MODIFIED GLYCOSYLATION OF CELLOBIOHYDROLASE-I FROM A HIGH CELLULASE-PRODUCING MUTANT STRAIN OF TRICHODERMA-REESEI

Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The natu re and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic asp ects of cellulolytic enzymes are well characterised. Using a combinati on of liquid chromatography, electrospray mass spectrometry, solid-pha se Edman degradation, and monosaccharide analysis we have identified e very site of glycosylation of CBHI from a high cellulase-producing mut ant strain of T. reesei, ALKO2877, and characterised each site in term s of its modifying carbohydrate and site-specific heterogeneity. The c atalytic core domain comprises three N-linked glycans which each consi st of a single N-acetylglucosamine residue. Within the glycopeptide li nker domain, all eight threonines are variably glycosylated with betwe en at least one, and up to three, mannose residues per site. All serin es in this domain are at least partially glycosylated with a single ma nnose residue. This linker region has also been shown to be sulfated b y a combination of ion chromatography and collision-induced dissociati on electrospray mass spectrometry The sulfate is probably mannose-link ed. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.