Enzymatic synthesis of a 2-O-alpha-D-glucopyranosyl cyclic tetrasaccharide by kojibiose phosphorylase

The glucosyl transfer reaction of kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 was examined using cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->} (CTS) as an acceptor. KPase produced four transfer products, saccharides 1-4. The structure of a major product, saccharide 4, was 2-O-alpha-d-glucopyranosyl-CTS, cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-[alpha-d-Glcp-(1-->2)]-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->}. The other transfer products, saccharides 1-3, were 2-O-alpha-kojibiosyl-, 2-O-alpha-kojitriosyl-, and 2-O-alpha-kojitetraosyl-CTS, respectively. These results showed that KPase transferred a glucose residue to the C-2 position at the ring glucose residue of CTS. This enzyme also catalyzed the chain-extending reaction of the side chain of 2-O-alpha-d-glycopyranosyl-CTS.     

Synthesis of 3-O-beta-N-acetylglucosaminyl cyclic tetrasaccharide through a lysozyme-catalyzed transfer reaction

Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS). Structural analysis showed that the transfer product was 3-O-beta-N-acetylglucosaminyl CTS, cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-GlcNAc-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. This branched saccharide is anticipated to be a model compound of the sugar chains of glycoproteins.     

Age-related changes in GM1, GD1a, GT1b components of gangliosides in Wistar albino rats

In this study, age-related changes of GM1, GD1a, GT1b fractions of gangliosides were investigated in whole brain of male Wistar albino rats. Insignificant increases were detected in GM1 values from the third to the 24th month, whereas GD1a and GT1b concentrations of ganglioside in 24-month-old rats decreased significantly as compared to 6-month-old rats. Although there were no significant differences in the GD1a/GT1b ratio of any groups, GM1/GD1a and GM1/GT1b ratios were significantly increased as compared to 6-month-old rats. The increase in the ratios of gangliosides are not due to an increase of GM1 fractions; they result from a decrease of GD1a and GT1b fractions of gangliosides. In conclusion, the concentration of ganglioside decreased with ageing.     

Enzymatic synthesis of a beta-D-galactopyranosyl cyclic tetrasaccharide by beta-galactosidases

The galactosyl transfer reaction to cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS) was examined using lactose as a donor and beta-galactosidases from Aspergillus oryzae and Bacillus circulans. The A. oryzae beta-galactosidase produced three galactosyl derivatives of CTS. The main galactosyl derivative produced by the A. oryzae enzyme was identified as 6-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-[beta-D-Galp-(1-->6)]-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. The B. circulans beta-galactosidase also synthesized three galactosyl-transfer products to CTS. The structure of main transgalactosylation product was 3-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-D-Galp-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These results showed that beta-galactosidase transferred galactose directly to the ring glucose residue of CTS.     

Purification and characterization of glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide in Bacillus globisporus C11

Glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide (CTS; cyclo [-->6]-alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->3)-alpha-D-glucopyranosyl-(1-->)) from alpha-1,4-glucan were purified from Bacillus globisporus C11. The former was a 1,6-alpha-glucosyltransferase (6GT) catalyzing the a-1,6-transglucosylation of one glucosyl residue to the nonreducing end of maltooligosaccharides (MOS) to produce alpha-isomaltosyl-MOS from MOS. The latter was an isomaltosyl transferase (IMT) catalyzing alpha-1,3-, alpha-1,4-, and alpha,beta-1,1-intermolecular transglycosylation of isomaltosyl residues. When IMT catalyzed alpha-1,3-transglycosylation, alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS was produced from alpha-isomaltosyl-MOS. In addition, IMT catalyzed cyclization, and produced CTS from alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS by intramolecular transglycosylation. Therefore, the mechanism of CTS synthesis from MOS by the two enzymes seemed to follow three steps: 1) MOS-->alpha-isomaltosyl-->MOS (by 6GT), 2) alpha-isomaltosyl-MOS-->alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS (by IMT), and 3) alpha-isomaltosyl-(1-->3)-alpha-isomaltosyl-MOS-->CTS + MOS (by IMT). The molecular mass of 6GT was estimated to be 137 kDa by SDS-PAGE. The optimum pH and temperature for 6GT were pH 6.0 and 45 degrees C, respectively. This enzyme was stable at from pH 5.5 to 10 and on being heated to 40 degrees C for 60 min. 6GT was strongly activated and stabilized by various divalent cations. The molecular mass of IMT was estimated to be 102 kDa by SDS-PAGE. The optimum pH and temperature for IMT were pH 6.0 and 50 degrees C, respectively. This enzyme was stable at from pH 4.5 to 9.0 and on being heated to 40 degrees C for 60 min. Divalent cations had no effect on the stability or activity of this enzyme.     

Studies on the Growth and Indole-3-Acetic Acid and Abscisic Acid Content of Zea mays Seedlings Grown in Microgravity

Measurements were made of the fresh weight, dry weight, dry weight-fresh weight ratio, free and conjugated indole-3-acetic acid, and free and conjugated abscisic acid in seedlings of Zea mays grown in darkness in microgravity and on earth. Imbibition of the dry kernels was for 17 h prior to launch. Growth was for 5 d at ambient orbiter temperature and at a chronic accelerational force of the order of 3 × 10⁻⁵ times earth gravity. Weights and hormone content of the microgravity seedlings were, with minor exceptions, not statistically different from seedlings grown in normal gravity. The tissues of the shuttle-grown plants appeared normal and the seedlings differed only in the lack of orientation of roots and shoots. These findings, based upon 5 d of growth in microgravity, cannot be extrapolated to growth in microgravity for weeks, months, and years, as might occur on a space station. Nonetheless, it is encouraging, for prospects of bioregeneration of the atmosphere and food production in a space station, that no pronounced differences in the parameters measured were apparent during the 5 d of plant seedling growth in microgravity.