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Extracellular beta-D-fructofuranosidase elaborated by Streptococcus salivarius strain 51: preparation, and mode of action on a levan and on homologues of inulobiose.

Streptococcus salivarius strain 51 produces an extracellular beta-D-fructofuranosidase when grown in a levan-containing medium. The enzyme hydrolyses levan, and inulo-biose, -tetraose, -pentaose, and -hexaose, but has negligible effects on inulin, inulotriose, and inuloheptaose. The branching in S. salivarius strain 51 levan is through a single (2 yields 1)-beta-D linkage.

Candida↗

Conformational analysis of levanbiose by molecular mechanics.

A relaxed conformational energy map for levanbiose, O-beta-D-fructofuranosyl-(2----6)-beta-D-fructofuranoside, was computed with the molecular mechanics program MM2(87). All torsion angles of the three linkage bonds were driven by 30 degrees increments while two primary alcohol groups were held at three staggered forms. The steric energy of all other parameters was optimized. The side groups were retained at the same relative positions on the two rings in this first part of the study so our results are directly applicable to the study of polymeric levan with identical repeating units. The low-energy dimers did not lead to viable polymers. The interresidue linkage torsion angles defined by C-6-O-2'-C-2'-C-1' (phi) and O-5-C-5-C-6-O-2' (omega) have minima at +60 degrees and -60 degrees, respectively, with accessible minima at other staggered forms. As observed in inulobiose, the preferred torsion angle at central linkage bond defined by C-5-C-6-O-2'-C-2' (psi) was antiperiplanar. An analysis of all conformations of staggered side groups showed that the C-1 and C-1' groups had little effect but the C-6' group showed a preference for chi-6'(O-5'-C-5'-C-6'-O-6') = -60 degrees. The fructofuranose rings were started at the low-energy 4(3)T conformation (angle of pseudorotation, phi 2 = 265 degrees) that was retained except when the linkage conformations created severe inter-residue conflict.

Carbohydrate Conformation↗

Microbial levan.

Levans are natural polymers of the sugar fructose found in many plants and microbial products. Like dextrans, they are formed as an undesirable by-product of sugar juice processing. On the other hand, levans, which can only be produced from sucrose, have potential industrial applications as thickeners and encapsulating agents and could provide additional, valuable products from sugarcane juice. A strain of B. polymyxa (NRRL B-18475) produced a high yield of polysaccharide when grown on sucrose solution. Hydrolysis and subsequent analyses showed the product to consist entirely of D-fructose. 13C-NMR and methylation analyses indicated the products to be a beta(2----6)-linked polymer of fructose, with 12% branching. The polysaccharide has a Mr of approximately 2 million and is readily soluble in water. Levan has not been utilized, but if developed, could be useful in food and other industrial applications.

Bacteria↗

Molecular weight and antitumour activity of Zymomonas mobilis levans.

Levans produced by several Zymomonas mobilis strains were classified by their viscosity average molecular weight and tested against sarcoma 180. Measurements of the samples' polydispersity were carried out to characterise the molecular weight distribution. The antitumour activities of levan samples were plotted against the viscosity average molecular weight and a maximum value of this activity was found at Mv = 210,000 (Mw = 456,900 and PD = 16.2). The results indicate that levan antitumour activities depend on the polysaccharide molecular weight and that a specific class of molecular weight may be responsible for this effect.

Animals↗

V lambda-light chain genes reconstitute immune responses to defined carbohydrate antigens or haptens by utilizing different VH genes.

The contribution of the lambda-light chain to the development of peripheral B cell repertoire and generation of specific antibodies to haptens and polysaccharide antigens was studied in genetically manipulated kappa-deficient and lambda 2-transgenic mice. The results clearly demonstrate a non-stoichiometric VH gene family expression in the absence of k-light chain and suggest a non-stochastic pairing between VH and V lambda genes, expressed in the peripheral B cell repertoire. A shift in VH gene utilization in the case of VI lambda + antibodies was evident in response to beta 2-6 fructosan and TNP hapten. These observations demonstrate the availability of compensatory mechanisms in the absence of VK genes and are consistent with the hypothesis that VH gene family expression is controlled by genetic factors from inside the VH locus. Furthermore, genetic factors from outside the VH locus, namely restricted available light chain diversity, may lead to a shift in VH gene utilization in the peripheral B cell repertoire.

