| Project/Area Number |
11556014
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | NAGOYA UNIVERSITY |
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Principal Investigator |
THUKAGOSHI Norihiro Nagoya University, Graduate School of Bioagricultural Sciences Professor, 大学院・生命農学研究科, 教授 (50115599)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANE Takashi Nagoya University, Graduate School of Engineering Professor, 大学院・工学研究科, 教授 (80030055)
KATO Masashi Nagoya University, Graduate School of Bioagricultural Sciences Assistant Professor, 大学院・生命農学研究科, 助手 (70242849)
KOBAYASHI Tetsuo Nagoya University, Graduate School of Bioagricultural Sciences Associate Professor, 大学院・生命農学研究科, 助教授 (20170334)
TAKAHASHI Mamoru Asahi Chemical Industry Co., Ltd., Department of Diagnostics Research and Development Assistant General Manager, 診断薬研究部, 副部長
SHIRAI Tsuyoshi Nagoya University, Graduate School of Engineering Assistant Professor, 大学院・工学研究科, 助手 (00262890)
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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 |
¥13,600,000 (Direct Cost: ¥13,600,000)
Fiscal Year 2000: ¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 1999: ¥7,700,000 (Direct Cost: ¥7,700,000)
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| Keywords | Acremonium sp.HI-25 / Ascorbate oxidaze / Taka-amylaseA promoter / site-specific mutagenesis / Upward pH shift / V193P / P190I / アルコルビン酸オキシダーゼ |
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| Research Abstract |
The ascorabate oxidase cDNA isolated from Acremonium sp, HI-25 was successfully expressed using Aspergillus nidulans as an intermediate host under the promoter of the Aspergillus oryzae Taka-amylaseA gene. Utilizing this newly developed enzyme production system, we attempted to increase the enzyme productivity, alter the enzyme properties especially pH profile of enzyme catalysis by protein engineering, determine the three dimensional structure and find novel ways to use the enzyme in clinical diagnostics and food storage
#l. To increase the enzyme productivity, Hap complex which enhances at least five times the expression level of Taka-amylaseA have been characterized in detail by reconstituting in vitro DNA binding complexes with various combinations of recombinat subunits.
#2. Substitution of Valine 193 for Proline in the Substrate Binding Region I(SRBI)was found to shift the pH optimum to neutrality by 1 unit. Then, many amino acid residues in this region was site-specificly mutated, followed by characterization of their pH optima, azide sensitivity and heat stability. The pH optima of V193P and P190I were shifted significantly upward without altering the other enzyme properties.
#3. Based on the ASOM structure deduced from computer graphics modelling, Glu192 forms ion pair with Arg309. Replacement of Val193 with Proline could disrupt this ion-pair and result in formation of new stable ion-pairing probably between Glu192 and Arg309. This alteration of the conformation of the specificity pocket influenced the optimum pH of enzyme catalysis.
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