Shigeo Shibatani

Selective enzymatic preparation of vinyl sugar esters using DMSO as a denaturing co-solvent

The protease-catalyzed transesterifications between hexoses and divinyladipate were examined. In dimethylformamide hexoses such as d-glucose, d-mannose, d-galactose and α-methyl d-galactoside were esterified with divinyladipate by alkaline protease from Streptomyces sp. to give corresponding 6-O-vinyl adipoyl sugars. When the denaturing cosolvent, DMSO, was added to the solvent, galactose was selectively esterified at only the C-2 position.

Production and characterization of active soluble human β1,4-galactosyltransferase in Escherichia coli as a useful catalyst in synthesis of the Gal β1→4 GlcNAc linkage

An active and soluble human beta1,4-galactosyltransferase (beta-GT) was produced in Escherichia coli using a maltose-binding protein fusion system. The purified recombinant beta-GT has a K(m) value of 0.035 mM for UDP-galactose and a V(max) of 643 x 10(3) nmol/mg/h. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-linked oligosaccharides. The properties of the purified enzyme were identical to those of bovine milk beta-GT.

Synthesis of vinyl arabinose ester catalyzed by protease from Streptomyces sp.

The transesterification of 0.5 M divinyladipate with 0.25 M arabinose in dimethylformamide for 7 days was catalyzed by Streptomyces sp. alkaline protease to give 5-O-vinyladipoyl-d-arabinofuranose at ca. 50% yield. Only enzymatic transesterification of primary hydroxyl group of arabinofuranose proceeded without esterification of arabinopyranose.

Regio-selective preparation of vinyl adenosine ester catalyzed by alkaline protease

The transesterification of divinyladipate with adenosine in DMF containing 20% (v/v) DMSO was catalyzed by Streptomyces sp. alkaline protease and esterification occurred exclusively at the 3′-position of hydroxyl group of ribofuranose in adenosine to give 3′-O-vinyladipoyl adenosine without other products.

Generation of Hydroxyl Radicals from Polymer Containing Reducing Sugar Branches in the Presence of Hydrogen Peroxide

The generation of hydroxyl radicals from poly (6-O-vinyladipoyl-D-glucose) [poly(VAG) in the presence of hydrogen peroxide was detected by oxidation of p-nitrosodimethylaniline (p-NDA). Several hydroxyl radical scavengers inhibited the reaction, but inhibition by superoxide dismutase (SOD) was low. This suggests that hydroxyl radicals from Harber-Weiss reaction made relatively small contributions to this reaction, and that the enediol moiety of reducing sugar branches in poly(VAG) reacted with hydrogen peroxide to produce hydroxyl radicals. The polymer degraded to a low molecular weight material after reaction with hydrogen peroxide.

Highly efficient oligosaccharide synthesis on water-soluble polymeric primers by recombinant glycosyltransferases immobilised on solid supports

Recombinant beta-1,4-galactosyltranferase (beta 1,4-GalT) and alpha-2,6-sialytransferase (alpha 2,6-SiaT) immobilised covalently with activated Sepharose beads were employed for the practical synthesis of a trisaccharide derivative, Neu-5Ac alpha(2-->6)Gal beta(1-->4)GlcNAc beta-O-(CH2)6-NH2, on a water-soluble primer having GlcNAc residues through a alpha-chymotrypsin-sensitive linker.

Hyaluronan Synthesis in Cultured Tobacco Cells (BY-2) Expressing a Chlorovirus Enzyme: Cytological Studies

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured‐cells (BY‐2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY‐2 cells harboring a vSPO‐cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N‐terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. Biotechnol. Bioeng. 2013; 110: 1174–1179.

Chemical and Enzymatic Synthesis of Polymer Containing Reducing Sugar Branches.

バチルス属由来のプロテアーゼを用いて, ジメチルホルムアミド (DMF) 中においてグルコースとアジピン酸ジビニルの間でエステル交換反応を行い, 重合性糖エステル, 6-ο-ビニルアジポイル-D-グルコースを合成した. ひき続きアゾ系の重合開始剤を用いてDMFあるいは水中で, 重合性糖エステルのラジカル重合を行いポリ (6-ο-ビニルアジポイル-D-グルコース) を得た. この還元糖の分岐を有するポリマーはニトロブルーテトラゾリウム法においてはモノマーよりも高い還元力を示したが, シトクロームc法においては変化はなく, 測定方法の違いにより異なった還元力を示した.