Creation of aromatic organic acid-reactive enzymes by rational design and their application to health examination for organic solvents
| Project/Area Number |
18K06616
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Review Section |
Basic Section 47020:Pharmaceutical analytical chemistry and physicochemistry-related
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| Research Institution | Setsunan University |
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Principal Investigator |
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Project Status |
Completed (Fiscal Year 2021)
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| Budget Amount *help |
¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
Fiscal Year 2020: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2018: ¥520,000 (Direct Cost: ¥400,000、Indirect Cost: ¥120,000)
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| Keywords | トルエン / キシレン / スチレン / 酵素的測定法 / 基質特異性 / 健康診断 / ハイスループット / タンパク質工学 / 有機溶剤作業 / 馬尿酸 / 加水分解酵素 / エンドポイントアッセイ / 芳香族有機酸 / 有機溶剤取扱作業 / 生体成分分析 / 自動分析 / ドッキングスタディ / 変異体酵素 / 有機溶剤検診 / プロテイン・エンジニアリング / 基質改変 |
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| Outline of Final Research Achievements |
To prevent health hazards, we developed high-throughput assay methods for urinary metabolites of toluene, xylene, and styrene, which are aromatic organic acids. First, we devised a new enzymatic assay to measure the total amount of hippurate and methyl hippurates, which are metabolites of toluene and xylene. The reaction mechanisms of the enzymes used were deduced based on structural informations, and mutants with improved specific activity and achieved low temperature optimization or disappearance of substrate inhibition were successfully developed. A new assay method for mandelate, an indicator of styrene exposure, was also devised. The enzyme used was rationally designed based on the tertiary structure of lactate oxidase to have multiple mutations. The reactivity of the prepared mutant to L-mandelate was improved about 200-fold compared with that of the wild type, and the development of mandelate oxidase was successfully achieved.
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| Academic Significance and Societal Importance of the Research Achievements |
有機溶剤取扱作業従事者は、健康被害予防のため特殊健康診断の受診が義務付けられている。現在の分析、診断手法は、操作が煩雑、スループットが低いなど課題が多い。本研究の成果は、これらの課題を解決する。既に馬尿酸・メチル馬尿酸測定の研究成果は検査企業に移管終了し、分析試薬が実用化の目処を得ている。マンデル酸測定の研究成果も企業移管中で、分析試薬開発が進められている。
臨床検査などで普及している酵素的測定法は、既存酵素の組合わせで測定系を構築している。本研究ではこのアプローチを逆転し、理想的な酵素的測定法を考案後、必要な新規酵素を合理的に開発した。今後、新たな開発手法となることを期待する。
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Report
(5 results)
Research Products
(63 results)
