| Project/Area Number |
12460147
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | Tokyo University of Agriculture and Technology |
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Principal Investigator |
SAKANO Yoshiyuki Tokyo University, Faculty of Agriculture, Professor, 農学部, 教授 (70014959)
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| Co-Investigator(Kenkyū-buntansha) |
SAKAI Hiroshi University of Shizuoka, Graduate School of Nutritional and Environmental Sciences, Professor, 大学院・生活健康科学研究科, 教授 (10092214)
KAMITORI Shigehiro Tokyo University, Faculty of Technology, Associate Professor, 工学部, 助教授 (00262246)
NISHIKAWA Atsushi Tokyo University, Faculty of Agriculture, Associate Professor, 農学部, 助教授 (30218127)
TONOZUKA Takashi Faculty of Agriculture, Assistant Professor, 農学部, 助手 (50285194)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥14,400,000 (Direct Cost: ¥14,400,000)
Fiscal Year 2002: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥8,000,000 (Direct Cost: ¥8,000,000)
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| Keywords | Pullulan / Cyclodextrin / α-amylase / TVA / Isopullulanase / Protein crystallography / パノシルパノース / 結構構造解析 |
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| Research Abstract |
In this study, highly specific enzymes which produce branched oligosaccharides, TVA I and TVA II from Thermoactinomyces vulgaris and isopullulanase from Aspergillus niger, have been studied, and the following results were obtained.
1. Crystal structures of TVA I and TVA II
Both TVA I and TVA II hydrolyze pullulan to produce panose. However, TVA I prefers polysaccharides, namely, starch and pullulan, while TVA II hydrolyzes oligosaccharides more strongly. The crystal structures of TVA I and also TVA II-substrate complex were determined and compared in this study.The results showed that : (1) the orientation of domain N of TVA I and TVA II are quite different, (2)TVA I is observed as a monomer in the crystal, while TVA II functions as a dimer, and these points appear to give the difference of substrate specificities between TVA I and TVA II.
2. Novel glucoamylase and cyclodextrin-binding protein from T. vulgaris
To obtain enzymes related to oligosaccharide metabolysis, the flanking region of TVA II was sequenced, and genes of glucoamylase and cyclodextrin-binding protein were found. Numerous glucoamylases from fungi have been studied, and they prefer starch to maltooligosaccharides. In contrast, the glucoamylase from T. vulgaris efficiently hydrolyzes maltooligosaccharides.
3. Construction of expression system and properties of isopullulanase
An expression system using a yeast, Pichia pastoris, has been established, and the enzyme was purified. The properties of the recombinant isopullulanase was also assessed, and the finding suggests that glycochains plays the essential role for the activity of isopullulanase.
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