Paul A. Sandford

Exocellular, Microbial Polysaccharides*

Publisher Summary This chapter emphasizes on the microbial exopolysaccharides that are produced commercially and those whose potential for commercialization is very likely. The general background of polysaccharides and microbial polysaccharides having antitumor activity is discussed in the chapter. A common feature of bacteria, fungi (yeasts and molds), and higher living organisms is the production of polysaccharides. Morphologically, there are three types: intracellular polysaccharides located inside or as part of the cytoplasmic membrane, cell-wall polysaccharides, and exocellular polysaccharides located outside the cell wall. Some exocellular polysaccharides are found covalently attached to the cell as a true capsule, whereas others are secreted unattached into the growth medium. In some cases, both capsular and unattached exopolysaccharides can be found associated with the same microbe. The expected rising cost of petroleum-derived, synthetic polymers has now elicited interest in natural polymers from replenishable resources. Microbial polysaccharides are also increasingly being used in the recovery of crude oil.

Reaction of sodium hypochlorite with amines and amides: A new method for quantitating amino sugars in monomeric form☆

Abstract Free and N-acetylated hexosamines can be determined spectrophotometrically by a three-stage assay: chlorinating the amide or amine with NaOCl; reducing unreacted NaOCl with NaNO2; and reacting chloroamide (-amine) with amylose-KI reagent to produce the blue amylosetriiodide complex, measured at 615 nm. Distinctive behavior of the common 2-amino-2-deoxy- d -hexoses and their 2-acetyl derivatives allows three types of measurements to be made: (a) identification and differentiation by characteristic behavior (assay individually in primary cacodylate buffers over the pH range 6.0–9.5, then adjust pH to 5.5 with secondary phthalate buffer and remeasure the color); (b) assay individually in a single buffer at the optimum pH; and (c) assay differently admixed 2-amino-2-deoxy- d -hexose · HCl and 2-acctamindo-2-deoxy- d -hexose (assay in primary buffer at pH 9.0–9.5 gives measure of only HexNAc (no reaction with ManNAc), then adjust pH to 5.5 with secondary buffer to measure additional color that results only from HexN · HCl).

Reaction of sodium hypochlorite with amines and amides: A new method for quantitating polysaccharides containing hexosamines

Abstract The NaOCl-amylose-KI spectrophotometric method for determining free and N -acetylated amino sugars has been applied to the analysis of PS Y-6272, which is composed of N -acetylglucosamine and N -acetylglucosaminuronic acid and produced extracellularly by the black yeast strain NRRL Y-6272 (6). PS Y-6272, as well as other amino sugar-containing polysaccharides such as chondroitin sulfate A, colominic acid, heparin, and hyaluronic acid, can be assayed readily in the range of 0.04 to 0.30 μmole N without previous depolymerization. Studies of the effects of pH and reaction time and temperature, carried out on these glycosaminoglycans as well as on various glucosamine derivatives and oligosaccharides, indicate that the color yield is influenced by the identity of the amino sugars present as well as by the position and anomeric type of linkages. However, since the color yield accords well with the nitrogen content of a particular amino sugar-containing polysaccharide, unknown quantities in solutions or column effluents can be determined readily and accurately by reference to calibration curves of the individual polysaccharide.

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.

Reaction of sodium hypochlorite with amines and amides: automation of the method.

Abstract The spectrophotometric NaOCl-amylose-KI method for analysis of free and N -acetylated hexosamines in monomeric and polymeric forms, amino acids, peptides, and proteins was automated, and the methods specificity was studied. Hexosamines react as in the manual assay. Significant differences in reactivity of amino acids led to a study of the effect of pH on the extent of the reaction. An automatic gradient titration method was devised to vary the pH of the primary reaction between 7.0 and 10.0. Results may be used to determine optimal reaction conditions for each amino acid and to differentiate amino acids; there are several reasons for differences in reactivity. The assay is especially useful for peptides, since amide N is generally more reactive than amine N. The method is suitable for continuous monitoring of column chromatographic effluents, as well as for analysis of individual samples as in the manual assay.

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.