Characterization of protein glycoforms in cellobiohydrolases and endoglucanases from Trichoderma reesei RUT-C30 and mutant strains using capillary isoelectric focusing and mass spectrometry

Trichoderma reesei is a filamentous fungus heavily used in the biotechnology industry due to its efficient secretion of cellulases. The enzymatic system of T. reesei consists primarily of four glycoproteins referred to as cellobiohydrolases (CBH I, CBH II) and endoglucanases (EG I, EG II). They exhibit microheterogeneity both in the N- and O-linked glycans. This thesis focuses on the method development to characterize the glycosylation profile and post-translational modifications present in these glycoproteins. Crude cellulase fermentation extracts RUT-C30 and its two mutant strains Iogen-M4, Iogen-B13 were initially analyzed by capillary isoelectric focusing (CIEF) to determine the cellulase composition in them. The major cellulase CBH I was purified from each strain and electrospray mass spectrometry (ESMS) was used to reveal the extent of overall glycosylation. To characterize the N-linked glycans and their attachment sites, CBH I from these strains were subjected to tryptic digest with and without PNGase F incubations followed by mass spectrometric detection. The O-linked glycans were released chemically by hydrazinolysis and were analyzed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The majority of O-linked glycans was di- and tri-saccharides. Two unusual posttranslational modification from strain RUT-C30 were observed: (1) both high mannose (predominantly Man 8GlcNAc2) and single GlcNAc in putative N-linked sites and (2) mannosylphosphorylation in a O-linked di-saccharide. Heterogeneity in putative N-linked sites was found consistently in CBH II, EG I and EG II from RUT-C30, however, no mannosylphosphorylation was observed at least on the proteins in the purified fractions. These results have led to the proposal of endogenous endoglycosidase H as well as mannosylphosphorylation activities possibly induced during fermentation.