2.4.1 Enzymology of Cellulose Degradation by Cellulases

Enzymic saccharification of pretreated biomass has gradually supplanted acid hydrolysis in pilot plant developments for ethanol production from lignocellulosic substrates (table 2.3). “Cellulase” is a deceptively complex concept, a convenient shorthand term for four enzyme activities and molecular entities, each with their Enzyme Commission (EC) identifying numbers, required for the complete hydrolytic breakdown of macromolecular cellulose to glucose:5960

1. Endoglucanases (1,4-P-D-glucan-4-glucanohydrolases, EC 3.2.1.4) decrease the degree of polymerization of macromolecular cellulose by attacking accessible sites and breaking the linear cellulose chain.

2. Cellodextrinases (1,4-P-D-glucan glucanohydrolases, EC 3.2.1.74) attack the chain ends of the cellulose polymers, liberating glucose.

3. Cellobiohydrolases (1,4-P-D-glucan cellobiohydrolases, EC 3.2.1.91) attack the chain ends of the cellulose polymers, liberating the disaccharide cellobiose, the repeating unit of the linear 1,4-linked polyglucan chain (figure 1.23).

4. Finally, P-glucosidases (EC 3.2.1.21) hydrolyze soluble cellodextrins (1,4- P-D-glucans) and cellobiose to glucose.

Plurals have been used for each discrete enzyme activity because cellulolytic organisms often possess multiple genes and separable enzymically active proteins; for example, the fungus Hypocrea jecorina[14] contains two cellobiohydrolases, five endoglucanases, and two P-glucosidases.61-63

Cellulases are widely distributed throughout the global biosphere because not only is cellulose the single most abundant polymer, but many organisms have also evolved in widely different habitats to feed on this most abundant of resources. Bacteria and fungi produce cellulases in natural environments and while contained in the diges­tive systems of ruminant animals and wood-decomposing insects (e. g., termites), but insects themselves may also produce cellulases, and other higher life forms — plants and plant pathogenic nematodes — certainly do.6064 Higher plants need to reversibly “soften” or irreversibly destroy cell wall structures in defined circumstances as part of normal developmental processes, including plant cell growth, leaf and flower abscis­sion, and fruit ripening; these are highly regulated events in cellular morphology.

The online Swiss-Prot database (consulted in December 2006) listed no fewer than 120 endoglucanases, 22 cellobiohydrolases, 4 cellodextrinases, and 27 P-glucosidases (cellobiases) in its nearly 250,000 entries of proteins fully sequenced at the amino acid level; most of these cellulase components are from bacteria and fungi, but higher plants and the blue mussel (Mytilus edulis) are also represented in the collection.65 The enormous taxonomic diversity of cellulase producers has aroused much speculation; it is likely, for example, that once the ability to produce cellulose had evolved with algae and land plants, cellulase producers arose on separate occasions in different ecological niches; moreover, gene transfer between widely different organisms is thought to occur easily in such densely populated microbial environments as the rumen.66

From the biotechnological perspective, fungal and bacterial cellulase producers have been foci of attention as potential industrial sources. More than 60 cellulolytic fungi have been reported, including soft-rot, brown-rot, and white-rot species — the last group includes members that can degrade both cellulose and lignin in wood samples.67 The penetration of fungal hyphae through the solid growth substrate represented by intact wood results in an enormous surface area of contact between the microbial population and lignocellulosic structures; the release of soluble enzymes then results in an efficient hydrolysis of accessible cellulose as different exo — and endoglucanases attack macroscopic cellulose individually at separate sites, a process often referred to as “synergy” — it is also highly significant that even different mem­bers of the same exo — or endoglucanase “family” have different substrate selectivities (table 2.4), thus ensuring a microdiversity among the catalytic population and a maxi­mized capacity to hydrolyze bonds in cellulosic glucans that may have different poly­mer-polymer interactions.59 Aerobic bacteria have a similar strategy in that physical adherence to cellulose microfibers is not a prerequisite for cellulose degradation, and a multiplicity of “cellulases” is secreted for maximal cellulose degradation — pres­ently, the most extreme example is a marine bacterium whose extraordinary meta­bolic versatility is coded by 180 enzymes for polysaccharide hydrolysis, including 13 exo — and endoglucanases, two cellodextrinases, and three cellobiases.68

