**17. Synergy between cellulases**

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‐

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‐

Also known as cellobiohydrolases, these enzymes catalyse the successive hydrolysis of resi‐ dues from the reducing and non-reducing ends of the cellulose, releasing cellobiose molecules as main product, which are hydrolysed by β-glucosidases. They account for 40 to 70% of the to‐

Exoglucanases have shown specificity on the ends of cellulose, such as*T. reesei* cellobiohy‐ drolase (CBH) I and II that act on the reducing and non-reducing cellulose chain ends, re‐

These enzymes are monomeric proteins with a molecular weight ranging from 50 to 65 kDa, although there are smaller variants (41.5 kDa) in some fungi, such as *Sclerotium rolfsii*. Low levels of glycosylation (around 12% to none at all) are found in these enzymes; and their op‐ timum pH is 4 to 5, with an optimum temperature from 37 to 60 °C, depending on the spe‐

Exoglucanases form part of the cellulolytic machinery of the fungi causing white and soft rot and they are found only in some of the basidiomycetes causing the brown rot, such as *Fomi‐*

Crystalline cellulose (Avicel, bacterial cellulose or filter paper), which is the main form of cellulose in most plant cell walls are good substrates for exoglucanase activity assay, be‐ cause it has a low DP and relatively low accessibility; however, some endoglucanases can

β-D-glucosidases hydrolyse soluble cellobiose and other cellodextrins with a DP up to 6 to

These enzymes have molecular weights ranging from 35 to 640 kDa, and they can be mono‐ meric or exist as homo-oligomers, as is the case β-glucosidase of the yeast *Rhodotorula minuta* [142]. Most β-glucosidases are glycosylated; in some cases, as that of the 300 kDa BGL from

produce glucose in the aqueous phase in order to eliminate cellobiose inhibition [13].

tal component of the cellulase system, and are able to hydrolyse crystalline cellulose.

teristics over time, resulting in rapid changes in hydrolysis rates [32].

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

cellulases and its hydrolysis does not require a CBD [110].

cific enzyme-substrate combination [137, 140].

release considerable reducing sugars from Avicel [13].

**15. Exoglucanases**

spectively [112].

*topsis palustris* [141].

**16. β-glucosidases**

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

Synergy endo-exo, occurs between endo and exoglucanases, where the action of endogluca‐ nases provide free ends of the cellulose chain to the exoglucanases.

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

Synergy between exoglucanases and β-glucosidases, the latter process cellobiose produced as final product of the action of the exoglucanases.

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, 1EG1).

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‐ lanases.

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 enzymes was reported in chitin degradation [28].

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‐ lose is still a slow process because the substrate is insoluble and poorly accessible to enzymes.

*Domain II*, at the C-terminal end, is homologous to group II pollen allergens from grasses. Some authors have speculated that this might be a polysaccharide-binding domain, due to the presence of aromatic and polar amino acids on the protein surface, where two trypto‐ phan and one tyrosine would form a planar platform of aromatic residues favouring this binding (149, 154). Domain II folds as a β-sandwich formed by two sheets of four antiparal‐ lel β strands each (Figure 8). In fact, a β-sandwich formed by 3 to 6 β strands per sheet is the most common fold in carbohydrate-binding modules of proteins binding substrates such as

Hydrolysis of Biomass Mediated by Cellulases for the Production of Sugars

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

139

(a G2A protein from *Phleum pratense*; PDB 1WHO). In (a), the domain I forms a barrel; amino-acid residues that are conserved in expansins are indicated in the single-letter amino-acid code. Images reproduced with publisher BioMed

**Figure 8.** a) Expansin proposed activity; b) Expansin domain I (the catalytic domain of a GH45 endoglucanase from

Expansins are classified in four families: α-expansins (EXPA), β-expansins (EXPB), α-expan‐

The *EXPA family* includes proteins participating in the relaxation and extension of plant cell walls through a pH-dependent mechanism; these proteins would participate in develop‐ mental processes such as organogenesis, the degradation of cell walls during the ripening of

The *EXPB family* includes group I pollen allergens from grasses. These proteins are secreted by pollen and have been suggested to soften the tissues of the stigma and style to facilitate

EXPB proteins, unlike EXPA members, relax specifically the cell walls of grass cells, proba‐ bly reflecting differences regarding the organization of cell walls between grasses and dicot‐ yledonous plants. Although an HFD motif, that is known to form part of the active site of endoglucanases, has been found in domain I of EXPA and EXPB family members, they do

The *EXLA and EXLB families* do not have this sequence motif, which suggests that their mode of action differs to that of the other expansins. The EXLA and EXLB families are com‐ prised of proteins identified by sequence analysis which, despite possessing the two- organi‐

fruits and other processes where relaxation of the cell wall is crucial [156-159].

crystalline cellulose or chitin [155].

*Humicolainsolens*; PBD, 2ENG); c) Expansin domain II

the penetration of the pollen tube [154].

not have hydrolytic activity [5, 160].

sin like-proteins (EXLA) and β-expansin like-proteins (EXLB) [5].

permission [5].

To overcome this situation scientists have optimized ratio of cellulolytic enzymes, and it was found that the best saccharification of crystalline cellulose is achieved with the enzyme blend: 60:20:20 (CBHI:CBHII:EGI) wherein a saturated level of BG was included to eliminate cellobiose inhibition [146]. In a different report, the impact of the cellulase mixture composi‐ tion on cellulose conversion was modelled, and the findings suggested different optimum ratios for substrates with different characteristics, specifically degrees of polymerization and surface area [147].

Also, researchers have pointed out the use of proteins that relax plant cell wall structure as a complementary activity before action of cellulases in order to improve saccharification.
