| Project/Area Number |
11660097
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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 | Kogakuin University |
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Principal Investigator |
OHTA Takahisa Kogakuin University, Department of Applied Chemistry, Professor, 工学部, 教授 (30011844)
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| Co-Investigator(Kenkyū-buntansha) |
SAKAGUCHI Masayoshi Kogakuin University, Department of Applied Chemistry, Assistant, 工学部, 助手 (80281351)
SUGAHARA Yasusato Kogakuin University, Department of Applied Chemistry, Lecturer, 工学部, 講師 (20133324)
OHKATSU Yasukazu Kogakuin University, Department of Applied Chemistry, Professor, 工学部, 教授 (20011009)
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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 |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Keywords | L-lactate dehydrogenase / organic synthesis by enzyme / substrate specificity / active site / site-directed mutagenesis / Bifidobacterium / バイオリアクター / タンパク質分解酵素 |
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| Research Abstract |
L-Lactate dehydrogenase (LDH) catalyzes stereo-specific reduction of pyruvate into L-lactate. Substerate specificity of Bifidobacterium LDH, Thermus aquaticus LDH and Thermus calophylus LDH, which were cloned in Esherichia coli, was measured. Thermus aquaticus LDHs can catalyze ther reduction of 2-keto acids having longer alkyl group than pyruvate, although Bifidobacterium LDH cannot.
Amino acid residues relating to substrate recognition in the active site of Bifidobacterium LDH were selected using computer analysis of 3-dimensional structure of the LDH.These residues were replaced to other amino acids by site-directed mutagenesis. Enzyme activity of the resulting modified LDHs were measured. k_<cat>/S_<0.5> Values of several modified LDHs were found to be 100 to 500 times higher than that of wild-type LDH.
Enzyme activity in several water-missible organic solvents of Bifidobacterium LDH and Thermus aquaticus LDHs was measured.
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