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Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi‐enzyme system is extremely difficult and relies on a combinatorial trial‐and‐error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96‐well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python‐based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four‐enzyme system to transform vinyl acetate into (S)‐3‐hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi‐enzyme pathways for chemical manufacturing. [ABSTRACT FROM AUTHOR]

Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi‐enzyme system is extremely difficult and relies on a combinatorial trial‐and‐error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96‐well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python‐based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four‐enzyme system to transform vinyl acetate into (S)‐3‐hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi‐enzyme pathways for chemical manufacturing. [ABSTRACT FROM AUTHOR]