Customized self-assembled bimetallic hybrid nanoflowers promoting the robustness of D-allulose 3-epimerase

Enzyme-based hybrid nanoflowers, a green biocatalyst technology employing metal ions as the main driving force of enzyme immobilization without participating in the catalytic reaction, have been extensively explored to increase enzyme robustness. Here, a customized self-assembled protein-bimetallic hybrid nanoflower system (HNF) was designed to immobilize D-allulose 3-epimerase (DAEase) with a hierarchical flower-like architecture. The tailored DAEase-HNFs were extremely stable in acidic conditions, retaining more than 70 % of their initial activity after incubation in aqueous solution at pH 3.0–6.0 for 4 h. They were also durable, retaining 92.4 % relative activity after 8 consecutive cycles, and maintaining 82.3 % relative activity after storage for 30 days at 4 °C. Molecular dynamics (MD) simulations were performed to probe the self-assembly processes related to the increased robustness of the encapsulated DAEase by investigating the conformational changes in the gating switch helix of the substrate entrance, hydrogen bond networks, and the catalytic cavity. Our data indicate that HNFs are a promising immobilization strategy to improve the robustness and durability of DAEase.