Marie-France Marais

Structure of extracellular polysaccharide produced by lignin-degrading fungus Phlebia radiata in liquid culture.

The extracellular material (EM) produced by the white rot fungus Phlebia radiata cultured in an N-limited liquid medium was studied. Carbohydrate analysis showed maximum concentration of glucose as the major monosaccharide component of EM was found on postinoculation day 9. Beyond day 9 of cultivation the proportion of glucose decreased suggesting that the glucan component of EM had been further metabolized. The analysis of EM at day 9 revealed the presence of the following monosaccharides (in relative %): glucose (62); galactose (16); mannose (13); xylose (4); and fucose (5). The carbohydrate analysis together with the presence of protein in EM corresponds to a mixture of glucan and glycoprotein. Purification by trypsin treatment yielded an enriched glucose-containing extracellular polysaccharide (EPS). Methylation analysis identified EPS as (1-3)-beta-D-glucan highly branched at C-6. The structure of the glucan was confirmed by 13C-NMR spectroscopy. The results suggest that P. radiatas EPS is entangled with a glycoprotein in a complex that makes the extracellular sheath surrounding the hyphae.

The use of bacteriophage depolymerization in the structural investigation of the capsular polysaccharide from Klebsiella serotype K3.

The structure of the repeating unit of the capsular polysaccharide from Klebsiella serotype K3 has been established from the results of n.m.r. (1H and 13C) spectroscopy and methylation analysis of P1, the pyruvic acetal-bearing pentasaccharide obtained on depolymerization of the polysaccharide with a bacteriophage-borne endogalactosidase, reduced deacetalated P1, and the native polysaccharide. The data permit the assignment of the following structure to the repeating unit: (formula see text)

Structure of the capsular polysaccharide of Klebsiella K- type 63

Structural investigation of the capsular polysaccharide from Klebsiella K type 63 by methylation analysis, periodate oxidation, and uronic acid degradation showed the repeating unit to consist of leads to 3)-alpha-D-Galp-(1 to 3)-alpha-D-GalpA-(1 TO 3)-ALpha-L-Fucp(1 to. This structure is identical to that of Escherichia coli serotype K-42 capsular polysaccharide. The 1H- and 13C-n.m.r. spectra of the original and modified polysaccharide are consistent with the foregoing structure.

A novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica

An apparently novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica has been characterized. The enzyme exhibits properties that differ markedly from those of the enzyme previously described [Fevre, M. & Dumas, C. (1977). J Gen Microbiol 103, 297-306] as it is active at alkaline pH, stimulated by the divalent cations Ca2+, Mg2+ and Mn2+, and appears to be located mainly in the apical part of the hypha. Taking into consideration the differences in pH optimum and effect of divalent ions, each enzyme activity could be assayed in the presence of the other. The insoluble polymeric product of the enzyme with alkaline pH optimum was characterized as a linear 1,3-β-glucan. Comparisons of the general properties of 1,3-β-glucan synthases suggest that enzymes from the oomycetes are more closely related to enzymes from higher plants than to those of true fungi, reflecting the fact that the oomycetes are highly divergent from chitinous fungi.

Changes in peroxidases in the suspension culture of Rubus fruticosus during growth

The growth parameters of a cell suspension culture of Rubus fruticosus L. were determined over a culture period including exponential growth, stationary phase and a glucose starvation period at the end of the normal culture cycle. Peroxidase activities were measured in the cytoplasm, in the cell wall, and in the culture medium by the guaiacol assay. There is a relationship between the activity found in the spent medium and the dry matter mass of the cells during the exponential growth. In the three compartments a bimodal repartition of peroxidase activities was observed, with the two peaks at day 4 and day 26, respectively. This suggests that the first peak corresponds to actively dividing cells whereas the second is associated with senescence, or stress due to starvation. Fractionation of the peroxidases from the culture mediuim revealed the presence of two sets of cationic isoenzymes, with minor amount of anionic peroxidases. Interestingly, the second peak of cationic enzymes which was of weak intensity at day 10 of the culture, becameprevalent at day 26. This indicates that not only the total amount of peroxidases varies as a function of culture time, but also that the nature of the peroxidases secreted into the medium changes during growth.

The molecular structure and solution conformation of an acidic heteropolysaccharide from Auricularia auricula-judae.

A water soluble acidic heteropolysaccharide named WAF was isolated from Auricularia auricula-judae by extracting with 0.9% NaCl solution. By using gas chromatography, gas chromatography-mass spectrometry, and NMR, its chemical structure was determined to be composed of a backbone of α-(1→3)-linked D-mannopyranose residues with pendant side groups of β-D-xylose, β-D-glucose, or β-D-glucuronic acid at position O6 or O2. Six fractions prepared from WAF with a weight-average molecular mass (M(w)) between 5.9 × 10⁴ and 64.7 × 10⁴ g/mol were characterized with laser light scattering and viscometry in 0.1M NaCl at 25°C. The dependence of intrinsic viscosity ([η]) and radius of gyration (R(g)) on M(w) for this polysaccharide were found to be [η] = 1.79 × 10⁻³ M(w) ⁰.⁹⁶ cm³ g⁻¹ and R(g) = 6.99 × 10⁻² M(w) (0.54) nm. The molar mass per unit contour length (M(L)) and the persistence length (L(p)) were estimated to be 1124 nm⁻¹ and 11 nm, respectively. The WAF exhibited a semirigid character typical of linear polysaccharides. Molecular modeling was then used to predict the ordered and disordered states of WAF; the simulated M(L) and L(p) were however much smaller than the experimental values. Taken altogether, the results suggested that WAF formed a duplex in solution.

The structural repeating-unit of the capsular polysaccharide from Klebsiella serotype K48

Investigation of the structure of the capsular polysaccharide from Klebsiella K48, using methylation analysis, periodate oxidation, Smith degradation, and 1H- and 13C-n.m.r. spectroscopy, indicated the repeating unit to be the pentasaccharide (formula; see text)