Yucesoy Deniz T, Akkineni Susrut, Tamerler Candan, Hinds Bruce J, Sarikaya Mehmet
Department of Bioengineering, Izmir Institute of Technology, Urla, Izmir 35430, Turkey.
Department of Materials Science and Engineering, University of Washington, Seattle, Washington 98195, United States.
ACS Omega. 2021 Oct 4;6(41):27129-27139. doi: 10.1021/acsomega.1c03774. eCollection 2021 Oct 19.
Biocatalysis is a useful strategy for sustainable green synthesis of fine chemicals due to its high catalytic rate, reaction specificity, and operation under ambient conditions. Addressable immobilization of enzymes onto solid supports for one-pot multistep biocatalysis, however, remains a major challenge. In natural pathways, enzymes are spatially coupled to prevent side reactions, eradicate inhibitory products, and channel metabolites sequentially from one enzyme to another. Construction of a modular immobilization platform enabling spatially directed assembly of multiple biocatalysts would, therefore, not only allow the development of high-efficiency bioreactors but also provide novel synthetic routes for chemical synthesis. In this study, we developed a modular cascade flow reactor using a generalizable solid-binding peptide-directed immobilization strategy that allows selective immobilization of fusion enzymes on anodic aluminum oxide (AAO) monoliths with high positional precision. Here, the lactate dehydrogenase and formate dehydrogenase enzymes were fused with substrate-specific peptides to facilitate their self-immobilization through the membrane channels in cascade geometry. Using this cascade model, two-step biocatalytic production of l-lactate is demonstrated with concomitant regeneration of soluble nicotinamide adenine dinucleotide (NADH). Both fusion enzymes retained their catalytic activity upon immobilization, suggesting their optimal display on the support surface. The 85% cascading reaction efficiency was achieved at a flow rate that kinetically matches the residence time of the slowest enzyme. In addition, 84% of initial catalytic activity was preserved after 10 days of continuous operation at room temperature. The peptide-directed modular approach described herein is a highly effective strategy to control surface orientation, spatial localization, and loading of multiple enzymes on solid supports. The implications of this work provide insight for the single-step construction of high-power cascadic devices by enabling co-expression, purification, and immobilization of a variety of engineered fusion enzymes on patterned surfaces.
生物催化因其高催化速率、反应特异性以及在环境条件下操作，是精细化学品可持续绿色合成的一种有用策略。然而，将酶可寻址地固定在固体载体上以进行一锅多步生物催化仍然是一个重大挑战。在天然途径中，酶在空间上相互耦合，以防止副反应、消除抑制性产物，并将代谢物依次从一种酶传递到另一种酶。因此，构建一个能够实现多种生物催化剂空间定向组装的模块化固定平台，不仅可以开发高效生物反应器，还能为化学合成提供新的合成路线。在本研究中，我们开发了一种模块化级联流动反应器，采用一种通用的固体结合肽导向固定策略，该策略允许融合酶以高位置精度选择性地固定在阳极氧化铝（AAO）整体柱上。在这里，乳酸脱氢酶和甲酸脱氢酶与底物特异性肽融合，以促进它们通过级联几何结构中的膜通道进行自我固定。使用这个级联模型，展示了两步生物催化生产L-乳酸的过程，同时伴随着可溶性烟酰胺腺嘌呤二核苷酸（NADH）的再生。两种融合酶在固定后都保留了它们的催化活性，表明它们在载体表面上得到了最佳展示。在与最慢酶的停留时间动力学匹配的流速下，实现了85%的级联反应效率。此外，在室温下连续运行10天后，仍保留了84%的初始催化活性。本文所述的肽导向模块化方法是一种控制多种酶在固体载体上的表面取向、空间定位和负载的高效策略。这项工作的意义在于，通过在图案化表面上实现多种工程融合酶的共表达、纯化和固定，为单步构建高功率级联装置提供了思路。
