With increasing attention directed towards lower severity pretreatments (e.g. Ammonia Fiber Expansion or AFEX), the identification of a suitable cocktail of cellulases and hemicellulases to completely hydrolyze the residual glucan and xylan becomes pertinent. A fundamental understanding of enzyme synergy in the hydrolysis of pretreated biomass requires a high-throughput combinatorial enzyme screening for lignocellulosic biomass and a rapid compositional assay for crude protein mixtures (e.g. quantitative proteomics).

Standard enzymatic hydrolysis protocols for cellulosic enzyme systems have several inherent disadvantages including long analysis times, excessive reagent and substrate usage, high labor inputs and non-realistic substrates (e.g.,. filter paper, purified xylans, and chromogenic substrates). The choice of an optimum enzyme cocktail depends largely on the substrate characteristics rather than standard enzyme-activities that are currently measured. Screening multi-enzyme systems directly on pretreated lignocellulosics would be a better way of identifying optimum synergistic cocktails of enzymes. The automated 96-well BCRL microplate method is a rapid hydrolytic assay technique (essentially a scaled down version of the NREL LAP 009 protocol) that is currently employed in our lab.

Commercially available cellulases and hemicellulases were partially purified based on their molecular weight and ionic properties. Quantification of the individual protein components for each crude mixture was performed using a high-throughput LC-MS/MS procedure. The goal of the current project was to develop an enzyme cocktail specifically tailored for AFEX treated corn stover to help reduce total protein loading employed during enzymatic hydrolysis.