The hydrolytic and transferase action of alternanase on oligosaccharides.

Alternanase is an enzyme which endo-hydrolytically cleaves the alpha-(1-->3), alpha-(1-->6)-linked D-glucan, alternan. The main products are isomaltose, alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc and the cyclic tetrasaccharide cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. It is also capable of acting on oligosaccharide substrates. The cyclic tetrasaccharide is slowly hydrolyzed to isomaltose. Panose and the trisaccharide alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-D-Glc both undergo transglycosylation reactions to give rise to the cyclic tetrasaccharide plus D-glucose, with panose being converted at a much faster rate. The tetrasaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc is hydrolyzed to D-glucose plus the trisaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc. Alternanase does not act on isomaltotriose, theanderose (6(Glc)-O-alpha-D-Glcp sucrose), or alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glc. The enzyme releases 4-nitrophenol from 4-nitrophenyl alpha-isomaltoside, but not from 4-nitrophenyl alpha-D-glucopyranoside, 4-nitrophenyl alpha-isomaltotrioside, or 4-nitrophenyl alpha-isomaltotetraoside.

Growth associated exopolysaccharide expression in Lactococcus lactis subspecies cremoris Ropy352.

A natural lactococcal isolate, Lactococcus lactis ssp. cremoris Ropy352, has been previously shown to express two phenotypically distinct exopolysaccharides (ropy and mucoid). This natural isolate was cultured on various media to explore the carbon requirements for exopolysaccharide expression. Ropy exopolysaccharide expression was optimal when grown in defined media rather than on M17-based media. Ropy352 was examined for inducible lysogenic phages. No lytic burst was observed in Ropy352 with ultraviolet light or mitomycin C for phage induction. The sugar compositions of the two phenotypically distinct exopolysaccharides were determined. The ropy exopolysaccharide is composed of galactose and glucose in the molar percents of 42 and 58%, respectively. The mucoid exopolysaccharide is composed of galactose, glucose, and mannose in the molar percents of 58, 29, and 13%, respectively. Mutational analysis revealed that mutations impairing ropy exopolysaccharide expression but not affecting mucoid exopolysaccharide expression could be isolated.

Some structural features of an insoluble alpha-D-glucan from a mutant strain of Leuconostoc mesenteroides NRRL B-1355.

Leuconostoc mesenteroides strain NRRL B-1355 produces two soluble extracellular alpha-D-glucans from sucrose: alternan and dextran. An unusual mutant strain derived from NRRL B-1355 has recently been isolated which produces practically no soluble polysaccharide, but significant amounts of an insoluble D-glucan. Methylation analysis shows it contains linear (1-->3) and (1-->6) linkages as well as (1-->2) and (1-->3) branch linkages. The insoluble glucan was partially digestible by endodextranase, giving rise to a series of oligosaccharides, a high-molecular weight soluble fraction and an insoluble residue. Treatment of the soluble dextranase-limit fraction with an alpha(1-->2) debranching enzyme led to further dextranase susceptibility. Methylation, FTIR and NMR analyses of the dextranase-treated fractions indicate a non-uniform structure with domains bearing similarities to L. mesenteroides strain NRRL B-1299 dextran and to insoluble streptococcal D-glucans.

Role of Streptococcus thermophilus MR-1C capsular exopolysaccharide in cheese moisture retention.

Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799-805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 and Lactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, the epsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticus LH100 plus the non-EPS-producing mutant S. thermophilus DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of D-galactose, L-rhamnose, and L-fucose in a ratio of 5:2:1.

Production of an extracellular polysaccharide by Agrobacterium sp DS3 NRRL B-14297 isolated from soil.

A bacterium isolated from soil and identified as Agrobacterium sp produced a water-soluble extracellular polysaccharide capable of producing highly viscous solutions. Gas chromatographic analysis revealed a sugar composition of glucose, galactose and mannose in the molar ratio of 7.5:2.4:1, together with 3.7% (w/w) pyruvic acid. Methylation analyses showed the presence of (1-->3)-, (1-->4)- and (1-->6)-linked glucose, (1-->3)- and (1-->4, 1-->6)-linked galactose and a small portion of (1-->3)-linked mannose residues. Succinic acid was not present. The molecular weight of the polysaccharide was estimated by light scattering to be 2 x 10(6) Da. The viscosity of solutions containing the polysaccharide remained constant from pH 3 to 11, and decreased by 50% when heated from 5 to 55 degrees C. Maximum yield of the polysaccharide, 20 g L-1, was reached in 48 h at 30 degrees C incubation.

Purification and characterization of a pyruvated-mannose-specific xanthan lyase from heat-stable, salt-tolerant bacteria.

A xanthanase complex secreted by a consortium of heat-stable, salt-tolerant bacteria includes a lyase that specifically removes terminal pyruvated beta-d-mannose residues from the side chains of xanthan gum. The enzyme was purified to homogeneity from the culture broth following ion-exchange chromatography and gel permeation chromatography. It consists of a single subunit of molecular weight 33,000. The enzyme is stable to 55 degrees C for more than 6 h in 20 mM sodium phosphate buffer (pH 5.0) containing 0.25 M NaCl. Optimal enzyme activity was observed at 0.05 M NaCl and a pH of 5. The enzyme has a pI of 3.7. It does not remove unsubstituted terminal beta-d-mannose residues from xanthan side chains nor does it hydrolyze p-nitrophenyl-beta-d-mannose. Treatment of xanthan with purified lyase results in a polysaccharide containing side chains terminating in an unsaturated 4,5-ene-glucuronic acid.

