| Project/Area Number |
11660161
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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 |
林産学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SAMEJIMA Masahiro The University of Tokyo, Graduate School of Agricultural and Life Sciences, Professor, 大学院・農学生命科学研究科, 教授 (30162530)
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| Co-Investigator(Kenkyū-buntansha) |
NISHINO Takeshi Nippon Medical School, Department of Biochemistry, Professor, 医学部, 教授 (40094312)
五十嵐 圭日子 東京大学, 大学院・農学生命科学研究科, 特別研究員
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | Cellulose / Cellobiose dehydrogenase / Biodegradation / Flavin / Heme / Oxidoreductase / Phanerochaefe chrysosporium / Humicola insolens |
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| Research Abstract |
Two different pH-dependent cellobiose dehydrogenases were purified from cellulolytic fungi Phanerochaete chrysosporium and Humicola insolens by column chromatographies. The purified enzymes were treated with papain to separate into flavin and heme domains. Redox potentials of both prosthetic groups were determined.
Furthermore, the pre-steady-state kinetics of inter-domain electron transfer in the extracellular flavocytochrome cellobiose dehydrogenase (CDH) from P. chrysosporium was studied under various conditions of pH and substrate concentration. Monitoring at the isosbestic point of each prosthetic group indicated that the reductive half-reactions of flavin and heme were biphasic and monophasic, respectively. The behavior of the second phase of the flavin reduction was almost identical to that of heme reduction at all substrate concentrations and pH values tested, suggesting that the formation of flavin semiquinone and heme reduction involve the same electron transfer reaction. Although flavin reduction by cellobiose was observed in the range of pH 3.0 to 7.0, the velocity of the next electron transfer step decreased with increase of pH and was almost zero above pH 6.0. The second phase of flavin reduction and the heme reduction were inhibited similarly by high concentrations of substrate, while the first phase of flavin reduction showed a hyperbolic relation to the cellobiose concentration. Increase of pH enhanced the substrate inhibition of heme reduction but not the initial flavin reduction.
Cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pasforis. The expression level reached 79 mg/L of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. The kinetic parameters of catalytic function for rCDH were nearly identical to those of wtCDH.
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