Research On The Composition And Structure Of Β -Glucans Isolated From Basidiomycete Raw Materials Inonotus Hispidus

This article highlights the conducted researches on the composition and structure of β-glucans isolated from the basidiomycete raw material Inonotus hispidus. By means of using the alditol acetate method, as well as by UV and IR methods, one-dimensional (13C NMR, 1H NMR), two-dimensional (1H-1H COSY, 1H-13C HSQC) NMR spectroscopy, the composition and molecular structure of polysaccharides were determined and their branching was proved. It was clarified that the composition of the polysaccharide fractions consists mainly of glucose residues (68-100%), as well as residues of fructose, xylose, mannose and galactose as minor monosaccharides. NMR spectroscopic studies of the specimens showed that the obtained polysaccharides consist of branched glucan structures linked by α- and β-glycosidic bonds. In the structure of β-glucans, the main chain is linked mainly through β-1,3-, partially β-1,4-glycosidic bonds, the branched parts consist of one or more β-D-glucose residues that are linked β-1,3- glycosidic bonds, as well as with the main chain, mainly through α- or β-1,6-glycosidic bonds. The results of the study of molecular parameters showed that the MW of β-glucans of basidiomycetes are in the range of 9100-9900 Da, MWD - 1.2-1.5.

Free amino acids and sugars in the flower of Carthamus tinctorius L.

Qualitative and quantitative analyses of free amino acids and sugars in the extracts from freshly collected florets of <em>Carthamus tinctorius</em> L. were performed by combination of thin-layer chromatography (TLC), automatic amino acid analysis and gas-liquid chromatography (GLC). Sixteen amino acids were detected and their quantitative relations were investigated. Alditol acetate derivatives of free sugars were examined by GLC. The retention time and resolution pattern of the following monosaccharides, rhamnose, arabinose, xylose, mannose and glucose, were ultimately investigated.

Methylation-GC-MS analysis of arabinofuranose- and galactofuranose-containing structures: rapid synthesis of partially O-methylated alditol acetate standards

Arabinose and galactose were treated with MeOH containing traces of H2SO4 or HCl at 25ºC to give mixtures of their methyl alpha- and beta-furanosides, as shown by 1D and 2D nuclear magnetic resonance (NMR). Oxidation of the Me alpha,beta-Araf mixture with NaIO4 preferentially oxidised the beta-isomer, to give pure Me alpha-Araf . Each product was progressively O-methylated using the Purdie reagent (MeI/Ag2O) at 25ºC and resulting mixtures of partially methylated glycosides (PMGs) were rapidly assayed by thin layer chromatography (TLC) first to favour higher yields of mono-O-methyl derivatives and later for products with higher degrees of methylation. The products were converted to complex mixtures of partially O-methylated alditol acetate derivatives (PMAAs) by successive hydrolysis, reduction with NaBD4, and acetylation. These can be used as gas chromatography-mass spectrometry (GC-MS) standards in methylation analysis of complex carbohydrates containing arabinofuranosyl and galactofuranosyl units. Of particular interest were the retention times and electron impact MS of the difficult to prepare alditol acetates of 5,6-Me2Gal, 2,5-Me2Gal, 2,5,6-Me3Gal, 3,5,6-Me3Gal, 5-MeAra, 2,5-Me2Ara, and 3,5-Me2Ara. The relative reactivities of hydroxyl groups for mixtures of Me alpha- and Me beta-Galf were HO-2 > HO-3 > HO-6 > HO-5, that of Me alpha and Me beta-Araf HO-2 > HO-3 > HO-5, and that of Me alpha-Araf HO-2 > HO-3 > HO-5.

Biosynthetic specificity of the rhamnosyltransferase gene of Mycobacterium avium serovar 2 as determined by allelic exchange mutagenesis

In prior studies, through recombinant expression in Mycobacterium smegmatis, the rtfA gene of Mycobacterium avium was shown to encode a rhamnosyltransferase that catalyses the addition of rhamnose (Rha) to the 6-deoxytalose of serovar 2-specific glycopeptidolipid (GPL). Whether RtfA also catalyses the transfer of Rha to the alaninol of the lipopeptide core is unknown. An isogenic rtfA mutant of M. avium serovar 2 strain TMC724 was derived using a novel allelic exchange mutagenesis system utilizing a multicopy plasmid that contained the katG gene of Mycobacterium bovis and the gene encoding green fluorescent protein (gfp). Overexpression of KatG in M. avium resulted in increased susceptibility to isoniazid, thus providing counter-selection by enriching for clones that had lost plasmid DNA. Plasmid loss was confirmed by screening for gfp-negative clones to select putative allelic exchange mutants. Two exchange mutants were created, confirmed by Southern hybridization, and demonstrated loss of serovar 2-specific GPL by thin-layer chromatography (TLC). Gas chromatography of alditol acetate derivatives revealed the loss of Rha and the terminal 2,3-O-Me-fucose and preservation of 3-O-Me-Rha and 3,4-O-Me-Rha substituents at the terminal alaninol of the lipopeptide core. Complementation of rtfA in trans through an integrative plasmid restored serovar 2-specific GPL expression identical to wild-type TMC724. This result shows that rtfA encodes an enzyme responsible only for the transfer of Rha to the serovar 2-specific oligosaccharide and provides a system of allelic exchange for M. avium as a tool for future genetic studies involving this species.