Purified SpyCatcher-ATA117 (5 µM) and SpyTag-ADH (5 µM) were covalently combined to form self-assembled dual-enzyme clusters (DENCs) at 4 °C for 3 h. The successful assembly of DENCs was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS). DENCs improved the storage stability by 36.2% and pH stability by 11.2% compared with those of free double enzymes. 56.89 g/L (R)− 1-[3,5-bis(trifluoromethyl)phenyl]ethanamine (R-BPA) was synthesized using DENCs with high yield (99.9%) and high enantiomeric excess (99.9%) at 45 °C and pH 9 for 24 h. Moreover, DENCs retained more than 78% of the initial activity after being reused in the preparation of R-BPA for five cycles. Thus, DENCs were successfully formed, and efficient amination was achieved, providing a new biocatalyst for efficient biosynthesis of chiral amines via asymmetric transamination cascade reaction.

纯化的 SpyCatcher-ATA117 (5 µM) 和 SpyTag-ADH (5 µM) 在 4 °C 下共价结合 3 小时，形成自组装双酶簇 (DENC)。使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、透射电子显微镜（TEM）、扫描电子显微镜（SEM）和动态光散射（DLS）验证了DENC的成功组装。与游离双酶相比，DENCs的储存稳定性提高了36.2%，pH稳定性提高了11.2%。使用 DENC 合成了56.89 g/L ( R )− 1-[3,5-双(三氟甲基)苯基]乙胺 ( R -BPA)，在 45 °C 下具有高产率 (99.9%) 和高对映体过量 (99.9%) pH 9 24 小时。此外，DENCs在R - BPA的制备中重复使用5个循环后，仍保留了78%以上的初始活性。由此，DENCs成功形成，并实现了高效胺化，为通过不对称转氨级联反应高效生物合成手性胺提供了一种新的生物催化剂。