In general, cellulase activities measured using insoluble substrates are more likely to be relevant to large-scale biomass deconstruction than chemically modified, soluble chains. A variety of different insoluble substrates are used to measure cellulase activ­ities. These substrates differ in their biological sources and pretreatment methods, and as a result, they have different structural and physical properties (Table 2). As a result, different substrates are more appropriate for different types of enzymes.

By pretreatment with acid (typically phosphoric acid), insoluble cellulose preparations can be obtained that have decreased crystallinity, and thus are typically more susceptible to enzymatic digestion than crystalline substrates [22] . Although published protocols differ in subtle but important ways, high concentrations of phosphoric acid can produce PASC (for phosphoric acid swollen cellulose, [78]). By prehydrating Avicel prior to acid treatment, Zhang et al. prepared an amorphous cellulose of even higher reactivity, which they term regenerated amorphous cellu­lose (RAC, [82] ).

Cellulose substrates with high crystallinity include bacterial cellulose (BC), which has high DP values of 2,000-8,000, and a high 60-90% crystallinity index [37]. Microcrystalline cellulose, or “Avicel” PH, which is prepared by acid hydro­lysis of wood pulp, has a lower DP value (150-300) [37], although it retains a high level of crystallinity. Because it has a high ratio of free ends to accessible b-gluco — sidic bonds due to its lower DP, Avicel is especially suited for the measurement of exoglucanase activity from a crystalline substrate [ 82]. Finally, Whatman No. 1 filter paper, manufactured from cotton linters, is highly heterogeneous and is often used for measuring total cellulase activity.