An online immobilized α-glucosidase microreactor for enzyme kinetics and inhibition assays

Abstract

In this work, a facile protocol for preparing an online immobilized α-glucosidase microreactor was described, and also the enzyme kinetics and inhibition kinetics of such an immobilized enzyme were studied. In this procedure, α-glucosidase was immobilized on the inner wall of the capillary treated with 3-aminopropyltriethoxysilane (3-APTES) and the homobifunctional linker glutaraldehyde (GA). With cross-linking technology, the immobilized α-glucosidase microreactor was fabricated. As the key point for studying the enzyme kinetics and inhibition kinetics of the immobilized α-glucosidase with the developed microreactor, pressure was applied as the driving force to push the reaction mixture to the detection window, and the detection wavelength was set to 405 nm at which only the formed p-nitrophenol (pNP) could be detected. More importantly, with such a detection wavelength it is unnecessary to separate the substrate from the reaction mixture by applying the alkaline inactivating background electrolyte (BGE, borate buffer, pH ≥ 9.0) which is favorable for the separation. So the incubation buffer (phosphate buffer, pH 7.0) could be used as the running buffer making the online study of the enzyme kinetics and inhibition kinetics of immobilized α-glucosidase come true. The inhibition assay was performed by using acarbose as a model inhibitor, and under the optimized conditions, the Michaelis–Menten constant (K_m), inhibition constant (K_i), and half-maximal inhibitory concentration (IC₅₀) for the immobilized α-glucosidase were determined as 0.43 mM, 30.65 μM and 99.48 μM, respectively. This study indicates, for the first time, that the immobilized α-glucosidase microreactor could be used for studying the enzyme kinetics and inhibition kinetics online.