5-10: Enzyme recycling as a strategy to reduce enzyme hydrolysis costs: The enzyme adsorption and activity profiles observed during hydrolysis of steam pretreated corn stover

Efficient enzymatic hydrolysis of cellulosic biomass typically requires high enzyme loadings (e.g. cellulases and xylanases). Consequently, enzyme costs are a major bottleneck in the commercialization of cellulosic ethanol. A promising cost reduction strategy, utilizing the enzymes’ high stability, is to recycle the enzymes. An efficient enzyme recycling strategy requires a good understanding of the distribution of the enzymes during hydrolysis. Unfortunately, as most past studies have simplified this complex interaction by using either model substrates (such as cotton or avicel) or by following the adsorption behavior of purified enzymes in a reconstituted mixture, our understanding of this fundamental enzyme-substrate interaction is still limited. The work that will be reported followed the adsorption profiles of the individual enzymes in a commercial cellulase mixture during hydrolysis of steam-pretreated corn stover (SPCS). Using zymograms, gel electrophoresis, enzyme activity assays, mass spectrometry, etc, the adsorption and activity profiles of 6 glycoside hydrolases were followed.  More than 80% of the initial protein could be recovered from the liquid (supernatant) and the solid phase (residual substrate) after 72h hydrolysis. The adsorption profiles varied with some enzymes found primarily in the liquid phase and others in both the solid and liquid phases during hydrolysis. Enzymes that did not bind to the substrate were generally less stable compared to enzymes that were distributed in both phases. Enzyme recycling carried out after short-term rounds of hydrolysis, with enzymes recovered from both the liquid and the solid phases, was found to be the most effective strategy.