The enzyme systems capable of digesting cellulose and hemicellulose are reviewed in this section. Although hemicellulose can be partly hydrolyzed by some thermo- chemical pretreatments, digestion of residual hemicellulose by enzymes can significantly improve overall conversion yields. Lignin, an aromatic-based biopoly- mer, is generally resistant to enzymatic digestion. While a few lignin-degrading enzymes have been discovered (Chen et al., 2011), lignin-degrading enzymes have not yet been sufficiently developed for use in a biorefinery, and are not included in the discussion below.
4.2.1 Biomass Recalcitrance
As our understanding of how the complex physiochemical structure of biomass contributes to recalcitrance has evolved, so has our understanding of how the assorted biomass-degrading enzymes work together to overcome biomass recalcitrance. It is now clear that the classical viewpoint of recalcitrance, which states that recalcitrance is primarily associated with the “physical presence of lignin and hemicellulose and the form of cellulose, with crystalline cellulose being more recalcitrant than the amorphous form,” is overly simplistic (Kohlmann et al., 1996). Himmel et al.
(2007) emphasize the importance of the phrase “biomass recalcitrance” and expand upon the traditionally terse definition of this term by discussing several barriers that biological systems face when performing degradation of lignocellulosic structural carbohydrates. They describe a number of anatomical features in plant tissues that contribute to recalcitrance. These include “the arrangement and density of the vascular bundles” and “sclerenchymatous” tissues, lignifications within the plant cell wall, and “the structural heterogeneity and complexity of cell wall constituents, such as microfibrils and matrix polymers.”
The major enzymes in all biomass conversion cocktails today are cellulases and hemicellulases. However, it is also important to recognize and consider the effects
TABLE 4.1 Enzyme Families and Their Activities on Lignocellulosic Substrates
Substrate Enzyme activity CAZy families
Cellulose β-1,4-Endoglucanase GH5,7,12,45,9,48
Cellobiohydrolase GH6,7,4,48,9
β-1,4-Glucosidase GH1,3,9
β-1,4-Endoglucanase/polysaccharide mono-oxygenase GH61, CBM33
Xylan β-1,4-Endoxylanase GH10,11,9,8
β-1,4-Xylosidase GH3,43
α-Glucuronidase GH67,115
α-Arabinofuranosidase GH51,54
Arabinoxylan arabinofuranohydrolase GH62
β-1,4-Galactosidase GH2,35
Acetyl xylan/feruloyl esterase CE1,4,5,16
Xyloglucan Xyloglucanβ-1,4-endoglucanase GH12,74
α-Arabinofuranosidase GH51,54
α-Xylosidase GH31
α-Fucosidase GH29,95
α-1,4-Galactosidase GH27,36
β-1,4-Galactosidase GH2,35
Source: Adapted from van den Brink and de Vries (2011).
of secondary accessory enzymes that aid the major cellulases and hemicellulases in performing enzymatic digestion. Also, when considering the carbohydrate-active enzymes (CAZy) glycoside hydrolase (GH) classification system, it is important to keep in mind that although enzymes within the same CAZy GH family share sequence similarity, many families can contain multiple activities. One example of this is the GH5 family, which contains many catalytic activities, including exoglucanases, endoglucanases, and endomannanases. Table 4.1 lists groups of several biomass- degrading enzyme families by function and substrate.
4.2.2 Cellulases
The primary target product of most traditional conversion processes is the fermentable monomer, glucose. Glucosein plantalargely exists in the form of crystalline cellulose, which consists of chains ofβ-1,4 linked glucosyl units that are stabilized by hydrogen bonds, van der Waals forces, and stacking of hydrophobic regions. These chains of glucose are the primary targets of processive cellobiohydrolases such as fungal GH6s (active on the nonreducing end) and fungal GH7s (active on the reducing end), but are aided by enzymes such as fungal family 5 endo-glucanases that create new reducing ends for the enzymes to function on (Nutt et al., 1998; van den Brink and de Vries, 2011). While fungal GH5,6,7’s are the most common cellulose-active enzymes in fun- gal cellulase mixtures, there also exist unique analogous families in bacteria, for exam- ple GH48’s are exo-acting cellulases (Zverlov et al., 1998; Irwin et al., 2000). GH9’s are also unique in that some are processive endo-cellulases (Table 4.1) (Li et al., 2007).
