17-28: Glycosylation Determination of Cel7A Enzymes

Cellobiohydrolases from filamentous fungi have become increasingly interesting for conversion of lignocellulosic biomass to ethanol.  Organisms such as Trichoderma reesei (Hypocrea jecorina) are known to secrete high concentrations of heterologous proteins and, as such, have become an industry standard for commercial-scale enzyme production.  While T. reesei’s cellulolytic proteins are well characterized, little is well know about the extent and specific level of glycosylation.  Additionally, recombinant expression utilizing differing carbon sources appears to result in varied glycosylation levels.  Cel7A enzymes from a variety of native and recombinant T. reesei strains, as well as from Penicillium funiculosum, were investigated for detailed biophysical characterization.  Proteins were enzymatically deglycosylated and the resultant glycans and trimmed enzymes were examined.  Both N- and O-linked glycosylation sites and their ensuing oligomers were characterized and the relative enzyme activities of the glycosylated variants and deglycosylated forms were determined.