Oxidoreductase-catalyzed reactions are a very high potential in the industry to synthesize valuable fine chemicals and drug precursors, but several reasons hamper their wide applications. 1) Oxidoreductase-catalyzed reactions consume expensive cofactors; 2) small molecule products should be separated from cofactors; 3) continuous flow or repeated batch reactions are not possible due to the loss of cofactors. Although enzymes are frequently immobilized for industrial processes, the immobilization of small molecule cofactors is challenging. In this study, we developed the multiple oxidoreductases and swing arm cofactor co-immobilized microreactor for overcoming these cofactor limitations. We chose the NAD-dependant cascade reaction of glucose dehydrogenase (GDH) and mannitol dehydrogenase (MDH) as a model system. As a genetically encoded swing arm linker, we designed a strep-tag II fused elastin-like polypeptide (ELP). The <i>Escherichia coli</i> expressed and purified swing arm linker was conjugated with the NAD to synthesize a cofactor swing arm (ELP-NAD+). The ELP-NAD+ was co-immobilized on a streptavidin-modified resin with two strep-tag II fused GDH and MDH to generate the microreactor. We observed substantial D-mannitol production from the microreactor. Furthermore, simple filtration could easily separate the product from the microreactor. In addition, the productivity was retained when applied for repeated batch reactions (&gt;seven cycles). These results demonstrate that the cofactor swing arm and multiple oxidoreductases were stably co-immobilized on the microreactor, which can maintain their reactivity because of the flexible ELP swing arm.

Cofactor swing arm co-immobilization with multiple oxidoreductases on the microreactor for <i>in situ</i> cofactor regeneration

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Cofactor swing arm co-immobilization with multiple oxidoreductases on the microreactor for in situ cofactor regeneration