Beloqui Ana, Kobitski Andrei Yu, Nienhaus Gerd Ulrich, Delaittre Guillaume
Institute of Toxicology and Genetics , Karlsruhe Institute of Technology (KIT) , Hermann-von-Helmholtz-Platz 1 , 76344 Eggenstein-Leopoldshafen , Germany . Email:
Preparative Macromolecular Chemistry , Institute for Technical Chemistry and Polymer Chemistry , Karlsruhe Institute of Technology (KIT) , Engesserstrasse 15 , 76131 Karlsruhe , Germany.
Chem Sci. 2017 Dec 1;9(4):1006-1013. doi: 10.1039/c7sc04438k. eCollection 2018 Jan 28.
We have established a simple one-step synthesis of single-enzyme nanogels (SENs), , nanobiocatalysts consisting of an enzyme molecule embedded in a hydrophilic, polymeric crosslinked nanostructure, as a most attractive approach to enhance the stability of enzymes. In contrast to earlier protocols, we demonstrate here that the addition of a small amount of sucrose makes the nanogel formation equally effective as earlier two-step protocols requiring enzyme pre-modification. This provides the dual advantage of skipping a synthetic step and preserving the surface chemistry of the enzymes, hence their native structure. Enzymes encapsulated in this way exhibit a high catalytic activity, similar to that of the free enzymes, in a markedly widened pH range. With our method, the thickness of the hydrogel layer can be finely tuned by careful adjustment of reaction parameters. This is most important because the shell thickness strongly affects both enzyme activity and stability, as we observe for a wide selection of proteins. Finally, a single-molecule analysis by means of two-color confocal fluorescence coincidence analysis confirms that our encapsulation method is highly efficient and suppresses the occurrence of nanoparticles lacking an enzyme molecule. The proposed method is therefore highly attractive for biocatalysis applications, ensuring a high activity and stability of the enzymes.
我们已经建立了一种简单的单步合成单酶纳米凝胶（SENs）的方法，单酶纳米凝胶是一种纳米生物催化剂，由嵌入亲水性聚合物交联纳米结构中的酶分子组成，是增强酶稳定性的最具吸引力的方法。与早期的方案相比，我们在此证明，添加少量蔗糖可使纳米凝胶的形成与早期需要酶预修饰的两步方案同样有效。这具有双重优势，既跳过了一个合成步骤，又保留了酶的表面化学性质，从而保留了它们的天然结构。以这种方式封装的酶在明显更宽的pH范围内表现出与游离酶相似的高催化活性。通过我们的方法，可以通过仔细调整反应参数来精细调节水凝胶层的厚度。这一点非常重要，因为正如我们对多种蛋白质的观察结果所示，壳层厚度强烈影响酶的活性和稳定性。最后，通过双色共聚焦荧光符合分析进行的单分子分析证实，我们的封装方法效率很高，并且抑制了缺乏酶分子的纳米颗粒的出现。因此，所提出的方法对于生物催化应用极具吸引力，可确保酶具有高活性和稳定性。
