Sweet potato storage root defensin and its tryptic hydrolysates exhibited angiotensin converting enzyme inhibitory activity in vitro

摘要

Sweet potato defensin (SPD1) overproduced in E. coli (M15) was purified by Ni2+-chelate affinity chromatography. The molecular mass of SPD1 is about 8,600 Da determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). Our previous paper showed that SPD1 had antimicrobial, dehydroascorbate reductase and monodehydroascorbate reductase activities. The activity of SPD1 to inhibit angiotensin converting enzyme (ACE) was shown using N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) as substrate in a dose-dependent manner (27.56 similar to 52.58 % inhibition). The 50% inhibition (IC50) of ACE activity required 190.47 mu g/mL SPD1 while that of Captopril was 10 nM (868 ng/mL). Thin layer chromatography (TLC) also identified SPD1 as an ACE inhibitor. SPD1 acted as a mixed type inhibitor against ACE using FAPGG as a substrate. When 200 mu g/mL SPD1 (10 mu g) were added, Vmax and Km were 0.01 Delta A/min and 0.69 mM, respectively; without SPD, 0.03 Delta A/min and 0.42 mM. Trypsin was used for SPD1 hydrolysis and part of the reaction mixture was removed and analyzed at set times. ACE inhibitory activity increased from 52.47% to about 74.38% after 24 h hydrolysis. The results suggested that small peptides increased by trypsin hydrolysis of the SPD1 ACE inhibitory capacity also increased up to 24 h, then decreased, which may be due to the disappearance of some active ingredients. Six peptides, namely GFR, FK, IMVAEAR, GPCSR, CFCTKPC and MCESASSK, were synthesized based on the simulated trypsin digest of SPD1, then tested for ACE inhibitory activity. IC50 values of individual peptides were 94.25 +/- 0.32, 265.43 +/- 1.24, 84.12 +/- 0.53, 61.67 +/- 0.36, 1.31 +/- 0.07 and 75.93 +/- 0.64 mu M, respectively, suggesting that CFCTKPC might represent the main domain for the ACE inhibition. The consumption of sweet potatoes may thus help alleviate hypertension and other diseases due to their SPD1 and hydrolysate content.