Abstract
A capillary electrophoresis (CE)-based beta-glucosidase (beta-Glu) immobilized enzyme microreactor (IMER) was constructed for enzyme kinetics study and inhibitor screening with the aid of polydopamine coating. The enzyme kinetic and inhibition studies of beta-Glu were comprehensively evaluated using p-nitrophenyl beta-d-glucopyranoside as a model substrate and castanospermine as a model inhibitor. The Michaelis-Menten constantvalue of the immobilized beta-Glu in the developed IMER was calculated to be 2.79 mmol/L. The half-maximal inhibitory concentration and inhibition constant of castanospermine were 13.22 μg/mL and 1.54 μg/mL, respectively. In addition, after 50 consecutive runs, the IMER activity was remained at 89.5% of the initial immobilized beta-Glu activity, which showed that the constructed IMER has good stability and repeatability. Finally, the developed method was successfully applied to screen beta-Glu inhibitors from twelveflavonoids. Four flavonoids include genistein, baicalein, epicatechin gallateand epigallocatechin gallate had significant inhibitory effect on beta-Glu, and their binding mode with enzyme was further verified via the molecular docking analysis. In summary, the developed CE based beta-Glu-IMER is a reliable method for screening beta-Glu inhibitors from natural products.
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Puerarin,
genistein,
quercetin,
dihydroquercetin,
curcumin,
jaundice,
apigenin,
catechin,
epicatechin,
epigallocatechin,
epicatechin gallate and
epigallocatechin gallate were all purchased from Chengdu
Biopurify Phytochemicals Ltd. (Chengdu, China) …
