Paenibacillus isolates possess diverse dextran-degrading enzymes.

AIMS: To isolate and identify dextran-degrading organisms from sugar mill and compost samples, and to examine the diversity of the dextranolytic enzymes produced. METHODS AND RESULTS: Fifteen dextranolytic prokaryotes were purified at various temperatures from sugar-mill or compost samples, using indicator plates containing blue dextran. A 16S rRNA gene sequence analysis showed that 12 isolates purified at 40, 50 or 70 degrees C were closely aligned to Paenibacillus spp. The three isolates purified at 60 degrees C had identical 16S rDNA sequences, with highest affinity to Bacillus spp. Liquid culture of the 11 isolates purified at 40 or 50 degrees C produced dextranolytic activity in the spent media with maximal activity at 40 or 45 degrees C under the assay conditions used. Hydrolysis of blue dextran in activity gels showed that the 12 Paenibacillus isolates produced from one to five dextranolytic proteins, ranging from 70 to 120 kDa. Based on 16S rDNA sequence, growth habit in liquid culture and dextranolytic enzyme pattern, the 12 Paenibacillus-like isolates could be differentiated into six distinct groups, one of which was capable of growth at 70 degrees C. CONCLUSIONS: The Bacillales, especially the Paenibacillus, are a valuable environmental repository for dextranolytic enzymes of diverse size and potentially diverse activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Dextranolytic enzymes produced by Paenibacillus spp. are an exploitable resource for those interested in modifying the structure of dextrans.

Analysis of starch structure using fluorophore-assisted carbohydrate electrophoresis.

The analysis of the fine structure of starches is important to the investigation of linkages between starch structure and function and to the investigation of the properties and roles of starch biosynthetic, modifying and degradation enzymes. Fluorophore-assisted carbohydrate electrophoresis has recently been introduced as a method for the analysis of the oligosaccharide populations released by the enzymatic digestion of starches, which has advantages in resolution and sensitivity over previously used methods, and provides the capacity for the facile analysis of oligosaccharide populations on either a molar or mass basis. The use of fluorophore-assisted carbohydrate electrophoresis for the analysis of oligosaccharides is reviewed with particular reference to the choice of label, efficiency of labeling and separation techniques. Examples of separations using slab gel electrophoresis, DNA sequencer analysis and capillary electrophoresis are presented and we conclude that on the basis of resolution and reproducibility, capillary electrophoresis is the method of choice for the separation of oligosaccharides of degree of polymerization from 1 to 100. Examples of isoamylase-debranched starches and glycogens analyzed by capillary electrophoresis are presented. The capillary electrophoresis analysis of starch structure through the analysis of oligosaccharides released by the debranching of limit dextrins derived from starches and glycogens is introduced as a useful diagnostic of starch structure. The potential for future development of novel diagnostics for starch structure using fluorophore-assisted carbohydrate electrophoresis is discussed.

High resolution slab gel electrophoresis of 8-amino-1,3, 6-pyrenetrisulfonic acid (APTS) tagged oligosaccharides using a DNA sequencer.

A novel electrophoretic method for the analysis of oligosaccharides using DNA sequencer technology is illustrated using malto-oligosaccharide distributions obtained following isoamylase digestion of glycogen, wheat starch and potato starch. The debranched starches were derivatized at the reducing and with the charged fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). This highly reproducible method provides baseline resolution of oligomers from chain lengths of 3 to more than 80 glucose units, and exhibits high sensitivity with detection thresholds of one femtomole per resolved band. In addition, the reductive amination procedure attaches a single fluorophore per oligosaccharide, allowing calculation of the results on either a mass or a molar basis. The efficacy of the method is illustrated through the determination of the profile of individual oligosaccharides of chain length with a degree of polymerization (DP) < 80, derived from loading less than 15 ng per analysis of glycogen, wheat and potato starches. While the results obtained were superior in resolution and sensitivity to previously reported observations using a range of techniques, they were nonetheless consistent with the overall differences between these polysaccharides. The resolution, sensitivity, reproducibility and high throughput of the method provides substantial advantages over existing methods for the analysis of linear oligosaccharide chain length distributions.

Bafilolides, potent inhibitors of the motility and development of the free-living stages of parasitic nematodes.

Three Streptomyces isolates were identified as producing macrolide antibiotics of the bafilomycin or leucanicidin types during an evaluation of Australian actinomyces for the production of inhibitors of larval development in the parasitic nematode, Haemonchus contortus. Bafilomycins A1, B1, C1, and D were obtained from culture A239 and the 2-O-methyl-L-rhamnosyl derivative of bafilomycin A1, leucanicidin, from cultures A223 and A240. All these 'bafilolides' gave similar patterns of inhibition typified by an initial paralysis of newly hatched L1 larvae and a lethal toxicity within 24 h. LD50 values for inhibition of larval development of McMaster H. contortus ranged from 0.23 micrograms ml-1 for leucanicidin to 2.5 micrograms ml-1 for bafilomycin D. The bafilolides had broad spectrum nematocidal activity, being equi-potent as inhibitors of H. contortus, Trichostrongylus colubriformis and Ostertagia circumcincta larval development. Further, all bafilolides caused some inhibition of H. contortus L3 motility, with the semi-synthetic analogue, bafilomycin B2, the most potent inhibitor (LP50 against McMaster H. contortus 1.9 microgram ml-1). Nematode strains resistant to the known benzimidazole, levamisole and avermectin anthelmintics showed no cross resistance to the bafilolides, supporting the hypothesis that the bafilolides act by an independent mechanism.