Purification of Intramineral Peptides from Cuttlebones and In Vitro Activity in CaCO₃ Biomineralization

Living organisms develop functional hard structures such as teeth, bones, and shells from calcium salts through mineralization for managing vital functions to sustain life. However, the exact mechanism or role of biomolecules such as proteins and peptides in the biomineralization process to form defect-free hierarchical structures in nature is poorly understood. In this study, we have extracted, purified, and characterized five major peptides (CBP1-CBP5) from the soluble organic materials (SOMs) of cuttlefish bone (CB) and used for the in vitro mineralization of calcium carbonate crystals. The SOMs induced nucleation of the calcite phase at low concentrations and the vaterite phase at high concentrations. The purified peptides nucleated calcite crystals and enhanced aggregation under laboratory conditions. Among five peptides, only CBP2 and CBP3 showed concentration-dependent nucleation, aggregation, and morphological changes of the calcite crystals within 12 h. Circular dichroism studies showed that the peptides CBP2 and CBP3 are in alpha helix and β-sheet conformation, respectively, in solution. CBP1 and CBP4 and CBP5 are in random coil and β-sheet conformation, respectively. In addition, the peptides showed different sizes in solution in the absence (∼27 nm, low aggregation) and presence (∼118 nm, high aggregation) of calcium ions. Aragonite crystals with needle-type morphologies were nucleated in the presence of Mg²⁺ ions in solution. Overall, exploring the activities of such intramineral peptides from CB help to unravel the mechanism of calcium salt deposition in nature.