Construction of Cellulose Binding Domain Fusion FMN-Dependent NADH-Azoreductase and Glucose 1-Dehydrogenase for the Development of Flow Injection Analysis with Fusion Enzymes Immobilized on Cellulose
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- Yano Shigekazu
- Department of Biochemical Engineering, Graduate School of Sciences and Engineering, Yamagata University
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- Hori Yukari
- Department of Biochemical Engineering, Graduate School of Sciences and Engineering, Yamagata University
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- Kijima Tatsuro
- Department of Biochemical Engineering, Graduate School of Sciences and Engineering, Yamagata University
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- Konno Hiroyuki
- Department of Biochemical Engineering, Graduate School of Sciences and Engineering, Yamagata University
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- Suyotha Wasana
- Department of Industrial Biotechnology, Faculty of Agro-industry
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- Takagi Kazuyoshi
- Department of Applied Chemistry, Faculty of Life Sciences, Ritsumeikan University
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- Wakayama Mamoru
- Department of Biotechnology, Faculty of Life Sciences, Ritsumeikan University
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説明
<p>The cellulose binding domain (CBD) of cellulosome-integrating protein A from Clostridium thermocellum NBRC 103400 was genetically fused to FMN-dependent NADH-azoreductase (AZR) and glucose 1-dehydrogenase (GDH) from Bacillus subtilis. The fusion enzymes, AZR-CBD and CBD-GDH, were expressed in Escherichia coli Rosetta-gami B (DE3). The enzymes were purified from cell-free extracts, and the specific activity of AZR-CBD was 15.1 U/mg and that of CBD-GDH was 22.6 U/mg. AZR-CBD and CBD-GDH bound strongly to 0.5 % swollen cellulose at approximately 95 and 98 % of the initial protein amounts, respectively. After immobilization onto the swollen cellulose, AZR-CBD and CBD-GDH retained their catalytic activity. Both enzymes bound weakly to 0.5 % microcrystalline cellulose, but the addition of a high concentration of microcrystalline cellulose (10 %) improved the binding rate of both enzymes. A reactor for flow injection analysis was filled with microcrystalline cellulose-immobilized AZR-CBD and CBD-GDH. This flow injection analysis system was successfully applied for the determination of glucose, and a linear calibration curve was observed in the range of approximately 0.16–2.5 mM glucose, with a correlation coefficient, r, of 0.998.</p>
収録刊行物
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- Journal of Applied Glycoscience
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Journal of Applied Glycoscience 66 (2), 65-72, 2019-05-21
日本応用糖質科学会
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詳細情報 詳細情報について
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- CRID
- 1390001288138145792
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- NII論文ID
- 40021901305
- 130007651714
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- NII書誌ID
- AA11809133
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- ISSN
- 18807291
- 13447882
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- NDL書誌ID
- 029700930
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- 本文言語コード
- en
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- データソース種別
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- NDLサーチ
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