IOCAS-IR  > 实验海洋生物学重点实验室
Thesis Advisor张全斌研究员
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name工程硕士


1. 从龙须菜胰蛋白酶的酶解多肽液(≤3 kDa的组分)中利用LC-MS/MS鉴定出两条具有特定序列的ACE抑制肽。经测定FQIN [M (O) ] CILRTGAPCRIC50值分别为9.64 ± 0.36 μM23.94 ± 0.82 μM。两条多肽具温度和pH稳定性,且能在模拟胃肠液中保持结构的稳定性并发挥稳定的ACE抑制作用。酶动力学考察显示两个多肽为ACE的非竞争性抑制剂。分子对接模拟显示两个多肽通过与ACE催化底物相关的关键氨基酸残基形成氢键来干扰ACE。此外,两个多肽在自发性高血压大鼠(SHR)体内仍能发挥显著的降压活性,两者能显著的降低SHR的收缩压(SBP)和舒张压(DBP),其中FQIN[M (O) ]CILRTGAPCRSHR体内能发挥更好的降血压效果。

2. 比较了实验室传统的提取方法及酶法对坛紫菜中蛋白质提取量的影响。通过比较得出超声提取法仍然是高效、经济的紫菜蛋白的提取方法。其次,纤维素酶可在一定程度上破坏紫菜细胞壁的结构,能够促进胞内蛋白质的溶出。

3. 考察了坛紫菜酶解肽段的抗氧化活性及ACE抑制活性。各蛋白酶的酶解坛紫菜肽段都有一定的抗氧化活性,且分子量≤1 kDa的组分相对于分子量≥1 kDa的组分能发挥更好抗氧化活性。5种蛋白酶的酶解多肽中,中性蛋白酶和胰蛋白酶的酶解坛紫菜多肽表现出更好的综合抗氧化能力。各蛋白酶酶解得到的坛紫菜多肽中仅部分肽段具ACE抑制活性。ACE的活性中心具有一定的空间结构。因此,分子量≤1 kDa的肽段能插入到ACE活性部位,干扰ACE催化底物,而发挥抑制活性。5种蛋白酶的坛紫菜酶解多肽中,胰蛋白酶和碱性蛋白酶的酶解多肽显示较好的ACE抑制活性。因此,在后期进一步筛选坛紫菜的生物活性多肽中可选择使用胰蛋白酶或碱性蛋白酶或者两者复合酶解来制备ACE抑制肽。

Other Abstract

Porphyra haitanensis and Gracilaria lemaneiformis are two important economic algae widely cultivated in coastal areas of China. The two algae are rich in nutrients for edible and medicinal purposes, especially P. haitanensis, which has the same amount of protein as terrestrial plant soybeans and is rich in essential amino acids. In this paper, the preparation of enzymatic peptides from P. haitanensis and G. lemaneiformis and the anti-oxidation and angiotensin-converting enzyme (ACE) inhibitory activities were studied.

1. Two ACE inhibitory peptides with specific sequences were identified by LC-MS/MS in the enzymatic hydrolysis peptide solution of trypsin of G. lemaneiformis (≤3 kDa). The IC50 values for FQIN [M (O) ] CILR and TGAPCR were determined to be 9.64 ± 0.36 μM and 23.94 ± 0.82 μM, respectively. Both peptides are temperature and pH stable and maintain structural stability and mimic ACE inhibition in simulated gastrointestinal fluids. Enzymatic kinetic studies revealed that the two peptides are non-competitive inhibitors of ACE. Molecular docking simulations revealed that two peptides interfere with ACE by forming hydrogen bonds with key amino acid residues associated with the ACE-catalyzed substrate. In addition, both peptides still exert significant antihypertensive activity in SHR, both of which significantly reduce SHR systolic blood pressure (SBP) and diastolic blood pressure (DBP). Among them, FQIN [M (O) ] CILR can exert better antihypertensive effect in SHR than TGAPCR.

2. Compare the traditional extraction methods of the laboratory and the effect of enzymatic method on the protein extraction in the P. haitanensis. By comparison, the ultrasonic extraction method is still an efficient and economical method for extracting P. haitanensis protein. Secondly, cellulase can destroy the structure of the cell wall of seaweed and promote the dissolution of intracellular proteins.

3. The antioxidant activity and ACE inhibitory activity of the enzymatic peptides of P. haitanensis were investigated. The enzymatic hydrolysate of each protease has certain antioxidant activity, and the component with molecular weight ≤1 kDa can exert better antioxidant activity than the component with molecular weight ≥1 kDa. Among the enzymatic peptides of the five proteases, the enzymatic hydrolysis of the peptide of the neutral protease and trypsin showed better comprehensive antioxidant capacity. Only a part of the peptides obtained by enzymatic hydrolysis of each protease have ACE inhibitory activity. The active center of ACE has a certain spatial structure. Therefore, a peptide having a molecular weight of ≤1 kDa can be inserted into an ACE active site, interfere with the ACE-catalyzed substrate, and exert an inhibitory activity. Among the enzymatically hydrolyzed peptides of the five proteases, trypsin and alkaline protease showed good ACE inhibitory activity. Therefore, in the further screening of the bioactive peptide of P. haitanensis, trypsin or alkaline protease or a combination of the two can be used to prepare the ACE inhibitory peptide.

Document Type学位论文
Corresponding Author邓真真
Recommended Citation
GB/T 7714
邓真真. 两种红藻多肽的抗氧化及降血压活性研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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