Isolation and characterization of non-labeled and 13C-labeled mannans from Pichia pastoris yeast

Abstract

Mannans from genetically modified Pichia pastoris yeast, used for overproduction of neural cell adhesion molecule protein, grown on normal media or on uniformly 13C-labeled glucose and methanol, were isolated and characterized by high-field (750 MHz) NMR spectroscopy. Fully 13C-labeled oligosaccharide fragments were prepared from mannans by acetolysis. According to the data obtained, the mannan is made up of a main chain of α-(1→6)-linked mannopyranosyl residues, substituted at O-2 with α-mannopyranosyl or a α- d-Man p-(1→2)-β- d-Man p-(1→2)-β- d-Man p-(1→2)-α- d-Man p- group, and with much lower content of substitution with β- d-Man p-(1→2)-α- d-Man p-. A fraction of these oligosaccharide side chains is again substituted with α- d-Glc p or α- d-Glc pNAc through a phosphodiester linkage to the 6 position of the first mannopyranosyl residue. Improved conditions of acetolysis, cleaving all α-(1→6) linkages, but not β-mannoside linkages, are proposed.