Nano-Entrapping Multiple Oxidoreductases and Cofactor for All-In-One Nanoreactors

Abstract

Oxidoreductases have been used for the synthesis of value-added chemicals via redox reactions under mild conditions. However, these enzymes often require cofactors, which are expensive and small molecules making them difficult to reuse in the popular immobilized enzyme processes. Here, we show that nicotinamide adenine dinucleotide-conjugated alginate (AlgNAD(+)) can overcome these cofactor limitations after in situ co-encapsulation with two oxidoreductases, formate dehydrogenase (FDH), and mannitol dehydrogenase (MDH), inside pluronic-based nanocarriers (PNCs). PNCs had high encapsulation yields for FDH and MDH (both >80%) and AlgNAD(+) (46.5%). AlgNAD(+)-co-encapsulated enzymes displayed enhanced catalytic activity compared with the free enzymes. Furthermore, we could control the amount of the encapsulated AlgNAD(+) in the PNC by varying its input, thereby enabling efficient construction of a cofactor regeneration system. Moreover, PNC-co-encapsulated FDH, MDH, and AlgNAD(+) produced triplicate batches of D-mannitol after separating the reaction products from the co-encapsulated complex, with no cofactor supplementation. These results demonstrate that PNC can efficiently co-encapsulate enzymes and cofactors in the desired amounts generating All-in-One nanoreactors for repeated enzymatic reactions.