A novel static affinity chromatography strategy for efficient isolation of cod bone calcium-binding peptide and...

The low yield of high-activity calcium-binding peptides constitutes a technical bottleneck restricting their application. Herein, a static affinity chromatography approach using needle-shaped hydroxyapatite was developed for the isolation of a novel cod bone calcium-binding peptide, GRGNEGPQ (CBP-8). The calcium-binding mechanism was investigated using experimental and computational methods, while its digestive behavior and calcium transport effects were evaluated using an in vitro digestion model and Caco-2 cell monolayer model. Results indicated that the carboxyl group of Gln served as a potential calcium-binding site and the CBP-8-Ca complex formed through multiple chelation modes. Electrostatic interactions were critical for coordination bond formation, with a formal charge transfer of -2.13 e from Ca²⁺ in the CBP-8-Ca complex. CBP-8-Ca exhibited good gastric stability but degraded intestinally. Additionally, CBP-8 promoted Ca²⁺ transport via non-specific calcium channels and TRPV6 channels. This study offers a promising and cost-effective strategy for the efficient production of highly active calcium-binding peptides.