Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies
Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies
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DOI：
10.1016/j.chroma.2003.11.080
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发表时间：
2004-03-26
影响因子：
4.1
通讯作者：
Andrisano, V
中科院分区：
文献类型：
--
作者：
Bartolini, M;Cavrini, V;Andrisano, V
报道了使用原位固定化程序制备的人重组乙酰胆碱酯酶（hrAChE）微固定化酶反应器（IMER）的开发和表征。 hrAChE 共价固定在乙二胺 (EDA) 整体对流相互作用介质 (CIM) 盘（12 mm x 3 mm W.）上，之前用戊二醛衍生化。固定的最佳条件是：将 12 jig 酶溶解在 800 RI 磷酸盐缓冲液（50 mM，pH 6.0）中。将混合物在4℃下轻轻搅拌过夜。所得席夫碱被氰基硼氢化物还原，剩余的醛基与单乙醇胺缩合。在这些条件下，0.22 U 的 hrAChE 被固定化，保留了 3.0% 的初始酶活性。固定化的hrAChE的活性可以稳定超过60天。通过将 micro-IMER 插入 HPLC 系统来研究 hrAChE micro-IMER 的活性和动力学参数，并证明该酶保留了其活性。微型 IMER 的特征在于固定化酶单位和固定化产率的最佳条件。比较了 IMER 的相对酶稳定性、固定单位、产量和非特异性基质相互作用。通过同时注射每种抑制剂和底物来评估 AChE 抑制剂的效果。发现相对 IC50 值与传统动力学分光光度法得出的值一致。与之前开发的基于 AChE 的 IMER 相比，AChE 整体式微型 IMER 在缩短分析时间（2 分钟）、降低非特异性基质相互作用和降低背压方面表现出优势。包含在 HPLC 系统中，可用于快速筛选新化合物的抑制效力。与传统方法相比，其优点是提高了酶稳定性和系统自动化，从而可以连续分析大量化合物。 (C) 2004 Elsevier B.V. 保留所有权利。
The development and characterization of a human recombinant acetylcholinesterase (hrAChE) micro-immobilized-enzyme reactor (IMER), prepared by using an in situ immobilization procedure is reported. hrAChE was covalently immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk (12 mm x 3 mm W.), previously derivatized with glutaraldehyde. The optimal conditions for the immobilization were: 12 jig of enzyme dissolved in 800 RI of phosphate buffer (50 mM, pH 6.0). The mixture was gently agitated overnight at 4degreesC. The resulting Schiff bases were reduced by cyanoborohydride and the remaining aldehydic groups were condensed with monoethanolamine. Under these conditions, 0.22 U of hrAChE were immobilized with retention of 3.0% of the initial enzymatic activity. The activity of the immobilized hrAChE was stable for over 60 days. The activity and kinetic parameters of the hrAChE micro-IMER were investigated by inserting the micro-IMER in a HPLC system and it was demonstrated that the enzyme retained its activity. The micro-IMER was characterized in terms of units of immobilized enzyme and best conditions for immobilization yield. IMERs were compared for their relative enzyme stability, immobilized units, yield and aspecific matrix interactions. The effect of AChE inhibitors was evaluated by the simultaneous injection of each inhibitor with the substrate. The relative IC50 values were found in agreement with those derived by the conventional kinetic spectrophotometric method. In comparison with previously developed AChE-based IMERs, AChE monolithic micro-IMER showed advantages in terms of reduction of analysis time (2 min), lower aspecific matrix interactions and lower backpressure. Included in a HPLC system, it can be used for the rapid screening of new compounds' inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analyzed in continuous. (C) 2004 Elsevier B.V. All rights reserved.