血管紧张素转化酶抑制肽的设计与抗酶解研究

设计了血管紧张素转换酶抑制肽(angiotensin converting enzyme inhibitory peptides,ACE抑制肽),并对设计的ACE抑制肽进行抗酶解研究。选取静电荷和疏水性这两个重要的结构参数,设计了一系列的ACE抑制肽。采用反相高效液相色谱(reversed-phase high performance liquid chromatography,RP-HPLC)来检测37℃下胃蛋白酶和胰蛋白酶对ACE抑制肽的酶解后的保留量,以研究静电荷和疏水性与肽的抗酶解之间的关系。结果表明,肽中疏水性氨基酸含量及蛋白质的平均疏水性均与ACE抑制肽的抗酶解活性呈正相关,而静电荷对ACE抑制肽抗酶解的影响较小。     

响应曲面法优化小黄鱼ACE抑制肽的酶解条件

以小黄鱼为原料,利用酶解技术制备高活性的ACE抑制肽。通过单因素及响应曲面分析,确定胰蛋白酶(PTN6.0S)的酶解工艺,利用反相高效液相色谱(RP-HPLC)法测定酶解产物(0.05g/L)的ACE抑制率,研究pH值、温度、时间、底物质量浓度、酶与底物比等因素对ACE抑制肽活性的影响。结果显示：最佳工艺条件为pH7.0、温度50℃、时间16.5h、底物质量浓度5.6g/100mL、酶与底物比9‰,酶解产物ACE抑制率达87.36%。     

龙须菜蛋白的提取工艺优化及降血压组分制备

目的 优化龙须菜蛋白的提取工艺,并制备降血压组分。方法 从胃蛋白酶、中性蛋白酶、碱性蛋白酶和胰蛋白酶中筛选能获得最佳蛋白提取率的蛋白酶,采用单因素试验考查pH、底物浓度、酶解温度、酶底比、酶解时间对蛋白提取率和ACE抑制率的影响,采用响应面法确定最佳工艺条件,采用超滤膜分离技术龙须菜蛋白酶解液中制备血管紧张素转换酶(angiotensin converting enzyme, ACE)抑制肽,并考察其ACE抑制率。结果 最佳酶解工艺条件为:pH 8.4、底物浓度18%、温度55℃、酶底比2.0%、碱性蛋白酶酶解3 h,酶解液的蛋白提取率为(19.54±0.56)%、ACE抑制率为(91.12±0.17)%;将酶解液分别通过10、5、1 kDa超滤膜,利用ACE抑制率来验证降血压活性, 1 kDa超滤膜的酶解液ACE活性最高,达到(71.37±0.22)%。结论 龙须菜可作为分离纯化制备龙须菜降血压肽的优质资源。     

马面鱼皮ACE抑制肽的制备、分离纯化及稳定性

为实现马面鱼皮的高值化利用,优化其胶原蛋白制备ACE抑制肽的酶解条件,通过超滤、凝胶色谱、高效液相及U-PLC-MS等方法分离、纯化及分析ACE抑制肽.探讨pH值、温度及体外模拟胃肠消化对分离制备多肽的ACE抑制活性的影响.结果表明:利用复合酶解法可制备高活性ACE抑制肽,即在蛋白酶N"天野"2G用量1200 U/g,...     

源于皱纹盘鲍裙边胶原蛋白ACE抑制肽的制备

为了提高皱纹盘鲍(Haliotis discus hannai)裙边的利用率,以其为原料纯化胶原蛋白,并通过胰蛋白酶和胃蛋白酶共酶解制备血管紧张素转换酶(angiotensin I-converting enzyme,ACE)抑制肽。酶解液经超滤、Superdex^TM peptide10/300GL凝胶柱和高效液相色谱分离纯化,获得了3条来源于皱纹盘鲍胶原蛋白的肽段SGEVGQ、QRGPAGAQGPQ和GPPGPAGAR。其中结合能力最强的肽为GPPGPAGAR,对ACE的IC50值为177.1μmol/L,分子对接结果显示其主要作用于ACE的S1活性口袋,抑制模式与赖诺普利类似,并且经模拟胃肠液消化后仍能发挥较强的ACE抑制作用。本研究通过酶解皱纹盘鲍裙边胶原蛋白制备ACE抑制肽,为鲍鱼裙边的精深加工和ACE抑制肽的开发提供了参考。     

