| Project/Area Number |
12839016
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物資源の変換と展開
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| Research Institution | Osaka Prefecture University |
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Principal Investigator |
KAWAGUCHI Takashi Grad. Sch. Agric. Biol. Sci., Osaka Prefecture University Assoc. Prof., 農学生命科学研究科, 助教授 (70195056)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | cellobiosephosphorylase / cellodextrinphosphorylase / Clostridium thermocellum / cellooligosaccharide / cellooligosaccharide analogue / セロビオースホスホリラ-ゼ / Clostridium themocellum / expression |
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| Research Abstract |
The expression vectors for cellobiose phosphorylase (CBPase) and cellodextrin phosphorylase (CDPase) genes from Clostridium thermocellum were constructed. E. coli transformed by these plasmid exhibited the corresponding enzyme activity. After optimizing the conditions such as cultivation temperature or time and a host strain, amounts of CBPase and CDPase produced by E. coli transformants were 130 and 930 mg/l, respectivery, which corresponding 30- and 440- fold higher than those by C. thermocellum. These enzymes were purified to homogeneity on electrophoresis. There was no defference on enzymatic and physicochemical properties between the recombinant and native enzymes.
The method of enzymatic conversion of starch to cellooligosaccharides were examined. Glucose-1-phosphate was prepared from starch by phsphorolysis reaction with potato α-glucan phosphorylase. Using reverse reaction of CBPase and CDPase with glucose-1-phosphate as a glucose donor and glucose as an acceptor, cellooligosaccharides mixture was produced with the total yield from starting material (starch) of 1.73%. Also, enzymatic synthesis of cellooligosaccharide labeled at its reducing end by chromophore was examined. DPase was able to synthesize p-nitrophenyl-cellooligosaccharides using glucose-1-phosphate as a glucose donor and p-nitrophyl-β-D-glucoside as an acceptor with the yield of 59%.
Here, we showed that CBPase and CDPase can be used for enzymatic synthesis of not only cellooligosaccharides but also cellooligosaccharide analogue which could be useful for the studies on cellulases.
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