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Title: Solid-binding peptide-guided spatially directed immobilization of kinetically matched enzyme cascades in membrane nanoreactors
Authors: Yücesoy, Deniz Tanıl
Akkineni, Susrut
Tamerler, Candan
Hinds, Bruce J.
Sarıkaya, Mehmet
Keywords: Biocatalysis
Issue Date: 2021
Publisher: American Chemical Society
Abstract: 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. © 2021 The Authors. Published by American Chemical Society.
ISSN: 2470-1343
Appears in Collections:Bioengineering / Biyomühendislik
PubMed İndeksli Yayınlar Koleksiyonu / PubMed Indexed Publications Collection
Scopus İndeksli Yayınlar Koleksiyonu / Scopus Indexed Publications Collection
WoS İndeksli Yayınlar Koleksiyonu / WoS Indexed Publications Collection

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