Characterisation of novel angiotensin-I-converting enzyme inhibitory tripeptide, Gly-Val-Arg derived from mycelium of Pleurotus pulmonarius

[Display omitted] •A novel ACE inhibitory tripeptide GVR was constructed through in silico analysis of Pleurotus pulmonarius protein sequence.•In silico analysis indicated that the tripeptide GVR was not cleaved by trypsin, pepsin and alpha-chymotrypsin.•Tripeptide GVR competitively inhibited ACE in vitro.•Molecular docking analysis confirmed the ability of the tripeptide GVR to bind at the ACE’s active site.•Oral administration of the tripeptide GVR to SHRs was able to significantly reduce the systolic blood pressure of the rats. It has been shown that fraction D6 from Pleurotus pulmonarius has the potential to inhibit ACE. After this discovery, additional studies were conducted to obtain peptides from that fraction, as ACE inhibitors. By size exclusion chromatography, single peak was resolved and termed as Psec. The IC50 of Psec was assessed to be 4.50μg/mL, which was 2.5 times lower than that of D6. When Psec was resolved by SDS-PAGE, three bands with estimated molecular weight of 63kDa, 55kDa and 11kDa were observed. The protein bands were subjected to MALDI-Tof MS/MS for protein identification. By using the BIOPEP database for predicting in silico digestion of gastrointestinal (GI) enzymes, four stable tripeptides with ACE inhibitor potential resulting from GI enzyme digestion were identified, namely GVR, VVR, NPR, and VVL. The IC50 was estimated to be 55μg/mL, 93μg/mL, 110μg/mL and >250μg/mL individually. Based on a Lineweaver-Burk plot, tripeptide GVR was determined to be a competitive inhibitor and this was confirmed by molecular docking analysis. At 100mg/kg of body weight (bw), the tripeptide GVR reduced SBP 33.5mm Hg in SHRs. The results suggested that this tripeptide is potentially beneficial as an antihypertensive agent.