Sequence analysis and molecular docking of antithrombotic peptides from casein hydrolysate by trypsin digestion

- Thrombin inhibitory peptides from casein were screened by bioinformatics analysis.
- Thirty-five peptides from casein digestion by trypsin were identified by UPLC-Q-TOF.
- One novel peptide with strong thrombin inhibitory activity was identified.
- The antithrombotic mechanism underlying the peptides by molecular docking was explored.

Casein has been recognized as a good source of bioactive peptides that can be used for the production of functional food. In this study, thrombin inhibitory peptides from casein were screened by bioinformatics based on sequences predicted from in silico enzymatic digestion with trypsin, and identified by UPLC-Q-TOF-MS/MS with Mascot analysis. Molecular docking was conducted using the software of Discovery Studio 2017 to provide the potential mechanism underlying the thrombin inhibitory activity of the peptides. A total of 35 novel peptides with thrombin inhibitory activity were identified. Moreover, FQSEEQQQTEDELQDK, derived from beta-casein (fragment 33-48), showed a remarkable sequence similarity with fragment 54-65 of hirudin, and exhibited strong inhibitory activity against thrombin. FQSEEQQQTEDELQDK had similar target domain (Lys36-Gln38-Arg73-Thr74-Lys81-Ile82-Lys110) on the stereo structure of thrombin compared with hirudin. This domain belongs to the partial structure of the active site 2 of thrombin (PDB: 2BVR), and it also located at the exosite I motif buried in the external surface of thrombin molecule, with potential inhibitory activity of the peptide.