fructooligosaccharide 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

The extracellular β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous mainly synthesizes the neo-fructooligosaccharides (neo-FOS) neokestose and neonystose. This enzyme is a glycoprotein with a content of 59-67% N-linked carbohydrates and an estimated molecular mass of 160-200 kDa. The extent level of glycosylation affects the thermal behaviour of the enzyme but not its hydrolase and transferase activities, which are optimal at 60-70 °C. The neo-FOS yield of this enzyme was increased from 40 to 168 g/L when the sucrose concentration increased from 420 to 600 g/L and when the reaction was carried out at 60 °C. The neo-FOS levels obtained (168 g/L) in this work are the largest reported for any microbial β-fructofuranosidase.

Topics: Basidiomycota; beta-Fructofuranosidase; Biocatalysis; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Extracellular Space; Glycosylation; Hydrolases; Kinetics; Oligosaccharides; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sucrose; Temperature; Time Factors; Transferases; 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