螺旋藻源血管紧张素转化酶抑制肽的纯化和鉴定

血管紧张素转化酶(ACE)抑制剂通过影响肾素-血管紧张素系统,对减缓和抑制高血压具有重要的作用.该研究通过超滤、凝胶过滤色谱、反相高效液相色谱等方法,从钝顶螺旋藻的木瓜蛋白酶水解液中分离、纯化得到一种血管紧张素转化酶(ACE)抑制肽,并利用基质辅助激光解吸电离-飞行时间质谱(MALDI-TOF-MS)和氨基酸测序对纯化肽进行鉴定.此外,对其抑制类型和体外模拟消化环境稳定性也进行了研究.结果表明,分子质量范围为0～3000ku的酶解液ACE抑制活性最高,IC50值为(1.03±0.04)g/L.该部分酶解液通过纯化获得ACE抑制肽,IC50值为(0.0094±0.0002)g/L,相当于(27.36±0.14)μmol/L,序列经鉴定为Val-Glu-Pro.Lineweaver-Burk图和Dixon图表明该ACE抑制肽为非竞争性抑制剂,Ki值为(23.59±0.54)μmol/L.体外稳定性实验显示,该抑制肽在胃蛋白酶、胰凝乳蛋白酶、胰蛋白酶等胃肠蛋白酶的消化下能够保持良好的抑制活性,表明螺旋藻源ACE抑制肽可以用于降血压功能食品和药剂方面,具有很好的发展前景.     

血管紧张素转化酶抑制肽的研究进展

血管紧张素转化酶 (angiotensin I convertingenzyme ,ACE)在血压调节方面起着重要的作用 ,当其受到抑制时血压就会降低。许多合成的ACE抑制剂被广泛地应用于临床 ,但会造成多种副作用。近年来 ,对天然ACE抑制肽的研究表明 ,一些来源于蛋白酶解产生的活性肽可以对ACE起到有效的抑制作用。综述了血管紧张素转化酶的抑制肽的降压原理 ,种类和来源以及结构特点等的研究进展 ,并对其应用前景进行了展望。     

新型抗高血压活性肽GAP-A的一级结构及生理活性研究

研究了新型乳酪蛋白源抗高血压活性肽GAP-A的分子量与一级结构,并检测了其对体外血管紧张素转化酶(ACE)的抑制活性及体内降血压效果。结果显示:抗高血压活性肽GAP-A分子量为M2,氨基酸序列为B1-B2-B3;GAP-A在体外对ACE有很强的抑制活性,抑制率为79.6%;GAP-A对自发性高血压大鼠(spontaneously hypertensive rats,SHR)有显著的降血压作用,而对血压正常的SD大鼠的血压没有影响。     

酶解鹿茸血ACE抑制活性及其与抗氧化活性关系的研究

实验采用木瓜蛋白酶和Alcalase碱性蛋白酶水解鹿茸血,在不同的时间取样,分别测定血管紧张素转化酶(ACE)抑制率、二苯代苦味肼基自由基(DPPH.)清除率和羟自由基(.OH)清除率。结果表明Alcalase蛋白酶水解产物ACE抑制活性和DPPH.清除活性较强,其水解3h的产物ACE抑制率为90.63%,DPPH.清除率为14.40%;木瓜蛋白酶水解5h的产物.OH清除率最高,为96.7%。用SPSS10.0软件分析结果,发现ACE抑制率与DPPH.清除率具有显著相关性。     

