(359b) Carrier-Free Enzyme-Assembled Hydrogel Based on Synergistic Weak Interactions to Boost Enzyme-Immobilized Microflow Biocatalysis

Immobilized enzymes can be potentially used as biocatalysts; however, their industrial applications are limited by the low specific activity and poor stability. We proposed a feasible strategy utilizing the synergistic bridging of triazoles and metal ions to induce the formation of porous enzyme-assembled hydrogels (EAGs) with increased activity. The catalytic efficiency of the prepared EAG toward acetophenone reduction was 6.3 times higher than that of the free enzyme, and the activity retention of 90.3% after 12 use cycles confirmed its reusability. A near-atomic resolution (2.1 Å) structure of the EAG enzyme was successfully analyzed via cryogenic electron microscopy, which indicated a structure–property relationship for the enhanced performance. In addition, the possible mechanism of EAG formation was elucidated, revealing the indispensability of triazoles and metal ions, which guided the use of two other enzymes to prepare EAGs capable of good reusability. The described strategy can pave the way for the development of new catalytic biomaterials and immobilized biocatalysts.

Furthermore, we demonstrated the microfluidic induction of alcohol dehydrogenase to form EAG in a microreactor, with controllable mechanical properties and porous architecture. Six chiral aryl alcohols were successfully synthesized by using this approach in flow asymmetric reduction. Moreover, the support-free EAG was capable of in situ flow immobilization and reversible de-immobilization without reactor disassembly, while exhibiting sufficient retention against cofactors NADP(H). The EAG monolithic microreactor presented an excellent space-time yield of 88.05 g L^-1 h^-1 and a high total turnover number of 62600 after 100 h of operation (over 1300 reactor volumes), highlighting its high activity and stability.