Streptavidin-Enzyme Linked Aggregates for the One-Step Assembly and Purification of Enzyme Cascades

Herein, we report enzyme aggregates assembled around covalently cross-linked streptavidin tetramers. The streptavidin oligomeric matrix (Sav(Matrix)) is produced by using SpyTag/SpyCatch technology and binds tightly to fusion proteins bearing a streptavidin-binding peptide (SBP). Fusing the SBPs to different enzymes leads to precipitation of the streptavidin-enzyme aggregates upon mixing the complementary components. This straightforward strategy can be applied to crude cell-free extracts, allowing the one-step assembly and purification of catalytically active aggregates. Enzyme cascade assemblies can be produced upon adding different SBP-fused enzymes to the Sav(Matrix). The reaction rate for lactate dehydrogenase (LDH) is improved tenfold (compared with the soluble enzyme) upon precipitation with the Sav(Matrix) from crude cell-free extracts. Additionally, the kinetic parameters are improved. A cascade combining a transaminase with LDH for the synthesis of enantiopure amines from prochiral ketones displays nearly threefold rate enhancement for the synthesis of (R)-alpha-methylbenzylamine compared with the free enzymes in solution.