| Project/Area Number |
12839005
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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 | Mie University |
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Principal Investigator |
KARITA Shuichi Mie University, Faculty of Bioresources, Lecturer, 生物資源学部, 講師 (90233999)
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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: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | Clostridium / Catabolite control protein / cellulase / xylanase / chitinase / ccpA gene / ccpA遺伝子 / Clostrdium / カタボライト調節蛋白質 / ヘリックスーターン-へリックスモチーフ / Bacillus subtilis / CcpA遺伝子 |
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| Research Abstract |
A gene encoding the catabolite control protein A (CcpA) having helix-turn-helix DNA binding motif was cloned from the cellulolytic anaerobe, Clostridium cellulovorans, using PCR and colony hybridization. A fusion ccpA gene with the promoter region of Bacillus subtilis ccpA gene was examined to complement test using ccpA defect mutant of B. subtilis. A gluconate kinase activity was measured to complement the mutant. As a result, the ccpA gene from C, cellulovorans was not able to complement the defect mutant. The CcpA protein expressed in Escherichia coli was purified using Ni-NTA and Resource Q column. The purified protein was examined for gel shift assay with the promoter region of C. cellulovorans scaffolding protein gene and B. subtilis amyE gene, being reglutated by catabolite. The DNA bands showed no shifting ; suggesting other factors need to bind the protein to DNA. And the protein sequence has similarity to B. subtilis CcpA, but the function is not same. In addition, cloning experiments of genes encoding phosphoenolpyruvate dependent phosphotransferase system (PTS) were done. PTS enzyme I and enzyme II Glc gene were cloned from C. cellulovorans, successfully. The presence of PTS genes suggest the catabolite control mechanism mediated PTS in C. cellulovorans. A ccpA gene from chitinolytic Clostridium paraputrificum was also cloned and sequenced. These have similar protein sequence.
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