1-kestose and neokestose

Under specific reaction conditions, levansucrase from Bacillus subtilis (SacB) catalyzes the synthesis of a low molecular weight levan through the non-processive elongation of a great number of intermediates. To deepen understanding of the polymer elongation mechanism, we conducted a meticulous examination of the fructooligosaccharide profile evolution during the levan synthesis. As a result, the formation of primary and secondary intermediates series in different reaction stages was observed. The origin of the series was identified through comparison with product profiles obtained in acceptor reactions employing levanbiose, blastose, 1-kestose, 6-kestose, and neo-kestose, and supported with the isolation and NMR analyses of some relevant products, demonstrating that all of them are inherent products during levan formation from sucrose. These results allowed to establish the network of fructosyl transfer reactions involved in the non-processive levan synthesis. Overall, our results reveal how the relaxed acceptor specificity of SacB during the initial steps of the synthesis is responsible for the formation of several levan series, which constitute the final low molecular weight levan distribution.

Topics: Bacillus subtilis; Catalysis; Disaccharidases; Disaccharides; Fructans; Hexosyltransferases; Kinetics; Molecular Weight; Oligosaccharides; Sucrose; Trisaccharides

An extracellular beta-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70 degrees C) and thermostability (with a T(50) in the range 66 to 71 degrees C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-beta-(2-->1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k(cat)/K(m) ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial beta-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter(-1). In addition, we isolated and sequenced the X. dendrorhous beta-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant beta-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.

Topics: Basidiomycota; beta-Fructofuranosidase; DNA, Fungal; Enzyme Stability; Fungal Proteins; Glycoproteins; Hydrogen-Ion Concentration; Molecular Sequence Data; Molecular Weight; Oligosaccharides; Phylogeny; Protein Structure, Tertiary; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Substrate Specificity; Sucrose; Temperature; Trisaccharides

Biosynthesis of fructo-oligosaccharides (FOS) was observed during growth of the thermophilic fungus Sporotrichum thermophile on media containing high sucrose concentrations. Submerged batch cultivation with the optimum initial sucrose concentration of 250 g/l allowed the production of 12.5 g FOS/l. The FOS mixture obtained was composed of three sugars, which were isolated by size-exclusion chromatography. They were characterized by acid hydrolysis and HPLC as 1-kestose, 6-kestose and neokestose. The mechanism of osmotic adaptation of S. thermophile was investigated and sugars and amino acids were found to be the predominant compatible solutes. The fungus accumulated glutamic acid, arginine, alanine, leucine and lysine, in order to balance the outer osmotic pressure. Fatty acid analysis of the membrane lipids showed a relatively high percentage of unsaturated lipids, which is known to be associated with high membrane fluidity.

Topics: Amino Acids; Carbohydrates; Cell Membrane; Chromatography, High Pressure Liquid; Culture Media; Cytoplasm; Hydrogen-Ion Concentration; Membrane Fluidity; Membrane Lipids; Oligosaccharides; Osmotic Pressure; Sporothrix; Sucrose; Trisaccharides

Topics: Carbohydrate Sequence; Carbon Isotopes; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Oligosaccharides; Plant Extracts; Protons; Sucrose; Trisaccharides; Vegetables