**7. Hemicellulose degradation**

**A**ctive En**Zy**mes) [32]. The fungal carbohydrate-active enzymes commonly (but not always) present a carbohydrate-binding module (CBM), which promotes the association of the en‐ zyme with the substrate. A review on fungal enzymes related to plant biomass degradation describes that such enzymes are assigned to at least 35 glycoside hydrolase (GH) families, three carbohydrate esterase (CE) families, and six polysaccharide lyase (PL) families [33]. Al‐ though the classification of CWDEs into families facilitates our view about a specific en‐ zyme, the activities of these enzymes are quite complicated to classify, because some families can contain several enzymatic activities. This is especially important because CA‐ Zymes usually act in a synergistic way, complementing the substrate specificity of each oth‐ er, in order to degrade complex polysaccharide matrices. For instance, GH5 comprises many catalytic activities, such as endoglucanases, exoglucanases and endomannanases [34]. This section will describe the fungal enzymatic set required for the main polysaccharides present in the plant biomass: cellulose, hemicellulose and pectin. A brief description of enzymes re‐ quired for lignin degradation will be depicted. Because most of the research in cellulase/ hemicellulase field is performed using the fungi *T. reesei* and *A. niger*, the focus of our dis‐ cussion about CWDEs will be conducted based on these microorganisms, although some as‐ pects related to other fungal species could be mentioned to demonstrate the diversity of

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

Cellulose, a polysaccharide consisted of linear β-1,4-linked D-glucopyranose chains, re‐ quires three classes of enzymes for its degradation: β-1,4-endoglucanases (EGL), exogluca‐ nases/cellobiohydrolases (CBH), and β-glucosidase (BGL). The endoglucanases cleave cellulose chains internally mainly from the amorphous region, releasing units to be degrad‐ ed by CBHs and/or BGLs. The cellobiohydrolases cleave celobiose units (the cellulose-de‐ rived disaccahride) from the end of the polysaccharide chains [6]. Finally, β-glucosidases hydrolise cellobiose to glucose, the monomeric readily metabolisable carbon source for fun‐ gi [35]. These three classes of enzymes need to act synergistically and sequentially in order to degrade completely the cellulose matrix. After endo- and exo-cleaving (performed by EGLs and CBHs, respectively), the BGLs degrade the remaining oligosaccharides to glucose.

The most efficient cellulose-degrading fungi is *Trichoderma reesei*. The highly efficient degra‐ dation of cellulose by *T. reesei* is mainly due to the highly effectiveness of cellulases acting synergistically in this specie, although *T. reesei* does not have the biggest number of cellulas‐ es in the fungi kingdom [36]. The *T. reesei* has five characterized EGLs, two highly expressed CBHs and two characterized BGLs, the latter being expressed at low levels [37, 38] reviewed in [33]. In addition to being expressed at very low levels in *T. reesei*, the BGLs are strongly subjected to product inhibition [39]. These features reduce the utilization of *T. reesei* for in vitro saccharification of cellulose substrates and, in industrial applications, cellulase mix‐ tures from *T. reesei* are often supplemented with BGLs from Aspergilli, which are highly ex‐

A schematic view of cellulose degradation is depicted in the Figure 3.

pressed and tolerant to glucose inhibition [33].

carbohydrate-active enzymes.

**6. Cellulose degradation**

Hemicellulose is a complex polysaccharide matrix composed of different residues branched in three kinds of backbones, named xylan, xyloglucan and mannan. The complexity of hemi‐ cellulose requires a concerted action of endo-enzymes cleaving internally the main chain, exo-enzymes releasing monomeric sugars, and accessory enzymes cleaving the side chains of the polymers or associated oligosaccharides, leading to the release of various mono- and disaccharides depending on hemicellulose type.

