainly found in the breakdown of lignocelluloses with examples such as Phanerochaete chrysosporium, Sporotrichum thermophile, and Trametes Versicolor.
  • Among the anaerobic cellulolytic fungi, the best-studied species are Neocallimastix frontalis, Piromyces (Piromonas) communis, and Orpinomyces.

Cellulolytic Bacteria

Cellulolytic bacteria often produce cellulases in small quantities, and the breakdown of cellulose appears to be through a group of multi-enzyme complexes.

  • Most of the bacterial cellulolytic enzymes come from Bacillus, Acinetobacter, Cellulomonas and Clostridium.
  • Approximately 90-95% of total bacterial cellulase activity is observed by aerobic bacteria under aerobic conditions. However, the remaining 10% is broken down by a diverse group of bacteria under anaerobic conditions.
  • In addition, some of the bacteria in the rumen are also known to produce cellulases, which can break down components of the cell wall.
  • Some of the examples include Fibrobacter succinogenes, Ruminococcus albus, Pseudomonas, Proteus, and Staphylococcus.
  • Some thermophilic bacteria such as Anoxybacillus sp, Geobacillus sp and Bacteroides also have cellulase activity.

Enzymes involved in the degradation of cellulose

The enzymes involved in the breakdown of cellulose are groups like cellulases. There are roughly five types of cellulases based on the reactions they catalyze.

1. Endoglucanase

  • Endoglucanases are a group of endocellulases that cleave the cellulose molecule at internal bonds on the non-crystalline surface of the molecule.
  • Endoglucanases accidentally attack the cellulose chain and cleave the β-1,4-glucosidic bonds present in the molecule.
  • Endoglucanases reduce the length of the cellulose so that other enzymes can act on the fragments.

2. Exoglucanases

  • Exogluconases are a group of exocellulases that hydrolyze the reducing or non-reducing ends of cellulose chains.
  • The main products of the enzymatic action are cellobiose, which are then hydrolyzed into monomeric units.
  • Exoglucanases act on the smaller tetrasaccharides and disaccharides that are formed after the action of endoglucanases.
  • Exoglucanases include both 1,4-β-D-glucanohydrolases, which release D-glucose from β-glucan and cellodextrins, and 1,4-β-D-glucan cellobiohydrolases, which release D-cellobiose from β-glucan in one process release.

3. Celobiasis

  • Cellobiases are enzymes that act on cellobiose units (disaccharides, trisaccharides, and tetrasaccharides) to form monomeric units.
  • Cellobiases are also called β-glucosidases because they form individual glucose units.

4. Oxidative cellulases

  • Oxidative cellulases are enzymes that depolymerize cellulose into smaller units through radical reactions.
  • Enzymes like cellobiose dehydrogenase catalyze the conversion of various forms into cellobiose so that cellobiases can act on it.

5. Cellobiose phosphorylases

  • Cellobiose phosphorylases are similar to cellobiases except that hydrolysis of polymer units occurs in the presence of phosphorus rather than water.

Aerobic and Anaerobic degradation of cellulose

1. Aerobic breakdown of cellulose

  • Aerobic cellulolysis is carried out by the synergistic action of three types of enzymatic activities: endoglucanases or 1,4-β-D-glucan 4-glucanohydrolases, exoglucanases and β-glucosidases or β-D-glucoside-glucohydrolases, resulting in the release of D -Glucose units from soluble cellodextrins and a variety of glycosides.
  • Aerobic cellulases are produced in high concentrations and also act sequentially.
  • Aerobic hydrolysis is quite simple and occurs in sequential steps, each of the steps being catalyzed by a different type of cellulase enzyme.
  • Endoglucanases attack the amorphous regions of cellulose fibers and form sites for exoglucanases, which can then hydrolyze the cellobiose units in the more crystalline regions of the fibers.
  • Finally, β-glucosidases lead to the formation of monomeric glucose units through hydrolysis of cellobiose.

2. Anaerobic degradation of cellulose

  • The mechanism by which cellulases from anaerobic bacteria catalyze the depolymerization of crystalline cellulose is ill-defined; however, it is known that the mechanism is markedly different from that of aerobic hydrolysis.
  • The cellulases of most anaerobic microorganisms are organized in large multiprotein complexes called cellulosomes.
  • Cellulosomes create close proximity between cell and substrate and thus minimize diffusion losses of hydrolytic products.

Factors affecting cellulose degradation

The breakdown of cellulose in soil or others is affected by a number of factors including:

1. Available minerals

  • The availability of nutrients and minerals influences the breakdown of cellulose, as these components are necessary for the production of biomolecules such as cellulases and other proteins.
  • The increase in nutrients and minerals increases the rate at which cellulose breaks down.

2. temperature

  • The degradation of cellulose takes place in the temperature range of 0 ° C and 65 ° C, since both psychrophilic and thermophilic organisms are able to hydrolyze cellulose.
  • However, cellulose degradation is optimal in the mesophilic temperature range of 25-30 ° C.

3. Ventilation

  • The availability of oxygen affects both the rate and mechanism of hydrolysis and the enzyme involved, and the mode of action differs in aerobic and anaerobic organisms.
  • In the presence of oxygen, there is a sequential process of hydrolysis of cellulose to glucose by three different groups of enzymes.
  • Cellulolytic enzymes form a comparatively slower multi-enzyme complex under anaerobic conditions.

4. pH

  • Cellulose degradation is slightly higher in acidic soils than in alkaline or neutral soils.
  • Under acidic conditions, fungi are the main group of organisms involved in cellulose breakdown, while bacteria and actinomycetes act as the dominant cellulose breakers under neutral to alkaline conditions.

