Summary: | 碩士 === 國立屏東科技大學 === 生物科技系所 === 101 === Peptides with angiotensin converting enzyme (ACE, EC 3.4.15.1) inhibitory activity have received considerable interest from the food industry due to their potential as antihypertensive and the consumer’s concern over the safety of the synthetic counterparts. The aim of this study is to optimize the hydrolysis condition of angiotensin inhibitory peptide production with the purpose of obtaining high yield and purity. Another aim is to investigate the mechanism of action of the active peptide. The previously identified angiotensin inhibitory peptide was derived from lysozyme C, the major protein in soft-shelled turtle egg white (SSTEW). The hydrolysis condition of the active peptide production using thermolysin were optimized based on enzyme to substrate ratio, hydrolysis time, and temperature. The SSTEW hydrolysates were ultrafiltrated by 3 kDa molecular weight cut-off (MWCO) membrane ultrafiltration. Analysis by RP-HPLC and peptide identification by LC-MS/MS showed that using optimized conditions (E/S ratio of 1:200 (w/w)and incubated for 3 h at 60oC), the target peptide was present in the SSTEW thermolytic hydrolysate as the most abundant peak. Moreover, using this optimized condition, the hydrolysate purity more than 80% with the yield of the target peptide more than 14 % can be readily obtained without any cumbersome purification procedure. The 50% inhibitory concentrations (IC50 value) of the active peptide was validated and determined at 4.39±0.31 μM. Inhibitory kinetic study showed that the peptide is competitively inhibit ACE. Molecular docking simulation between peptide and ACE proposed that the inhibition mainly due to interaction of the isoleucine residue at N-terminus with Lys 118, Tyr 523, Asp 121, and Glu123 via hydrogen bonds.Simulation of gastrointestinal digestion showed that active peptide was partly hydrolysed by the action of pancreatic enzyme, trypsin, resulted in two fragments. The decreasing IC50 value to 2.42±0.09 μM after simulated gastrointestinal digestion suggested that it might has antihypertensive activity. In vivo assay and clinicals trials are necessary to prove their physiological effect. Based on this remarkable activity, production of ACE-inhibitory peptide from SSTEW is promising to be upscaled to industrial production. To the best of our knowledge, this is the first report on angiotensin inhibitory peptide from soft-shelled turtle egg white.
|