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ID 115487
Author
Mine, Masanori Tokushima University
Matsumoto, Naoya Tokushima University
Content Type
Journal Article
Description
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.
Journal Title
Analytical Methods
ISSN
17599679
Publisher
The Royal Society of Chemistry
Volume
12
Issue
48
Start Page
5846
End Page
5851
Published Date
2020-10-26
Remark
論文本文は2021-10-26以降公開予定
EDB ID
DOI (Published Version)
URL ( Publisher's Version )
language
eng
TextVersion
その他
departments
Science and Technology