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I W Sutherland

Publications and source records attributed to I W Sutherland.

At least 19 recordsLinked to original sources

Influence of acetyl and pyruvate substituents on the solution properties of xanthan polysaccharide.

Xanthan, an exocellular polysaccharide produced by the plant pathogenic bacterium Xanthomonas campestris has been the subject of considerable interest in recent years because of its unusual rheological properties in solution ('weak gel') and consequent range of applications. The polymer consists of a cellulosic backbone with trisaccharide side chains linked to alternate backbone residues; acetyl and pyruvate substituents are carried in variable amounts on these side chains. In this study a series of xanthans differing in the percentage of substituent groups and in molecular weight range have been prepared by culturing a variety of different strains of X. campestris. All of the xanthans have been characterized by a range of physicochemical techniques. In particular, the intrinsic viscosities at low shear rates, and at a range of ionic strengths, have been determined and the geometric persistence lengths evaluated by the Smidsrød-Haug method. Intensity light scattering measurements have been made using the procedure of Coviello and co-workers to promote molecular dispersion. Despite significant differences in the acetyl and pyruvate contents, the molecular weight vs mean square radius behaviour of our samples did not differ substantially from each other or from those reported for other xanthan samples in the literature. The persistence length, determined by the method of Schmidt et al. (120 +/- 8 nm) was also, within experimental error, the same for all the samples measured. These values differed considerably from those calculated from the ionic strength dependence of intrinsic viscosity (the Smidsrød-Haug method) was reported by Tinland and Rinaudo and calculated for our samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates

Long-term storage of xanthan in seawater at elevated temperature: physical dimensions and chemical composition of degradation products.

Commercial xanthan and xanthan from Xanthomonas strain 646 produced in the laboratory have been subjected to heat treatment for various periods of time in oxygen depleted, high salinity, aqueous solutions. Both the viscosity and the carbohydrate content decreased with increasing incubation time at a specified temperature. The losses increased with increasing temperature. Data from electron micrographs and dialysable sugar content indicate that random cleavage of the double-stranded xanthan chain is the main mechanism responsible for the decreasing viscosity. Removal of pyruvate and acetate substituents on the side chains was apparently not related to the change in physical dimensions. The mannose/glucose ratio in the non-diffusible fraction decreased with incubation time, apparently not related to change in physical dimensions. Electron micrographs showed that one of the samples appeared as highly aggregated in the native condition. After 1 month at 80 degrees C, we observed that the aggregates had dissolved and that the viscosity had increased fivefold. This suggests that heat treatment can be used to avoid microgels and to obtain higher viscosifying power of the native xanthan.

Carbohydrates

Monoclonal antibodies reacting with the exopolysaccharide xanthan from Xanthomonas campestris.

We have prepared murine hybridomas secreting monoclonal antibodies against the exopopolysaccharide xanthan from Xanthomonas campestris pv. campestris 646 after fusing NSO myeloma cells and spleen cells from BALB/c mice immunized with xanthan. Four hybridomas, secreting antibodies designated A6 (IgM kappa), B3 (IgM kappa), D1 (IgM kappa), and D3 (IgG2A kappa), were selected for further studies. All antibodies reacted with a range of different xanthans. Competition studies using variants of the exopopolysaccharide as competitors suggested that specificity was mainly against the side-chain. One of the antibodies (B3) appeared to require the fully acylated side-chain with the pyruvylated terminal mannose as the immunodominant part. The three others were assumed to be directed against the nonsubstituted trisaccharide with the inner mannose-glucuronic acid being immunodominant. None of the antibodies reacted with cellulose (the xanthan backbone). Using immunoblotting techniques on nitrocellulose paper both a mixture of monoclonal antibodies, and also polyclonal ascitic fluid, could detect xanthan quantities of approximately 0.1 microgram.

Animals

Microbial polysaccharides--a comparison with eukaryotic polymers.

