James S.S. Gray

The glycans of horseradish peroxidase.

Horseradish peroxidase (E.C. 1.11.1.7) isozyme c (HRPc) is a glycoprotein found to contain 21.8% carbohydrate with the average composition: 2 mol GlcNAc, 2.6 mol Man, and 0.8 mol each of Fuc and Xyl. The oligosaccharides of HRPc were investigated by a combination of High pH Anion-Exchange Chromatography with Pulsed Amperometric Detection, methylation analysis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The structure of the major oligosaccharide released by digestion with glycopeptidase A, accounting for between 75 and 80% of the total, was confirmed to be [sequence: see text]. Most of the remaining oligosaccharides were found to belong to the (Xyl)xManm(Fuc)fGlcNAc2 (m = 2, 4, 5, 6; f = 0 or 1; x = 0 or 1) family. Less than 5% of the oligosaccharides were of the ManmGlcNAc2 (m = 4 to 7) type. Methylation analysis of holo- and apo-HRPc and its tryptic glycopeptides support the structures proposed for the oligosaccharides. Furthermore, methylation analysis of the tryptic glycopeptides provides evidence for the heterogeneity of the oligosaccharides occurring at each of the N-linked sites.

Heterogeneity of glycans at each N-glycosylation site of horseradish peroxidase

The tryptic glycopeptides of horseradish peroxidase isozyme c (HRPc) were studied by methylation linkage analysis, exoglycosidase degradation, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOFMS). Over 90% of the predicted tryptic peptides and glycopeptides of HRPc could be identified in the unfractionated digest. Four glycans, namely (Xyl)Man3(Fuc)GlcNAc2 (major species), (Xyl)Man2(Fuc)GlcNAc2, (Xyl)Man3GlcNAc2, and Man3(Fuc)GlcNAc2 (minor species), were observed at all of the N-glycosylation sites and account for greater than 95% of the carbohydrate. Other members of this glycan family, namely (Xyl)xManm(Fuc)f GlcNAc2 (x = 0 or 1, f = 0 or 1, m = 4, 5, 6, or 7), account for the rest of the glycans. Only traces of high mannose-type glycans were detected in HRPc. Two sites, namely those at Asn-57 and Asn-267, were found to be more heterogeneous than the sites at Asn-13, Asn-158, Asn-186, 198 (doubly glycosylated peptide), Asn-214, and Asn-255. Two of the glycopeptides were observed as part of disulfide-linked species. MALDITOFMS confirmed the N-glycosylation sites previously reported [K.G. Welinder, Eur. J. Biochem., 96 (1979) 483-502] and was used to determine the heterogeneity of the glycan pool at each site.

Extracellular polysaccharide of Erwinia chrysanthemi Ech6

Many strains of Erwinia chrysanthemi, which are Gram-negative bacterial phytopathogens, produce copious amounts of extracellular polysaccharides. The extracellular polysaccharide from E. chrysanthemi pv. zeae strain SR 260, a phytopathogen of corn, is a branched-chain glucomannorhamnan of proven structure (Gray et al., Carbohydr. Res. 1993, 245, 271-287). The extracellular polysaccharide from E. chrysanthemi Ech6 is different, containing no rhamnose or mannose. It is composed of L-fucose, D-galactose, D-glucose and D-glucuronic acid in the ratio 2:2:1:1. The structure of the polysaccharide is as follows: [sequence: see text]

Structure of an extracellular polysaccharide produced by Erwinia chrysanthemi

Erwinia chrysanthemi pv zeae strain SR260, a phytopathogen of corn, produced from lactose an acidic extracellular polysaccharide which was purified and found to consist of L-rhamnose, D-mannose, D-glucose, and D-glucuronic acid in the ratio of 3:1:1:1. A combination of chemical (carboxyl-group reduction, methylation analysis, periodate oxidation, Smith degradation, and lithium-ethylenediamine degradation) and physical (1 and 2D NMR spectroscopy) methods revealed that the polysaccharide is composed of a hexasaccharide repeating unit 1: [formula: see text]

Extracellular polysaccharide of Erwinia chrysanthemi A350 and ribotyping of Erwinia chrysanthemi spp.

