| Project/Area Number |
12650819
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
工業物理化学
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| Research Institution | Kanagawa Institute of Technology |
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Principal Investigator |
SATOU Ikuo Kanagawa inst. Tech., Faculty of Engineering, Department Applied Chemistry, Professor, 工学部, 教授 (20148125)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | Heavy metals / Microanalysis / Immobilized enzyme / Thermolysin / Flow-injection analysis / Calorimatry / Zinc ions / Apoenzyme reactivation method / 重金属イオン / 亜鉛(II)イオン / アポ酵素 / 金属酵素 |
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| Research Abstract |
○ Construction of a photometric flow-injection biosensing system and its characteristics Thermolysin, one of the most typical zinc-enzymes and thermostable neutral protease, was dissolved in borate buffer solution containing 40 % glycerol and immobilized onto porous glass beads with controlled pore size(24.2 nm). The immobilization yield was about 30 %. The immobilized preparations were packed into a small polymer column and then, attached to a photometric sensing system. Zinc (II) ions ranging from 1. 0 nM to 1. 0 mM could be determined, but the enzyme column did not always retain excellent operational stability due to exposure to chelating agents (0.1 M EDTA, pH 4.0).
○ Construction of a calorimetric flow-injection biosensing system and its performance Sodium bromide as good solvent for thermolysin was added to the enzyme-borate buffer solution, and thereby, the enzymes could be immobilized in a satisfactory yield. The preparations packed in the same size of the column was applied to a calorimetric sensing system. The enzyme column exhibited high catalytic activity for 1.0 mL-injections of 4.0 mM synthetic substrate (N- (3-[2-furyl]acryloyl-gly-leu amide), pH 8.0) and the temperature response was about 4.0 mK. Introduction of 2.5 mL of the chelating agent to the column caused significant decrease in the catalytic activity. The once chelator-exposed column was readily reactivated by injecting zinc)II) ions solution. Reactivation of the almost zinc-free column was related to concentrations of zinc)II) ions added. Thus, calorimetric detrmination of zinc(II) ions in a concentration range of 0.01 to 1.0 mM was positively achieved based on.the apoenzyme reactivation method.
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