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Immobilized Kallikrein Microreactor Based on Capillary Electrophoresis for Online Enzyme Kinetics Analysis and Inhibitor Screening
Chen, Guo Ying1; Zhang, Hao1; Zhang, Chun Yan1; Wang, Yuan1; Zhao, Cong Peng1; Chen, Hua1; Yang, Feng Qing1; Hu, Yuan Jia2
2021-12-01
Source PublicationChromatographia
ISSN0009-5893
Volume84Issue:12Pages:1141-1150
Abstract

In this study, an online capillary electrophoresis-based immobilized enzyme microreactor (CE-IMER) was developed for evaluating inhibitory activity of small-molecule compounds and tea polyphenol extracts on kallikrein (KLK). KLK was immobilized on the inner wall at the inlet of capillary to prepare the KLK-IMER with the aid of chitosan and glutaraldehyde. The immobilized KLK activity and other kinetic parameters were evaluated by measuring the peak area of hydrolysate of chromogenic substrate S-2302 (H-D-Pro-Phe-Arg-pNA). The Michaelis–Menten constant (K) was determined to be 1.82 mM, and the half-maximal inhibitory concentration (IC) and inhibition constant (K) of nicotinamide (positive control drug) were measured to be 12.07 and 4.31 mM, respectively. The negative control drug tinidazole (5.00 mM) had no inhibitory effect on KLK. Moreover, the activity of the immobilized KLK remained approximately 80.0% of the initial immobilized enzyme activity after 30 runs. The CE-IMER was applied to investigate the inhibitory activity of 10 small-molecule compounds and six tea polyphenol extracts on KLK. The results show that four small-molecule compounds (epicatechin gallate, epigallocatechin, epicatechin and isochlorogenic acid C) (0.25 mM) and six polyphenol extracts (Raw Pu-erh, Mingqian Mao Feng, Fuding white tea, Dark green tea, Biluochun and Black tea) (0.25 mg mL) have high inhibitory activity on KLK. In addition, these four small-molecule compounds have binding energies below − 5.0 kcal mol on KLK according to the molecular docking. In short, this study reports a time saving method (0.75 h) for KLK immobilization under mild conditions (25 °C). Graphic abstract: [Figure not available: see fulltext.]

KeywordCapillary Electrophoresis Enzyme Assay Immobilized Enzyme Microreactor Kallikrein Molecular Docking
DOI10.1007/s10337-021-04098-9
URLView the original
Indexed BySCIE
Language英語English
WOS Research AreaBiochemistry & Molecular Biology ; Chemistry
WOS SubjectBiochemical Research Methods ; Chemistry, Analytical
WOS IDWOS:000706026800001
Scopus ID2-s2.0-85116728932
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Cited Times [WOS]:0   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionUniversity of Macau
Corresponding AuthorYang, Feng Qing; Hu, Yuan Jia
Affiliation1.School of Chemistry and Chemical Engineering, Chongqing University, Chongqing, 401331, China
2.State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, 999078, Macao
Corresponding Author AffilicationInstitute of Chinese Medical Sciences
Recommended Citation
GB/T 7714
Chen, Guo Ying,Zhang, Hao,Zhang, Chun Yan,et al. Immobilized Kallikrein Microreactor Based on Capillary Electrophoresis for Online Enzyme Kinetics Analysis and Inhibitor Screening[J]. Chromatographia,2021,84(12):1141-1150.
APA Chen, Guo Ying,Zhang, Hao,Zhang, Chun Yan,Wang, Yuan,Zhao, Cong Peng,Chen, Hua,Yang, Feng Qing,&Hu, Yuan Jia.(2021).Immobilized Kallikrein Microreactor Based on Capillary Electrophoresis for Online Enzyme Kinetics Analysis and Inhibitor Screening.Chromatographia,84(12),1141-1150.
MLA Chen, Guo Ying,et al."Immobilized Kallikrein Microreactor Based on Capillary Electrophoresis for Online Enzyme Kinetics Analysis and Inhibitor Screening".Chromatographia 84.12(2021):1141-1150.
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