A Study Of Immobilized Enzyme Microreactor For On-Line Screening Enzyme Inhibitors By Capillary Eletrophoresis

Natural extract has been the source of drug discovery. Enzymes are important drug screening targets. Many drugs of important diseases, such as acquired immune deficiency syndrome (AIDS), tumor, cancer, hypertension, Alzheimer’s disease (AD) and so on, are all sorts of enzyme inhibitors. During the drug’s high throughput screening (HTS), reagent consumption accounts for three-fourths in all the cost of screening, which will reduced heavily when screening technology miniaturized. Capillary electrophoresis (CE) technique is a very good enzyme inhibitors screening platform, due to its advantages of high separation efficiency, quick analytical speed and less reagent consumption. With the right immobilized enzyme method, enzyme molecular can be self-assembly on the inlet of capillary to create an immobilized capillary enzyme microreactor (ICEMR) for on-line studying enzyme kinetics and enzyme inhibition kinetics by CE, which will not only reduce the consumption of reagent, but also simplify measuring procedure. During inhibitors screening, firstly the mixture of screening compounds and substrate was injected in ICEMR. After full reaction, inhibition degree contrasting blank was determined by the peak area or peak height of product, from which whether the inhibitors of known enzyme exist in the screening compounds can be judged. The screening method is simple, high automation and shows potential use for high throughput screening of enzyme inhibitors from natural extracts.Based on the previous studies, two kind ICEMRs were developed for studying enzyme kinetics and enzyme inhibition kinetics by CE. And the screening results indicated that the method can be successfully applied to screening enzyme inhibitors from natural extracts. In this paper, two innovative works were carried out in this paper.(1) The present study developed a capillary electrophoresis (CE) method to prepare online capillary immobilized acetylcholinesterase (AChE) microreactor. The enzyme kinetics and enzyme inhibition of immobilized AChE was studied, and the inhibitors of AChE were successfully screened from natural extracts. After activated by NaOH, an approximately2.9cm long plug of PEI solution was injected into the capillary to create a positively charged coating. Then negatively charged enzyme solution was adsorbed at the inlet of capillary via electrostatic interaction. Afterwards, the surface of AChE was coated by a biological layer through the gel function of calcium alginate and film role of chitosan. Finally about2.9cm length immobilized AChE microreactor was created in the inlet of capillary. The rest of capillary was used as a channel for separating the generated product and unreacted substrate of the enzymatic reaction. The peak area of product was measured as quantitative criteria to monitor the enzyme kinetics and enzyme inhibition kinetics of AChE.10mmol L-1substrate (no inhibitors or inhibitors) was injected into microreactor and incubated for2min at37℃. Then buffer of20mmol L-1Tris-HCl (pH8.0), voltage of25kV and temperature of37℃was applied and the separation of the product and substrate was achieved within3.0min by CE. The parameters influencing the activity of immobilized AChE, such as type and concentration of buffer, incubation time, concentration of substrate were evaluated systematically. Under the optimal conditions, obtained values of Michaelis constant (Km), inhibition kinetics constant (Ki) and half maximal inhibitory concentration (ICso) for immobilized AChE using Huperzine-A as a model inhibitor were0.66mmol L-1,0.551μmol L-1and1.52μmol L-1, respectively. The proposed method was shown to be a simple, efficient and dependable approach for screening a small compound library containing two known AChE inhibitors and30natural extracts.(2) A capillary immobilized trypsin microreactor was successfully created for on-line studying the enzyme kinetics and enzyme inhibition of immobilized trypsin and screening its inhibitors from natural extracts by CE. After pretreated by NaOH, HCl, deionized water and methanol in sequence, the capillary was blowed dry with vacuum and heated up to40℃. Then an about1.0cm long plug of3-aminopropyltri-methoxysilane methanol solution was injected into the capillary, after full reaction the glutaraldehyde solution with the same plug length was also injected into the capillary. After reaction, the same plug length of trypsin solution was injected into capillary, adequately crosslinking at25℃for creating an approximately1.0cm long immobilized trypsin microreactor. The rest of capillary was chosen as a channel for separating the generated product and unreacted substrate of the trypsin enzymatic reaction. The peak height of product was measured as quantitative criteria to monitor the enzyme kinetics and enzyme inhibition kinetics of trypsin.10mmol L"1substrate (no inhibitors or inhibitors) was injected into microreactor and incubated for2min at37℃. Then buffer of50mmol L-1Tris-HCl (pH8.0), voltage of25kV and temperature of37℃was applied and the separation of the product and substrate was achieved within3.5min by CE. The factors influencing the separation of product and substrate and activity of immobilized trypsin, such as type and concentration of buffer, pH, voltage, incubation time, concentration of substrate were evaluated systematically. Under the optimal conditions, obtained values of Michaelis constant (Km), inhibition kinetics constant (Ki) and half maximal inhibitory concentration (ICso) for immobilized trypsin using benzamidine hydrochloride hydrate as a model inhibitor were3.16mmol L-1,12.9mmol L-1and3.98mmol L-1, respectively. This method was simple, efficient, reliable and used successfully for screening trypsin inhibitors from19kinds of natural extracts.