Matrix polysaccharides (i. e., hemicelluloses), are the second most abundant polymer in nature, and, as has been discussed, consist of heterogeneous polymers of pentoses (xylose, arabinose), hexoses (mannose, glucose, galactose), and sugar acids (Girio et al. 2010). Due to the abundance and functional importance of xylan in the cell walls of switchgrass, we focus here on the GHs that degrade xylan. However, enzymes that target every class of cell wall polysaccharide have been characterized (Table 3). Many microorganisms, such as Penicillium capsulatum and Talaromyces emersonii, possess complete degradation systems for grass glucuronoarabinoxylans (Filho et al. 1991). Like cellulose biodecomposition, total degradation of xylan also requires diverse enzymes for depolymerization and side-group cleavage. Endo-xylanases attack internal bonds in the main chains of xylans; exo-xylanases hydrolyze p-1,4-xylose linkages at chain ends to release xylobiose; and then p-xylosidase further hydrolyzes xylo-oligosaccharides and xylobiose to xylose (Gilbert et al. 2008). Side chains on xylose units block the action of some xylanases, leading to the evolution of diverse accessory enzymes to remove the side-chains and render the xylan backbone accessible for complete hydrolysis (Perez et al. 2002; Gilbert et al. 2008). Hydrolases with a-arabinofuranosidase and a-glucuronidase activities are responsible for removing arabinose and 4-O-methyl glucuronic acid substituents, respectively, from the xylan backbone. Furthermore, esterases hydrolyze linkages between xylose units and acetic acid (acetylxylan esterase) or between arabinose side chain residues and the hydroxycinnamic acids, ferulic acid (feruloylesterases) and p-coumaric acid (p-coumaric acid esterase).

Cellulosomes

The individual classes of hydrolases described above function within both non-complexed and complexed cellulase systems (Fig. 6) (Fontes et al. 2010). The non-complexed systems consist of individual polypeptides that can have multiple catalytic and CBM domains, but that otherwise act without interacting physically with other classes of hydrolases. In contrast, the complexed systems, also known as cellulosomes, are superstructural, multi-polypeptide enzyme complexes that adhere to cell walls of lignocellulolytic bacteria and fungi (Fontes et al. 2010). They consist of a multi-functional integrating subunit, called a scaffoldin, that is composed of multiple cohesion modules, and diverse enzymatic subunits with dockerin modules that interact with the scaffoldin. For example, the cellulosomes of C. cellulolyticium have the potential to contain numerous cellulases, xylanases, mannases, and even protease inhibitors (Blouzard et al. 2010).

Well-studied cellulosome-producing anaerobic bacteria include Clostridium species and Ruminococcus species (Doi et al. 2004). Cellulosome composition is dynamic and heterogenous, depending on the bacteria and composition of extracellular polysaccharides, and the relative amounts of the available dockerin-containing modules consistent with this (Raman et al. 2009). Cellulosomes have higher cellulose degradation efficiency compared with non-complexed enzymes since their adhesion to the cell surface prevents their products from being lost via diffusion or uptake by neighboring bacteria (Schwarz 2001). In vitro construction of mini — cellulosomes and self-assembly of cellulosomes on the surface of yeast significantly enhances cellulose hydrolysis compared with free enzymes (Wen et al. 2010; Fan et al. 2012; You et al. 2012).

Cellulosome-generating microorganisms also exhibit diversity in cellulosomal composition and architecture. For example, the Ruminococcus flavefaciens FD-1 genome encodes over 200 dockerin-containing proteins (Berg Miller et al. 2009); whereas, the Bacteroides cellulosolvens cellulosomes may possess more than 100 enzymes (Ding et al. 2000; Xu et al. 2004). This genomic diversity is likely functional. Proteomics of isolated cellulosomes from C. cellulolyticum confirmed the expression of 50 dockerin-containing proteins out of 62 predicted by bioinformatics (Blouzard et al. 2010). The complexity of the cellulosome is related to the availability and abundance of cellulosomal components, the expression of which is influenced by substrate induction and catabolite repression. For example, C. cellulolyticum grown on cellulose, expresses 36 cellulosome component enzymes. A partially distinct set of 30 cellulosome enzymes are detected on xylan; and 48 are expressed on wheat straw (Blouzard et al. 2010). Thus, cellulosomes are heterogeneous with varied components and stoichiometries. Moreover, some microbes exhibit even more diverse cellulosomes due to the presence of multiple types of scaffoldins within a single genome (Fontes et al. 2010). C. thermocellum contains four type II cohesion-containing anchoring scaffoldins (Bayer et al. 1998). For example, the cellulosomes assembled by the type II dockerin domain of CipA are further organized into a larger complex, called a polycellulosome, via type II cohesion-containing anchoring scaffoldins (Bayer et al. 1986; Raman et al. 2009). In short, cellulosomes generally have diverse content with heterogenous composition and architecture.