MORI Haruhide Hokkaido Univ., Grad. School of Agr., Inst., 大学院・農学研究科, 助手 (80241363)
CHIBA Seiya Hokkaido Univ., Grad. School of Agr., Pro., 大学院・農学研究科, 教授 (30001449)
|Budget Amount *help
¥3,600,000 (Direct Cost : ¥3,600,000)
Fiscal Year 1999 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1998 : ¥2,000,000 (Direct Cost : ¥2,000,000)
In microorganisms there are the N- and O-linked sugar chains which are not found in animals and plants. Their biological function and synthesis have not been elucidated. In this project the structural determination of novel O-linked sugar chains was done, and the enzymes concerned with the formation of α-galactofuranosyl structure (Galf) in N-linked oligosaccharide, UDP-Galf synthetase and Galf transferase, were analyzed. (1) Five kinds of O-linked sugar chains, which were separated chemically from Aspergillus niger α-glu-cosidase, were purified, and the following structures were determined by monosaccharide analysis, exo-glycosidase treatment, and MS, 1D- and 2D-NMR: I) mannose, ii) mannobiose having α-1,2-linkage, iii) glucosylmannobiose of branched type, iv) two mannotrioses of branched (iv-a) and linear (iv-b) structures. Sugar chains of iii and iv-b were novel ones. (2) The substrate and product for Galf transferase were prepared from A .niger α-gluco-sidase. Since the activities of UDP-Galf synthetase and Galf transferase in cell extract of A .niger were low, the cultivation conditions were examined. The addition of maltose or starch to culture broth increased the both enzyme activities with induction of amylases (secretory proteins). When disruption of cells, the loss of activities was observed. It was found that the detergent stabilized the Galf transferase. The transferase preparation of high purity, which did not give a single band in electophoretic analysis, was obtained after several chromatographies. The purification is now doing to figure out the amino acid sequence and to separate the enzyme gene.