Xylan, a polymer composed by ß-1,4-linked D-xylose units, is degraded through the action of ß-1,4-endoxylanase, which cleaves the xylan backbone into smaller oligosaccharides, and ß-1,4-xylosidase, which cleaves the oligosaccharides into xylose. Fungal ß-1,4-endoxylanase are classified as GH10 or GH11 [40], differing from each other in substrate specificty [41]. Endoxylanases belonging to family GH10 usually have broader substrate specificity than en‐ doxylanases from family GH11 [33]. GH10 endoxylanases are known to degrade xylan back‐ bones with a high degree of substitutions and smaller xylo-oligosaccharides in addition to degrade linear chains of 1,4-linked D-xylose residues. Thus, GH10 endoxylanases are neces‐ sary to degrade completely substituted xylans [42]. ß-Xylosidases are highly specific for small unsubstituted xylose olygosaccharides and they are important for the complete degra‐ dation of xylan. Some ß-xylosidases have been shown transxylosylation activity, suggesting a role for these enzymes in the synthesis of specific oligosaccharides [43, 44].

ylan arabinofuranohydrolase from *A. niger*, is not able to release arabinobiose from xylan or substitued L-arabinose from D-xylose residues adjacent to D-glucuronic acid residues [54].

Another type of substituent present in hemicellulose is D-xylose. Hydrolases responsible for the release of D-xylose residues from the xyloglucan backbone are referred to α-xylosidases. These enzymes can differ with respect to the type of glycoside they can hydrolize. For in‐ stance, α-xylosidase II (AxhII) from *Aspergillus flavus* hydrolyzes xyloglucan oligosacchar‐ ides and AxhIII is most active on *p*-nitrophenyl α-L-xylose residues and does not hydrolyze

There are many other possible substituents in hemicellulose, such as L-fucose, α-linked Dgalactose, D-glucuronic acid, acetyl group and *p*-coumaric and ferulic acids (Figure 1). A list containing the respective fungal accessory enzyme responsible for the release of each of these residues is shown in Table 1. An overview of fungal enzymatic complex for hemicellu‐

**Hemicellulose polymer Residue released Enzyme**

**Table 1.** Fungal accessory enzymes for the cleavage of hemicellulose-derived residues.

Xyloglucan/xylan L-arabinose α-arabinofuranosidases

Xyloglucan D-xylose α-xylosidases Xyloglucan L-fucose α-fucosidases Xylan/galactomannans D-galactose α-galactosidases

Xylan D-glucuronic acid α-glucuronidases Xylan acetyl group acetyl xylan esterases Xylan *p*-coumaric acid *p*-coumaroyl esterases Xylan ferulic acid feruloyl esterases

Pectins are composed of a main backbone of α-1,4-linked D-galacturonic acid, and consist of two regions: the "smooth" region and the "hairy" region. The "smooth" region contains resi‐ dues of D-galacturonic acids that can be methylated or acetylated, while in the "hairy" re‐ gion, the backbone of D-galacturonic acids residues is interrupted by α-1,2-linked Lrhamnose residues. Moreover, in the hairy region, long side chains of L-arabinose and Dgalactose residues can be attached to the rhamnose residues (Figure 2). As observed for cellulose and hemicellulose, degradation of pectins also requires a set of hydrolytic enzymes to degrade completely the polymer. Glycoside hydrolases (GHs) and polysaccharide lyases (PLs) are the two classes of hydrolytic enzymes required for pectin backbone degradation.

arabinoxylan arabinofuranohydrolases

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xyloglucan [55, 56].

lose degradation is shown in Figure 4.

**8. Pectin degradation**

Xyloglucan consists of ß-1,4-linked D-glucose backbone substituted mainly by D-xylose and therefore requires endoglucanases (xyloglucanases) and ß-glucosidases action in or‐ der to be degraded. Some endoglucanases are specific for the substituted xyloglucan backbone, and they are not able to hydrolise cellulose [45]. Xyloglucan-active endogluca‐ nases have specific modes of action. For instance, a xyloglucanase from *T. reesei* cleaves at branched glucose residues, whereas the GH12 xyloglucanase from *A. niger* prefers xy‐ logluco-oligosaccharides containing more than six glucose residues with at least one nonbranched glucose residue [46]-[47].

