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

Hendrik Mallin; Thomas R. Ward

Index: 10.1002/cctc.201800162

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Abstract

Herein, we report on enzyme aggregates assembled around covalently cross‐linked streptavidin tetramers. The streptavidin oligomeric matrix (SavMatrix) is produced using the 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 extract, 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 SavMatrix. The reaction rate for lactate dehydrogenase (LDH) is improved tenfold (compared to the soluble enzyme) upon precipitation with the SavMatrix 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)‐α‐methylbenzylamine compared to the free enzymes in solution.