利用鲍鱼性腺制备ACE抑制肽

采用碱性蛋白酶和木瓜蛋白酶分步酶解鲍鱼性腺蛋白,以酶解产物分子质量及ACE抑制率为考核指标,用正交试验及响应面分析法对其制备工艺参数进行优化。结果显示,第1步碱性蛋白酶最佳工艺参数为反应p H 8.5,反应温度55℃,加酶量0.5%,酶解时间6 h;第2步木瓜蛋白酶最佳工艺参数为反应p H 6.5,反应温度57℃,酶解时间36 min,加酶量1.5%。将酶解产物用10 ku及3 ku膜超滤,采用凝胶过滤色谱法测得超滤组分中分子质量在1 ku以下的小肽含量为98.5%,IC50为0.44 mg/m L。本研究制备的低分子质量、高活性ACE抑制肽,为鲍鱼加工下脚料的高值化利用提供了理论参考。     

酶解酪朊酸钠制备ACE抑制肽研究

目的：探讨利用Alcalase2．4L酶解酪朊酸钠制备高活性ACE抑制肽的工艺条件；方法：以ACE抑制活性、肽得率和水解度（DH）为主要评价指标，通过正交试验优化制备ACE抑制肽的工艺条件；结果：在最佳工艺条件为pH8．0、反应温度印℃、底物浓度6％、酶用量1000U／g底物、酶解时间10h条件下测得ACE抑制率为40．38％，肽得率为53．76％，DH为22．07％，高效体积排阻色谱（HPSEC）对酶解产物的分析结果表明分子量小于3kDa的肽占总蛋白的16．38％。     

谷朊粉酶解物的制备及其ACE抑制肽的分离鉴定

以谷朊粉为原料,以血管紧张素转换酶（ACE）抑制率为指标,比较4种蛋白酶的酶解效果。采用超滤、葡聚糖凝胶色谱、反相高效液相色谱（RP-HPLC）的方法分离纯化ACE抑制肽并用电喷雾飞行时间质谱联用(ESI-TOF-MS)鉴定其结构。结果表明：碱性蛋白酶酶解3 h的谷朊粉酶解物具有较高的ACE抑制活性;超滤后,分子质量Mr<1 ku的组分具有较高的ACE抑制率;分离纯化后得到小肽的ACE抑制率高达（81.03±1.20）%;由ESI-TOF-MS质谱图得出该小肽的m/z为630.89,氨基酸序列为Trp-Phe-Gln-Pro（WFQP）。     

酶法制备刺参内脏ACE抑制肽的工艺优化及产物鉴定

刺参内脏作为海参初加工的主要副产物，具有良好的营养和开发利用价值。文章研究了利用酶解法从刺参内脏中制备ACE抑制肽的工艺。通过单因素实验和响应面优化法确定了胰蛋白酶酶解制备海参内脏ACE抑制肽的最佳条件为：温度37℃、pH8.5、料液比1:1.98、加酶量2.56%、酶解时间1.0 h，所得内脏多肽ACE抑制率为56.80%。通过超滤分级、Sephadex G-25分子筛层析柱层析，对活性肽进一步纯化，得到最优组分ACE抑制率为50%时抑制浓度0.535 mg/mL，通过高效液相色谱-串联质谱(HPLC-MS/MS)分析鉴定，得到多肽氨基酸序列组成和分子质量等信息。该研究为刺参内脏综合利用和食源性ACE的开发提供了基础。     

