Characterization of the granule-associated protein AzPhaP from Azotobacter sp. FA8 as a promising protein-based...

Phasin proteins are attractive candidates for the development of bio-based emulsifiers because they naturally localize at the interface between hydrophobic polyhydroxyalkanoate granules and the bacterial cytoplasm. For most phasins, however, surfactant activity has been described mainly as a macroscopic property, with limited information on which structural elements contribute to emulsion stability after formation. We addressed this question using AzPhaP from Azotobacter sp. FA8 and a C-terminally truncated variant, AzPhaPΔC, lacking the final 27 residues predicted to be intrinsically disordered. The deletion had little effect on the overall secondary structure in solution, with both proteins exhibiting melting temperatures near 54 °C and partial structural recovery after heating to 90 °C. Likewise, surface activity was unaffected, as both proteins reduced surface tension to 43-45 mN m^-1 and exhibited an operational critical aggregation concentration of approximately 2.0 mg/mL. The difference, however, emerged after emulsification. Full-length AzPhaP maintained droplet size between 24 and 72 h, whereas droplets stabilized by AzPhaPΔC increased from 2.27 ± 0.26 to 4.30 ± 1.49 μm. In a 21-day refrigerated benchmark assay, AzPhaP produced smaller droplets than bovine serum albumin on days 7 and 14, although Triton X-100 remained superior. These results distinguish initial surface-tension reduction from long-term emulsion stabilization and indicate that the disordered C-terminal region of AzPhaP is required for emulsion persistence after interface formation, uncoupling surface activity from droplet stabilization and providing insight into phasin interfacial function.