一株高ACE抑制活性乳杆菌的筛选鉴定、培养条件优化及其基因组分析

【背景】乳杆菌是人体肠道益生菌,其发酵乳中可检测到血管紧张素转换酶(Angiotensin converting enzyme,ACE)抑制肽。海洋蕴藏着丰富的微生物种质资源,分布着大量的乳杆菌。【目的】从高通量测序结果中发现渤海沉积物中分布着乳杆菌资源。为了进一步开发具有ACE抑制活性的海洋乳杆菌资源,提高乳杆菌发酵乳的ACE抑制活性,筛选瑞士乳杆菌(Lactobacillus helveticus)并对其特性进行研究。【方法】采用高通量测序技术从渤海沉积物中检测乳杆菌,并对其进行富集分离,对筛选出的乳杆菌进行16S rRNA基因鉴定和全基因组测序分析,测定该菌发酵乳的ACE抑制活性,并采用正交实验优化发酵条件。【结果】渤海沉积物中含有乳杆菌并成功筛选出一株瑞士乳杆菌GY-3,其发酵乳具有较高的ACE抑制活性。该菌在发酵温度37°C,接种量3%,且在脱脂乳培养基中添加1.0%葡萄糖,0.6%大豆蛋白胨,1.0%酵母浸粉,0.04%MnSO_4·4H_2O时,抑制活性最高,可达79.52%。通过对该菌基因组进行测序研究,发现其产ACE抑制肽涉及蛋白酶系统、多肽转运系统和肽酶系统。【结论】为扩大海洋源产ACE抑制肽的乳杆菌种质资源、开发高产ACE抑制活性的发酵菌株奠定了基础,进一步研究了如何提高乳杆菌产ACE抑制肽的水平,并对其基因组进行了研究,为今后生物学特性和ACE抑制活性机理的研究奠定了基础,并对降血压相关产品的开发具有重要意义。     

血管紧张素转换酶的结构功能及相关抑制剂

血管紧张素转化酶(angiotensin converting enzyme, ACE, EC 3.4.15.1)是一种位于细胞膜上, 依赖锌离子的羧二肽酶, 催化水解十肽血管紧张素I羧基末端两个氨基酸, 生成具有血管收缩作用的八肽血管紧张素II。ACE在血压调节系统renin - angiotensin system (RAS系统)中具有重要作用, 从ACE的结构功能、基因多态性及其抑制剂等方面进行了详细综述。发现体细胞ACE两个活性中心催化血管紧张素I和缓激肽的机制不同, 因此以体细胞ACE单个活性中心为靶点的研究, 将会为研制开发副作用更少, 安全性更高的ACE抑制剂提供新的途径。     

N^＋注入Lactobacillus bulgarius诱变选育高产ACE抑制剂的研究

采用N^＋注入技术对实验室保存的4株保加利亚杆菌L601、L602、L603、L606进行诱变处理,旨在选育出高产血管紧张素转换酶（angiotensin converting enzyme,ACE）抑制剂菌株。结果表明,在离子注入能量为20keV,剂量为3．0×0．5×10^15 ions／cm^2条件下,诱变效果较好。从正突变菌株中反复筛选,获得1株对ACE抑制活性较高的菌株M602,其ACE抑制率为82．49％。经过培养条件优化,其对ACE抑制率进一步提高为85．12％,经连续传代实验,其性状遗传稳定。     

食品蛋白质中血管紧张素转化酶抑制肽的研究

血管紧张素转化酶Ⅰ (angiotensinIconvertingenzyme ,简称ACE)在人体血压调节过程中起重要的生理作用。源于食品蛋白质中的血管紧张素转化酶抑制肽 (angiotensinIconvertingenzymeinhibitorypeptides ,简称ACEIP)有明显的降血压作用 ,这些肽是通过抑制ACE的活性起降血压作用。文章中综述了来源于各种食品蛋白质的ACEIP的最新研究进展以及两种主要制备方法和评价方法 ,并对食品蛋白质中ACEIP的应用前景进行了展望     

鹿茸血制备ACE抑制肽的酶解条件优化

以天山马鹿的鹿茸血为原料,通过体外检测方法检测不同酶及酶解条件下的产物的ACE抑制肽活性及抗氧化活性。结果表明,以A lcalase碱性蛋白酶水解,底物浓度6%、加酶量40μl/g蛋白、pH 8.0、温度55℃的条件下,酶解3 h,水解度为28.0%,酶解产物的血管紧张素转化酶(angiotensin converting enzym e,ACE)抑制活性可达93.55%,与抗氧化活性呈正相关关系。     

