Synthetic enzyme mixtures for biomass deconstruction: Production and optimization of a core set

The high cost of enzymes is a major bottleneck preventing the development of an economically viable lig- nocellulosic ethanol industry. Commercial enzyme cocktails fortheconversion ofplantbiomass tofermentablesugarsare 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 þ/� Multifect Xylanase, and Spezyme CP þ/� Novozyme 188) were used to test the system and serve as comparative benchmarks. Using ammonia-fiber expansion (AFEX) pre- treated corn stover ground to 0.5mm and a glucan loading of 0.2%, an enzyme loading of 15mg protein/g glucan, and 48h digestion at 508C, 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 opti- mization of glucose alone, xylose alone, and the average of glucose þxylose for two digestion durations, 24 and 48h. 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-b1,4-glucanase 1, 14% (5%) b-glucosidase, 22% (34%) endo-b1,4-xylanase 3, and 5% (17%) b-xylosidase in 48h at a protein loading of 15mg/g glucan. Comparison of two AFEX-treated corn stover preparations ground to different particle sizes indi- cated that particle size (100 vs. 500 mm) 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 devel- opment of superior enzyme mixtures for biomass conversion.