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

Mannans from genetically modified Pichia pastoris yeast, used for overprodu ction of neural cell adhesion molecule protein, grown on normal media or on uniformly C-13-labeled glucose and methanol, were isolated and characteriz ed by high-field (750 MHz) NMR spectroscopy. Fully C-13-labeled oligosaccha ride fragments were prepared from mannans by acetolysis. According to the d ata obtained, the mannan is made up of a main chain of alpha-(l-->6)-linked mannopyranosyl residues, substituted at O-2 with alpha-mannopyranosyl or a lpha-D-Manp-(1-->2)-beta-D-Manp-(1-->2)-beta-D-Manp-(1 -->2)-alpha-D-Manp- group, and with much lower content of substitution with beta-D-Manp-(1-->2) -alpha-D-Manp-. A Fraction of these oligosaccharide side chains is again su bstituted with alpha-D-Glcp or alpha-D-GlcpNAc through a phosphodiester lin kage to the 6 position of the first mannopyranosyl residue. Improved condit ions of acetolysis, cleaving all alpha-(1-->6) linkages, but not beta-manno side linkages, are proposed. (C) 2000 Elsevier Science Ltd. All rights rese rved.