Discovery of a novel nanomolar angiotensin-I converting enzyme inhibitory peptide with unusual binding mechanisms derived from Chlorella pyrenoidosa.

Chlorella pyrenoidosa (C. pyrenoidosa) has been cultivated in large quantities and proven to be antihypertensive when consumed orally. However, the antihypertensive peptides derived from C. pyrenoidosa remains scarce. In this study, trypsin was chosen to prepare the hydrolysate of C. pyrenoidosa, which was then fractionated by column chromatography. And ninety-nine peptides were identified by LC-MS/MS, after which 10 peptides were chosen by docking-based virtual screening and demonstrated their ability to inhibit ACE. Among them, LVAKA (LV-5) had the lowest IC50 (26.66 μM). LV-5, LKKAP, and PGLRP were identified as non-competitive ACE inhibitory peptides with significant stability under extreme pH and high temperatures conditions. Both in silico and in-vitro simulated gastrointestinal digestion revealed that these three peptides could release ACE inhibitory peptide fragments upon digestion. Sequence optimization of LV-5 led to the discovery of LRAKA (LR-5), which was identified as a novel nanomolar ACE peptide with an IC50 of 350 nM in-vitro and a potent antihypertensive peptide in-vivo. Moreover, molecular dynamic simulation indicated that LR-5 interacted with an unconventional binding site on ACE. These findings underscore the potential of Chlorella as a source of antihypertensive peptides and suggest a promising future for the use of Chlorella-derived peptides in hypertension management.