Self-assembling p40 inclusion bodies enable direct functionalisation of diverse materials

Conventional enzyme immobilisation often relies on chemical crosslinkers that can compromise biocompatibility and activity. This study introduces a crosslinker-free strategy for biomaterial functionalisation that exploits the reversible self-aggregation of the p40 domain from Caldibacillus cellulovorans. Inclusion bodies of p40-fusion proteins were solubilised with guanidinium hydrochloride and reaggregated onto diverse matrices, including polypropylene fibres, cellulose fabrics, and porous beads, forming stable enzyme-functionalised surfaces under mild aqueous conditions. Fluorescent mCherry_p40 fusions confirmed uniform reaggregation and matrix attachment, demonstrating the versatility of the approach across material types. The method achieved functionalisation efficiencies of 82-100%, while catalytically active p40-enzyme inclusion bodies retained 75-100% of their initial activity following matrix functionalisation. α-Amylase_p40-functionalised polypropylene fibres maintained full catalytic activity for twelve reaction cycles at 70 °C and approximately 50% at 80 °C, while tagatose 4-epimerase_p40-functionalised matrices demonstrated proof-of-concept applicability in a SpinChem® rotating bed reactor, supporting D-tagatose formation over ten cycles. Fourier transform infrared analyses indicated β-sheet-rich secondary structures consistent with ordered, functional aggregates. These findings show that β-sheet-mediated, reversible aggregation of p40 inclusion bodies provides a robust, scalable, and sustainable route for producing highly stable enzyme-matrix assemblies, offering a general platform for industrial biocatalysis and other biofunctional material applications.