Many microorganisms secrete exopolysaccharides, most of which are composed of monosaccharides widely found in non-microbial sources. Bacterial polysaccharides are frequently acylated or may contain phosphate esters but, with the exception of cyanobacterial material, appear to lack sulphate. They may contain a number of unusual or methylated sugars. The majority of microbial exopolysaccharides are composed of repeating units ranging in size from disaccharides to heptasaccharides. Some of these polymers are either identical to, or very similar to, polysaccharides found in eukaryotic tissues. In the microorganism, however, the function of the material is very different to that in the eukaryote.

Animals

Structural studies of the capsular polysaccharides from Klebsiella types 8 and 82, a reinvestigation.

The structures of the capsular polysaccharides elaborated by Klebsiella types 8 (K8) and 82 (K82) have been reinvestigated. N.m.r. spectroscopy of the original and chemically modified polysaccharides was the principal method used. It is concluded that the polysaccharides are composed of repeating units having the following structures. (Formula: see text). The presence of L-glutamic acid, linked as an amide to the carboxyl group of a uronic acid, has not been observed hitherto in bacterial polysaccharides.

Carbohydrate Conformation

Xanthan lyases--novel enzymes found in various bacterial species.

Xanthan lyases, cleaving the terminal beta-mannosidic linkage of the side-chain of the exopolysaccharide xanthan from Xanthomonas campestris, have been obtained from several sources. These include a Bacillus species, a Corynebacterium species and a mixed culture. The lyases were initially associated with endo-beta-glucanases cleaving the main chain of xanthan. Partial purification of the enzymes was achieved and the Bacillus preparation was separated by FPLC into material free of endoglucanase and glycosidase activities. The lyase was active on polysaccharides with and without acetate and pyruvate. The optimal size of the substrate appeared to be in the range of degree of polymerization (DP) 25-35, i.e. 5-7 repeat units of the polysaccharide. No activity was found against xanthan modified by reduction of the carboxyl groups or by the addition of amine or hydroxyethyl groups. The combined action of the lyase and the endoglucanase yielded a series of oligosaccharides, each with a side-chain terminating in an unsaturated uronic acid and containing the molar ratio of D-glucose to D-mannose, 2:1.

Bacillus

Analysis of bacterial exopolysaccharides.

Extracellular polysaccharides have been isolated from cultures of freshwater and marine bacteria originally isolated from material adhering to surfaces and underivatized hydrolysates have been analyzed by high-performance liquid chromatography methods. A scheme has been developed whereby the uronic acids can be identified on strong anion-exchange columns, while neutral monosaccharides can be separated and identified using aminobonded columns or cation-exchange adsorbent loaded with a heavy metal ion. The methods permit rapid and accurate comparison of polysaccharides with differing chemotype. The strains studied show a range of different chemotypes, all containing a uronic acid and several neutral monosaccharides. Some of the polysaccharides isolated from marine bacteria possessed a very high acetyl content.

Chromatography, High Pressure Liquid

Structure of the extracellular gelling polysaccharide produced by Enterobacter (NCIB 11870) species.

The gelling polysaccharide produced by a species of Enterobacter (NCIB 11870) contains L-fucose, D-glucose, and D-glucuronic acid in the ratios 1:2:1. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide revealed terminal non-reducing glucose, (1----3)-linked fucose, (1----3,1----4)-linked glucose, and (1----4)-linked glucuronic acid in the ratios 1:1:1.2:0.8. From the results of Smith degradation of the polysaccharide and spectroscopic studies of the acidic tetra- and octa-saccharides produced by bacteriophage-induced enzymic depolymerization of the polysaccharide, the following tetrasaccharide repeating-unit is proposed. (Formula: see text). This repeating-unit is identical to that of the capsular polysaccharide produced by Klebsiella aerogenes serotype K54 except for the absence of O-acetyl groups. The effects of the O-acetyl groups on the secondary structure and rheological properties of these polysaccharides are discussed.

