Immobilized Kallikrein Microreactor Based on Capillary Electrophoresis for Online Enzyme Kinetics Analysis and Inhibitor Screening

• Published in: Chromatographia 2021-12, Vol.84 (12), p.1141-1150
• Main Authors: Chen, Guo-Ying, Zhang, Hao, Zhang, Chun-Yan, Wang, Yuan, Zhao, Cong-Peng, Chen, Hua, Yang, Feng-Qing, Hu, Yuan-Jia
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Black tea; Capillary electrophoresis; Chemistry; Chemistry and Materials Science; Chitosan; Chromatography; Electrophoresis; Enzyme activity; Enzyme kinetics; Evaluation; Green tea; Hydrolysates; Laboratory Medicine; Microreactors; Molecular docking; Nicotinamide; Original; Pharmacy; Proteomics; Substrates
• ISSN: 0009-5893; 1612-1112
• DOI: 10.1007/s10337-021-04098-9

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 m ) was determined to be 1.82 mM, and the half-maximal inhibitory concentration (IC 50 ) and inhibition constant ( K i ) 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 −1 ) have high inhibitory activity on KLK. In addition, these four small-molecule compounds have binding energies below − 5.0 kcal mol −1 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