The major bottleneck in the production of soluble sugars from cellulosic biomass involves the high cost of the enzymes necessary for this process. In theory, the solution should be simple: either to find cheaper ways of producing the enzymes or to find more active enzymes!

Actually, to improve the catalytic efficiency of cellulases may pose a formidable task, since these enzymes are, arguably, already of the most efficient in nature. Even if we were to improve the efficiency or thermal tolerance of one enzyme (acting alone), the question remains whether this improved enzyme will now work better in a synergistic mixture with the other enzymes. An exception to this consideration seems to be the case of the cellobiohy — drolase I and II enzymes (GH7 and GH6, respectively) from fungi. In the free cellulose system, these enzymes are the star performers producing the majority of the soluble sugars from cellulose, aided primarily by only a small addition of endoglucanase activity.

Nevertheless, current strategies focus on the identification of new and improved enzymes, with the hope that those of highest activities will work best together. Since the improvements in producing industrial quantities of enzymes are more of a technical, engineering feat, we will concentrate here on strategies designed for improving enzyme activities or combinations thereof rather than improvements of processes for their production. In order to assemble improved enzyme systems for biomass conversion, a series of methodologies is required (Figure 5.4) . New enzymes are identified by mining established enzyme databases, newly sequenced microbial genomes, and/or relevant cellulosic microenvironments. The newly identified enzymes are evaluated by genomic, proteomic, or metabolic profiling, and their activities are assessed. The properties of these enzymes can then presumably be improved by rational mutagenesis or directed evolution, leading to improved enzyme cocktails for enhanced conversion of cellulosic biomass to soluble sugars.