ORCID Profile
0000-0001-7026-0287
Current Organisation
京都大学 / Kyoto University
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Publisher: Portland Press Ltd.
Date: 15-06-2004
DOI: 10.1042/BJ20040145
Abstract: Oligo- and poly-saccharides have a large number of important biological functions, and they occur in natural composite materials, such as plant cell walls, where they self-assemble during biosynthesis in a poorly understood manner. They can also be used for the formation of artificial composite materials with industrial applications. Fundamental and applied research in biology and nanobiotechnology would benefit from the possibility of synthesizing tailor-made oligo- oly-saccharides. In the present paper, we demonstrate that such syntheses are possible using genetically modified glycoside hydrolases, i.e. glycosynthases. The ability of the endoglycosynthase derived from Bacillus (1→3,1→4)-β-d-glucanase to catalyse self-condensation of sugar donors was exploited for the in vitro synthesis of a regular polysaccharide. The specificity of the enzyme allowed the polymerization of α-laminaribiosyl fluoride via the formation of (1→4)-β-linkages to yield a new linear crystalline (1→3,1→4)-β-d-glucan with a repeating 4βG3βG unit. MS and methylation analyses indicated that the in vitro product consisted of a mixture of oligosaccharides, the one having a degree of polymerization of 12 being the most abundant. Morphological characterization revealed that the (1→3,1→4)-β-d-glucan forms spherulites which are composed of platelet crystals. X-ray and electron diffraction analyses allowed the proposition of a putative crystallographic structure which corresponds to a monoclinic unit cell with a=0.834 nm, b=0.825 nm, c=2.04 nm and γ=90.5°. The dimensions of the ab plane are similar to those of cellulose Iβ, but the length of the c-axis is nearly twice that of cellulose I. It is proposed that four glucose residues are present in an extended conformation along the c-axis of the unit cell. The data presented show that glycosynthases represent promising enzymic systems for the synthesis of novel polysaccharides with specific and controlled structures, and for the analysis in vitro of the mechanisms of polymerization and crystallization of polysaccharides.
Publisher: Elsevier BV
Date: 10-2002
Publisher: American Chemical Society (ACS)
Date: 29-04-2003
DOI: 10.1021/BI0340550
Abstract: Detergent extracts of microsomal fractions from Saprolegnia monoïca and blackberry (Rubus fruticosus) cells were incubated with UDP-glucose to yield in vitro (1-->3)-beta-d-glucans. The insoluble products were analyzed by conventional and cryo transmission electron microscopy, X-ray diffraction, and (13)C CP/MAS NMR, and their molecular weights were determined by light scattering experiments. All the products were microfibrillar, but for the detergent extracts from S. monoïca, important morphological differences were observed when the pH of the synthesizing medium was modified. At pH 6, the product had a weight average degree of polymerization () exceeding 20 000 and consisted of endless ribbon-like microfibrils. The microfibrils obtained at pH 9 had a length of only 200-300 nm, and their was approximately 5000. Of all the in vitro (1-->3)-beta-d-glucans, the one from R. fruticosus had the shortest length and the smallest. Crystallographic and spectroscopic data showed that the three in vitro s les consisted of triple helices of (1-->3)-beta-d-glucans and contained substantial amounts of water molecules in their structure, the shortest microfibrils being more hydrated. In addition, the long microfibrils from S. monoïca synthesized at pH 6 were more resistant toward the action of an endo-(1-->3)-beta-d-glucanase than the shorter ones obtained at pH 9. These results are discussed in terms of molecular biosynthetic mechanisms of fungal and plant (1-->3)-beta-d-glucans, and in relation with the possible existence of several (1-->3)-beta-d-glucan synthases in a given organism. The interpretation and discussion of these observations integrate the current knowledge of the structure and function of (1-->3)-beta-d-glucans.
Publisher: Elsevier BV
Date: 08-2002
No related grants have been discovered for Tomoya Imai.