The immobilization of multi-enzyme systems on solid materials is rapidly gaining interest for the construction of biocatalytic cascades with biotechnological applications in industry. The heterogenization and control of the spatial organization across porous materials of the system components are essentials to improve the performance of the process providing higher robustness, yield, and productivity. In this chapter, the co-immobilization and co-localization of a bi-enzymatic bio-redox orthogonal cascade with in situ cofactor regeneration are described. An NADH-dependent alcohol dehydrogenase catalyzes the asymmetric reduction of 2,2,2 trifluoroacetophenone using an NADH regeneration system consisting of a glutamate dehydrogenase and glutamic acid. Three different spatial organizations of the enzymes were compared in terms of cofactor-recycling efficiency. Furthermore, we demonstrated how the co-localization and uniform distribution (by controlling the enzyme immobilization rate) of the main and recycling dehydrogenases inside the same porous particle lead to enhance the cofactor-recycling efficiency of the bi-enzymatic bio-redox systems.
Keywords: Co-localization; Cofactor regeneration; Enzymes; Heterogeneous biocatalysis; Immobilization; Porous materials.