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Real-time assay of invertase activity using isoquinolinylboronic acid as turn-on fluorescent sensor

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Abstract

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. Km and υmax 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 IC50 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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Funding

This work was supported by the Central University Basic Research Fund of China.

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Authors and Affiliations

  1. Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu, 610065, China

    Qinfei Chen, Mengqi Han, Hong Zhou, Jiadong Chen & Wenbin Liu

  2. Emergency Department, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, 610065, China

    Yang Yang

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  1. Qinfei Chen
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  4. Hong Zhou
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Correspondence to Wenbin Liu.

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Chen, Q., Han, M., Yang, Y. et al. Real-time assay of invertase activity using isoquinolinylboronic acid as turn-on fluorescent sensor. Anal Bioanal Chem 415, 5297–5309 (2023). https://doi.org/10.1007/s00216-023-04841-1

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  • DOI: https://doi.org/10.1007/s00216-023-04841-1

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