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ID 115487
著者
Mine, Masanori Tokushima University
Matsumoto, Naoya Tokushima University
資料タイプ
学術雑誌論文
抄録
An enzymatic hydrolysis of p‐nitrophenyl acetate with carboxyesterase was analyzed by capillary electrophoresis/dynamic frontal analysis (CE/DFA). A plateau signal was expected with the anionic product of p‐nitrophenol by the CE/DFA applying in‐capillary reaction and the continuous CE resolution of the product from the substrate zone. However, the plateau height was not sufficient, and/or the plateau signal fluctuated and drifted. Therefore, a pressure assist was utilized in the CE/DFA to detect the product zone fast and to average the fluctuated plateau signal by mixing in a laminar flow. The plateau signal became relatively flat and its height was developed by the pressure‐assisted capillary electrophoresis/dynamic frontal analysis (pCE/DFA). The plateau height was used for the Michaelis‐Menten analysis, and a Michaelis‐Menten constant was determined as KM = 0.83 mmol L−1. An enzyme inhibition was also examined with bis (p‐nitrophenyl) phosphate by adding it in the separation buffer. The height of the plateau signal decreased by the inhibition, and a 50% inhibitory concentration was determined as IC50 = 0.79 μmol L−1. The values of KM and IC50 obtained in this study agreed well with the reported values. Since the proposed pCE/DFA includes electrophoretic migration of the substrate zone in a capillary, it is also noticed that the deactivation of the enzyme by ethanol on the preparation of the substrate solution can be avoided, as well as the exclusion of the inhibition by the product.
掲載誌名
Analytical Methods
ISSN
17599679
出版者
The Royal Society of Chemistry
12
48
開始ページ
5846
終了ページ
5851
発行日
2020-10-26
EDB ID
出版社版DOI
出版社版URL
フルテキストファイル
言語
eng
著者版フラグ
著者版
部局
理工学系