Animals↗

Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature.

A thermoactive and thermostable levansucrase was purified from a newly isolated thermophilic Bacillus sp. from Thailand soil. The purification was achieved by alcohol precipitation, DEAE-Cellulose and gel filtration chromatographies. The enzyme was purified to homogeneity as determined by SDS-PAGE, and had a molecular mass of 56 kDa. This levansucrase has some interesting characteristics regarding its optimum temperature and heat stability. The optimum temperature and pH were 60 degrees C and 6.0, respectively. The enzyme was completely stable after treatment at 50 degrees C for more than 1 h, and its activity increased four folds in the presence of 5 mM Fe(2+). The optimum temperature for levan production was 50 degrees C. Contrary to other levansucrases, the one presented in this study is able to produce high molecular weight levan at 50 degrees C.

Bacillus↗

Transfer of T or CD8+ cells from hemorrhaged mice produce alterations in bacterial antigen specific plasma cell repertoires in normal syngeneic recipients.

Hemorrhage has multiple effects on immunologic response, including alteration of B cell repertoires and T cell function. This study examined possible relationships between these two phenomena by determining the effects of T cells and T cell subsets transferred from hemorrhaged donors into normal, unhemorrhaged syngeneic recipients on B cell repertoires. Mice given total T or CD8+ cells from hemorrhaged animals then immunized with the bacterial polysaccharide antigen levan had a decreased percentage of plasma cells producing antibody to levan compared to that in mice given T or CD8+ cells from unhemorrhaged animals. These effects of post hemorrhage CD8+ cells were not seen after transfer into nu/nu mice, indicating that these cells did not directly affect B cell function, but rather required other T cell populations in order to alter the B cell repertoire. These results demonstrate that hemorrhage-induced alterations in bacterial antigen specific B cell repertoires may result from T and CD8+ cell mediated changes in T-B interactions.

Animals↗

Regulatory idiotopes, parallel sets and internal image of the antigen within the polyfructosan-A48 idiotypic network.

The study of the A48 idiotype anti-beta 2----6-polyfructosan immune response was used as a tool to investigate the various postulates of the immune network theory. The results of this study have shown: that only certain idiotopes, designated as regulatory idiotopes, play a role in idiotype-regulated immune responses; that some of the A48 idiotopes were expressed on a myeloma protein with unknown binding activity (MOPC173) and on MOPC167, a phosphorylcholine-binding myeloma protein; that one out of six monoclonal antiidiotypic antibodies tested carried the internal image of the antigen and could elicit an antibacterial levan immune response in mice treated at birth with bacterial levan or polyclonal anti-A48Id antibodies.

Animals↗

Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes.

The accumulation of compatible solutes, such as betaines, proline and sugar alcohols, is a widespread response that may protect plants against environmental stress. It is not yet fully understood how these compounds are involved in the stress tolerance of whole plants. Some plants have been genetically engineered to express enzymes that catalyze the synthesis of various compatible solutes. Some interventions have increased the tolerance of some crop plants to abiotic stress. Furthermore, analysis of such transgenic plants has begun to clarify the roles of compatible solutes in stress tolerance.

Adaptation, Physiological↗

Levan fructotransferase from Arthrobacter oxydans J17-21 catalyzes the formation of the di-D-fructose dianhydride IV from levan.

