Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 415, Issue 22, Pages 5297-5309Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-023-04841-1
Keywords
Fructose; 8-IQBA; Invertase; Fluorescent sensor
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In this study, a continuous fluorometric method was developed for real-time detection of invertase activity. The method used 8-isoquinolinylboronic acid as a fluorescent sensor to selectively recognize fructose. By monitoring the fluorescence change during the conversion process, the activity characteristics of invertase were obtained. This method has broad applications.
Invertase is the key enzyme involved in several crucial biological processes by hydrolyzing sucrose for production of glucose and fructose. Invertase plays important roles in the fields of food, pharmacy, cosmetics, biofuels, and agriculture. Detection of invertase activity is urgently necessary for scientific research and industrial processes. Herein, a continuous fluorometric method was developed for real-time detection of invertase activity. 8-Isoquinolinylboronic acid responded to fructose by formation of a fluorescent complex in turn-on manner, and served as a fluorescent sensor to selectively recognize fructose in ternary enzymatic mixture containing sucrose and glucose. The limit of detection (LOD) for fructose was 0.07 mM. Progress curve for fructose production was established by directly and continuously monitoring the fluorescence for invertase reaction with sucrose as substrate. Initial velocity was obtained to characterize invertase activity. LOD for invertase assay was 0.10 U.mL(-1). K-m and D-max for invertase were determined as 7.70 mM and 0.86 mM.min(-1), respectively. Copper ion was demonstrated to inhibit the invertase activity with CIC50 of 33.61 mM. Applicability in high-throughput screening for inhibitor was demonstrated. The proposed method allows for real-time, simple, and rapidly monitoring the invertase activity. It has a broad range of potential applications for kinetics and screening inhibitor.
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