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

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.