Temperature triggered switching of enzymatic activity via polymer bound, thermoresponsive inhibitor motifs

主要目的是建立温度可控酶活性的新概念。这个概念，类似于“开/关”开关原理，依赖于一个可逆的，但几乎完全失活的酶通过跨越特定的阈值温度。为此，我们想利用某些低分子量有机物对选定酶的抑制作用。这些抑制结构基序应该衍生的方式，他们可以共价连接到含有硫内酯，热响应聚合物。通过达到各自聚合物的云点，这些基序可以从溶液体积中可逆地去除，从而可以使用温度触发器开关酶活性。所提出的概念可用于温度指示系统，但也可能用于生物催化中酶级联的温度控制调节（反应级联中单个酶的选择性，可逆失活）。同时，它拓宽了功能性、反应性聚合物的应用范围，从而强调了聚合后改性新策略的重要性。在拟议的项目中，应该利用至少三种酶/抑制剂组合，这些组合是通过事先筛选各自的数据库选择的，以便证明这一新概念的广泛适用性。需要完成的任务包括所选酶的提供、衍生化抑制剂的合成、衍生化和与热敏聚合物结合后的抑制效果的验证以及通过所利用聚合物的结构变化来研究和优化温度开关效应。最后，至少要利用先前优化的系统中的一个来生成光度指示器，该指示器显示超出某个定义的阈值温度以及该超出的持续时间。

The main goal is to establish a new concept for temperature controllable enzymatic activity. This concept, comparable to an "on/off"-switch principle, relies on a reversible but almost complete deactivation of an enzyme by the crossing of a particular threshold temperature. For that, we want to make use of the inhibition effect of certain low molecular weight organic substances towards selected enzymes. These inhibiting structural motifs are supposed to be derivatized in a way, that they can be covalently attached to a thiolactone containing, thermoresponsive polymer. By reaching the cloud point of the respective polymer, these motifs can be reversibly removed from the solution volume, making it possible to switch enzymatic activity on and off using a temperatur trigger. The proposed concept can be used in temperature indicator systems but is also potentially useful for a temperature controlled regulation of enzyme cascades in biocatalysis (selective, reversible deactivation of single enzymes within the reaction cascade). At the same time, it widens the application spectrum of functional, responsive polymers and thereby emphasizes the importance of novel strategies for post polymerization modification. Within the proposed project, at least three enzyme/inhibitor combinations, that have been selected by a prior screening of respective databases, are supposed to be utilized in order to demonstrate the broad applicability of this new concept. Tasks to be accomplished include the provision of the selected enzymes, the synthesis of the derivatized inhibitors, the verification of the inhibtion effect after derivatization and binding to the thermoresponsive polymer and investigations on and optimization of the temperature switch effect by structural variation of the utilized polymers. Finally, at least one of the previously optimized systems is supposed to be utilized to generate of photometric indicator that displays the exceedance of a certain, defined threshold temperature as well as the duration of this exceedance.