| Project/Area Number |
16K08116
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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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| Research Field |
Environmental agriculture(including landscape science)
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| Research Institution | Osaka Prefecture University |
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Principal Investigator |
ueda mitsuhiro 大阪府立大学, 生命環境科学研究科, 准教授 (50254438)
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| Co-Investigator(Kenkyū-buntansha) |
玉田 太郎 国立研究開発法人量子科学技術研究開発機構, 高崎量子応用研究所 東海量子ビーム応用研究センター, 上席研究員(定常) (50391248)
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Project Status |
Completed (Fiscal Year 2018)
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| Budget Amount *help |
¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2017: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2016: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
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| Keywords | 低温適応酵素 / 糖質分解酵素 / 塩橋 / X線結晶構造解析 / バイオ燃料 / 連続糖化発酵法 / 低温適応性酵素 / マンナン分解酵素 / 生デンプン分解酵素 / ミミズ / セルラーゼ / アミラーゼ / バイオエネルギー / 糖質加水分解酵素 / 食品系廃棄物 / セルラーぜ / バイオエタノール / 低温糖化・低温発酵 |
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| Outline of Final Research Achievements |
It revealed that the structure of Ef-Man is similar to that of the cold-active mannanase (Ca-Man) from Cryptopygus antarcticus. However, the optimum temperatures of Ef-Man and Ca-Man were 60°C and 35°C each other. The weakening of salt bridge is one of the methods by which high flexibility and catalytic efficiency can be conferred on cold-active enzymes at lower temperatures, but the weakening of salt bridge causes a decrease in thermostability. It is reported that the weakening of salt bridge can change property of enzymes.However, there is no report about the weakening of salt bridge . In this study, we applied site-directed mutagenesis combined with rational design to generate a cold-adapted enzyme with improved thermostability and catalytic efficiency at lower temperatures. We found the structure and function of the salt bridge weakening mutant Ef-Man (R302K). The specific activity of R302K was higher than that of WT at lower temperature.
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| Academic Significance and Societal Importance of the Research Achievements |
研究期間内に実施した研究及び成果：ミミズ由来のマンナン分解酵素の低温活性を向上させるために，塩橋に着目し構造と機能に関する研究を行った．塩橋を弱めることで低温活性を向上させるとともに，熱に安定な変異酵素を得ることに成功した．次にシマミミズ由来の2種類の生デンプン分解酵素を酵母を用いて異種宿主発現に成功した．さらにアミラーゼI の高次構造を明らかにした．
学術的意義や社会的意義：低温適応酵素の構造と機能を明らかにすれば，糖化プロセスにエネルギーを必要としないことからCO2ガスの排出量が減少する．低炭素社会構築に向けて今後さらに研究を発展させることは学術的・社会的意義がある．
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