Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich GmbH, Wilhelm Johnen Strasse, D-52425 Jülich, Germany.
IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich GmbH, Wilhelm Johnen Strasse, D-52425 Jülich, Germany.
ACS Synth Biol. 2022 May 20;11(5):1881-1896. doi: 10.1021/acssynbio.2c00035. Epub 2022 May 2.
In industries, enzymes are often immobilized to obtain stable preparations that can be utilized in batch and flow processes. In contrast to traditional immobilization methods that rely on carrier binding, various immobilization strategies have been recently presented that enable the simultaneous production and in vivo immobilization of enzymes. Catalytically active inclusion bodies (CatIBs) are a promising example for such in vivo enzyme immobilizates. CatIB formation is commonly induced by fusion of aggregation-inducing tags, and numerous tags, ranging from small synthetic peptides to protein domains or whole proteins, have been successfully used. However, since these systems have been characterized by different groups employing different methods, a direct comparison remains difficult, which prompted us to benchmark different CatIB-formation-inducing tags and fusion strategies. Our study highlights that important CatIB properties like yield, activity, and stability are strongly influenced by tag selection and fusion strategy. Optimization enabled us to obtain alcohol dehydrogenase CatIBs with superior activity and stability, which were subsequently applied for the first time in a flow synthesis approach. Our study highlights the potential of CatIB-based immobilizates, while at the same time demonstrating the robust use of CatIBs in flow chemistry.
在工业中，酶通常被固定化以获得稳定的制剂，这些制剂可用于分批和流动过程。与依赖载体结合的传统固定化方法相比，最近提出了各种固定化策略，这些策略能够同时生产和体内固定化酶。催化活性包涵体 (CatIB) 是此类体内酶固定化的一个很有前途的例子。CatIB 的形成通常通过聚集诱导标签的融合来诱导，并且已经成功使用了许多标签，范围从小的合成肽到蛋白质结构域或整个蛋白质。然而，由于这些系统由不同的小组采用不同的方法进行了表征，因此直接比较仍然很困难，这促使我们对不同的 CatIB 形成诱导标签和融合策略进行基准测试。我们的研究表明，重要的 CatIB 特性，如产率、活性和稳定性，强烈受到标签选择和融合策略的影响。通过优化，我们获得了具有优异活性和稳定性的醇脱氢酶 CatIB，随后首次将其应用于流动合成方法中。我们的研究强调了基于 CatIB 的固定化剂的潜力，同时展示了 CatIB 在流动化学中的强大应用。
