Intracellular Assembly of Interacting Enzymes Yields Highly‐Active Nanoparticles for Flow Biocatalysis

Abstract

All-enzyme hydrogel (AEH) particles with a hydrodynamic diameter of up to 120 nm were produced intracellularly with an Escherichia coli-based in vivo system. The inCell-AEH nanoparticles were generated from polycistronic vectors enabling simultaneous expression of two interacting enzymes, the Lactobacillus brevis alcohol dehydrogenase (ADH) and the Bacillus subtilis glucose-1-dehydrogenase (GDH), fused with a SpyCatcher or SpyTag, respectively. Formation of inCell-AEH was analyzed by dynamic light scattering and atomic force microscopy. Using the stereo-selective two-step reduction of a prochiral diketone substrate, we show that the inCell-AEH approach can be advantageously used in whole-cell flow biocatalysis, by which flow reactors could be operated for > 4 days under constant substrate perfusion. More importantly, the inCell-AEH concept enables the recovery of efficient catalyst materials for stable flow bioreactors in a simple and economical one-step procedure from crude bacterial lysates. We believe that our method will contribute to further optimization of sustainable biocatalytic processes.

Nano‐ and Microscale Confinements in DNA‐Scaffolded Enzyme Cascade Reactions

“…To gain detailed insight into the mechanisms of nanoscale enzyme cascades, the components involved were precisely quantified and extensively characterized. The investigation of the kinetic parameters of the individually immobilized biocatalysts of the cascade is particularly important, as it was known from previous studies that DON immobilization of individual KREDs can lead to significantly altered enzymatic activity, [ 46 ] and that the increase in activity of one enzyme should increase the productivity of the entire cascade. [ 27 ] Therefore, both cascade enzymes were first examined individually with respect to their kinetic parameters on the microbeads and DNA origami before analyzing the activity of the co‐assembled cascade.…”

Biocatalytic Foams from Microdroplet‐Formulated Self‐Assembling Enzymes

“…[14] Later work indicated that AEH can be produced by genetically programmable selfassembly of arbitrary enzymes, [15][16][17][18] however, monolithic AEH materials have some limitations in terms of diffusion and mass transport within the materials, calling for improved formulations of these powerful biocatalysts. [19] In order to simultaneously maintain the solid anchorage and high enzyme concentration of AEH while still realizing a high surface-to-volume ratio for efficient perfusion and mass transport, we reasoned that these two aspects could be reconciled by foam formulations of AEH. Liquid foams consist of gas bubbles densely packed in a liquid carrier matrix to form a network of gas/liquid interfaces in which merging of the bubbles is prevented by the addition of stabilizing agents such as low molecular weight surfactants, polymers, proteins, nanoparticles or mixtures thereof.…”

“…[ 14 ] Later work indicated that AEH can be produced by genetically programmable self‐assembly of arbitrary enzymes, [ 15–18 ] however, monolithic AEH materials have some limitations in terms of diffusion and mass transport within the materials, calling for improved formulations of these powerful biocatalysts. [ 19 ]…”