AbstractThe high cost of enzymes is a major bottleneck preventing the development of an economically viable lignocellulosic ethanol industry. Commercial enzyme cocktails for the conversion of plant biomass to fermentable sugars are complex mixtures containing more than 80 proteins of suboptimal activities and relative proportions. As a step toward the development of a more efficient enzyme cocktail for biomass conversion, we have developed a platform, called GENPLAT, that uses robotic liquid handling and statistically valid experimental design to analyze synthetic enzyme mixtures. Commercial enzymes (Accellerase 1000 +/− Multifect Xylanase, and Spezyme CP +/− Novozyme 188) were used to test the system and serve as comparative benchmarks. Using ammonia‐fiber expansion (AFEX) pretreated corn stover ground to 0.5 mm and a glucan loading of 0.2%, an enzyme loading of 15 mg protein/g glucan, and 48 h digestion at 50°C, commercial enzymes released 53% and 41% of the available glucose and xylose, respectively. Mixtures of three, five, and six pure enzymes of Trichoderma species, expressed in Pichia pastoris, were systematically optimized. Statistical models were developed for the optimization of glucose alone, xylose alone, and the average of glucose + xylose for two digestion durations, 24 and 48 h. The resulting models were statistically significant (P < 0.0001) and indicated an optimum composition for glucose release (values for optimized xylose release are in parentheses) of 29% (5%) cellobiohydrolase 1, 5% (14%) cellobiohydrolase 2, 25% (25%) endo‐β1,4‐glucanase 1, 14% (5%) β‐glucosidase, 22% (34%) endo‐β1,4‐xylanase 3, and 5% (17%) β‐xylosidase in 48 h at a protein loading of 15 mg/g glucan. Comparison of two AFEX‐treated corn stover preparations ground to different particle sizes indicated that particle size (100 vs. 500 µm) makes a large difference in total digestibility. The assay platform and the optimized “core” set together provide a starting point for the rapid testing and optimization of alternate core enzymes from other microbial and recombinant sources as well as for the testing of “accessory” proteins for development of superior enzyme mixtures for biomass conversion. Biotechnol. Bioeng. 2010;106: 707–720. © 2010 Wiley Periodicals, Inc.