Using glycome profiling on plant biomass for functional characterization of cell wall hydrolytic enzymes

Microbes, which are responsible for the degradation of plant biomass in the biosphere, produce wide arrays of glycosylhydrolases in order to mobilize the sugars in biomass.  However, the hydrolytic activity and specificity of most of these hydrolases remain unknown.  Most assays for delimiting the activity of glycosylhydrolases rely upon isolated polysaccharides as substrates for assays, and thus multiple assays are required to fully characterize newly identified putative hydrolases.  We describe the use of intact biomass as substrate for glycosylhydrolases, followed by antibody-based Glycome Profiling to reveal the activities of hydrolases capable of degrading biomass components.  The enzymes examined are:  CjXyl10B (family GH10), NpXyl11A (family GH11), CelEcc_CBM3a (an engineered multifunctional GH5) and XynY (a potentially processive hemicellulase from the GH10 family).  In general, Glycome Profiling of the enzyme-treated plant biomass and/or screening of enzyme-treated base extracts prepared from native biomass showed the depletion of xylan epitopes as a result of the action of four enzymes.  Removal of lignin from the biomass significantly increased the efficiency of xylan degradation in poplar and switchgrass biomass by CjXyl10B and NpXyl11A.  Treatment of native or AFEX pretreated switchgrass and corn stover biomass with CelEcc_CBM3a and XynY showed that, in addition to the removal of xylans from the biomass, xyloglucan epitopes were also depleted, suggesting multifunctionality in the activities of these two enzymes.  Thus, the biomass/Glycome Profiling-based assay system shows promise as a rapid, moderate through-put method for characterization of putative enzymes for use in biomass deconstruction.