11-6
Advanced immunological methods for comprehensive analysis of recalcitrant cell wall components in AFEXTM pretreated corn ztover using glycan-directed monoclonal antibodies
Wednesday, April 27, 2016: 10:35 AM
Key Ballroom 9-10, 2nd fl (Hilton Baltimore)
S. Xue* and V. Balan, DOE Great Lakes Bioenergy Research Center, Lansing, MI, USA; S. Pattathil, Bioenergy Science Center (BESC) Oak Ridge National Laboratory, Oak Ridge, TN and Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA; N. Uppugundla, C. Gunawan and B. Dale, DOE Great Lakes Bioenergy Research Center, Michigan State University, Lansing, MI, USA; L. Alexander, Michigan State University, Lansing, MI, USA; M.J. Bowman, United States Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL, USA; L.D.C. Sousa, Department of Chemical Engineering and Materials Science, Michigan State University, Lansing, MI, USA
Ammonia-fiber expansion (AFEX
TM) pretreatment has improved enzyme digestibility of plant cell walls. Although complex enzymes were identified to effectively break down and generate simple sugars from pretreated lignocellulosic biomass, enzyme-resistant linkages remain in the cell walls; unhydrolyzed polysaccharides in residual solids (UHS) and oligosaccharides in the hydrolysate accumulate, causing a 7-10% yield loss of inlet untreated biomass. Fundamental understanding of these recalcitrant cell wall structures after enzyme hydrolysis help identify missing enzyme activities, thereby enabling more effective enzyme cocktails for better sugar release.
Use of glycan-directed monoclonal antibodies (glycome profiling) is a powerful tool for in-depth characterization of most major cell-wall glycans. Glycome profiling, however, only facilitates comprehensive characterization of larger cell-wall glycans (>20 kDa) as majority of oligosaccharide’s immobilization to ELISA plate is inefficient. In this study, AFEX-pretreated corn stover was subjected to enzymatic hydrolysis at high solid-loading. Glycome profiling was employed to determine the composition/extractability of large glycans present in UHS and hydrolysate. Analysis of smaller oligosaccharides (<15 kDa) in the hydrolysate, however, necessitating additional tools for efficient oligosaccharides immobilization. Herein, we report a novel method to immobilize oligosaccharides by combining oligosaccharide-biotinylation and subsequent ELISA screening on NeutrAvidinTM-coated plates. The immobilized biotinylated-oligosaccharides showed great affinity to antibodies enabling rapid-and-efficient structure elucidation. MALDI and glycosyl-residue composition analysis substantiated this novel immuno-screening method. These studies thus demonstrate that combining oligosaccharide-biotinylation and subsequent ELISA screening on NeutrAvidinTM-coated plates with glycan-directed mAbs is useful in identifying cross-linkages in oligosaccharides and can be widely applied to other biochemical studies of oligosaccharides structure characterization.