高效液相色谱法测定可口革囊星的ACE活性

目的利用高效液相色谱法测定可口革囊星虫酶解物中具有抑制血管紧张素转化酶(ACE)活性的活性肽。方法用HPLC测定血管紧张素转换酶抑制肽活性,在该色谱条件下,可通过测定由ACE水解马尿酰组氨酰亮氨酸后产生的马尿酸峰面积或含量得到酶解液的活性。结果酶解物在质量浓度为0.48mg/ml时对ACE的抑制率为17.62%。结论可口革囊星虫蛋白经碱性蛋白酶水解后得到的酶解物活性较高。     

Latent production of angiotensin I-converting enzyme inhibitors from buckwheat protein.

The latent production of angiotensin I-converting enzyme (ACE) Inhibitors from tartary buckwheat (BW) was investigated, and the peptides responsible for ACE inhibition characterized. Intact buckwheat was found to exhibit ACE inhibitory activity having an IC50 value of 3.0 mg/ml. The activity of the protein fraction (IC50: 0.36 mg protein/ml) was not enhanced by pepsin treatment. Pepsin, followed by chymotrypsin and trypsin hydrolysis, resulted in a significant increase in the ACE inhibitory activity (IC50: 0.14 mg protein/ml). The rutin contained in the buckwheat did not exhibit any ACE inhibition. A single oral administration of BW digest lowered the systolic blood pressure of a spontaneously hypertensive rat. Thus, BW proteins offer a potential resource for producing ACE inhibitory peptides during the digestion process. From the di-/tri-peptide fraction (DTPF) of the BW digest, inhibitory peptides were identified. The magnitude (%) of the total ACE inhibitory contribution of each identified peptide, relative to the overall inhibition of the DTPF, was about 41%.     

Intestinal transport of the lactokinin Ala-Leu-Pro-Met-His-Ile-Arg through a Caco-2 Bbe monolayer.

ACE inhibitory peptides are biologically active peptides that play a role in blood pressure regulation. When derived from food proteins during food processing or gastrointestinal digestion, these peptides could function as efficient agents in treating and preventing hypertension. However, in order to exert an antihypertensive effect by inhibition of the ACE enzyme, they have to reach the bloodstream intact. The aim of this research was to assess if the known ACE inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, derived from a tryptic digest of beta-lactoglobulin, could be absorbed through a Caco-2 Bbe cell monolayer in an Ussing chamber and reach the serosal side undegraded. Samples of the mucosal compartment showed high ACE inhibitory activity. No or only little ACE inhibitory activity was detected in the serosal compartment. However, when the serosal sample was concentrated three-fold, a substantial ACE inhibitory activity was registered. Concomitantly, HPLC and MS clearly showed the presence of Ala-Leu-Pro-Met-His-Ile-Arg in the mucosal compartment, whereas in the serosal compartment only MS was able to detect the heptapeptide. In conclusion. under the observed experimental conditions, the ACE inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg was transported intact through the Caco-2 Bbe monolayer, but in concentrations too low to exert an ACE inhibitory activity.     

Insight into the binding of ACE-inhibitory peptides to angiotensin-converting enzyme: a molecular simulation

Angiotensin-I-converting enzyme (EC 3.4.15.1; ACE) plays an important physiological role in the regulation of blood pressure by converting angiotensin I to angiotensin II, a potent vasoconstrictor. Therefore, ACE inhibition has become a major target control for hypertension. Pipefish, or hailong, is an essential traditional Chinese medicine that is widely used in anti-fatigue and anti-cancer. A recent study has found two ACE-inhibitory peptides (TFPHGP and HWTTQR) purified from the seaweed pipefish by Alcalase enzymatic hydrolysis. Two peptides exhibited different ACE-inhibitory activities; however, the molecular mechanism involved is poorly understood. Further investigations are necessary to elucidate the relationship between the inhibition mechanism and the peptides. The current study is focused on investigating the interactions between each ACE-inhibitory peptide and ACE by performing molecular docking and molecular dynamics (MD) simulations. ACE protein remained stable throughout the simulations. Furthermore, ACE-TFPHGP complex showed lower binding energy as compared to ACE-HWTTQR complex, which is in good agreement with the experimental results. Glu384 and Glu411 of ACE are key residues upon the ACE-inhibitory peptides binding. Molecular basis generated by this attempt could provide valuable information towards designing new medicine for ACE inhibitor.     

