Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States.
Department of Chemistry, University of California Irvine, Irvine, California 92697, United States.
ACS Synth Biol. 2022 Aug 19;11(8):2857-2868. doi: 10.1021/acssynbio.2c00256. Epub 2022 Jul 25.
Here, we describe a stress-tolerant, recyclable, and renewable biocatalyst platform based on T7 RNA polymerase-enabled high-density protein display on bacterial spores (TIED). TIED uses high-level T7 RNA polymerase-driven expression of recombinant proteins specifically in sporulating cells to allow spontaneous assembly of recombinant fusion proteins on the spore surface. TIED enables high loading density in the range of 10 to 10 recombinant enzymes per spore, robust catalytic activity of displayed enzymes comparable to the respective free enzymes, and enhanced kinetic stability of displayed enzymes in methanol and elevated temperatures. Furthermore, we demonstrate TIED enzymes to be not only recyclable but also fully renewable after the loss of activity through induction of germination and sporulation, enabling perpetual regeneration of these immobilized biocatalysts.
在这里，我们描述了一种基于 T7 RNA 聚合酶的耐应激、可回收和可再生的生物催化剂平台，该平台可实现细菌孢子表面高密度的蛋白质展示（TIED）。TIED 利用高水平的 T7 RNA 聚合酶在产孢细胞中特异性表达重组蛋白，从而允许重组融合蛋白自发组装在孢子表面。TIED 可实现每粒孢子 10 到 100 个重组酶的高负载密度，展示酶具有与相应游离酶相当的强大催化活性，并且在甲醇和高温下展示酶的动力学稳定性得到增强。此外，我们证明 TIED 酶不仅可回收，而且在通过诱导萌发和产孢丧失活性后还可以完全再生，从而使这些固定化生物催化剂能够永久再生。