酶解胶原蛋白制备血管紧张素转化酶抑制肽的研究

血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制剂是目前用于治疗高血压的重要药物.本实验采用胃蛋白酶(pepsin)和胰蛋白酶(trypsin)组合酶解胶原蛋白制备水解液.使用1kD超滤膜对水解液进行超滤,超滤液采用葡聚糖凝胶Sephadex G-15凝胶过滤层析得到多种组分.各组分经酶活力和蛋白浓度测定,筛选出有较高 ACE抑制率的两个样品.继续使用反相高效液相色谱进一步分离筛选出样品,对分离出的不同组分进行蛋白浓度和抑制率测定,通过分析实验数据,筛选出具有最高活性组分.最终样品经脱盐和冷冻干燥后进行高抑制活性的血管紧张素转化酶抑制肽的分子量的测定.     

Evaluation of angiotensin I-converting enzyme (ACE) inhibitory activities of hydrolysates generated from byproducts of freshwater clam

Solid wastes of freshwater clam in food processing, including mainly mantle, were used as a raw material for the recovery of bioactive peptides related to angiotensin I-converting enzyme (ACE) inhibitor. Among the primary hydrolysates of dried mantle (DM), the peptides corresponding to hydrolysates using 2 crude peptidases exhibited a strong ACE inhibitory activity (IC₅₀, 0.23 mg/mL), and recovery efficiency of soluble materials and their protein content were considerably large with 42.65% and 468.6 mg/g, respectively. The ACE inhibitory activity of all secondary hydrolysates digested by pepsin and trypsin was significantly increased as compared to primary hydrolysates. Furthermore, the peptic secondary hydrolysates were fractionated by gel filtration and reverse phase-HPLC (RP-HPLC) and characterized by matrix-assisted laser desorption-ionization time of flight/mass spectrometry (MALDI-TOF/MS). These peptides with molecular weight of less than 1 kDa possessed the stronger ACE inhibitory effect, and their inhibitory pattern was found to be competitive. The results showed that the DM hydoplysates might be utilized as a rich source of bioactive peptide.     

Production and characterization of angiotensin converting enzyme (ACE) inhibitory peptides from apricot (Prunus armeniaca L.) kernel protein hydrolysate

Six different proteases (Flavourzyme^®, Neutrase^®, Protamex^®, Alcalase^® 2.4L, Proleather^® FG-F, and papain) were employed to hydrolyze apricot kernel protein (AKP). Alcalase^® is an inexpensive and non-specific protease that has been shown to be useful for the generation of bioactive peptides from AKP. Alcalase^® 2.4L was selected for further study on enzymatic preparation of ACE inhibitory peptide from AKP. After 60-min hydrolysis, the highest ACE inhibition was 82 ± 0.14%. Results of molecular weight distribution revealed that most of ACE inhibition activity was probably attributed to low-molecular weight peptide fraction ranging from 200 to 900 Da. Ultrafiltration on membranes with several molecular weight cutoffs (MWCFs) demonstrated that most of the ACE inhibitory activity was due to peptides with a less than 1,000 Da molecular weight: the IC₅₀ value of the 1-kDa ultrafiltrate was 0.15 ± 0.007 mg mL^?1, while it was 0.378 ± 0.015 mg mL^?1 before ultrafiltration. Additionally, further separation and purification of the ACE inhibitory peptides were carried out using gel filtration and C₁₈ RP-HPLC. The result of research can be used to optimize AKP enzymatic hydrolysis for producing ACE inhibitory peptides which could be used for food industry and nutraceuticals.     

