Most cellulase assays measure the production of reducing sugars from a high molecular weight form of cellulose, as every cleavage event produces a newreducing end. Endocellulases reduce the viscosity of carboxymethylcellulose (CMC), so another way to assay them is to measure the decrease in viscosity of CMC (12). Cellulases also can be assayed by measuring the increase in the number of cellulose particles produced by a cellulase incubated with a cellulose preparation of uniform particle size (13). This assay gave different kinetics of hydrolysis than measuring reducing end increase as it was linear with time and enzyme. It needed particles that were larger than 100 ^m in diameter, as smaller particles did not show an increase in number even though they gave an increase in reducing ends (14).

There are two known cellulase mechanisms: hydrolysis with retention of the stereochem­istry of the anomeric hydroxyl group, and hydrolysis with inversion of the anomeric hydroxyl group (15). One important difference between these mechanisms is that most retaining enzymes can catalyze transglycosylation as well as hydrolysis, while no known inverting enzymes catalyze transglycosylation (16). Cellulases are named by the family number asso­ciated with their catalytic domain (CD) followed by a capital letter that is assigned based on the order in which family members were discovered in a given organism, with A being used for the first (17).

Cellulases are currently the third largest industrial enzyme product worldwide, by dollar volume, because of their use in cotton processing, paper recycling, as detergent enzymes, in juice extraction, and as animal feed additives. However, cellulases will become the largest volume industrial enzyme, if ethanol, butanol, or some other fermentation product of sug­ars produced from biomass becomes a major transportation fuel, as seems likely. Currently, industrial cellulases are almost all produced from aerobic cellulolytic fungi, such as Hypocrea jecorina (Trichoderma reesei) or Humicola insolens. This is due to the ability of these organ­isms to produce extremely large amounts of crude cellulase (about 130 g/L), the relatively high specific activity of their crude cellulase on crystalline cellulose, and the ability to ge­netically modify these strains to tailor the set of enzymes they produce, so as to give optimal activity for specific uses.

Most aerobic cellulolytic microorganisms secrete a set of individual cellulases, which contain a carbohydrate-binding module (CBM) joined by a flexible linker peptide to the CD, and additional domains are often present. In some cellulases, the CBM is N-terminal to the CD, while in others it is C-terminal and the location probably does not affect its function. In contrast, most anaerobic microorganisms produce large (>1 million MW) multienzyme complexes, called cellulosomes, which are usually bound to the outer surface of the microorganism (18, 19). Only a few of the enzymes in cellulosomes contain a CBM, but the scaffoldin protein to which they are attached does contain a CBM, which binds the complex to cellulose. In both aerobic and anaerobic organisms, certain cellulases can act synergistically on crystalline cellulose with the specific activity of some mixtures being more than ten times that of any single cellulase in the mixture (20). Even though cellulose is a homopolymer of glucose, with only a single type of linkage (p-1-4), and with the disaccharide cellobiose being the repeating unit, cellulases are very diverse in their structures, mechanisms, and sequences.