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

Area of Science:

• Biomaterials Engineering
• Biocatalysis
• Protein Engineering

Background:

• Conventional enzyme immobilization often uses chemical crosslinkers, potentially reducing biocompatibility and enzyme activity.
• There is a need for robust, scalable, and sustainable methods for enzyme functionalization of materials.

Purpose of the Study:

• To develop and validate a crosslinker-free enzyme immobilization strategy using the reversible self-aggregation of the p40 domain.
• To demonstrate the versatility and efficiency of this method across various matrices and enzyme types.

Main Methods:

• Utilized reversible self-aggregation of p40 inclusion bodies from Caldibacillus cellulovorans for biomaterial functionalization.
• Reaggregated solubilized p40-fusion proteins onto polypropylene fibers, cellulose fabrics, and porous beads.
• Confirmed uniform attachment and stability using fluorescent protein fusions and Fourier transform infrared spectroscopy.

Main Results:

• Achieved high functionalization efficiencies (82-100%) with retained enzyme activity (75-100%).
• Demonstrated excellent stability of functionalized enzymes at elevated temperatures (70-80°C) and over multiple reaction cycles.
• Successfully applied the method in a SpinChem® reactor for D-tagatose formation, showcasing industrial biocatalysis potential.

Conclusions:

• The p40 domain's reversible, β-sheet-mediated aggregation offers a robust, scalable, and sustainable platform for creating stable enzyme-matrix assemblies.
• This crosslinker-free approach provides a generalizable strategy for industrial biocatalysis and biofunctional material applications.