The polypeptide chain of human lactotransferrin possesses two glycosylation sites to which glycans are linked through an N-(P-asparty1)-N-acetylglucosaminylamine bond and which are structurally heterogenous. After chymotryptic or pronase digestions, glycopeptides with five different glycan structures could be isolated. For three of them, the structure has been determined by the application of methanolysis, methylation analysis, hydrazinolysis/nitrous deamination, enzymatic cleavage and 'H-NMR spectroscopy at 360 MHz: Glycopeptides A and B : NeuAc(cr2-6)Gal(~1-4)GlcNAc(~l-2)Man(crl-3) shows that important differences exist between the number and the structure of the glycans present in these four kinds of transferrins.In the case of human lactotransferrin, in 1966, the presence of two glycans linked by a 4-N-(2-acetamido-2-deoxy-B-~-glucopyranosy1)-L-asparagine linkage was described as well as one 0-glycosidically-linked glycan [17]. The presence of two N-glycosidically-linked glycans was confirmed in 1973 and for one of them the structure was proposed [18].Microheterogeneity of the carbohydrate moieties was later demonstrated and partial results concerning different glycan structures were given in general reviews [19-211. In the present paper we describe the complete primary structure of three types of glycans as determined by methanolysis, methylation analysis, hydrazinolysis/nitrous deamination, mass spectrometry, enzymatic cleavage and 'H-NMR spectroscopy at [111._.___

Recently, the novel C-glycosidic linkage of a hexopyranosyl residue to the indole ring of tryptophan residue 7 of human RNase U(s) was reported [Hofsteenge, J., Müller, D. R., de Beer, T., Löffler A., Richter, W. J., & Vliegenthart, J. F. G. (1994) Biochemistry 33, 13524-13530]. Identification of this monosaccharide is a prerequisite for studies of its biosynthesis and its biological relevance. Using vicinal proton-proton coupling constants and rotating-frame nuclear Overhauser enhancements, ewe demonstrate that the C-linked substituent is alpha-mannopyranose. Furthermore, the nuclear magnetic resonance (NMR) data indicate that the mannopyranose moiety in a glycopeptide derived from RNase U(s) adopts several conformations on the NMR time scale.

Lactobacillus reuteri LB 121 cells growing on sucrose synthesize large amounts of a glucan (d-glucose) and a fructan (d-fructose) with molecular masses of 3,500 and 150 kDa, respectively. Methylation studies and 13C or1H nuclear magnetic resonance analysis showed that the glucan has a unique structure consisting of terminal, 4-substituted, 6-substituted, and 4,6-disubstituted α-glucose in a molar ratio of 1.1:2.7:1.5:1.0. The fructan was identified as a (2→6)-β-d-fructofuranan or levan, the first example of levan synthesis by a Lactobacillus species. Strain LB 121 possesses glucansucrase and levansucrase enzymes that occur in a cell-associated and a cell-free state after growth on sucrose, raffinose, or maltose but remain cell associated during growth on glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of sucrose culture supernatants, followed by staining of gels for polysaccharide synthesizing activity with sucrose as a substrate, revealed the presence of a single glucansucrase protein of 146 kDa. Growth of strain LB 121 in chemostat cultures resulted in rapid accumulation of spontaneous exopolysaccharide-negative mutants that had lost both glucansucrase and levansucrase (e.g., strain K-24). Mutants lacking all levansucrase activity specifically emerged following a pH shiftdown (e.g., strain 35-5). Strain 35-5 still possessed glucansucrase and synthesized wild-type glucan.

A novel exopolysaccharide (EPS) produced by Lactobacillus sake 0-1 (CBS 532.92) has been isolated and characterized. When the strain was grown on glucose, the produced EPS contained glucose and rhamnose in a molar ratio of 3:2 and the average molecular mass distribution (M m) was determined at 6 ؋ 10 6 Da. At a concentration of 1%, the 0-1 EPS had better viscosifying properties than xanthan gum when measured over a range of shear rates from 0 to 300 s ؊1 , while shear-thinning properties were comparable. Rheological data and anion-exchange chromatography suggested the presence of a negatively charged group in the EPS. Physiological parameters for optimal production of EPS were determined in batch fermentation experiments. Maximum EPS production was 1.40 g ⅐ liter ؊1 , which was obtained when L. sake 0-1 had been grown anaerobically at 20؇C and pH 5.8. When cultured at lower temperatures, the EPS production per gram of biomass increased from 600 mg at 20؇C to 700 mg at 10؇C but the growth rate in the exponential phase decreased from 0.16 to 0.03 g ⅐ liter ؊1 ⅐ h ؊1. EPS production started in the early growth phase and stopped when the culture reached the stationary phase. Growing the 0-1 strain on different energy sources such as glucose, galactose, mannose, fructose, lactose, and sucrose did not alter the composition of the EPS produced.

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