Mannans, also referred to galacto(gluco)mannans, consist of a backbone of ß-1,4-linked Dmannose (mannans) and D-glucose (glucomannans) residues with D-galactose side chains. The degradation of this type of hemicellulose is performed by the action of ß-endomanna‐ nases (ß-mannanases) and ß-mannosidases, commonly expressed by aspergilli [48]. The ßmannanases cleave the backbone of galacto(gluco)mannans, releasing mannooligosaccharides. Several structural features in the polymer determine the ability of ß-mannanases to hydrolise the mannan backbone, such as the ratio of glucose to mannose and the number and distribution of substituents on the backbone [49]. It has been shown that ß-mannanase is most active on galactomannans with a low substitution of the backbone [50], and the presence of galactose residues on the mannan backbone significantly prevents ß-mannanase activity [51]. The main products of ß-mannanase activity on mannan are man‐ nobiose and mannotriose. ß-Mannosidases act on the nonreducing ends of mannooligosac‐ charides, releasing mannose. As shown by substrate specificity studies, ß-Mannosidase is able to completely release terminal mannose residues when one or more adjacent unsubsti‐ tuted mannose residues are present [52].

The complete degradation of hemicellulose is only achieved after release of all substitutions present on the main backbone. The high degree of substitution in the hemicellulose poly‐ mers requires the action of various accessory enzymes able to release all these substitutions from the polysaccharide. At least nine different enzyme activities distributed along 12 GH and 4 CE families are required to completely degrade the hemicellulose substituents [33].

Arabinose is one of the most common sugar residues in hemicellulose and is present in ara‐ binose-substituted xyloglucan and (arabino-)xylan. The release of arabinose from the poly‐ mer is performed by α-arabinofuranosidases and arabinoxylan arabinofuranohydrolases. α-Arabinofuranosidases are mainly found in GH 51 and 54 families, and the differences in the substrate specificity between these enzymes could be exemplified by two arabinofuranosi‐ dases of *A. niger*, AbfA and AbfB. AbfA (GH 51) releases L-arabinose from arabinan and sugar beet pulp, while AbfB (GH 54) also releases L-arabinose from xylan [48]. The arabi‐ noxylan arabinofuranohydrolases act in the L-arabinose residues of arabinoxylan, specifical‐ ly against the α-1,2- or α-1,3-linkages [53]. Moreover, arabinoxylan arabinofuranohydrolases appear to be sensitive to the substitutions of adjacent D-xylose residues. AxhA, an arabinox‐ ylan arabinofuranohydrolase from *A. niger*, is not able to release arabinobiose from xylan or substitued L-arabinose from D-xylose residues adjacent to D-glucuronic acid residues [54].

Another type of substituent present in hemicellulose is D-xylose. Hydrolases responsible for the release of D-xylose residues from the xyloglucan backbone are referred to α-xylosidases. These enzymes can differ with respect to the type of glycoside they can hydrolize. For in‐ stance, α-xylosidase II (AxhII) from *Aspergillus flavus* hydrolyzes xyloglucan oligosacchar‐ ides and AxhIII is most active on *p*-nitrophenyl α-L-xylose residues and does not hydrolyze xyloglucan [55, 56].

There are many other possible substituents in hemicellulose, such as L-fucose, α-linked Dgalactose, D-glucuronic acid, acetyl group and *p*-coumaric and ferulic acids (Figure 1). A list containing the respective fungal accessory enzyme responsible for the release of each of these residues is shown in Table 1. An overview of fungal enzymatic complex for hemicellu‐ lose degradation is shown in Figure 4.


**Table 1.** Fungal accessory enzymes for the cleavage of hemicellulose-derived residues.