三斑海马蛋白ACE抑制肽的制备及其二级结构的研究

以三斑海马为原料,经碱性蛋白酶酶解,获得富含血管紧张素转化酶(angiotensin I-converting enzyme,ACE)抑制肽的酶解液。酶解液经透析、Sephadex G-25凝胶过滤色谱和反相高效液相色谱得到进一步分离纯化。结果表明,透析产物的IC50值为(0.44±0.26)mg/mL,相比酶解液(0.81±0.12)mg/mL,其ACE抑制活性更强。透析产物经Sephadex G-25凝胶柱分离纯化后,得到3种组分,其中组分F2的ACE抑制活性最强,IC50值为(0.11±0.08)mg/mL。F2经反相高效液相色谱进一步分离后,得到6个具有ACE抑制活性的组分峰,其中组分F2-4的ACE抑制活性最强,IC50值为(0.005 7±0.000 9)mg/mL。经过3步分离纯化后,成功从三斑海马蛋白中分离得到一种活性较强ACE抑制肽:ProAla-Gly-Pro-Arg-Gly-Pro-Ala(PAGPRGPA),多肽分子量为721.39 Da。圆二色谱分析多肽二级结构表明其主要含无规则卷曲。因此,从三斑海马蛋白中分离得到的多肽可能成为营养保健品和抗高血压药品及相关疾病的一种有益成分,且对海马蛋白资源的开发利用具有重要意义。     

Purification and characterization of a novel extracellular beta-1,3-glucanase produced by Bacillus clausii NM-1 isolated from ezo abalone Haliotis discus hannai

A novel extracellular alkaline stable beta-1,3-glucanase produced by Bacillus clausii NM-1 isolated from the ezo abalone Haliotis discus hannai was purified by ammonium sulfate precipitation, DEAE-Sepharose FF ion exchange chromatography and Sephacryl S-200HR gel filtration. The molecular weight of the purified enzyme was estimated to be 71 kDa from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was very stable at pH 5.3 to 11.5 but unstable at pH 4.0 to 4.5. The optimum temperature and thermostability of the enzyme increased in the presence of CaC1, The enzyme hydrolyzed R-1,3-glucan from marine organisms, but did not show activity against any other beta-1,3-glucans. The major hydrolysis products of beta-1,3-glucan from Laminaria digitata and Eisenia bicyclis were laminaritriose and laminaritetraose, respectively. The N-terminal amino acid sequence of the purified enzyme was similar to that of several beta-1,3-glucanases in the glycoside hydrolase family 16.     

重组鲍鱼肌肉脯氨酰内肽酶的性质研究

为研究皱纹盘鲍（Haliotis discus hannai）中脯氨酰内肽酶（Prolyl endopeptidase,Hdh-PEP)的酶学特性与结构特性,利用基因工程技术重组并在大肠杆菌中高效表达了皱纹盘鲍PEP。原核表达的Hdh-PEP分子量为85 kDa,在pH2～6、温度20～60 ℃条件下,Hdh-PEP的表面疏水性明显升高。氨基酸序列同源性分析结果表明,Hdh-PEP催化结构域中有三个高度保守的氨基酸序列:Seq 1:K-D-G-T-K/R-I-P、Seq 2:Y-G-Y-G-G-F和Seq 3:I-R-G-G-E-Y/F。酶动力学研究表明,Hdh-PEP的米氏常数K_m为5.32 μmol/L,催化常数k_cat值为15.7 s?1。PEP的特异性抑制剂SUAM-14746和ZPP对Hdh-PEP酶活力具有强抑制作用,丝氨酸蛋白酶抑制（PMSF）对Hdh-PEP酶活力也有较大程度的抑制作用。本实验制备了高特异性抗Hdh-PEP多克隆抗体,可检测鲍鱼肌肉中天然PEP的存在情况。Hdh-PEP的体外高效表达和特异性多克隆抗体制备为后续深入研究Hdh-PEP的性质提供了重要参考。     

源于鸡蛋清溶菌酶ACE抑制肽的分离纯化及结构鉴定

利用胃肠消化酶系水解疏水性氨基酸含量高的蛋清溶菌酶蛋白,筛选能抗胃肠消化的高活性ACE抑制肽。通过超滤、制备型反相高效液相色谱(RP-HPLC)、分析型RP-HPLC纯化、氨基酸组成分析及基质辅助激光解吸电离飞行时间串联质谱(MALDI-TOF-TOF-MS),从胃肠消化水解液中分离鉴定出两种ACE抑制肽Lys-Val-Phe(KVF)和Trp-Ile-Arg(WIR),化学合成该两种三肽后,ACE抑制活性测定结果显示其IC50值分别为14μmol/L与88.5μmol/L。研究结果表明,蛋清溶菌酶蛋白来源的ACE抑制肽有望用于高血压的预防与治疗。     