5. Organic material

  • The presence of organic material also increases the rate of cellulose degradation, as much of the organic material serves as a substrate.
  • However, when cellulose is the only constituent of the fabric, the rate of hydrolysis will decrease.
  • The rate of degradation increases with the addition of a small amount of easily degradable organic matter because it allows microorganisms to grow.

6. Lignin

  • The presence of lignin reduces the rate of cellulose degradation.
  • Lignin is closely related to cellulose, which affects the breakdown of cellulose.

Process (Simple Steps) of cellulose degradation

The breakdown of cellulose takes place in three simple steps;

1. Hydrolysis by endoglucanases

  • The first step in cellulose breakdown is the action of endoglucanases, which accidentally attack cellulose fibrils.
  • This step leads to a reduction in the size of the cellulose chains as it breaks the polymer into smaller fragments.
  • The enzyme works internally at random locations on the polymer.

2. Hydrolysis by exoglucanases

  • Exoglucanases act on the smaller fragments, resulting in even smaller tetrasaccharide or disaccharide units.
  • Exoglucanases act at the reducing end of the fragments to form dimeric or cellobiose units.

3. Hydrolysis by β-glucosidase

  • The β-glucosidase or cellobiose acts on the dimeric glucose units of cellobiose to form monomeric units, glucose.
  • This is the last step in cellulose breakdown, which leads to the formation of individual free units of the glucose molecule.

Mechanisms of microbial degradation of cellulose

  • Cellulolytic microorganisms use two well-studied mechanisms to break down the cellulose present, and brown rot fungi are known to use a third, less well-studied oxidative mechanism.
  • Both of the well-studied mechanisms of cellulose breakdown are generated by the enzymatic action of cellulases to break β-1 bonds, 4; however, the way in which cellulases are presented to the environment is very different.
  • Many aerobic microorganisms studied to use the free cellulase mechanism to digest cellulose, although brown rot fungi appear to use a different oxidative mechanism to break down cellulose.

A. Hydrolytic mechanism of cellulose degradation

  • In glycosyl hydrolases, the enzymatic hydrolysis of the glycosidic bond generally occurs through general acid/base catalysis that requires two critical residues: a proton donor (HA) and a nucleophile/base (B-).
  • This catalytic activity is provided by two residues of aspartic or glutamic acid.
  • Mechanically, it is known that the reactions catalyzed by all cellulases involve general acid-base catalysis by a carboxylate pair at the active site of the enzyme, even if they have a different structure.
  • One of the residues acts as a general acid and protonates the oxygen of the o-glycosidic bond, while the other residue acts as a nucleophile.
  • On the basis of the distance between the two carboxyl groups, reversal mechanisms (distances of 10 Å) or retention (distances of 5) are observed in cellulases.

1. Investment Mechanism

  • In the case of the reverse cellulase mechanism, two enzyme residues, typically carboxylate residues, act as an acid and a base.
  • The reverse mechanism is generated by the attack of a water molecule on the C1 carbon of the glucose ring in an Sn2 displacement reaction, which leads to the inversion of the configuration at the anomeric C1 carbon.

2. Storage mechanism

  • In the case of the cellulase retention mechanism, hydrolysis occurs in a two-step mechanism, with each step involving an inversion. As with inversion, two enzyme residues are involved, one acting as a nucleophile and the other as an acid or base.
  • In the first step, the nucleophile attacks the anomeric center, which leads to deprotonation. The deprotonated carboxylate then acts as a base in the next step, which helps the nucleophilic water to form the hydrolyzed product.

3. Mechanism of glucosidase

  • Recently, a fundamentally different glycosidase mechanism for the divalent metal ion-dependent glycosidases GH4 and NAD + was discovered.
  • In this case, hydride extraction at C3 produces a ketone, followed by deprotonation of C2, accompanied by acid-catalyzed removal of glycosidic oxygen and the formation of an unsaturated 1,2-intermediate.
  • This α-β-unsaturated species undergoes base-catalyzed attack by water to generate a 3-keto derivative, which is then reduced by NADH to complete the reaction cycle.

Example of hydrolytic mechanism

The hydrolytic mechanism is observed in most aerobic and anaerobic microorganisms including Bacillus, Acinetobacter, Cellulomonas, Clostridium, Aspergillus niger, Fusarium oxysporum, Neurospora crassa, and Trichoderma reesei.

B. Oxidative mechanism of cellulose degradation

  • Although most aerobic bacteria break down cellulose through the synergistic effect of different cellulases, some filtrates from cellulolytic fungal cultures break down cellulose more quickly in an oxygen atmosphere than under anaerobic conditions.
  • The enzyme cellobiose dehydrogenase (CDH) plays an important role in this.
  • In a ping-pong-like reaction, CDH catalyzes the oxidation of cellobiose (the main product of the cellulase effect) to cellobionolactone with the reduction of various electron acceptors such as quinones, chelated Fe (III), O2 (which produces hydrogen peroxide) and phenoxy. Radical.
  • In addition, cellobiose dehydrogenase has other functions in the breakdown of cellulose.
  • CDH oxidizes the free ends produced by endo-acting cellulases and prevents condensation of the cellulose chain.
  • Inhibition of the product is avoided by removing cellobiose as high concentrations of this disaccharide inhibit many cellulases.
  • CDH can produce Fe2 + and H2O2 by reducing Fe3 + and O2. Together they form hydroxyl radicals in a Fenton-like reaction, which depolymerize or modify cellulose.

Example of oxidative mechanism

Examples of oxidative mechanisms can be observed in fungal species such as Phanerochaete chrysosporium, Sporotrichum thermophile, Poria placenta, Lenzites trabea, etc.

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Define cellulose, Structure of cellulose, What are cellulases?

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