**11. Mechanisms of cellulose biodegradation**

**10. Cellulases structure**

tamic and aspartic acid [1, 110-111].

which are joined by unstructured linker sequences [109].

be both, C or N terminal and occasionally is centrally positioned.

somes dramatically decrease its enzymatic activity (revised in [109]).

interaction is stabilized by polar residues that form hydrogen bonds [61].

hemicellulases, endomannanases and acetilxylanesterases [113].

independent function of the domains (Figure 6) [115-116].

Proteins with hydrolytic activity such as cellulases and hemicellulases comprises a complex molecular architecture of discrete modules (a catalytic domain (CD) and one or more CBDs),

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

The *catalytic domain* spans more than 70% of protein sequence. A sequence analysis of these domains in different cellulases shows a significant variability between them, in fact, active site of the enzyme has distinct three dimensional arrangements: in tunnel shape for a proc‐ essive exo degradation or in a cleft shape for an endo degradation. This domain is N-glyco‐ sylated and is responsible of the cleavage of the glycosidic bond, which occurs through an acid hydrolysis mechanism, using a donor of protons and a nucleophyle or base such as glu‐

The *cellulose binding domain* facilitates hydrolysis by keeping the catalytic domain nearby the substrate, therefore the presence of CBD is important for cellulases starting and processivity [112]. The CBDs, which is usually O-glycosylated, contain from 30 to about 200 amino acids, and exist as a single, double, or triple domain in a protein. Their location in the protein can

The CBDs bring the enzyme into a closer and prolonged association with the substrate, in‐ creasing the rate of catalysis, this domain was found to function more efficiently in substrate degradation, and removing the CBM from the enzyme or from the scaffolding in cellulo‐

In the union of CBD and cellulose, some non polar residues left exposed, mostly tyrosines and tryptophans, showing the flat face of their aromatic ring towards the pyranose ring, this

Besides cellulases, CBDs have also been found in other polysaccharides degrading enzymes:

The *linker peptide* is a sequence of amino acids connecting the cellulose binding domain and the catalytic domain. This linker contains from 6 to 59 amino acids and functions as a flexi‐ ble hinge that allows the independent function of each domain [114]. The sequence of the linker varies between enzymes, however, the composition is typically rich in proline, treo‐ nine and serine, like in the sequence PTPTPTPTT(PT)7 of the endoglucanase of *C. fimi* and NPSGGNPPGGNPPGTTTTRRPATTTGSSPG of the cellobiohydrolase CBHI of *T. reesei*.

Treonine and serine residues of the peptide linker are highly O-glycosylated to be protected from proteolysis; if the linker is completely absent or is too short then both domains, CBD and CD, obstruct each other and the affinity reduces. Based on the similarities of the linker between cellulases it has been suggested that it could be acting as a flexible hinge facilitating

Once the cellulase has recognized a free chain end, it threads the chain into the tunnel to form a catalytically active complex (CAC). Because cellulose decrystallization in water is free-energetically unfavourable, the tunnels or clefts of cellulase CDs contain hydrophobic and polar residues that form favourable contacts with the chain.

Several studies have mutated hydrophobic residues in the CD tunnels of cellulases and chi‐ tinases (structurally similar to cellulases), and have demonstrated that hydrophobic residues need to be present in the CD tunnels for digestion of crystalline cellulose to occur [117].

Additionally, in [27] have shown that removal of hydrophobic residues in cellulase and chi‐ tinase tunnels can increase processivity rates on more accessible polymers.

Once a cellulase forms a CAC with a cellodextrin chain, the hydrolysis reaction occurs usu‐ ally via a retaining or inverting mechanism, depending on the directionality of the enzyme. After the reaction occurs, the product must be expelled and another CAC formed by thread‐ ing another cellobiose unit into the CD (Figure 6) [117].

In most cases, the hydrolysis of the glycosidic bond is catalysed by two amino acid residues of the enzyme: a general acid (proton donor) and a nucleophile/base [111]. Depending on the spatial position of these catalytic residues, hydrolysis occurs via overall retention or overall inversion of the anomeric carbon. Recently, a completely unrelated mechanism has been demonstrated for two families of glycosidases utilizing NAD+ as a cofactor [118-119].

The *retaining glycoside hydrolase mechanism* leads to a net retention of the configuration at the anomeric carbon (C1] of the substrate after cleavage, since the hydrolysis of a glycosidic bond creates a product with the same configuration at the anomeric carbon as the substrate had before hydrolysis.

The *inverting glycoside hydrolase mechanism* leads to a net inversion of the configuration at the anomeric carbon (C1] of the substrate after cleavage. This is performed via a single nucleo‐ philic displacement mechanism, where the hydrolysis of a -glycosidic bond creates a prod‐ uct with the -configuration, and vice-versa [120].
