Continuous-Flow Asymmetric Synthesis of (3<i>R</i>)-3-Hydroxyl-5-hexenoates with Co-Immobilized Ketoreductase and <i>Lactobacillus kefir</i> Dehydrogenase Integrating Greener Inline Microfluidic Liquid–Liquid Extractors and Membrane Separators

Abstract

The dual-enzyme system composed of Ketoreductase (KRED) and Lactobacillus kefir Dehydrogenase (LkADH) is powerful in converting 3-oxohex-5-enoates into (3R)-3-hydroxyl-5-hexenoates, but its practical applications are hindered by low stability, difficult recovery, and poor reusability in the free state. In this work, KRED/LkADH was co-immobilized into polyvinyl alcohol (PVA) particles via the entrapment approach as the resulting KRED/LkADH@PVA not only exhibited the highest catalytic activity but also had superior physical and mechanical stability. Notably, it was observed that the KRED/LkADH@PVA could be recycled for at least six consecutive cycles, and its maximum tolerable substrate concentration was 2.34 times as high as that of the free KRED/LkADH. A greener continuous-flow process was subsequently developed by loading the KRED/LkADH@PVA into a fixed bed reactor that was coupled with inline microfluidic liquid–liquid extraction and membrane separation units. The model substrate tert-butyl 3-oxohex-5-enoate can be completely transformed into tert-butyl (3R)-3-hydroxyl-5-hexenoate in 10 min residence time in the continuous-flow process, which afforded a 93% isolated yield with excellent stereoselectivity (>99.9% ee) and a space time yield of 22.03 g L–1 h–1 (i.e., 528.72 g L–1 day–1) after 24 h of continuous operation. A low value of process mass intensity of 31.1 was achieved.