Heterogeneous Systems Biocatalysis: The Path to the Fabrication of Self‐Sufficient Artificial Metabolic Cells

Abstract

Industrial biocatalysis is playing a key role in the development of the global bio-economy that must change our current productive model to pair the socio-economical development with the preservation of our already harmed planet. The exploitation of isolated multi-enzyme systems and the discovery of novel biocatalytic activities are leading us to manufacture chemicals that were inaccessible through biological routes in the early past. These endeavors have been grouped under the concept of systems biocatalysis. However, by using isolated biological machineries, fundamental features underlying the protein confinement found inside the living cells are missed. To re-gain these properties, such concepts can be expanded to a new concept; heterogeneous systems biocatalysis. This new concept is based on the fabrication of heterogeneous biocatalysts inspired by the spatial organization and compartmentalization that orchestrate metabolic pathways within cells. By assembling biological machineries (including enzymes and cofactors) into artificial solid chassis, one can fabricate self-sufficient and robust cell-free systems able to catalyze orchestrated chemical processes. Furthermore, the confinement of enzymes and and "artificial cofactor" inside solid materials has also attracted our attention because these self-sufficient systems exert de novo and non-natural functionalities. Here, we intend to go beyond immobilization of multi-enzyme systems, discussing only those enzymatic systems that have been co-immobilized with their cofactor or exogenous partners to enhance their cooperative action. In this article, we review the latest architectures developed to fabricate self-sufficient heterogeneous biocatalysts with application in chemical manufacturing, biosensing or energy production.

Increasing the Enzyme Loading Capacity of Porous Supports by a Layer-by-Layer Immobilization Strategy Using PEI as Glue

“…This may be achieved using polyethylenimine (PEI) [18][19][20][21] (Figure 1). This polymer is multicationic, has primary, secondary and tertiary amino groups and has been broadly used in biocatalysis design, in some instances to coimmobilize enzymes [22,23] or enzymes and cofactors [24][25][26]. The only problem (with the second and further enzyme layers) of this new strategy is that, after immobilizing the enzyme via ion exchange on PEI, the new coating with PEI of the previously ionically exchanged enzyme could produce some enzyme leaching from the support by competition among the PEI coating the previously immobilized enzyme layer, the immobilized enzyme molecules layer and the new doses of free PEI.…”

Sensors Based on Bio and Biomimetic Receptors in Medical Diagnostic, Environment, and Food Analysis

“…Thus, the receptor located in the polymer pores, on the one hand, is reliably retained on the transducer’s surface, and on the other hand, its reactivity is preserved. This method, as well as physical sorption, is most often used for the immobilization of enzymes [ 53 , 54 ] and living cells [ 55 ]. In addition, the method is simple and it significantly prolongs the receptor’s activity.…”

Engineered enzymes that retain and regenerate their cofactors enable continuous-flow biocatalysis

“…Continuous-flow chemistry offers advantages to biocatalysis, avoiding process issues caused by substrate/product inhibition, equilibrium controlled limitations on yield and allosteric control . Modular continuous-flow biochemistry would also allow the flexible assembly of different complex multistep reactions [3][4][5][6] . Here we tackle some technical challenges that currently prohibit the wide-spread use of continuous flow biocatalysis; cofactor immobilization and site-specific immobilization.…”

“…Cofactors, such as nicotinamide adenine dinucleotide (NAD + ) and adenosine triphosphate (ATP), are used stoichiometrically unless recycled, typically by a second enzyme: without recycling, cofactors become prohibitively expensive for most industrial syntheses. As cofactors require diffusion for recycling, they are ill-suited for use in continuous-flow reactors and the lack of a practical engineering solution for the issue has stymied the use of cofactor-dependent enzymes in continuous-flow applications ; although, growing interest in immobilized biocatalysts for cell-free metabolic engineering has led to the development of a variety of enzyme-cofactor-carrier combinations 5 .…”