Erwinia chrysanthemi spp. are gram-negative bacterial phytopathogens causing soft rots in a number of plants. The structure of the extracellular polysaccharide (EPS) produced by the E. chrysanthemi strain A350, which is a lacZ- mutant of the wild type strain 3937, pathogenic to Saintpaulia, has been determined using a combination of chemical and physical techniques including methylation analysis, low-pressure gel-filtration and anion-exchange chromatography, high-pH anion-exchange chromatography, partial acid hydrolysis, mass spectrometry and 1- and 2D NMR spectroscopy. In contrast to the structures of the EPS reported for other strains of E. chrysanthemi, the EPS from strain A350 contains D-GalA, together with L-Rhap and D-Galp in a 1:4:1 ratio. Evidence is presented for the following hexasaccharide repeat unit: [structure: see text] All the Erwinia chrysanthemi spp. studied to date have been analyzed by ribotyping and collated into families, which are consistent with the related structures of their EPS.

Extracellular polysaccharide of Erwinia chrysanthemi CU643.

Erwinia chrysanthemi are gram-negative bacterial phytopathogens causing soft rots in a number of plants. The structure of the extracellular polysaccharide (EPS) produced by E. chrysanthemi strain CU643, pathogenic to Philodendron, has been determined using a combination of chemical and physical techniques including methylation analysis, high- and low-pressure gel-filtration and anion-exchange chromatography, high-pH anion-exchange chromatography, partial acid hydrolysis, mass spectrometry, and 1- and 2-D NMR spectroscopy. In contrast to the structures of the EPS reported for other strains of E. chrysanthemi, the EPS from strain CU643 is a linear polysaccharide containing L-Rhap, D-Galp, and D-GlcAp in the ratio 4:1:1. Evidence is presented for the following hexasaccharide repeat unit: -->3)-beta-D-Galp-(1-->2)-alpha-L-Rhap-(1-->4)-beta-D-GlcAp- (1-->2)-alpha-L- Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->(1 ).

Extracellular polysaccharide of Erwinia chrysanthemi

Abstract The extracellular polysaccharide (EPS) produced by E. chrysanthemi pv. zeae strain SR260, a strain pathogenic to corn, has a branched hexasaccharide repeat unit consisting of a glucomannorhamnan backbone [ → 3)-β- d - Glc p-(1 → 4)-α- d - Man p-(1 → 3)-α- l - Rha p-(1 → ] and a trisaccharide side-chain [ α- l - Rha p-(1 → 3)-α- l - Rha p-(1 → 4)?- GlcA p-(1 → ] attached to O-3 of the mannose in the backbone [1]. All the anomeric configurations, except that of the rhamnosyl-glucosyluronic linkage in the side chain, were unambiguously assigned by a combination of 1 and 2D NMR spectroscopy. The tentative assignment of the rhamnosyl-glucosyluronic glycosyl linkage as α- l is now proven.

Comparison of extracellular polysaccharides of Erwinia chrysanthemi spp

Two extracellular polysaccharides from strains of Erwinia chrysanhemi Ech 1 and 9, phytopathogens of potatoes, have been isolated and purified. Both have similar compositions and other properties to that produced by strain SR 260, a phytopathogen of corn. These polysaccharides are composed of L-rhamnose, D-mannose, D-glucose, D-glucuronic acid in the ratio 3:1:1:1. Initial structural aspects of these polysaccharides are reflected in the 600 MHz 1H NMR spectra and the products of partial acid hydrolysis, which are presented for comparison.

Extracellular polysaccharides of modified strains of Erwinia spp.

The structure of the extracellular polysaccharide (EPS) produced by Erwinia chrysanthemi strain A2148 has been determined using low pressure size-exclusion and anion-exchange chromatographies, high pH anion-exchange chromatography, glycosyl-linkage analysis, and 1D 1H NMR spectroscopy. The polysaccharide is structurally similar, if not identical, to the EPS produced by E. chrysanthemi strain A350. A streptomycin-resistant strain of E. chrysanthemi Ech6 (Ech6S(+)) has been generated and has an elevated production of EPS, as does a streptomycin-resistant strain (Ech9Sm6) of E. chrysanthemi Ech9. These modified E. chrysanthemi spp. have been ribotyped and found to be closely related to their parent strains.

TREHALOSE AS A COMMON INDUSTRIAL FERMENTATION BYPRODUCT

Abstract During the analysis of several industrial fermentation byproduct streams significant amounts of α,α-trehalose and α,β-isomaltose were found. These disaccharides were purified from one of the fermentation-byproduct streams and identified by chromatography and 600-MHz 1 H NMR. Examination of fermentation streams before and after the addition of active organisms established that α,α-trehalose is a product of the fermentation and α,β-isomaltose is a component of the starting nutrients. Analysis of a number of agribusiness industrial-byproduct streams has had the objective of identifying compounds of potential value for isolation, or determing those that may be toxic to microorganisms in the anaerobic methanogenesis of organic compounds in the streams. The frequent occurrence of two disaccharides was noted in those streams from fermentation processes and prompted this study.