The primary factor in the high cost of enzymes for biomass hydrolysis is sim­ply the amount of enzyme that must be used. Compared to starch hydrolysis, 40- to 100-fold more enzyme protein is required to produce an equivalent amount of ethanol (Novozymes data). It was recognized very early on that efficient cellulose hydrolysis requires a complex, interacting collection of en­zymes during initial characterization of the T. reesei cellulase system [35]. To significantly reduce the amount of these enzymes requires that either more efficient component enzymes are identified or that additional enzymes can be added that reduce the total enzyme loading. Synergy, the ability of two or more enzymes to work simultaneously more effectively than in succession, was first described in cellulases more than 30 years ago when describing the action of CBH I and EG activities [36]. In this case, the synergy can be mech­anistically explained by the production of new cellulose ends by the action of the endoglucanase, creating new sites of exoglucanase attack by the CBH. Similarly, studies of the observed synergism between CBH I and CBH II from Humicola insolens, revealed that this CBH II, although capable of acting pro — cessively from non-reducing chain ends, does also cleave the cellulose chains in an endo fashion [37]. To drive enzyme loading down, we needed to search for similar synergistic enzyme pairs that could complement the preferred T. reesei cellulase system.

4.2.1