Spectrophotometric assay for protease activity in ionic liquids using chromogenic substrates.
Spectrophotometric assay for protease activity in ionic liquids using chromogenic substrates.
复制标题
DOI：
10.1016/j.ab.2007.11.015
复制
发表时间：
2008-03
影响因子：
2.9
通讯作者：
K. Nakashima;T. Maruyama;N. Kamiya;M. Goto
中科院分区：
文献类型：
--
作者：
K. Nakashima;T. Maruyama;N. Kamiya;M. Goto
开发了一种新的分光光度测定法来评估离子液体 (IL) 中的蛋白酶活性。该测定由两种策略组成，可对 IL 中的酶促反应进行实时光谱分析。首先，用梳状聚乙二醇 PM13 修饰酶，以获得透明的 IL 酶溶液。其次，使用显色底物来跟踪 IL 中的酶促反应。对硝基苯胺衍生的底物经过蛋白酶催化醇解，释放出显色的对硝基苯胺，可以通过紫外-可见分光光度计进行定量检测。通过使用这种方法，我们可以很容易地评估 IL 中的蛋白酶活性，而无需从反应混合物中分离产物。在不同反应条件下，对 IL 1-乙基-3-甲基咪唑鎓双(三氟甲磺酰基)亚胺 ([Emim][Tf2N]) 中枯草杆菌蛋白酶催化反应的动力学分析证明了新型测定系统的可用性。由于两种不同的丝氨酸蛋白酶（枯草杆菌蛋白酶和 α-胰凝乳蛋白酶）在 IL 中基本上保留了其原始底物特异性，因此通过使用合适的显色底物，可以将该测定扩展到其他酶。
A new spectrophotometric assay has been developed to evaluate protease activity in ionic liquids (ILs). The assay consists of two strategies to enable real-time spectrometric analysis of enzymatic reaction in ILs. First, enzymes are modified with a comb-shaped poly(ethylene glycol), PM13, to obtain a transparent enzyme solution in IL. Second, a chromogenic substrate is used to follow the enzymatic reaction in IL. p-Nitroaniline-derivatized substrates are subjected to protease-catalyzed alcoholysis to release chromogenic p-nitroaniline that can be quantitatively detected by a UV–Vis spectrophotometer. By using this method, we can evaluate protease activity in ILs quite easily without separation of products from the reaction mixture. The availability of the novel assay system was demonstrated in a kinetic analysis of subtilisin-catalyzed reaction in the IL 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([Emim][Tf2N]) under different reaction conditions. Because two different serine proteases, subtilisin and α-chymotrypsin, substantially retained its original substrate specificity in the IL, the assay can be extended to other enzymes by using suitable chromogenic substrates.