| Project/Area Number |
06660108
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Shimane University |
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Principal Investigator |
SAWA Yoshihiro Shimane, Univ., Life and Environmental Science, Professor, 生物資源科学部, 教授 (70127489)
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| Co-Investigator(Kenkyū-buntansha) |
ASHIDA Hiroyuki Shimane Univ., Genetic Research, Research Associate, 遺伝子実験施設, 助手 (70231909)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1994: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | Glutathione production / ATP regeneration / GSH-I / GSH-II / Cyanobacteria / Phormidium / 好温性ラン藻 / Phormidium lapideum / gshI / gshII / Synechococcus sp.PCC7002 / pQCx / GSH-I / GSH-II |
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| Research Abstract |
1. PCR Clonings of gsh-I and gsh-II from E.coli MV1184 strain and E.coli cells with plasmid containing those genes : PCR clonings were carried out with the primer set designed from the terminal regions of gsh-I and gsh-II,respectively, and a template DNA from MV1184 strain. The amplified genes were inserted to pUC18 vector and the plasmids containing gsh-I and II were introduced into MV1184 strain. The MV1184 with the plasmid showed significantly high GSH-I (21.5-fold) and GSH-II (475-fold) activities compared to the strain without plasmid. 2. Treatment of the cloned E.coli cells with toluene : The MV1184 strains with plasmids were treated with toluene (0-10 %) at 25ﾟC for 1 hr. The optimum concentration of toluene in the accumulated glutathione level was around 4 %. The treated cells were stable at least for 1.5 year at -20ﾟC.3. Glutathione production by the mixture of the cloned E.coli cells and cyanobacterial cells (Phormidium lapideum) : The optimum ratio (based on wet weight) of gsh-I cloned cells and gsh-II cloned cells was 30 : 1 (GSH-I activity : GSH-II activity =1 : 3). Under the optimum condition, the glutathione level was 7mumol/wet g for 6 hr. 4. Transformation of E.coli gsh-I and II genes into cyanobacterial cells : To develop an efficient cyanobacterial glutathione production, the E.coli gsh-I and II genes were preliminary transformed into cyanobacterial cells.
To increase intracellular supply of the substrates (glutamate, cysteine, glycine), the enzymes involving biosynthesis of those amino acids were also studied.
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