Freezing avoidance and the distribution of antifreeze glycopeptides in body fluids and tissues of Antarctic fish.

The distribution of antifreeze glycopeptides (AFGPs) in the body fluids and tissues of antarctic notothenioid fish was determined. In Dissostichus mawsoni (Norman), the peritoneal, pericardial and extradural fluid, like the blood, contained all eight AFGPs and in concentrations sufficient to depress freezing points below that of sea water (-1.9 degree C). Secreted fluids including urine, endolymph and aqueous and vitreous humour either lack all AFGPs or have very low concentrations of only the low molecular weight forms and have freezing points of about -1.0 degree C, and are therefore undercooled with respect to environmental temperature. Fluids with high concentrations of AFGPs also contain high levels of proteins similar to plasma proteins. Systemic administration of tritiated AFGPs in the closely related species Trematomus bernacchii (Boulenger) yielded a distribution pattern similar to that of the native AFGPs in D. mawsoni. This suggests passive distribution of AFGPs into the various fluid compartments following secretion from the liver; a pattern typical of secreted blood proteins. Tissue distribution of AFGPs was determined by comparison with that of the extracellular space marker [14C]polyethylene glycol. AFGPs were found in the interstitial fluid of all body tissues examined except brain tissue. No tissue showed any intracellular accumulation of tritiated AFGPs from the blood.

Rhizobins, a group of peptides in the free-amino-Acid pool of the soybean-Rhizobium system.

Free-living Rhizobium (according to Bergey's Manual of Systematic Bacteriology, [1984, The Williams & Wilkins Co., Baltimore], Bradyrhizobium) japonicum was found to release a peptide into the nutrient media. Soybean nodules contained this peptide and exuded it into the soil. The name "rhizobin A" is suggested for this peptide. Nodules also contained another peptide, rhizobin B, as well as an unidentified, ninhydrin-positive compound, rhizobin C. The three peptides were confined to the free-amino-acid pool of the soluble fraction and eluted consecutively from a cation-exchange column. Rhizobin A was isolated in a highly purified form; its molecular mass was approximately 1,600 daltons as determined by Sephadex gel filtration and mass spectrometry. The amino-acid composition could be determined only approximately, because a long time was necessary for acid hydrolysis, possibly due to unusual linkages. The rhizobin concentration in soybean nodules continually increased during 50 days of growth, from 2 to approximately 400 mug/g (fresh weight). When combined nitrogen was added to nodulated soybean and subsequently removed, nitrogenase activity, nodulation, and nodule growth first decreased and then recovered. The relative amount of rhizobin A followed a similar pattern. Rhizobins were not detected in the roots, stems, and leaves of nodulated soybean plants. They were present in Lupinus nodules, but absent in alder nodules.

Methyl esterification of glutamic acid residues of methyl-accepting chemotaxis proteins in Bacillus subtilis.

The amino acid residue modified in the reversible methylation of Bacillus subtilis methyl-accepting chemotaxis proteins was identified as glutamic acid; methylation results in the formation of glutamate 5-methyl ester. Identification was made by comparing the behaviour of a 3H-labelled compound isolated from proteolytically hydrolysed methyl-accepting chemotaxis proteins labelled in vivo with that of authentic methylated amino acids by chromatographic and electrophoretic techniques. Also, the isolated compound on mild alkaline hydrolysis shows behaviour identical with that of authentic glutamate 5-methyl ester. [3H]Methanol released by mild alkaline hydrolysis was made to react with 3,5-dinitrobenzyl chloride to form [3H]methyl 3,5-dinitrobenzoate, which was identified by reverse-phase high-pressure liquid chromatography.

Membrane-wall interrelationship in Gaffkya homari: sulfhydryl sensitivity and heat lability of nascent peptidoglycan incorporation into walls.

Membrane-walls from Gaffkya homari require a specific interrelationship between membrane and wall that functions in the incorporation of nascent peptidoglycan into the preexisting peptidoglycan of the wall. Two different methods were used to inhibit selectively this incorporation process: (i) sensitivity to sulfhydryl reagents and (ii) heat inactivation. Of the sulfhydryl reagents tested, 2.2 mM iodoacetamide inhibited the synthesis of wall peptidoglycan 50%, whereas greater than 100 mM was required to inhibit the synthesis of sodium dodecyl sulfate (SDS)-soluble peptidoglycan. Heat treatment at 37 degrees C (t 1/2 = 5.7 min) inhibited wall peptidoglycan synthesis without affecting SDS-soluble peptidoglycan synthesis. Inhibition of LD-carboxypeptidase by iodoacetamide and heat gave 50% inhibition and t 1/2 values similar to those observed for the incorporation process. Thus, it is suggested that the LD-carboxypeptidase may be one of the enzymes responsible for the sulfhydryl sensitivity and heat lability and that this enzyme may play a role in the relationship between membrane and wall in G. homari.