血管紧张素转化酶(angiotensin I-converting enzyme,ACE)在血压调控中发挥着重要作用。从食源蛋白中筛选具有ACE抑制活性的多肽已经成为研究热点。螺旋藻作为最早被食用的藻类,目前其蛋白质含量在藻类食物中最高,因此该研究利用碱性蛋白酶、中性蛋白酶、胰蛋白酶、胃蛋白酶水解螺旋藻蛋白制备ACE抑制肽。首先,通过水解度、DPPH清除率、ACE抑制率筛选得到最佳水解酶为碱性蛋白酶。联合肽组学与计算模拟发现水解组分中2条新型ACE抑制肽分别为His-Ile-Ile-Ala-Arg-Pro-His(HIIARPH,IC50=461.5 μmol/L)和Leu-Arg-Leu-Lys-Glu(LRLKE,IC50=155.8 μmol/L),其酶抑制动力学表现形式为非竞争抑制模式。进一步采用分子对接分析其非竞争性抑制的分子机制,发现HIIARPH和LRLKE与ACE的S1、S2口袋形成氢键发挥抑制作用,此外,LRLKE能与S3口袋以及Zn2+结合,表现出更佳的抑制活性。最后,评价了HIIARPH和LRLKE的稳定性和细胞毒性。其中,LRLKE具有较好的热稳定性,在酸性和弱碱性条件下都能保持较高的活性。HIIARPH和LRLKE在≤1 mmol/L时对HUVEC细胞无明显毒性。综上,该研究提供了一种快速筛选ACE抑制肽的方法,为开发螺旋藻源降血压肽提供理论参考。
Angiotensin I-converting enzyme (ACE) plays an important role in the regulation of blood pressure. Screening peptides with ACE inhibitory activity from food source proteins has become a research hotspot. Spirulina is the earliest algae to be eaten with the highest protein content in food. In this study, ACE inhibitory peptides were prepared by hydrolyzed Spirulina protein with alkaline protease, neutral protease, trypsin, and pepsin. Alkaline protease was selected as the best protease according to hydrolysis degree, DPPH scavenging activity, and ACE inhibitory activity. Two new ACE inhibitory peptides were found from the hydrolyzed fractions using peptidomics, and computational simulation: His-Ile-Ile-Ala-Arg-Pro-His (HIIARPH, IC50=461.5 μmol/L) and Leu-Arg-Leu-Lys-Glu (LRLKE, IC50=155.8 μmol/L). The enzyme kinetics of two new peptides are shown as non-competitive inhibition mode. The results of molecular docking showed that HIIARPH and LRLKE could form hydrogen bonds with S1 and S2 pockets of ACE to play an inhibitory role. Moreover, LRLKE could bind with S3 pockets and Zn2+, which contributed to its better inhibitory activity. Stability study showed that LRLKE had good thermal stability and maintains high activity under acidic or mildly alkaline conditions. Cytotoxic study showed that HIIARPH and LRLKE had no significant influence on HUVEC cells viability under the 1 mmol/L concentration. In summary, this study provides a method for rapid screening of ACE inhibitory peptides and provides theoretical reference for the development of Spirulina-derived antihypertensive peptides.
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