Fabrication of a 3D nano-bienzyme cascade reactor based on a Dps protein scaffold for chiral amine synthesis.

Based on the self-assembling properties of the SpyCatcher/SpyTag system and the structural advantages of Dps protein, this study successfully constructed a three-dimensional nano-enzyme cascade reactor (3DNECR) through the covalent self-assembly of SpyTag-ADH and SpyCatcher-Dps-ATA117 fusion proteins. The 3DNECR exhibited significantly enhanced catalytic efficiency compared to the two-dimensional control, attributed to optimized spatial organization promoting substrate channeling. The reactor exhibited remarkable storage, pH, and thermal stability. It maintained over 80% activity after 9 days of storage, showed superior pH tolerance across pH 8-10, and remained stable in the temperature range of 4-40 ℃. Molecular docking confirmed strong interfacial binding (- 17.3 kcal/mol) between assembly components and favorable substrate binding (- 7.4 kcal/mol) within the active site. Furthermore, the 3DNECR was applied to the asymmetric synthesis of (R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanamine (R-BPA) in an oil-water biphasic system. Under optimized conditions, the 3DNECR consistently achieved high yields (99.9%) and excellent enantioselectivity (99.9%). The 3DNECR maintained a relative enzyme activity as high as 92% even after six cycles of reuse. This integrated platform showcases substantial potential for efficient and sustainable biocatalytic applications.