A new levan fructotransferase (LFTase) isolated from Arthrobacter oxydans J17-21 was characterized for the production of difructose dianhydride IV (DFA IV). LFTase was purified to apparent homogeneity by Q-Sepharose ion exchange chromatography, Mono-Q HR 5/5 column chromatography, and gel permeation chromatography. The enzyme had an apparent molecular mass of 54000 Da. The optimum pH for the enzyme-catalyzed reaction was pH 6.5, and the optimum temperature was observed at 45 degrees C. The LFTase was activated by the presence of CaCl(2) and EDTA-2Na but inhibited strongly by MnCl(2) and CuSO(4) at 1 mM and completely by FeSO(4) and Ag(2)SO(4) at 1 mM. A bacterial levan from Zymomonas mobilis was incubated with an LFTase; final conversion yield from the levan to DFA IV was 35%. Neither inulin, levanbiose, sucrose, dextran, nor starch was hydrolyzed by LFTase. DFA IV was very stable at acidic pH and high temperature, thus indicating that DFA IV may be suitable for the food industry and related areas.

Arthrobacter↗

The biological and chemical variability of yacon.

This paper focuses on the biological and chemical variability of four yacon (Smallanthus sonchifolius) accessions cultivated under field conditions. Significant variations in tuber shape, weight, content of oligofructans, as well as in leaf isozymes, phenolics, and relative DNA contents were found. Accessions 6 and 88 were the most productive (up to 3.01 and 3.74 kg/plant); accession 48 was the most balanced from the yield aspect in three vegetative periods. A significantly higher content of beta-(2-->1) oligofructans was noted in accessions 48 and 88 as compared to 6 and 60. No difference in sucrose, glucose, and fructose level was observed. Only accession 6 exhibited separate acid phosphatase and esterase isoforms. Accessions 6 and 60 had the highest content of phenolics, and accession 88 had the lowest relative DNA content. Large yacon intraspecific variation may be useful in future detailed research as a good background for breeding, growing, and utilization in industrial processing.

Asteraceae↗

selective production and characterization of levan by Bacillus subtilis (Natto) Takahashi.

To meet the industrial need of an efficient microbial method for increased levan production, Bacillus subtilis (natto) Takahashi, a commercial natto starter for preparing fermented soybeans (natto), was used to produce levan. After cultivation for 21 h, 40-50 mg of levan mL(-1) was produced in medium containing 20% (w/w) sucrose, which was approximately 50% yield on available fructose. The product consisted of two fractions with different molecular masses (1794 and 11 kDa), which were easily separated by fractionation using an ethanol gradient. The products were well characterized by GPC, 13C NMR, and 1H NMR. The various sugars and concentrations, initial pH, fermentation temperature, and agitation speed affected the levan production by B. subtilis (natto) Takahashi. Takahashi strain is the most efficient levan-producing strain among all of the B. subtilis strains tested and, as previously reported, it produced the highest yield of levan in the least time (21 h) under the common cultivation condition.

Bacillus subtilis↗

Chemical syntheses of inulin and levan structures.

A fructofuranosyl thiglycoside donor, ethyl 6-O-acetyl-3-O-benzyl-1,4-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2-thio-beta-D-fructofuranoside (11), designed to yield stereospecifically beta-linkages and also to allow subsequent elongation in the 6- and/or 1-positions, was prepared and used in syntheses of levan and inulin structures. DMTST-promoted glycosylation between 11 (1.3 mol equiv) and methyl beta-D-fructofuranoside 6-OH and 1-OH acceptors (3 and 6) gave stereospecifically the protected methyl levanobioside 12 and inulinobioside 17 in excellent yields (80 and 86%), respectively. Protecting group manipulations on these afforded new disaccharide 6'-OH and 1'-OH acceptors (13 and 19), which were coupled again with donor 11 (1.0 mol equiv) to yield methyl levanotrioside 14 and inulinotrioside 20 in high yields, 65 and 67%, respectively. These were transformed into new acceptors and also fully deprotected to afford the methyl glycosides of levanotriose and inulinotriose, all structures that have earlier not been accessible by chemical synthesis.

Carbohydrate Sequence↗

Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity.

Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues.

Amino Acid Sequence↗