Capillary electrophoresis applied to screening of trypsin inhibitors using microreactor with trypsin immobilized by glutaraldehyde

Abstract

An efficient and durable online capillary immobilized trypsin microreactor was successfully established to study the enzyme kinetics of trypsin and screen its inhibitors from natural extracts through capillary electrophoresis (CE). In this procedure, trypsin was immobilized on the inner wall at the inlet of the capillary treated with 3-aminopropyltrimethoxy silane (3-APTES), producing a trypsin microreactor via cross-linking of glutaraldehyde with 3-APTES and trypsin. The rest of the capillary was selected as a channel for separating the generated product and unreacted substrate of the trypsin enzymatic reaction. The parameters affecting the separation efficiency and activity of immobilized trypsin were evaluated systematically. The optimized conditions were as follows: 50mM Tris–HCl (pH 8.0), 15kV, 37°C, 10mM substrate, incubation for 2min. Under optimal conditions, separation of the product and substrate was achieved through CE within 3.5min. The obtained results of Michaelis constant, inhibition kinetics constant, and half-maximal inhibitory concentration for the immobilized trypsin using benzamidine hydrochloride hydrate as a model inhibitor were 1.56, 1.79 and 3.98mM, respectively. The proposed method was successfully applied for screening of trypsin inhibitors from 19 kinds of natural extracts.