**8. Pectin degradation**

small unsubstituted xylose olygosaccharides and they are important for the complete degra‐ dation of xylan. Some ß-xylosidases have been shown transxylosylation activity, suggesting

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 non-

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‐

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‐

a role for these enzymes in the synthesis of specific oligosaccharides [43, 44].

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

branched glucose residue [46]-[47].

tuted mannose residues are present [52].

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.

The GHs involved in pectin backbone degradation include endo- and exo-polygalacturonas‐ es, which cleave the backbone of smooth regions, while the intricate hairy regions are fur‐ ther degraded by endo- and exo-rhamnogalacturonases, xylogalacturonases, αrhamnosidases, unsaturated glucuronyl hydrolases, and unsaturated rhamnogalacturonan hydrolases [33]. Endo- and exo-polygalacturonases are able to cleave α-1,4-glycosidic bonds of α-galacturonic acids. Rhamnogalacturonases cleave α-1,2-glycosidic bonds between Dgalacturonic acid and L-rhamnose residues in the hairy region of the pectin backbone [57]. An endo-xylogalacturonase from *Aspergillus tubingensis* has been shown to cleave the xy‐ lose-substituted galacturonic acid backbone [58]. The other GHs required for the degrada‐ tion of main chain of pectin, α-rhamnosidases, unsaturated glucuronyl hydrolases, and unsaturated rhamnogalacturonan hydrolases, are not well-characterized biochemically [33].

move the side chains, providing access for the main-chain pectinolytic enzymes. The accessory enzymes endoarabinanases, exoarabinanases, β-endogalactanases, and several es‐ terases are specific for pectin degradation, while α-arabinofuranosidases, β-galactosidases,

Microbial Degradation of Lignocellulosic Biomass

http://dx.doi.org/10.5772/54325

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Lignin, a highly insoluble complex branched polymer of substituted phenylpropane units joined by carbon-carbon and ether linkages, provides an extensive cross-linked network within the cell wall, and it is known to increase the strength and recalcitrance of the plant cell wall. Microbial lignin degradation is often complicated, once the microbe needs to cope with three major challenges related to lignin structure: (i) enzymatic system to degrade the lignin polymer needs to be essentially extracellular, because lignin is a large polymer, (ii) the mechanism of enzymatic degradation should be oxidative and not hydrolytic, since the lig‐ nin structure comprises carbon-carbon and ether bonds, and (iii) lignin stereochemistry is ir‐ regular, requiring enzymes with less specificity than hydrolytic enzymes required for cellulose/hemicellulose degradation [61]. The most well characterized enzymes able to de‐ grade the lignin polymer are lignin peroxidase (LiP), laccase (Lac), manganese peroxidase (MnP), versatile peroxidase, and H2O2-generating enzymes such as glyoxal oxidase (GLOX)

Lignin and manganese peroxidases (LiP and MnP, respectively) catalyse a variety of oxida‐ tive reactions dependent on H2O2. LiP oxidizes non-phenolic units of lignin (mainly Cα-Cβ bonds) by removing one electron and creating cation radicals that decompose chemically [62]. MnPs differ significantly from LiPs, once they cannot oxidize directly non-phenolic lig‐ nin-related structures [63]. In order to oxidize non-phenolic lignin-related components, the oxidizing power of MnPs is transferred to Mn3+, a product of the MnP reaction: 2 Mn(II) +

+ H2O2 → 2 Mn(III) + 2H2O [64]. In this way, Mn3+ diffuses into the lignified cell wall,

Laccases oxidize phenolic compounds and reduce molecular oxygen to water. Lac catalyses the formation of phenoxyl radicals and their unspecific reactions, leading finally to Cα-hy‐ droxyl oxidation to ketone, alkyl-aryl cleavage, demethoxylation and Cα-Cβ cleavage in phenolic substructures [61]. Versatile peroxidases (VPs) are able to oxidize phenolic and

In order to degrade lignin, microbes require sources of extracellular H2O2, to support the ox‐ idative turnover of LiPs and MnPs responsible for ligninolysis. The hydrogen peroxide is provided by extracellular oxidases that reduce molecular oxygen to H2O2, with the synergis‐ tic oxidation of a cosubstrate. The most well characterized extracellular H2O2-generating en‐

Most studies on enzymatic lignin degradation rely on white-rot fungi, which can miner‐ alise lignin to CO2 and H2O in pure cultures [65, 66]. Among these fungi are *Phanero‐*

and β-xylosidases are also required for hemicellulose degradation.

**9. Lignin degradation**

and aryl alcohol oxidase (AAO).

attacking it from the inside [63].

non-phenolic aromatic compounds, as well as Mn2+ [64].

zymes are glyoxal oxidase (GLOX) and aryl alcohol oxidase (AAO).

2H+

**Figure 4.** Schematic view on a hemicellulolytic system, degradation of arabinoxylan is depicted. The arrows represent each enzyme active for a determined substrate.

The fungal PLs pectin and pectate lyases hydrolyze α-1,4-linked D-galacturonic acid resi‐ dues in the smooth regions of pectin backbone [59]. Pectin lyases have preference for sub‐ strates with a high degree of methylesterification, whereas pectate lyases prefer substrates with a low degree of esterification. Moreover, pectate lyases require Ca2+ ions for catalysis while pectin lyases lack such ion requirement to catalysis [60]. The PL rhamnogalacturonan lyase cleaves within the hairy region of pectin and appears to be structurally different from pectin and pectate lyases. As presented by nailing reviews [33, 48], the pectin structures xy‐ logalacturonan and rhamnogalacturonan require a repertoire of accessory enzymes to re‐ move the side chains, providing access for the main-chain pectinolytic enzymes. The accessory enzymes endoarabinanases, exoarabinanases, β-endogalactanases, and several es‐ terases are specific for pectin degradation, while α-arabinofuranosidases, β-galactosidases, and β-xylosidases are also required for hemicellulose degradation.
