초록
<P>The construction of artificial multienzyme nanodevices with desired spatial arrangements have shown great promise for improving the overall performance of targeted enzyme cascades. However, it is a challenge to rationally design and construct multiple oligomeric enzyme assemblies that can be used as stable and reusable catalysts. Herein, we report a novel approach to rapidly achieve ultrastable multimeric enzyme nanoclusters (MENCs) based on enzymes property of oligomerization and the SpyTag/SpyCatcher system of orthogonally reactive split peptides. The SpyCatcher peptide and its binding partner SpyTag were fused to a dimeric cytochrome P450 monooxygenase mutant (P450BM3m) and a tetrameric glucose dehydrogenase (GDH), respectively. The fusion proteins self-assembled into the MENCs, forming a covalently coupled supramolecular multienzyme nanodevices that facilitated NADPH regeneration and converted indole into a pigment indigo. We investigated the morphology of the MENCs and found these multimeric enzymes assembled into two-dimensional layerlike nanoscale architecture, ranging from a few to several hundred square microns in size. Importantly, enzymatic analysis revealed that the MENCs not only increased the initial rate by more than three times for the indigo synthesis, but also achieved significant improvements on stability and reusability compared to unassembled enzyme mixtures. This work demonstrates a versatile and efficient strategy to construct stable and multifunctional biocatalysts with potential applications in metabolic engineering and synthetic biology.</P><BR>[FIG OMISSION]</BR>