Development of a versatile multicomponent glycoside hydrolase mixture from thermophilic bacteria for high temperature biomass saccharification

Highly active multicomponent mixtures of glycoside hydrolases are required for the production of cellulosic biofuels. These enzymatic mixtures have usually been obtained from filamentous fungi, which secrete large amounts of glycoside hydrolases for cellulose/hemicellulose hydolysis. These fungal glycoside hydrolases have limited temperataure and pH ranges and are sensitive to chemicals used during biomass pretreatment. Bacterial glycoside hydrolases have broader temperature/pH ranges, but bacterial glycoside hydrolase mixtures for biomass saccharification are not well-developed. A highly active bacterial cocktail that operates at temperatures up to 80 C and in a pH range of 5-8 was formulated by recovering supernatants from thermophilic microbial communities adapted to grow on biomass substrates and supplementing these supernatants with purified proteins from thermophiles obtained by expression in E. coli. The components of this bacterial cocktail, called Jtherm, were prodced at pilot scale (300 L) and used to to scale biomass saccharifications in the presence of 20% ionic liquid. Purification of the active component of the supernatant base of Jtherm permitted an four-fold lowering of protein concentration for saccharification and demonstrated that the active Jtherm component was expressed by an uncultivated population present at low abundance in the microbial community. Heterologous expression of the Jtherm components in Aspergillus niger has provided a pathway to high titer protein expression of the cocktail for industrial application.