Self-Sufficient Reusable Biocatalytic System Outfitted with Multiple Oxidoreductases and Flexible Polypeptide-Based Cofactor Swing Arms

Abstract

Oxidoreductases are widely used in value-added chemical synthesis. Since many oxidoreductases require the consumption of expensive cofactors, multi-enzyme cascade reactions, including cofactor regeneration, have been developed. Although enzymes are frequently immobilized for industrial processes, the immobilization of the small molecule cofactor is challenging. Here, we demonstrate that the co-immobilization of a swing arm cofactor and multiple oxidoreductases can overcome these cofactor limitations. We chose a very flexible protein, elastin-like polypeptide (ELP), fused to a strep-tag II as a genetically encoded swing arm. The Escherichia coli expressed and purified swing arm was conjugated with NAD+ to generate a cofactor swing arm (ELP-NAD+). The cofactor swing arm was co-immobilized on a streptavidin resin with two oxidoreductases fused to a strep-tag II: glucose dehydrogenase (GDH) and mannitol dehydrogenase (MDH). We observed a substantial d-mannitol production (about 1.6 mM) in the three-component (GDH, MDH, and ELP-NAD+) co-immobilized microreactor. The three-component co-immobilized system showed 2 and 3 times higher d-mannitol production than the one with free three components and another with the immobilized enzymes and free cofactor, respectively. Moreover, the product could be easily separated from the co-immobilized reactor and the activity was retained when applied for repeated batch reactions (> seven cycles). These results demonstrate that the genetically encoded cofactor swing arm can be stably co-immobilized with multiple oxidoreductases on a solid support and it can maintain reactivity as a cofactor because of the flexible ELP swing arm.

Real flue gas CO2 hydrogenation to formate by an enzymatic reactor using O2- and CO-tolerant hydrogenase and formate dehydrogenase

“…However, the enzymatic reactor system developed in this study is expected to enable the utilization of CO 2 and H 2 obtained from other sources, such as industrial waste gases, biomass and gasified plastics. We plan to improve the stability of the enzymes for the long-term operation of the reactor and improve the cofactor co-immobilization system ( Cha et al, 2023b ) for implementation as a continuous reactor.…”

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

“…A handful of sophisticated approaches have emerged for cofactor coimmobilization where the cofactor is irreversibly bound through swing arms. , However, the complexity of these strategies limits the universality of the methodology. For this reason, the coimmobilization of cofactor-dependent enzymes and their cofactors more commonly exploits the reversible or irreversible attachment of enzymes on a support, which is then coated with cationic polymers (i.e., polyethylenimine-PEI, polyallylamine-PAH) for the ionic adsorption of the cofactors on the same support (i.e., porous materials). − Such electrostatic interactions occur between the positively charged amine moieties of the polymeric coating and the negatively charged phosphate groups of phosphorylated cofactors (i.e., NAD­(P)­H, PLP, FAD).…”