2.6 Maize stover utilization by herbivores
2.7.2 Cellulosomes
The second theory explains a greater level of complexity observed in some anaerobic microbes where several cellulases are grouped into an enzymatic complex, called cellulosome (Bayer et al., 1998a). Cellulosomes are extracellular super molecular machines that can efficiently degrade crystalline cellulosic substrates and associated plant cell wall polysaccharides (Shoham et al., 1999). The cellulosomal concept was first established by Lamed et al. (1983), in their study on the antigenic active cellulose-binding factor (CBF) of the bacterium Clostridium thermocellum. In an attempt to isolate and characterized the antigenic CBF, also called cellulose binding domain (CBD), they realized that the molecular weight was higher than predicted. Electron microscopic analyses of the CBF complex revealed a particulate, multi-subunit entity of a complicated quaternary structure. This complex molecule was resolved into 14 polypeptide bands on a polyacrylamide gel (Laemmli, 1970).
Eight of the polypeptide components exhibited celullolytic activity. They concluded that not
-1, 4 exocellulase -1, 4 cellobiase
only was the CBF complex molecule responsible for cell adherence to cellulose, but contain polypeptide chains that also hydrolysis cellulose.
The cloning and sequencing of cellulase genes from Clostridium thermocellum led to the prediction of the modular structure and multi-subunits of cellulosome (Bayer and Lamed, 1986; Shoham et al., 1999; Madkour and Mayer, 2003; Desvaux, 2005). Although anaerobic clostridial species were the only species with full genetic information available, signal sequences have been identified in other species including anaerobes and fungi (Table 2.6).
The different types of glycosyl hydrolases involved with cellulosome complexes are cellulases, hemicellulases and some carbohydrate esterases. All the enzymes are held
Figure 2.6 Schematic representation of the hydrolysis of amorphous and microcrystalline cellulose by a non-complexed (A) and a complexed (B) cellulase system. Solid squares represent reducing ends and open squares represent non-reducing ends (Lynd et al., 2002) modified.
together by a major polypeptide called scaffoldin, also known as cellulosome integrating protein A (CipA). The different activities of scaffoldin are dictated by its functional modules, dokerin domains, cohesion domains and CBD (Bayer et al., 1998a; Shoham et al., 1999;
Desvaux, 2005). Scaffoldin interacts with the following processes, cellulose binding, cell anchorage and the organization of enzyme subunits in the complex as well as promoting the cellulosomal enzyme activities. Cohesin facilitates the interaction of cellulosomal complex with enzymes and cell surface. Dockerin allows the interaction of catalytic domain and cohesin domains. The molecular weights of cellulosome complexes range from 40 to 180 KDa. Cell-free cellulosome clusters coating substrate surface and solubilizing celluloses has been reported by Bayer and Lamed (1986), which implies that not all cellulosomes are cell bound.
Table 2.6 Evidence for cellulosomes in cellulolytic microorganisms
Organism Cellulosome signature sequence
Protein Domain
Anaerobic bacteria Clostridium thermocellum
Clostridium cellulovorans, Clostridium cellulolyticum, Clostridium josui
Acetivibrio cellulolyticus
Bacteroides cellulosolvens
Ruminococcus albus, Ruminococcus flavefaciens
Scaffoldin Surface-anchoring proteins
Enzymes Scaffoldin Enzymes
Scaffoldin and surface- anchoring protein Scaffoldin or surface anchoring protein Enzymes
CohI1CBD1DocII CohII
DocI CohI1 CBD DocI
CohI1CBD1DocII CohII
CohII1CBD DocI
Aerobic bacteria
Vibrio sp. Enzyme Fungal-type dockerin
Anaerobic fungi
Neocallimastix, Piromyces,
Enzymes Fungal dockerins
Abbreviations: CBD, cellulose-binding domain, CohI, type-I cohesin domain; CohII, type-II cohesin domain;
DocI, type-I dockerin domain; DocII, type-II dockerin domain, (Shoham et al., 1999).
Shoham et al. (1999)
Figure 2.7 A schematic view of cellulosome subunits and its interaction between cellulose interaction and microbes
The intriguing question is, why is the arrangement of cellulases in a cellulosome complex more efficient than free enzyme systems in the solubilisation of crystalline cellulose?
Cellulosomes allow optimum cooperative activity and synergism of cellulase, avoids non- productive adsorption of cellulases, limits competition between cellulases for the site of adsorption and optimal processing of the cellulose all along the cellulose fibre (Schwarz, 2001). Johnson et al. (1982) compared crystalline cellulose solubilisation by cellulosome from C. thermocellum to that of free enzyme systems of Trichoderma reesei and found a much lesser amount of protein from C. thermocellum was required to completely solubilize the same amount of substrate. Boisset et al. (1999) also demonstrated that cellulosomes from C.
thermocellum were more efficient in solubilizing substrates of high crystalline content. Cell- density dependent growth is affected by the availability of solubles released from cellulose.
Cellulosome-mediated attachment of microbial cells to celluloses has an urge over microbes secreting free enzymes with respect to solubles utilization. This is because solubles generated by free enzymes away from the cells are not readily available. Therefore, limiting the concentration of solubles available to support growth at very low cell densities as compared to
cellulosomal systems where cells are available at the site of solubilisation. Cellulose solubilisation is a step wise process involving more than one enzyme. Therefore, the concentration of these enzymes in a cellulosome suggests an even stronger synergism among the catalytic units. Further research on the structural arrangement of cellulosomes and its benefit would be beneficial in the feed industries.
Although there is evidence supporting the cellulosome concept as a major break-through for the solubilisation of cellulose, many questions still need to be readdressed; (i) more evidence on the advantages of cellulosome over free cellulases, (ii) the overall structure of cellulosome, (iii) cellulosome secretion, attachment and assembly on the bacteria surface, (iv) whether a particular microbial species can produce both cellulosome and free cellulases, (v) what are the functions and structures of the accessory domains?, and (vi) its stability and survival in the rumen when applied as feed additives.