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.
Highlights
IntroductionEnzymatic pathways that perform multistep reactions in biological organisms are the key processes that enable life.[1−4]Biomimetic reconstruction of these metabolic pathways by incorporating multiple enzymes and relevant cofactors in a confined environment is a highly valuable strategy for sustainable green synthesis of fine chemicals e.g., pharmaceuticals, biofuels, and consumer products, as well as developing efficient biomolecular devices.[5−9] The presence of multiple enzymatic components, brings unique challenges, such as (i) controlling the spatial distribution of enzymes on the support surface; (ii) enabling efficient transport of reactive intermediates from one enzyme to another; and (iii) kinetically matching enzymes with different turn-over rates.[10−12]An anodic aluminum oxide (AAO) membrane is a suitable platform to reconstruct enzyme assemblies due to the dominance of convective flow through their highly oriented monolithic channels, which facilitate efficient transport of reactive intermediates sequentially from one enzyme site to another.[13−16] Using the well-established anodization processes, the size of the monolithic channels can be modified to control enzyme load and the flow behavior.[17−19] Numerous efforts have been devoted to construct multienzyme assemblies on AAO using a variety of approaches.[20−23] One major shortcoming of these approaches, is the utilization of the same coupling chemistry for the entire system, which limits the immobilization of the cascading components in the correct sequence.[20,22,24,25] With their exquisite material recognition and self-assembly properties, solid-binding peptides are appealing engineering tools, which can be utilized as molecular linkers to selectively immobilize biomolecules, e.g., enzymes, on a variety of solid surfaces.[26−29] In particular, by Received: July 15, 2021 Accepted: September 20, 2021 Published: October 4, 2021ACS Omega http://pubs.acs.org/journal/acsodfArticle constructing AAO membranes functionalized with diverse materials, mixtures of fusion enzymes can be self-directed to immobilize on a desired target material layer, in a single step with high positional precision
The design of the enzymatic flow reactor is schematically described in Figure 1, where the engineered lactate dehydrogenase (LDH) and formate dehydrogenase (FDH) fusion enzymes are self-immobilized by the genetically conjugated peptide tags to sequentially catalyze two-step regeneration of the NADH cofactor under continuous flow
We developed a modular cascade flow reactor using a generalizable strategy based on the peptide-directed immobilization approach that enables selective self-immobilization of fusion enzymes on anodic aluminum oxide (AAO) monoliths with high positional precision
Summary
Enzymatic pathways that perform multistep reactions in biological organisms are the key processes that enable life.[1−4]Biomimetic reconstruction of these metabolic pathways by incorporating multiple enzymes and relevant cofactors in a confined environment is a highly valuable strategy for sustainable green synthesis of fine chemicals e.g., pharmaceuticals, biofuels, and consumer products, as well as developing efficient biomolecular devices.[5−9] The presence of multiple enzymatic components, brings unique challenges, such as (i) controlling the spatial distribution of enzymes on the support surface; (ii) enabling efficient transport of reactive intermediates from one enzyme to another; and (iii) kinetically matching enzymes with different turn-over rates.[10−12]An anodic aluminum oxide (AAO) membrane is a suitable platform to reconstruct enzyme assemblies due to the dominance of convective flow through their highly oriented monolithic channels, which facilitate efficient transport of reactive intermediates sequentially from one enzyme site to another.[13−16] Using the well-established anodization processes, the size of the monolithic channels can be modified to control enzyme load and the flow behavior.[17−19] Numerous efforts have been devoted to construct multienzyme assemblies on AAO using a variety of approaches.[20−23] One major shortcoming of these approaches, is the utilization of the same coupling chemistry for the entire system, which limits the immobilization of the cascading components in the correct sequence.[20,22,24,25] With their exquisite material recognition and self-assembly properties, solid-binding peptides are appealing engineering tools, which can be utilized as molecular linkers to selectively immobilize biomolecules, e.g., enzymes, on a variety of solid surfaces.[26−29] In particular, by Received: July 15, 2021 Accepted: September 20, 2021 Published: October 4, 2021ACS Omega http://pubs.acs.org/journal/acsodfArticle constructing AAO membranes functionalized with diverse materials, mixtures of fusion enzymes can be self-directed to immobilize on a desired target material layer, in a single step with high positional precision. Enzymatic pathways that perform multistep reactions in biological organisms are the key processes that enable life.[1−4] Biomimetic reconstruction of these metabolic pathways by incorporating multiple enzymes and relevant cofactors in a confined environment is a highly valuable strategy for sustainable green synthesis of fine chemicals e.g., pharmaceuticals, biofuels, and consumer products, as well as developing efficient biomolecular devices.[5−9] The presence of multiple enzymatic components, brings unique challenges, such as (i) controlling the spatial distribution of enzymes on the support surface; (ii) enabling efficient transport of reactive intermediates from one enzyme to another; and (iii) kinetically matching enzymes with different turn-over rates.[10−12]. The authors and other groups have identified multitudes of solid-binding peptides that are specific to metals, ceramics, and gmrianpehriatles.,35e,3.7g−.,43ATu,heAirg,utPiltit,yTaisOa2n, chSioOri2n,ghmydorioetxiyeaspfaatciitleit,atainndg immobilization of functional proteins onto solid surfaces has been demonstrated at large length scales.[29,32,44−46] With their exclusive specificity and binding properties, solid-binding peptides offer a superior alternative to traditional surface functionalization and activation approaches that utilize nonspecific physical adsorption or covalent bonding.[11,32,47,48] The ease of genetic insertion of these short sequences to a permissive site or to the C- or N-terminus of proteins makes them highly useful heterofunctional molecular constructs.[11,47,49,50] By providing addressable self-organization when combined with other functional biomolecules, e.g., DNA, RNA, enzymes, etc., in chimera, these peptides, are well suited for immobilization of bioactive molecules providing much greater control over their binding, assembly, and orientation control on solid surfaces.[34,51,52]
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