马氏珍珠贝肉蛋白水解特征及其ACE抑制肽的筛选

Hydrolysis Characteristics of Pinctada fucata Meat Protein and Screening for Angiotensin-Converting Enzyme Inhibitory Peptides

为探索马氏珍珠贝肉的水解规律及快速鉴定血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制活性肽,本研究以不同时间(2、4、6、8、10 h)的酶解物为对象,采用三羟甲基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、分子质量分布、反相高效液相色谱(reversed-phase high performance liquid chromatography,RPHPLC)表征其水解规律。另外,对10 h酶解物进行初步分离纯化,鉴定高活性组分中的肽序列,并采用分子对接的方法对鉴定的肽进行筛选,验证筛选肽的活性,阐释其抑制机理。结果表明,马氏珍珠贝肉在酶解过程中,大分子质量蛋白逐渐水解为小分子肽,在RP-HPLC上出现某一特征峰的富集。初步分离后,发现F2、F3组分具有较高的ACE抑制活性,从这2个组分中共获得了可信度较高的54个肽序列。分子对接筛选出6个潜在的ACE抑制肽,在1 mg/mL时,5个肽具有不同程度的ACE抑制活性,其中,WFHAVFW和WHAFLW显示了最高的ACE抑制活性,分别为(95.57±0.37)%和(98.59±0.08)%,进一步测定六肽WHAFLW的半抑制浓度(IC50)值,为52.39μmol/L。分子对接显示肽的抑制机理可能是通过氢键、范德华作用以及Pi-Pi作用形成稳定的肽-酶复合物。本研究既揭示了珍珠贝肉蛋白不同酶解时间的规律变化,又将活性实验跟踪与计算机辅助筛选相结合,提供了一种快速筛选酶解物中高活性ACE抑制肽的方法。

This study aimed to explore the hydrolysis characteristics of Pinctada fucata meat and quick identification of angiotensin-converting enzyme(ACE) inhibitory peptides from its hydrolysates. Hydrolysates at different times(2, 4,6, 8 and 10 h) were collected to characterize the hydrolyis process by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and molecular mass distribution measurement, and reversed-phase high performance liquid chromatography(RP-HPLC). In addition, the 10 h hydrolysate was separated and purified, and then the fractions with a high ACE inhibitory activity were identified and screened by molecular docking. Moreover, their activities were verified and their mechanisms of action were elucidated. The results showed that as the hydrolysis proceeded, large-molecular-mass proteins were gradually digested into low-molecular-mass peptides, and that a characteristic peak appeared in the RP-HPLC profile. A total of 54 peptide sequences with high average local confidence(ALC) were obtained from fractions F2 and F3,which had stronger ACE inhibitory activities. Six potential ACE inhibitory peptides were determine by molecular docking and five of them exhibited different ACE inhibitory activities at 1 mg/mL, among which, WFHAVFW and WHAFLW had the strongest ACE inhibitory activity with inhibition percentages of(95.57 ± 0.37)% and(98.59 ± 0.08)%, respectively.The half-maximal inhibition concentration(IC50) value of the hexapeptide WHAFLW was determined to be 52.39 μmol/L.The molecular docking indicated that the peptides could interact with ACE through hydrogen bonding, van der Waals interaction, and Pi-Pi interaction to form a stable peptide-enzyme complex. In conclusion, the combined use of bioactivityguided fractionation and computer-aided screening can provide a rapid method for screening hydrolysate for strong ACE inhibitory peptides.