| Project/Area Number |
11660071
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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 |
応用微生物学・応用生物化学
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| Research Institution | Chiba University |
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Principal Investigator |
FUJII Takaaki Chiba University, Faculty of Horticulture, Professor, 園芸学部, 教授 (50125952)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥3,100,000 (Direct Cost: ¥3,100,000)
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| Keywords | D-lactate dehydrogenase / alcohol dehydrogenase / FAD-D-lactate dehydrogenase / cytochrome c_2 / metabolism of lactate / Rhodopseudomonas palustris / 紅色非硫黄細菌 / Rhodopseudomonas.palustris / PMS-依存乳酸脱水素酵素 |
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| Research Abstract |
Information of electron gain system from organic compounds in purple nonsulfur bacteria is limited. NAD- dependent alcohol dehydrogenase (ADH), and NAD- independent (PMS-dependent) D-and L-lactate dehydrogenases (D-LDH and L-LDH) were detected in the cell-free extracts of Rhodopseudomonas palustris No.7. From these enzymes, D-LDH was purified as an electrophoretically homogeneous protein. The molecular weight of D-LDH and its subunit were estimated to be approximately 235 kDa and 57 kDa by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis, respectively. The pI was 5.0. The optimum pH for the enzyme activity was 8.5 and the optimum temperature was 50℃. The Km against D-lactate was 0.8 mM.The substrate specificity of the enzyme was narrow, and it was inert to L-lactate. The enzyme was reversibly inhibited by oxalate (Ki, 0.12 mM). The prosthetic group of the enzyme was clarified to be FAD.Furthermore, cytochrome in the cell-free extracts of strain No.7 was purified as an electrophoretically homogeneous protein. Its molecular weight was estimated to be approximately 12.4 kDa. It was identified as cytochrome c_2 (Cyt c_2). The reduction of Cyt c_2 was observed with the oxidation of D-lactate by D-LDH.From this, it was suggested that the electron from lactate was utilized in flowing to D-lactate→D-LDH (FAD)→Cyt c_2→photosystem→NAD.L-DH and ADH were unstable. These enzymes have not been purified as a homogeneous protein.
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