Are biomass-degrading enzymes working maximally?

Biomass-degrading enzyme preparations must work to convert as much of the polysac­charides in the cell wall as possible to monomers. Currently, high loadings of cellulases are needed to reach 95% conversion of cellulose in pretreated biomass to sugars in 3-5 days using simultaneous saccharification and fermentation. Cellulase preparations are expensive in the biorefinery context for two reasons historically: 1) the source of the enzymes, usually Trichoderma reesei, was costly to grow and induce and 2) the specific performance (or activity) are low compared to other polysaccharide degrading enzymes.

However, a significant breakthrough in reducing the cost to produce and use T. reesei cellulases in the biorefinery was achieved by the DOE Office of the Biomass Program funded subcontracts awarded to Genencor International andNovozymes Biotech (2000-2005). Over the period of performance of these subcontracts, this cost was reduced about 10-fold from the starting cost of about $5 per gallon of ethanol produced. We note that only a small percentage of the final cost reduction came from actually improving enzyme structure/function. The question is often asked: Howlow-cost must cellulases be to enable a new biorefinery industry? One answer may be based in considering the current cost of starch-degrading enzymes, which are about $0.01-$0.05 per gallon of ethanol produced. New amylase and glucoamylase technology introduced in 2005 will further lower these costs. For cellulase costs to approach that of starch-degrading enzymes, we must focus on considering the resistance of cellulose in plant microfibrils to deconstruction.

A deep understanding of the structure/function principles governing cell wall polysaccha — ridase action is critical. The study of cellulase action is especially challenging, considering that these enzymes function to first decrystallize cellodextrins and then hydrolyze the extracted chains to cellobiose and glucose. This process is not currently understood at the kinetic or thermodynamic level. Fundamental questions exist on the limits of enzyme activity and the action of soluble enzymes or enzyme aggregates on insoluble polymeric substrates in aqueous environments. It is possible, for example, that enzymes acting on microcrystalline cellulose are already working at the maximal rate!

The areas of poor scientific understanding presented above have clearly deterred past re­search programs aiming to reduce cellulase cost by improving performance. To summarize, the task of improving the specific activity of cellulases is complicated by our poor under­standing of 1) cellulase natural diversity, 2) cellulase active-site architecture, 3) cellulase processivity, 4) cellulose decrystallization, and 5) the cellulose structure in plants.