Carbohydrate Conformation

Industrially useful microbial polysaccharides.

A number of microbial exopolysaccharides possess properties of industrial importance, especially as viscosifiers and gelling agents. This group of biopolymers is diverse in its chemical composition and structure; it includes both anionic and neutral polysaccharides, most of which are of bacterial origin.

Bacteria

The role of cell wall carbohydrates in binding of microorganisms to mouse peritoneal exudate macrophages.

The recognition by macrophages of unopsonized bacteria was studied, employing a binding assay, performed at 4 degrees C. Various Gram positive and Gram negative bacteria were shown to bind to glass-adherent mouse peritoneal exudate cells under these conditions, Str. pneumoniae being the only exception. The binding could be inhibited by pretreatment of the macrophage monolayers with various monosaccharides. The role of particular components of the bacterial cell wall in binding was examined further using different strains of K. aerogenes and S. typhimurium with a known cell wall composition and mutant strains deficient in certain sugars. The ability of a particular constituent to inhibit binding was found to correlate closely with its presence in the bacterial cell wall. It is concluded, that this form of binding, mediated by cell wall carbohydrates represents a primitive recognition mechanism enabling phagocytes to bind microorganisms.

Animals

An alginate lysate from Azotobacter vinelandii phage.

The alginate depolymerase associated with bacteriophage infection of Azotobacter vinelandii has been used in the analysis of sodium alginate. The enzyme degraded the polysaccharide to a series of oligouronides each containing a terminal 4-deoxy-alpha-L-erythro-hex-4-enopyranuronosyl residue. Analysis of these oligouronides, together with kinetic information, indicated that the enzyme was specific for mannuronic acid-containing regions of the polyuronide. The specificity of the enzyme made it possible to determine the primary structure of the macro-molecule. The phage-induced enzyme was shown to be distinct from the alginate lyase elaborated by the host organisms by its pH optimum, molecular weight, Michaelis constant and stability.

Alginates

Glucan common to the microcyst walls of cyst-forming bacteria.

Chemical analysis indicated that D-glucose is tha major neutral monosaccharide present in the microcysts of a range of gram-negative bacteria. Varying amounts of other neutral sugars were found. The glucose was mainly present as a glucan that could be extracted from microcysts of representative strains with alkali or mild acid treatment. The glucan could be identified as an alpha-1,3-linked polymer on the basis of (i) periodate resistance of the extracted polymer and the material present in microcysts; (ii) lectin agglutination of the microcysts; (iii) lectin precipitation of the extracted glucans; and (iv) susceptibility of the glucan either in the walls or after extraction to a specific alpha-1,3-glucanase from Aspergillus nidulans, yielding glucose as the sole hydrolysis product. The galactosamine found in microcysts of Myxococcus xanthus by other workers is clearly a component of another polymer, distinct from the glucan. The presence of an alpha 1,3-linked glucan, common to microcyst walls of various bacterial genera, probably contributes to the rigidity of the walls of these forms and, inter alia, to their resistance to ultrasonic treatment. Preliminary experiments indicate that the gulcan is discarded on germination of the microcysts rather than being broken down by specific enzymes.

Agglutination Tests

Purification and properties of an alginate lyase from a marine bacterium.

An unidentified pseudomonad isolated by enrichment procedures from decomposing seaweed was grown in defined medium containing sodium alginate as the sole carbon source. The alginate lyase recovered from disrupted bacterial cells was purified by a procedure of (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. From sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis experiments a mol.wt. of about 50 000 was determined. The enzyme was active against both algal and bacterial alginate preparations. Kinetic studies together with analysis of the unsaturated oligouronide products of alginate lyase action indicated the enzyme was specific for guluronic acid-containing regions of the macromolecular substrate. The specificity of the enzyme can be used to give information about the primary composition of alginate samples.

Alginates