Catch-and-Release: The Assembly, Immobilization, and Recycling of Redox-Reversible Artificial Metalloenzymes
Catch-and-Release: The Assembly, Immobilization, and Recycling of Redox-Reversible Artificial Metalloenzymes
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DOI：
10.1021/acscatal.3c05294
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发表时间：
2024-02-15
期刊：
影响因子：
12.9
通讯作者：
Duhme-Klair，Anne-K.
中科院分区：
文献类型：
--
作者：
Miller，Alex H.;Blagova，Elena V.;Duhme-Klair，Anne-K.
提高人工金属酶（ArMs）的适用性的技术正在获得相当大的兴趣;其中一种方法是将这些生物混合催化剂固定在载体材料上，以提高稳定性并使其保留，回收和再利用。在这里，我们描述了多组氨酸标记的ARM的固定化，其允许氧化还原控制的已经失去活性的催化辅因子的替换，例如，由于中毒或分解，在固定化金属亲和色谱树脂上。通过使用来源于嗜热细菌（GstCeuE和PthCeuE）的周质铁载体结合蛋白支架与铁载体连接的亚胺还原催化剂组合，实现了比先前用空肠弯曲菌的类似蛋白CjCeuE获得的反应速率快约3.5倍的反应速率。固定化后，theGstCeuE衍生的ArM表现出减少周转频率在脱氢salsolidine减少3.4倍，同时保留对映体选择性（36%），并表现出改善的稳定性，允许重复回收和循环。在最初的四个循环中保持了催化活性。在随后的周期中，活动逐渐减少是明显的。一旦初始活性降低至初始活性的约40%（第23次再循环循环），氧化还原触发的人工辅因子释放允许随后用新鲜的活性辅因子对固定的蛋白质支架进行再装填，从而恢复固定的ArM的初始催化活性并允许其再使用几个循环。此外，ArM可以直接从粗细胞提取物中存在的蛋白质组装，避免了耗时且昂贵的蛋白质纯化步骤。总的来说，这项研究表明，氧化还原可逆的ARM的固定化有利于他们的“捕获和释放”的组装和拆卸，以及回收他们的组件，提高其潜在的商业可行性和环境足迹。
Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilization of these biohybrid catalysts on support materials to enhance stability and enable their retention, recovery, and reuse. Here, we describe the immobilization of polyhistidine-tagged ArMs that allow the redox-controlled replacement of catalytic cofactors that have lost activity, e.g., due to poisoning or decomposition, on immobilized metal affinity chromatography resins. By using periplasmic siderophore-binding protein scaffolds that originate from thermophilic bacteria (GstCeuE andPthCeuE) in combination with a siderophore-linked imine reduction catalyst, reaction rates were achieved that are about 3.5 times faster than those previously obtained withCjCeuE, the analogous protein ofCampylobacter jejuni. Upon immobilization, theGstCeuE-derived ArM showed a decrease in turnover frequency in the reduction of dehydrosalsolidine by 3.4-fold, while retaining enantioselectivity (36%) and showing improved stability that allowed repeat recovery and recycling cycles. Catalytic activity was preserved over the initial four cycles. In subsequent cycles, a gradual reduction of activity was evident. Once the initial activity decreased to around 40% of the initial activity (23rd recycling cycle), the redox-triggered artificial cofactor release permitted the subsequent recharging of the immobilized protein scaffold with fresh, active cofactor, thereby restoring the initial catalytic activity of the immobilized ArM and allowing its reuse for several more cycles. Furthermore, the ArM could be assembled directly from protein present in crude cell extracts, avoiding time-consuming and costly protein purification steps. Overall, this study demonstrates that the immobilization of redox-reversible ArMs facilitates their “catch-and-release” assembly and disassembly and the recycling of their components, improving their potential commercial viability and environmental footprint.