Self-Sufficient Heterogeneous Biocatalysis through Boronic Acid-Diol Complexation of Adenylated Cofactors

Abstract

Self-sufficient heterogeneous biocatalysts (ssHB) are promising candidates for implementing cofactor-dependent enzymes in chemical biomanufacturing. Most strategies for coimmobilizing cofactors with dehydrogenases on porous agarose microbeads involve the use of cationic polymers (i.e., polyethylenimine, PEI) that interact electrostatically with the phosphate groups of their corresponding phosphorylated cofactors. Although the latter is a powerful and versatile approach, ionic bonds are disrupted in biotransformations operating at a high ionic strength, where screening of bonded ions takes place. Harnessing the ribose groups present in adenylated cofactors, we immobilize a selection of these (NAD(P)H, NAD(P) + , FAD, and ATP) on agarose microbeads functionalized with boronic acid to establish reversible covalent bonds between the cis-diol of the ribose in the cofactor backbone and the boronic acid. To do so, we functionalize cobalt-activated porous agarose beads with boronic acid (AG-B/Co 2+ ) for the coimmobilization of adenylated cofactors with the corresponding cofactor dependent His-tagged dehydrogenases. First, we demonstrate that the adenylated cofactor-support interactions are reversible but show resistance against high salt concentrations, overcoming the main limitation of the current selfsufficient heterogeneous biocatalysts. Then, we coimmobilized several His-tagged enzymes with their corresponding adenylated cofactors and investigated the functionality and stability of these ssHBs in reductive aminations performed under high ionic strength in both batch and flow reactors. As a result, we manage to reuse the immobilized enzymes and the cofactors 3.5 × 10 5 and 167 times, respectively. This work expands the usefulness of ssBHs for hitherto bioprocess regardless of the ionic strength of the media.

Single‐Particle and Single‐Molecule Characterization of Immobilized Enzymes: A Multiscale Path toward Optimizing Heterogeneous Biocatalysts

“…In contrast SP studies can inform about larger areas (μm-cm) [30] in wider operational times (from s to days). [31] A potential limitation of SM approaches is the mandatory use of fluorophore-labeled enzymes. In contrast, SP can be performed with unlabeled enzymes if particles are labeled, minimizing the effects of enzyme modification on the property under study.…”

Single‐Particle and Single‐Molecule Characterization of Immobilized Enzymes: A Multiscale Path toward Optimizing Heterogeneous Biocatalysts

“…However, SM methods are generally limited to small surface areas (1–10 μm) and short reaction times (from ms to s) due to photobleaching issues. In contrast SP studies can inform about larger areas (μm‐cm) [30] in wider operational times (from s to days) [31] . A potential limitation of SM approaches is the mandatory use of fluorophore‐labeled enzymes.…”

Coencapsulating Anionic Cofactor and Silica Nanoparticle-Bound Enzymes in Cationic Microgel for Multienzyme Cascade Reaction with Cofactor Recycling

“…In the past few decades, substantial efforts have been made to construct efficient coimmobilization systems of enzymes and cofactor. − Electrostatic interaction/ion-exchange interaction through the negative-charged phosphate group of cofactors and positive-charged supports/polymers is an effective method for immobilizing cofactors since there is a dynamic association/dissociation equilibrium in ionically bound cofactor systems, guaranteeing the immobilized cofactors to access enzymes with high mobility. Lopez-Gallego and colleagues constructed a coimmobilization system with NAD + ionically adsorbed and enzymes immobilized and cross-linked .…”