**14. Endoglucanases**

lar wood chips and hardwood forest leading to the identification of new genes and

134 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

To have a better impression of the latest developments regarding fungal carbohydrate-active enzymes, the following sections will discuss the enzymes needed for cellulose degradation.

(*a*) Cel7A binding to cellulose, (*b*) recognition of a reducing end of a cellulose chain, (*c*) initial threading of the cellu‐ lose chain into the catalytic tunnel, (*d* ) threading and formation of a catalytically active complex, (*e*) hydrolysis in a processive cycle and ( *f )* product expulsion and threading of another cellobiose (shown in yellow in *e* and *f*). Image

**Figure 6.** Activity on substrate of cellulase (exoglucanase, Cel7A) of *T. reesei.* The enzyme has a small carbohydratebinding domain (CBD) of 36-amino acid, a long flexible linker with O-glycan (dark blue), and a large catalytic domain

Multiple types of modular cellulases formed by catalytic and carbohydrate binding domains have been discovered, including at least two exo-β-glucanases, or cellobiohydrolases (CBHs,CBH I and CBH II), four endoglucanases (EG; EG I, EG II, EG III, EG V), and one β-

Cellulases are O-glucoside hydrolases (GH, EC 3.2.1.), a widespread group of enzymes which hydrolyse the β-1,4 linkages or glycosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. GH are classified into cellulases

A classification of glycoside hydrolases in families based on amino acid sequence similari‐ ties has been proposed a few years ago. Because there is a direct relationship between se‐ quence and folding similarities, this classification reflects the structural features of these enzymes better than their sole substrate specificity, and helps to reveal the evolutionary re‐ lationships between these enzymes, which represent a convenient tool to deduce informa‐

families on the basis of amino acid sequence similarity [31, 132].

(CD) with N-linked glycan (pink) that can thread a single chain of cellulose into the catalytic tunnel of 50 Å.

organisms with cellulolytic activities [107, 121-130].

reproduced with publisher´s permission [131].

**13. Cellulases**

glucosidase (BG) [1].

tion of the mechanism [132-133].

These enzymes cleave internal linkages in amorphous cellulose filaments, generating oligo‐ saccharides with different sizes and creating new chain ends that can in turn be attacked by exoglucanases (135). The cellulolytic process is initiated by endoglucanases that randomly cleave internal linkages at amorphous regions of the cellulose fibre and creating new reduc‐ ing and non reducing ends that are susceptible to the action of cellobiohydrolases [136].

Endoglucanases are monomeric enzymes with a molecular weight that ranges from 22 to 45 kDa, although some fungi such as *Sclerotium rolfsii* and *Gloeophyllum sepiarium* have endo‐ glucanases twice this size [137]. In general, endoglucanases are not glycosylated; however, they sometimes may have relatively low amounts of carbohydrate (from 1 to 12%) [2]. Un‐ like other endoglucanases reported with optimum pH 4 to 5; the only known endoglucanase with a neutral pH optimum is that from the basidiomycete *Volvariella volvacea,* expressed in recombinant yeast. Basically, their optimum temperature ranges from 50 to 70 °C [138-139].

Exhaustively hydrolysing cellulose also requires the action of β-glucosidases (BGL) (EC 3.2.1.21), which hydrolyse cellobiose, releasing two molecules of glucose and thereby pro‐ vide a carbon source that is easy to metabolize. Fungi causing white and brown rot, mycor‐ rhizal fungi and plant pathogens produce these enzymes [2, 135].

According to [13], primary hydrolysis occurs on the surface of solid substrates and releases soluble sugars with a degree of polymerization (DP) up to 6 into the liquid phase upon hy‐ drolysis by endoglucanases and exoglucanases. This depolymerisation step performed by endoglucanases and exoglucanases is the rate-limiting step for the whole cellulose hydroly‐ sis process. The second hydrolysis involves primarily the hydrolysis of cellobiose to glucose by β-glucosidases, although some β-glucosidases also hydrolyse longer cellodextrins. The combined actions of endoglucanases and exoglucanases modify the cellulose surface charac‐ teristics over time, resulting in rapid changes in hydrolysis rates [32].

*Trametes versicolor*, glycosylation may be superior to 90%. Their optimum pH ranges from 3.5 to 5.5, and their optimum temperature ranges from 45 to 75 °C (3). β-D-glucosidase activ‐ ities can be measured using cellobiose, which is not hydrolysed by endoglucanases and exo‐

Hydrolysis of Biomass Mediated by Cellulases for the Production of Sugars

http://dx.doi.org/10.5772/53719

137

Synergistic cooperation between cellulases is a prerequisite for efficient degradation of cellu‐ lose, but its molecular mechanisms are not fully understood. Synergistic action has been ob‐ served between two different cellobiohydrolases and between endoglucanases. However,

Synergy endo-exo, occurs between endo and exoglucanases, where the action of endogluca‐

Synergy exo-exo, exoglucanases progressively act on reducing and non-reducing ends of the

Synergy between exoglucanases and β-glucosidases, the latter process cellobiose produced

Intramolecular synergy between catalytic domain and cellulose binding domain of cellulases.

**Figure 7.** Cellulases activities. Exoglucanases act on reducing and non-reducing ends degrading crystalline cellulose, while Endoglucanase act on amorphous cellulose. Structures: CBHI (PBD, 1CB2), CBHII (PDB, 3CBH) and EGL (PDB,

As a whole system, plant cell wall polysaccharides should be degraded efficiently not only by synergy between cellulases but with participation of the other degrading enzymes as xy‐

In (145) a synergistic mechanism between cellulases and xylanases in order to saccharify wheat straw for bioethanol production is reported. More recently, a new type of synergism between enzymes that employ oxidative reactions to break glycosidic bonds and hydrolytic

Although a significant amount of information has been generated related to the action of cel‐ lulases and their mechanisms to degrading cellulose, the biodegradation of crystalline cellu‐

glucanases [13].

cellulose chain.

1EG1).

lanases.

**17. Synergy between cellulases**

more synergistic mechanisms have been proposed [143-144]:

as final product of the action of the exoglucanases.

enzymes was reported in chitin degradation [28].

nases provide free ends of the cellulose chain to the exoglucanases.

To assay endoglucanase activity, there are substrates that are used, such as carboxymethyl‐ cellulose (CMC), a soluble amorphous cellulose form that is an excellent substrate for endo‐ cellulases and its hydrolysis does not require a CBD [110].
