Capillary Electrophoresis Based Immobilized Enzyme Micro Reactor For Enzyme Assay And Inhibition Study

Enzyme reactions are the basic of the metabolism of all living systems. Any malfunction, including mutation, overproduction, underproduction or deletion, of a single critical enzyme can lead to a severe human disease. In addition, enzymes are important targets in research and development of drugs and play a role in clinical diagnosis of diseases. A rapid, accurate and efficient method to determine the activity, kinetics and inhibition of enzymes is therefore of significant importance for understanding the mechanism of enzyme catalyzed reactions in cell, for drug development, and for treatment of human diseases.Capillary electrophoresis (CE) technique, which offers several advantages, such as high separation efficiency and sensitivity, extremely small sample requirement, rapid analysis and utilization of various detection modes, has become a powerful approach for quantitative studies of enzymatic reactions. As a widely used approach for online enzyme assay, CE-based immobilized enzyme micro reactor (IMER) has been applied in almost all aspects of enzyme assays during the past two decades, including evaluation of the enzymatic activity and kinetics, screening of inhibitor, investigation of enzyme-mediated metabolic pathways, and proteome analysis. This thesis is mainly focused on the development and application of CE-based IMER for rapid, accurate and efficient enzymatic assays in different analytical tasks.(1) We have conducted an online CE assay for measuring glucose-6-phosphate dehydrogenase (G6PDH) enzyme activity and inhibition. G6PDH-based open tubular immobilized enzyme micro reactor (OT-IMER) was constructed by the easy-to-operate but reliable layer-by-layer strategy for rapid and efficient enzyme assay and inhibition studies. The micro reactor exhibited good stability and reproducibility for online assay of G6PDH enzyme. Both the activity as well as the inhibition of the G6PDH enzyme was investigated using online assay of the CE-based IMERs.(2) Since natural herbal medicines are an important source of enzyme inhibitors for the discovery of new drugs. A number of natural extracts such as green tea have been used in prevention and treatment of diseases due to their low-cost, low toxicity and good performance. We have conducted an online assay of the activity and inhibition of the green tea extract of the G6PDH enzyme using multilayer CE-IMERs. The multilayer CE-IMERs were produced by increasing the layer number in the previous OT-IMER. The use of multilayer OT-IMER could not only increase the enzyme loading amount of the micro reactor but could also enhance its stability. The activity of the G6PDH enzyme was determined and the enzyme inhibition by the inhibitors from green tea extract was investigated using online assay of the multilayer CE-IMERs.(3) We developed a novel OT-IMER by modifying the inner surface of the capillary with silica brush, which largely enhanced the surface area for enzyme immobilization. The fabrication procedure is rapid and simple. Performance and feasibility of the OT-IMER were investigated using trypsin digestion as the model enzyme reaction, and the digested products were quantitatively determined by CE. With N-?-Benzoyl-L-arginine ethyl ester hydrochloride (BAEE) as the substrate, high reproducible enzyme assay was demonstrated with RSD less than 3.45% for the concentration of produced Benzoyl-L-arginine (BA) over 50 consecutive runs. The enzyme can still maintain 79.05% activity after 20-day usage, indicating a remarkable stability of the CE-IMERs. The CE-IMERs were successfully applied for the digestion of standard proteins (cytochrome C, lysozyme).(4) A novel IMER and on-plate enzyme assay was developed based on thin layer chromatography (TLC) technique. The essential idea of the proposed TLC-based IMER is on-plate mixing of the substrate and the enzyme, which was immobilized on the plate, in a short-contact format to start the reaction, followed by TLC separation of the products and the un-reacted substrates. The developed TLC plate is then densitometrically evaluated to determine the peak area of the products for quantitative measurement of the enzyme reaction. Using G6PDH as the test enzyme, we evaluated the performance of the present method for on-plate TLC enzyme assay as well as the inhibition kinetics study.