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 mCherryp40 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-Amylasep40-functionalised polypropylene fibres maintained full catalytic activity for twelve reaction cycles at 70 °C and approximately 50% at 80 °C, while tagatose 4-epimerasep40-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.
Vijayakumar et al. (Fri,) studied this question.