Paul R. Watson

Characterization and properties of the phosphomannan from Hansenula hostii NRRL Y-2448

Abstract The phosphorylated mannan produced in good yield from glucose by the bisexual diploid yeast, Hansenula holstii NRRL Y-2448, appears to be the first significantly phosphorylated polysaccharide to be obtained from a yeast or a nonpathogen. Isolated as the potassium salt, this water-soluble polysaccharide derivative has constituents in the proportion, d -mannose:phosphorus:potassium::5:1:1. The product has [ α ] D 25 + 103 ° (in 0.1 M potassium chloride), M N (reducing power) 103,000 ± 10,000, M W (light scattering) of the order of 16 × 10 6 , S 20, w = 44, and an unusually homogeneous molecular distribution for an unfractionated, native polymer. The single titration equivalence-point of the polyacid at pH 7.2 indicates a phosphodiester structure. Weak acid liberates secondary hydrogen ions and causes molecular degradation; dilute alkali appears to cause no structural change. Aqueous solutions show exceptional resistance to microbial attack. The brilliantly clear aqueous solutions have properties characteristic of a plastic, thixotropic, polyelectrolyte. The viscosity-concentration curve shows a viscosity maximum of 2500 cp. at 1.5% polysaccharide concentration and a minimum of 1700 cp. at 3%. At suitable concentrations of phosphomannan and borax, complexing and cross-linking occur; the presence of potassium chloride augments these effects.

An extracellular fungal polysaccharide composed of 2-acetamido-2-deoxy-d-glucuronic acid residues☆

The black yeast-like fungus NRRL YB-4163, now tentatively identified as Rhinocladiella elatior Mangenot, has been found to produce an extracellular microbial polysaccharide composed mainly of 2-acetamido-2-deoxy-D-glucuronic acid residues. Polysaccharide (PS) YB-4163, when isolated in good yield as the neutral potassium salt, dissolves readily in water to produce extremely viscous solutions, which form stable foams and emulsions. By depolymerizing PS YB-4163 with [14C]methanol-HCl, the polysaccharide can be both identified and quantitated radiochemically by determining the individual [14C]methyl glycosides after their separation by paper chromatography. When the methyl glycosides of PS YB-4163 were reduced with NaB3H4, only the methyl glycosides of 2-acetamido-2-deoxy-D-[6-3H]glucose were found. Analysis of the monosaccharide released from carboxyl-reduced PS YB-4163 by acid hydrolysis or methanolysis also showed 2-acetamido-2-deoxy-D-glucuronic acid to be the main constituent. Previously, the only polysaccharides known to be composed entirely or hexosaminuronic acid have been cellular products from pathogens. Of these, the antigenic polysaccharide (SPSA) from Staphylococcus aureus is composed entirely of 2-amino-2-deoxy-D-glucuronic acid, but its amino groups are substituted equally with acetyl and N-acetylalanyl groups. The specific optical rotation of PS YB-4163, [alpha]20D -75 degrees (c 0.5, water), is similar to that of SPSA (-91 degrees), and suggests beta-D-linkages that must be either (1 leads to 3) or (1 leads to 4).

An extracellular microbial polysaccharide composed of 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucuronic acid: Radiochemical and gas-chromatographic analysis of the products of methanolysis

Abstract When the polysaccharide from the black yeast NRRL Y-6272, composed of 2-acetamido-2-deoxy- D -glucose ( 1 ) and 2-acetamido-2-deoxy- D -glucuronic acid ( 2 ), is hydrolyzed, extensive humin formation occurs by decomposition of component residues, especially the hexosaminuronic acid. Methanolysis avoids this decomposition by forming stable methyl glycosides amenable to quantitation by both radiochromatographic techniques and gas chromatography. Unlike hydrolysis, which results in incomplete depolymerization, refluxing methanol-HCl ( M , 16–24 h) completely depolymerizes polysaccharide Y-6272 to the methyl glycosides of its component sugars. Use of 14 C-methanol-HCl allows quantitation of 1 and 2 by counting the individual 14 C-methyl glycosides after separation by paper chromatography. As the methyl glycosides derived from the hexosaminuronic acid in polysaccharide Y-6272 consist of both a methyl ester and a lactone, for quantitation it was necessary to convert these two glycoside forms into a common derivative of known 14 C-methyl content by treatment with mild alkali. Methanolysis by using radioisotopes affords an extremely valuable method for detecting and quantitating amino sugars in polysaccharides; it is rapid and sensitive and it should be especially applicable for analyzing other polysaccharides and proteins that contain constituents labile to normal hydrolytic conditions.

Liquid scintillation counting of radioactive monomeric and polymeric carbohydrates on paper chromatograms.

Abstract A simple, improved scintillation counting procedure was developed for the assay of radioactive mono- and polysaccharides on paper chromatograms. Segments of chromatograms are placed in scintillation vials and soaked in water to completely elute the carbohydrate before addition of Aquasol, a xylene-based scintillation fluid. The resulting water-Aquasol solution is counted in a liquid scintillation counter. Evaluation of numerous experimental variables revealed optimal conditions for complete elution of mono- and polysaccharides with water before counting in Aquasol. The water elution-Aquasol procedure allows water-soluble substances (14C- and 3H-labeled) on paper to be assayed with increased accuracy and sensitivity (three- to fivefold improvement in counting efficiency of tritiated samples). The simplicity of the procedure allows entire radiochromatograms to be assayed readily.