Discovery and Functional Determinations of Biomass-Degrading Enzymes by Robotic Cell-Free Translation

Our use of a platform for functional determination of enzymes will be presented. Robotic wheat germ cell-free protein translation system was used for protein synthesis and enzymatic assays were performed using the translation reactions obtained directly from the robot. Fluorescent sugar analogs and purified polysaccharides were used for initial characterizations. Nanostructure initiator mass spectrometry (NIMS)-based assay provided additional functional assignments and also revealed diagnostic differences in time-dependent cascades of products given by different enzymes. The approach was used to evaluate a majority of secreted cellulosomal proteins from C. thermocellum in different enzyme formats, their comparative single reactivities, and their synergistic behavior for hydrolysis of various polysaccharide-containing materials. Combinatorial enzyme activity assays revealed synergistic activities between enzymes with different assigned functions