The crystalline nature of the cellulose chains in plant cell walls has always been regarded as one barrier to conversion, though a variety of cellulose-degrading enzyme systems have evolved to digest the crystalline form of cellulose at various rates (Fan et al., 1981; Kohlmann et al., 1996; Himmel et al., 2007). However, native cellulose in plants is composed of both crystalline and amorphous components. Some researchers have reported increases in the binding of a cellobiohydrolase (Cel7A) to the amorphous form of cellulose, and resulting 24 hour extents of conversion, com- pared with the highly crystalline form (Schroeder et al., 1986; Jeoh et al., 2007). One possible benefit of an acidic thermo-chemical pretreatment process may be to modify cellulose to contain more extensive regions of disordered and reduced crystallinity.
If the disordered cellulose is also amorphous-like, this would explain the enhanced cellulase action observed on dilute acid pretreated cellulose (Matthews et al., 2010).
The classical endo–exo deconstruction model of pure cellulose is undergoing revi- sion in light of the recent discovery of a family of copper-dependent polysaccharide mono-oxygenases from the GH61 family (Table 4.1) (Harris et al., 2010; Quinlan et al., 2011). These enzymes represent a novel mechanism to deconstruct cellulose in that they cleave cellulose oxidatively, unlike traditional acid/base catalyzed enzyme systems. Family GH61 enzymes can also provide powerful synergistic benefits to cocktails of traditional GH5, GH6, and GH7 enzyme mixtures (Harris et al., 2010).
4.2.3 Hemicellulases
The second most abundant sugar found in lignocellulosic feedstocks (excepting soft- woods) is xylose that comprises the xylan backbone of hemicellulose. In general, it is thought that cellulose microfibrils are held together loosely by a network of various cross-linking glycans (hemicelluloses) using hydrogen bonding and carbohydrate–
carbohydrate interactions (Gorshkova et al., 2010). The primary enzymes required for removal of the xylan backbone, which comprises the bulk of xylan found in plant materials, are endo-xylanases from diverse families (GH5, GH8, GH10, and GH11) that act onβ-D-(1,4) linkages. In particular, GH10 enzymes degrade linear chains of β-D-(1,4)-linkages, xylan backbones with high degrees of substitutions, and smaller xylo-oligosaccharides, while GH11 xylanases function primarily onβ-D-(1,4) link- ages (Collins et al., 2005). A smaller body of work to date has focused on the impact of other less abundant hemicelluloses, particularly those associated with primary cell walls, including xyloglucans, glucomannans, and mixed linkage glucans, that is, β-D-(1,3-1,4) glucan (Scheller and Ulvskov, 2010). All of these carbohydrate linkages are known to exist at low levels in cell walls. Finally, the released xylo- oligosaccharides, typically xylobiose, must also be degraded byβ-xylosidases. Most fungalβ-xylosidases belong to the GH3 family, but several putativeβ-xylosidases are assigned to GH43 (Table 4.1) (van den Brink and de Vries, 2011).
4.2.4 Accessory Enzymes
While cellulose and xylan comprise the vast majority of sugars available in plant cell walls, other polysaccharides are also present at much lower levels. Depending on the
plant species, these secondary polysaccharides include xyloglucans, glucomannans, and mixed linkage glucans such asβ-1,3 glucans or mixed linkageβ-(1,3);β-(1,4) glucans, (Matthews et al., 2006; Scheller and Ulvskov, 2010). Thus, these small pools of secondary sugars are largely inaccessible to traditional cellulases and require β-(1,3) debranching activities provided by family GH55 enzymes. One example of a glucose releasing accessory enzyme is a xyloglucanase obtained fromTrichoderma reesei. When added to simple cellulase mixtures, this enzyme improves the extent of conversion, though the modest nature of these improvements makes it difficult to discern whether the benefit is synergistic or merely additive (Benko et al., 2008).
