Abstract
Immobilized enzyme has been gradually applied to the screening of enzyme inhibitors owing to its retained catalytic activity and reusability. In this work, the cheap and available cellulose filter paper (CFP) was used as a carrier for the immobilization of α-glucosidase (α-Glu). In virtue of the self-polymerization-adhesion behavior of dopamine, CFP was coated with a polydopamine composite layer and then α-glucosidase is covalently bound to the modified CFP through Schiff base reaction and Michael addition reaction. Combined with capillary electrophoresis (CE) analysis, enzyme reaction kinetics, inhibition kinetics and other performance of the prepared immobilized enzyme (CFP/Dopa/α-Glu) were examined and verified. Its Michaelis constant (Km) was calculated to be 0.83mM. And the inhibition constant (Ki) and half-maximal inhibitory concentration (IC50) for acarbose were determined to be 0.16 and 0.17μM, respectively. CFP/Dopa/α-Glu had the same optimum working pH value (7.0) as free α-Glu and slightly higher working temperature (65°C) than free α-Glu. In addition, it exhibited good batch-to-batch reproducibility with an RSD value of 4.4% (n=10), and excellent reusability with 71% of the initial enzyme activity after being recycled 11 times. Finally, the CFP/Dopa/α-Glu was applied to screen α-glucosidase inhibitors from 11 traditional Chinese medicines, and Terminalia chebula possessed the strongest inhibition effect on α-glucosidase.
Published Version
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