**9. Lignin 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].

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

**Figure 4.** Schematic view on a hemicellulolytic system, degradation of arabinoxylan is depicted. The arrows represent

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‐

each enzyme active for a determined substrate.

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) and aryl alcohol oxidase (AAO).

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) + 2H+ + H2O2 → 2 Mn(III) + 2H2O [64]. In this way, Mn3+ diffuses into the lignified cell wall, attacking it from the inside [63].

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

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

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‐* *chaete chrysosporium*, *Ceriporiopsis subvermispora*, *Phlebia subserialis*, and *Pleurotus ostreatus*, which are able to metabolize the lignin in a variety of lignocellulosic biomass [62, 67, 68]. In addition, other species of fungi, such as *Postia placenta* (a brown-rot fungus), and some bacteria (such as *Azospirillum lipoferum* and *Marinomonas mediterranea*), are able to metabolize lignin. The saprotrophic homobasidiomycete *Pycnoporus cinnabarinus* is recog‐ nized by its high lignocellulolytic potential [69] overproducing high redox potential lac‐ case, and a variety of studies have been performed in order to increase the ability of this specie to produce laccases for biotechnological applications, including heterologous ex‐ pression in other species such as *A. niger* [70, 71, 72]. In addition, white-rot fungi such as *Cyathus cinnabarinus* and *Cyathus bulleri* demonstrated potential to degrade lignin [73, 74].

**11. Induction of cellulases**

Although the biochemistry of the process behind lignocellulosic degradation has been stud‐ ied in detail, the mechanism by which filamentous fungi sense the substrate and initiate the overall process of hydrolases production is still unsolved. Some researchers have been pro‐ posed that a low constitutive level of cellulase expression is responsible for the formation of an inducer from cellulose, amplifying the signal. Another group of scientists suggest that the fungus initiates a starvation process, which could in turn activates cellulase/hemicellulase expression. Also, it is possible that an inducing sugar derived from carbohydrates released somehow from the fungal cell wall could be the derepressing molecules for hydrolase in‐ duction. Despite of the fact that the true mechanism behind natural cellulase/hemicellulase induction is still lacking, some individual molecules are known to induce these hydrolases.

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The fungus *Trichoderma ressei* is an impressive producer of cellulases and most of studies concerning the regulation of cellulase genes have been performed in this specie. The most powerful inducer of cellulases in *T. reesei* is sophorose, a disaccharide composed of β-1,2 linked glucose units. Sophorose appears to be formed from cellobiose through transglycosy‐ lation activity of β-glucosidase [76 - 78]. In addition to *T. reesei*, sophorose is known to

Cellobiose (two β-1,4-linked glucose units) appears to induce cellulase expression in many species of fungi. Cellobiose is formed as the end product of cellobiohydrolases activity, and it has been show to induce cellulase expression in *T. reesei*, *Volvariella volvacea*, *P. janthinellum* and *A. nidulans* [81 - 84]. However, studies concerning the inducing effect of cellobiose on cellulase expression are controversial [6]. For instance, cellobiose can be transglycosylated by β-glucosidases, producing sophorose, which could be the true inducer of cellulases. Be‐ sides, β-glucosidases are able to cleave the cellobiose into glucose, which may cause repres‐ sion by CCR. Therefore, the outcome in cellobiose cultures appears to be dependent on the balance between hydrolysis and transglycosylation, as well as the subsequent uptake of the

Lactose (1,4-*O*- β-D-galactopyranosyl-D-glucose) is a disaccharide that has been shown eco‐ nomically viable to induce cellulase expression in *T. reesei*. Interestingly, lactose is not a com‐ ponent of plant cell wall polymers and the mechanism through which this sugar induces cellulase expression appears to be complex. In filamentous fungi, lactose is cleaved by ex‐ tracellular β-galactosidase into glucose and galactose. Lactose induction of cellulase genes requires the β-anomer of D-galactose, which can be converted to fructose by an alternative pathway in addition to the Leloir pathway [85]. In this alternative pathway, D-xylose reduc‐

Moreover, induction of cellulase genes could be achieved in *T. reesei* cultures after addition of various other oligosaccharides such as laminaribiose, gentiobiose, xylobiose, L-sorbose and δ-cellobiono-1,5-lactone. L-arabitol and different xylans also have been show to induce

induce cellulase expression in *A. terreus* and *P. purpurogenum* [79 - 80].

generated sugars and the intracellular signals they initiate.

tase (encoded by *xyl1*) is the enzyme catalyzing the first step [86].

expression of cellobiohydrolase 1 (*cbh1*) in *T. reesei* (reviewed in reference [6].

In summary, microbial degradation of lignocellulosic material requires a concerted action of a variety of enzymes arranged in an enzymatic complex, depending on the biomass to be degraded. The gene expression, production and secretion of plant cell wall-degrading en‐ zymes demand energy from the microbial cells and therefore the overall process is highly regulated. There is an intense cross-talk in induction of expression of the genes encoding dif‐ ferent classes of enzymes. The control of the regulation of CWDEs production could be the key for the development of new microbial strains that efficiently produce and secrete CWDEs. The regulation of genes encoding polysaccharide-degrading enzymes will be the subject of the next section of this chapter.