When considering the conversion of the xylan backbone, one feature that is notable is its decoration with additional sugars and other moieties (e.g., acetyl), adding to its complexity. Accessory enzymes hydrolyze some of the linkages of these com- plex xylooligomers. Xylose can also be present in other hemicellulose forms, such as in xyloglucans. Substitutions on the individual xylose units comprising these materials can includeacetyl,arabinofuranosyl, andmethyl glucuronosylgroups that vary in placement and abundance widely depending on the plant material. Addi- tionally,arabinofuranosyl units often cross-link the xylan backbones to lignins in the plant cell wall viaferuloyl esterunits, which are thought to be ether-bonded to the lignin (Jeffries, 1990; Scheller and Ulvskov, 2010). In corn stover specifically, chemically isolated xylans have been shown to be primarily decorated with 2-O- and 3-O-monoacetyl, [MeGlcA-α-(1,2)][3-OAc], and arabinofuranosyl units (Naran et al., 2009). It is primarily these “decorations” on xylose and various cross-links that are the targets of accessory enzymes. To target the acetyl decorations, acetyl xylan esterase (AXE) enzymes are employed. AXEs inhabit several carbohydrate esterase (CE) families, the key differences being the specific O-linked acetyl linkage to be hydrolyzed. CE families 1, 4, and 5 have a strong preference for 2-O-linked residues, the most common linkage in hemicellulose, while CE16 prefers 3-O- and 4-O-linked residues (Margolles-Clark et al., 1996; van den Brink and de Vries, 2011; Zhang et al., 2011). Cleavage of arabinofuranosyl linkages typically requires fungal α-arabinofuranosidases mainly found in GH families 51 and 54, although some bifunctional enzymes from GH3 and GH43 have also been shown to haveα- arabinofuranosidase activity (Numan and Bhosle, 2006; van den Brink and de Vries, 2011). For the deconstruction of methyl-glucuronosyl linkages there are two primary families forα-glucuronidases: the family GH67α-glucuronidases are active on short oligosaccharides, while some of the GH115α-glucuronidases are active on polymeric xylan (Table 4.1) (Nagy et al., 2002, 2003; van den Brink and de Vries, 2011).
4.2.5 Synergy with Xylan Removal and Cellulases
The synergy between cellulases, hemicellulases, and accessory enzymes has been explored in some depth but is still not fully understood. Studies utilizing purified cellulases and hemicellulases have shown that when some of the hemicellulose component in lignocellulose remains intact following pretreatment, the enzymatic hydrolysis of the remaining xylan and substituted xylan moieties improves cellulose hydrolysis by cellulases (Selig et al., 2008, 2009). This improvement in cellulose
conversion by hemicellulose removal has been shown to be a linear relationship.
One proposed hypothesis for this improvement is that hemicellulose provides a steric hindrance for cellulases, but there is not yet any conclusive evidence for this. To improve both rates and total extents of conversion of hemicellulose to monomers, one needs to effectively cleave side-chain sugars from the xylan backbone using enzymes such as AXE,α-glucuronidase, andα-L-arabinofuransidase (Naran et al., 2009). The impact on xylan hydrolysis by removing such side groups was initially observed by Biely et al. (1986), who in their early studies on AXEs noted that using them with xylanases improved the overall rate of conversion. A similar improvement using arabinofuranosidases was also reported that indicated arabinofuranosyl side groups impeded hydrolysis of the xylan backbone in arabinoxylans (Sứrensen et al., 2003). Furthermore, acetyl side groups, and feruloyl esters linked to arabinofuranosyl side chains, also have a direct impact on the hydrolysis of residual xylan remaining after chemical pretreatment (Selig et al., 2008, 2009). It has also been reported that commercially available hemicellulase preparations have been shown to be effective in providing adequate supplementation of debranching enzyme activities (Sứrensen et al., 2007).
One possible alternative to the steric hindrance hypothesis of synergistic action is the recent work by Qing et al. (2010), in which they have demonstrated that complex xylooligomers, much more so than monomeric xylose and insoluble xylan, are strongly inhibitory toward hydrolysis of pretreated lignocelluloses when using commercial cellulase complexes. This may be another explanation for the improve- ments seen when using cellulase–xylanase mixtures. Furthermore, Qing et al. (2010) also noted that pre-hydrolysis of xylan and xylooligomers prior to cellulase addition was more effective than simultaneous hydrolysis of these carbohydrates, which may suggest that the hemicellulosic carbohydrates may somehow irreversibly bind to and inactivate the cellulases.
Our understanding of the interaction of biomass-degrading enzymes on lignocel- lulosic biomass has advanced considerably in the last decade. Nonetheless, further work in this area will help point the way toward optimized systems of enzymes tailored for specific feedstocks and pretreatment methods, as further discussed in section 4.5.