- Large Scale Production of Recombinant α-1,2-Mannosyltransferase from E. coli for the Study of Acceptor Specificity and Use of the Recombinant Whole Cells in Synthesis
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We report the first large scale heterologous expression of a recombinant yeast α-1,2-mannosyltransferase in E. coli.The enzyme was isolated from 10-L and 50-L fermentations, purified and used for mannosylation reactions.The specificity of the recombinant enzyme was extensively studied by using mannose derivatives, oligosaccharides, and analogs as acceptors, and the results show that the enzyme exhibits high activities toward ManOMe and disaccharides connected by an α-1,2-mannosidic linkage.The recombinant E. coli cells were also used as a catalyst for glycosylation reaction, and mannosylation of saccharides and glycopeptides proceeded in moderate to good yields.
- Herrmann, Guido F.,Wang, Peng,Shen, Gwo-Jenn,Garcia-Junceda, Eduardo,Khan, Shaheer H.,et al.
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- The extracellular polysaccharide of Pichia (Hansenula) holstii NRRLL Y-2448: The phosphorylated side chains
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The exopolysaccharide produced by Pichia (Hansenula) holstii NRRL Y-2448 is composed of a phosphomannan core to which oligosaccharide diester phosphate side chains are appended. The oligosaccharides of the side chains were released as oligosaccharide phosphates and neutral oligosaccharides by mild hydrolysis with aqueous acetic acid and aqueous hydrogen fluoride, respectively. The liberated oligosaccharide phosphates were studied by NMR spectroscopy and by electrospray and fast atom bombardment mass spectrometry. The structures of the neutral oligosaccharides were determined by 1D and 2D NMR spectroscopic experiments. Further insight into the length of the side chains was obtained from a matrix assisted laser desorption ionisation-time of night mass spectrometric study of high and low molecular weight fragments obtained from partial acid hydrolysis of the native polysaccharide.
- Parolis, Lesley A.S.,Parolis, Haralambos,Kenne, Lennart,Meldal, Morten,Bock, Klaus
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