Anaerobic bacteria, however, contain many examples of a quite different biochemical approach: the construction of multienzyme complexes (cellulosomes) on the outer surface of the bacterial cell wall; anaerobic cellulolytics grow optimally when attached to the cellulose substrate, and for some species, this contact is obligatory.60 The ability of such anaerobic organisms to break down cellulose and to ferment the resulting sugars to a variety of products including ethanol has prompted several investigators to promote them as ideal candidates for ethanol production from lignocellulosic biomass.69

The drive to commercialize cellulases — in applications as diverse as the stonewashing of denims, household laundry detergent manufacture, animal feed

TABLE 2.4

Substrate Selectivities of Trichoderma Cellulase Components

Endoglucanase

Cellobiohydrolase Cellobiohydrolase

3

2

5

1

3

1

1

1

Note: Numbers represent relative activity: 0 inactive, 5 maximum activity production, textile “biopolishing,” paper deinking, baking, and fruitjuice and beverage processing70 — has ensured that the biochemistry of the exo — and endoglucanases that attack macromolecular cellulose has been extensively researched. Most of these enzymes share a fundamental molecular architecture comprising two “domains” or “modules”: a cellulose-binding region (CBD or CBM) and a catalytic module or core.71 As more cellulase enzymes have been sequenced at the levels of either amino acids or (almost invariably now) genes, families of conserved polypeptide structures for CBD/CBM have been recognized; they form part of the 34 presently recognized carbohydrate-binding modules collated in a continuously updated data — base.72 All proteins in three families (CBM1, CBM5, and CBM10) bind to crystalline cellulose, whereas proteins in the CBM4 and CBM6 families bind to cellulose as well as to xylans and other polysaccharides using different polysaccharide binding sites.73 Removal of the portion of the cellulase responsible for binding to cellulose reduces cellulase activity with cellulose as the substrate but not with cellodextrins; conversely, the isolated binding domains retain their affinity for cellulose but lack catalytic action.74 The contribution of cellulose binding to overall cellulase activ­ity has more recently been elegantly demonstrated in a more positive manner: the endoglucanase II from Hypocrea jecorina has five amino acid residues in a topo­graphically distinct planar surface in the CBM; selectively altering the amino acids at two positions increased and decreased cellulose binding affinity and cellulose hydrolysis rate in synchrony (table 2.5) and demonstrated that the native enzyme could be catalytically improved by combinatorial mutagenesis.75

• The Cip units are anchored to the cell wall via other cohesin domains.

• Large and stable complexes are formed with molecular weights in the range of 2-16 MDa, and polycellulosomes occur with molecular weights up to 100 MDa.

The elaboration of such complex structures is thought to aid the energy-deficient anaerobes by maximizing the uptake of cellodextrins, cellobiose, and glucose by the spatially adjacent bacteria and ensuring a greatly increased binding affinity to the

cellulose.59,60

The cellulases of cellulolytic anaerobes may also be more catalytically effi­cient than those of typical aerobes and, in particular, the soluble cellulases secreted by fungi — but this is controversial because enzyme kinetics of the cellulose/cel — lulase system are problematic: the equations used for soluble enzyme and low — molecular-weight substrates are inadequate to describe the molecular interactions for cellulases “dwarfed” and physically binding to macroscopic and insoluble cel­luloses.59 Nevertheless, some results can be interpreted as showing that clostridial cellulases are up to 15-fold more catalytically efficient (based on specific activity measurements, i. e., units of enzyme activity per unit enzyme protein) than are fun­gal cellulases.60 Similarly, a comparison of fungal (Hypocrea, Humicola, and Irpex [Polyporus]) and aerobic bacterial (Bacillus, Pseudomonas, and Thermomonospora) cellulases noted a higher specific activity of more than two orders of magnitude with the bacterial enzymes.67