| Project/Area Number |
09660087
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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 | KYOTO UNIVERSITY |
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Principal Investigator |
KATAOKA Michihiko Kyoto Univ.Agr.Assistant Prof., 農学研究科, 助手 (90252494)
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| Co-Investigator(Kenkyū-buntansha) |
OGAWA Jun Kyoto Univ.Agr.Assistant Prof., 農学研究科, 助手 (70281102)
KOBAYASHI Michihiko Kyoto Univ.Agr.Senior Lecturer, 農学研究科, 講師 (70221976)
SHIMIZU Sakayu Kyoto Univ.Agr.Prof., 農学研究科, 教授 (70093250)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1997: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | Lactone-ring-cleaving enzyme / stereo specificity / Fusarium oxysporum / Brevibacterium / Lacfonase / Fusarium / 結晶構造解析 / パントラクトン / ラクトン / 開裂酵素 / Brevibacterium protophormiae / 立体選択的 |
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| Research Abstract |
Elucidation of the reaction and stereospecificity-control mechanisms of microbial stereospecific lactonohydrolases were carried out through structure-function analyses of the enzymes from a fungal strain Fusarium oxysporum and a bacterial strain Brevibacterium protophormiae.
As to the enzyme of F.oxysporum, the amino acid sequences of the NH_2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR.An approximate 1,000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. The enzyme was crystallized by the vapor-diffusion procedure in the presence of PEG 4,000 as a precipitant. The crystals belong to the monoclinic space group P2_1 with unit-cell parameters a = 156, b = 100, c = 94.1 A, beta = 91.7。.
The enzyme of B.protophormiae was isolated and characterized in some detail. The amino acid sequences of NH_2 terminus and internal peptide fragments of the enzyme were determined. On based on these sequence data, synthetic oligonucleotides were prepared as primers for the PCR.Genomic DNA fragment was amplified, and cloning of genomic DNA for the enzyme using this amplified fragment as the probe is now in progress.
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