A Simple Strategy for Co‐Immobilization of NADH‐Dependent Enzymes and NADH for Efficient Biocatalysis in Continuous Flow Reactors

The application of nicotinamide adenine dinucleotide (NAD)(H)-dependent enzymes in continuous flow reactors poses significant challenges due to the high cost of NAD(H) and its inevitable loss during reactor operation. To overcome these limitations, a novel platform comprising Cry3Aa-enzyme fusion particles modified with polyethylenimine (PEI) is developed and its application for two catalytic processes is demonstrated. In the first system, two NAD(H)-dependent enzymes, formate dehydrogenase (FDH), and leucine dehydrogenase (LDH) are genetically co-immobilized within Cry3Aa particles and then modified with PEI to facilitate NADH co-immobilization. To adapt the system for continuous flow reactors, PEI-modified particles are entrapped within agarose beads, loaded with NADH, and then used to catalyze L-tert leucine production under continuous flow conditions for 30 days. This configuration achieved LDH and NADH turnover numbers of 22,196 and 7,202, respectively, and a space-time yield (STY) of 0.0262 g L^-1 h^-1. This platform is then validated for another NAD(H)-dependent multienzyme system comprised of FDH and alcohol dehydrogenase (ADH) and used for the continuous conversion of ethyl acetoacetate to ethyl-(R)-3-hydroxybutyrate. ADH and NADH turnover numbers of 15,074 and 3,256 are obtained after 30 days with a STY of 0.02 g L^-1 h^-1. These examples illustrate significant potential of this technology for facilitating NADH-dependent enzyme-mediated biocatalysis in continuous flow reactors.