Behavior of the Bionanoreactor with Coencapsulated Cofactors and Enzymes in HOF-101 for Cascade Chiral Amine Synthesis

Constructing heterogeneous cofactor-dependent multienzyme cascades could promote the stability and recyclability of enzymes and cofactors, which would benefit the economic feasibility and sustainability of biocatalytic processes. Recently, we have reported a polyallylamine (PAH)-assisted encapsulation approach to coimmobilize cofactor-dependent multienzyme cascade systems within hydrogen-bonded organic frameworks (HOFs), such as BioHOF-1 and HOF-101, with increased cascade activities over the free counterparts. However, the cause behind the increased performance remains unclear. Hence, the catalytic behaviors of nicotinamide cofactor (NAD)-dependent alcohol and amine dehydrogenase (ADH and AmDH) cascade trapped within PAH-HOF-101 are simultaneously investigated and observed through evaluation of the molecular events occurring during the biocatalytic process. It reveals that the enhanced cascade activity in the bionanoreactor is primarily attributed to the improved cofactor transfer by its coimmobilization with the enzymes. This is demonstrated by two substrates with significantly different molecular sizes and structures, which is evidenced through comparing the cascade activities in biocatalytic systems employing different immobilization strategies and structure-sized PAH-HOF-101s. Furthermore, the structure stability and recyclability of the coimmobilized NAD-dependent ADH-AmDH system are further enhanced by designing a PAH-HOF-101 shell outside the biocatalysis system. This study provides valuable insights into the underlying mechanisms driving the enhanced performance of coimmobilized cofactor-dependent multienzyme cascades in PAH-HOFs, contributing to the rational design of more efficient and sustainable biocatalytic systems.