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

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: 50 mM Tris-HCl (pH 8.0), 15 kV, 37 °C, 10 mM substrate, incubation for 2 min. Under optimal conditions, separation of the product and substrate was achieved through CE within 3.5 min. 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 3.98 mM, respectively. The proposed method was successfully applied for screening of trypsin inhibitors from 19 kinds of natural extracts.

On-line immobilized acetylcholinesterase microreactor for screening of inhibitors from natural extracts by capillary electrophoresis.

In this study we developed a simple capillary electrophoresis (CE) method with an on-line acetylcholinesterase (AChE) microreactor at the inlet of capillary for inhibitor screening. The fused-silica capillary surface was modified with a polycationic polyethylenimine coating. Solutions of the enzyme and chitosan were then injected to immobilize the enzyme in approximately 2.9 cm of the capillary inlet (total length of capillary 60.2 cm) by electrostatic interaction and the film overlay technique. Separation of enzyme reaction product (thiocholine, ThCh) and unreacted substrate (acetylthiocholine, AThCh) was achieved within 3.0 min. The conditions affecting the efficiency of reaction of the enzyme were optimized by measuring the peak area of ThCh. Under the optimum conditions, using Huperzine-A as model inhibitor, K (i) and IC (50) were 0.551 µmol L(-1) and 1.52 µmol L(-1), respectively, for immobilized AChE. Finally, screening of a small compound library containing two known AChE inhibitors and 30 natural extracts was conducted, and species with inhibition activity were directly identified. Compared with previous publications on screening for AChE inhibitors in natural products based on CE methods, the method developed in this work has the advantages of lower cost per analysis, less leakage, and better bioaffinity for the immobilized enzyme because of the unique properties of sodium alginate and chitosan.