[Fusion expression and immobilization of an imine reductase and a formate dehydrogenase]

Chiral amines as pivotal intermediates in organic synthesis are widely utilized in the production of pharmaceuticals, fine chemicals, and bioactive molecules, holding significant industrial value. The asymmetric synthesis of chiral amines has always gained great attention. NAD(P)H-dependent imine reductases (IREDs) with wide substrate ranges, high activity, and high enantiomeric selectivity can be used for asymmetric reduction of imines to chiral amines. Our study aims to develop an efficient and stable immobilized dual-enzyme system through the fusion expression of imine reductase and formate dehydrogenase, in order to address the challenge of coenzyme regeneration and enhance catalytic efficiency, thereby providing a novel strategy for the green synthesis of chiral amines. The constructed system was applied to catalyze the asymmetric reduction of 1-methyl-3,4-dihydroisoquinoline to synthesize (S)-1-methyl-1,2,3,4-tetrahydroisoquinoline. Comparative analysis indicated that the catalytic efficiency of this fusion expression system exceeded that of both co-expression and standalone dual-enzyme systems. Furthermore, mesoporous silica nanoflowers were utilized as carriers to immobilize the fusion-expressed dual enzymes through a covalent method. In the case of covalent binding duration of 1.5 h and an initial enzyme concentration of 2.5 mg/mL, the protein loading achieved 193.2 mg/g. The immobilized enzymes demonstrated excellent pH, thermal, and storage stability. When the immobilized enzymes were employed to catalyze asymmetric reduction reactions of other cyclic imines, such as 1-ethyl-3,4-dihydroisoquinoline, 5-phenyl-3,4-dihydro-2H-pyrrole, 2,3,3-trimethyl-3H-indole, 2,3,3,5-tetramethylindole, and myosmine, the conversion rates exceeded 95%, and the values of e.e. surpassed 96%. The data confirm the application potential of the immobilized fusion enzymes in the green and efficient synthesis of chiral amines. Our study provides a novel strategy for the industrial biosynthesis of chiral amines, and the developed fused-enzyme immobilization approach holds significant theoretical and practical value for addressing common technical challenges in cofactor-dependent biocatalytic processes.