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

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-Manp-(1→2)-β-d-Manp-(1→2)-β-d-Manp-(1→2)-α-d-Manp- group, and with much lower content of substitution with β-d-Manp-(1→2)-α-d-Manp-. A fraction of these oligosaccharide side chains is again substituted with α-d-Glcp or α-d-GlcpNAc 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.