Analysis of novel angiotensin-I-converting enzyme inhibitory peptides from protease-hydrolyzed marine shrimp Acetes chinensis.

Acetes chinensis is an underutilized shrimp species thriving in the Bo Hai Gulf of China. In a previous study, we had used the protease from Bacillus sp. SM98011 to digest this kind of shrimp and found that the oligopeptide-enriched hydrolysate possessed antioxidant activity and high angiotensin I-converting enzyme (ACE) inhibitory activity with an IC50 value of 0.97 mg/ml. In this paper, by ultrafiltration, gel permeation chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC), five peptides with high ACE inhibitory activity were purified from the shrimp hydrolysates and their sequences were identified by amino acid composition analysis and molecular weight (MW) analysis. Three of them, FCVLRP (a), IFVPAF (f) and KPPETV (j), were novel ACE inhibitory peptides. Their IC50 values were 12.3 microM, 3.4 microM and 24.1 microM, respectively, and their recoveries were 30 mg/100 g (solid basis of shrimp), 19 mg/100 g and 33 mg/100 g, respectively. Lineweaver-Burk plots for the three novel peptides showed that they are all competitive inhibitors. To test the ACE inhibitory activity of peptide a, f, j after they were digested by digestive enzymes in vivo, 12 derived peptides from FCVLRP and IFVPAF were synthesized based on their amino acid sequences and the cleavage sites of digestive enzymes. No digestive enzyme cleavage site was found in KPPETV. The IC50 values of the derived peptides were determined and the result showed that except for VPAF, FC and FCVL, the ACE inhibitory activity of the other nine derived peptides did not significantly change when compared with their original peptides. Surprisingly, five peptides had lower IC50 values than their original peptides, particularly for RP (IC50 value = 0.39 microM), which is about 30 times lower than its original peptide and almost the lowest IC50 value for ACE inhibitory peptides reported. Therefore, the novel peptides identified from A. chinensis hydrolysates probably still maintain a high ACE inhibitory activity even if they are digested in vivo. This is the first report about novel ACE inhibitory peptides from hydrolysates of marine shrimp A. chinensis. The novel peptides from hydrolysate of A. chinensis and some of their derived peptides with high ACE inhibitory activity probably have potential in the treatment of hypertension or in clinical nutrition.     

In vitro and in silico studies of novel synthetic ACE-inhibitory peptides derived from Saccharomyces cerevisiae protein hydrolysate

The structure-function relation of YR-10 (YGKPVAVPAR) was investigated by synthesizing four structural analogs of that including YHR-10 (YGKHVAVHAR), GA-8 (GKPVAVPA), GHA-8 (GKHVAVHA), and PAR-3 (PAR). GA-8 (GKPVAVPA) was synthesized on the basis of simulated enzymatic gastrointestinal digestion performed by bioinformatics tools (expasy-peptide cutter). This study explains the molecular mechanisms for the interaction of synthetic peptides with ACE. The IC₅₀ values of each were 139.554 ± 2.3, 61.91 ± 1.2, 463.230 ± 3.56, 135.135 ± 2.1, 514.024 ± 5.86 µM, respectively. Results indicated that Pro replacement with His in YR-10 and GA-8 increased ACE inhibitory activity respectively, by 55.63% and 70.82%. Removal of Tyr and Arg from respectively N and C terminal positions of YR-10, following in silico simulated gastrointestinal digestion caused the 3.31 fold decrease in ACE inhibitory activity. YHR-10 showed the best docking poses, and GHA-8 exhibited interaction with Zn²⁺. Lineweaver–Burk plots of most active peptides suggest that they act as noncompetitive inhibitors against ACE.     

