Recycling of Cofactors in Crude Enzyme Hydrogels as Co-immobilized Heterogeneous Biocatalysts for Continuous-Flow Asymmetric Reduction of Ketones.

Flow biocatalysis involving oxidoreductase is limited by the difficulty in recycling expensive cofactors. In this study, an enzyme-rich hydrogel monolithic microreactor was developed via in situ microfluidic assembly of inexpensive crude enzymes. This porous gel biocatalyst exhibited good tethering functions to nicotinamide cofactors; thus, they were retained by the hydrogel to controllably form a novel heterogeneous biocatalyst with self-sufficient cofactors. The flow asymmetric production of a chiral alcohol in this cofactor-entrapped gel microreactor achieved >99 % enantioselectivity and a high space-time yield of 46.3 g L-1  h-1 at 94.8 % conversion. Moreover, the turnover number of cofactors reached as high as 4800 after continuous operation of 160 reactor volumes, realizing significantly higher utilization of the cofactors compared with many reported strategies. Furthermore, this engineered heterogeneous biocatalyst exhibited improved performance in terms of product tolerance and storage stability, paving the way for a green, cost-effective, and sustainable continuous-flow production of enantiopure alcohols.