Novel Angiotensin I-Converting Enzyme Inhibitory Peptides Found in a Thermolysin-Treated Elastin with Antihypertensive Activity

Angiotensin I-converting enzyme (ACE) inhibitory activity was generated from elastin and collagen by hydrolyzing with thermolysin. The IC₅₀ value of 531.6 µg/mL for ACE inhibition by the elastin hydrolysate was five times less than 2885.1 µg/mL by the collagen hydrolysate. We confirmed the antihypertensive activity of the elastin hydrolysate in vivo by feeding spontaneously hypertensive rats (male) on a diet containing 1% of the elastin hydrolysate for 9 weeks. About 4 week later, the systolic blood pressure of the rats in the elastin hydrolysate group had become significantly lower than that of the control group. We identified novel ACE inhibitory peptides, VGHyp, VVPG and VYPGG, in the elastin hydrolysate by using a protein sequencer and quadrupole linear ion trap (QIT)-LC/MS/MS. VYPGG had the highest IC₅₀ value of 244 µM against ACE and may have potential use as a functional food.     

In vitro and in vivo ACE inhibitory of pistachio hydrolysates and in silico mechanism of identified peptide binding with ACE

The ACE inhibitory activity of pistachio (Pistacia vera L.) kernel's hydrolysates by gastrointestinal enzymes was studied. Results indicated that hydrolysate successively hydrolyzed by pepsin and trypsin, Pe–Tr–H, presented in vitro ACE inhibitory activity as IC₅₀ 0.87 ± 0.04 mg/ml. The Pe–Tr–H can in vivo decrease around 22 mmHg in systolic blood pressure (SBP) and 16 mmHg in the diastolic blood pressure (DBP) at 4 h after the oral administration, however the pistachio kernel powder can slightly lower SBP and DBP. The Pe–Tr–H with the highest activity was then separated by ultrafiltration membrane of 3 kDa, size exclusion chromatography on Sephadex G-15 and G-10 columns and reversed phase high-performance liquid chromatography (RP-HPLC) consecutively. A novel ACE inhibitory peptide, ACKEP, with the IC₅₀ value of 126 μM, was identified by MALDI–TOF/TOF system. ACKEP has the same C-terminal residue as Lisinopril and Enalapril, which plays a key role in binding with ACE. The binding mechanism was explored at a molecular basis by docking experiments, which revealed that seven residues from ACE active site (His383, His387, Glu384, Arg522, Asp358, Ala356 and Asn70) and two atoms of ACKEP (O5, H60) greatly contributed to the combinative stabilization.     

Antihypertensive effect and purification of an ACE inhibitory peptide from sea cucumber gelatin hydrolysate

Gelatin from the sea cucumber (Acaudina molpadioidea) was hydrolyzed sequentially with bromelain and alcalase. The hydrolysate was fractionated into three ranges of molecular weight (GH-I, <10 kDa; GH-II, <5 kDa; GH-III, <1 kDa) using an ultrafiltration membrane bioreactor system. The GH-III brought about a high angiotensin-I-converting enzyme (ACE) inhibitory activity, with IC₅₀ value of 0.35 mg/ml. An ACE inhibitory peptide was isolated from the GH-III, using the chromatographic methods including ion-exchange chromatography, gel filtration and reversed phase high-performance liquid chromatography. The purified ACE inhibitory peptide with a molecular weight of 840 Da consisted of five main amino acids (Glu, Asp, Pro, Gly and Ala), and its IC₅₀ value was 0.0142 mg/ml. This sea cucumber gelatin hydrolysate (GH-III) was used as drinks administered to renal hypertensive rats (RHR) for 1 month. The systolic blood pressure and diastolic blood pressure of the RHR were significantly reduced, which indicates an antihypertensive effect by oral administration.