豆粕血管紧张素转化酶抑制肽的结构鉴定及作用机制解析

以大豆粕为原材料,利用超声辅助酶解技术、超滤-?KTA层析相结合的方法分离纯化获取豆粕酶解产物中血管紧张素转化酶（angiotensin-converting enzyme,ACE）抑制肽,对其分子质量分布进行研究,后通过质谱分析与分子对接技术鉴定并筛选出ACE抑制活性肽的氨基酸序列,经固相合成肽序列,检测其ACE抑制肽的活性并基于分子对接技术探索其抑制机制。结果表明：经超声辅助酶解提取获得的豆粕肽分子质量主要分布在6 000 Da以下;根据分子对接的最低预测自由能筛选出的GVRP（－8.44 kcal/mol）和IIVTP（－9.04 kcal/mol）可以抑制ACE活性,半抑制浓度（50% inhibitory concentration,IC50）分别为（84±0.06）、（77±0.08）μmol/L;分子对接结果表明：GVRP、IIVTP能够与ACE的活性口袋S1、S1′、S2形成氢键相互作用,共有的过近接触（3.5 ?范围内）ACE氨基酸残基为His513、Ala354和Glu384。本研究基于串联质谱与分子对接技术,建立从混合多肽中快速鉴定、筛选活性多肽的方法,探究活性多肽与ACE稳定结合并体现其ACE活性的抑制机制,为后续的深入研究提供一定参考。     

ACE-inhibitory activity of tilapia protein hydrolysates

Fish processing wastes can be used for preparing bioactive peptides with various functionalities. Our objective was to evaluate the in vitro angiotensin converting enzyme (ACE) inhibitory activity of tilapia protein hydrolysates and its corresponding fractionates. Tilapia protein was alkali-solubilised at pH 11.0 and recovered at pH 5.5 to obtain a stable substrate. This substrate was hydrolysed using two enzymes, Cryotin-F or Flavourzyme, to 7.5% and 25% degree of hydrolysis (DH). The hydrolysates were ultra-filtered into three fractions: >30 kDa fraction, 10–30 kDa fraction, and <10 kDa fractions. Both hydrolysates and fractionates were tested for ACE inhibition. Results showed that both Cryotin and Flavourzyme hydrolysates with 25% DH gave maximum ACE inhibitory activity. Low MW peptides showed higher ACE inhibition than high MW peptides. The inhibitory activity of fractionates was lower than that of the corresponding hydrolysates, possibly due to separation and removal of synergistic peptides by ultrafiltration. Amongst fractionates, all the 7.5% DH Cryotin fractions and 25% DH Flavourzyme fractions exhibited optimum % ACE inhibition. The results of this research could be used for optimising enzyme parameters to obtain peptides from tilapia with optimum in vitro ACE inhibitory activity.     

Angiotensin-converting enzyme (ACE) inhibitory peptides from fermented sausages inoculated with Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201

Natural ACE inhibitory peptides derived from food are considered to be an effective supplement for lowering blood pressure. This study investigated the effects of Lactobacillus plantarum CD101 and Staphylococcus simulans NJ201 on proteolysis and the sequence composition of ACE inhibitory peptides in fermented sausages. The ACE inhibitory activity of the inoculated group reached the maximum values on day 35 during the production of fermented sausages. A more positive effect in the hydrolysis of the sarcoplasmic and myofibrillar protein was observed in the inoculated group through sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The free amino acid content of the inoculated group was 865.21 ± 12.55 mg/100 g, which was significantly higher (P < 0.05) than that of the control group. The molecular weight distribution showed a significant increase (P < 0.05) in peptides with a molecular weight less than 1 kDa in the inoculated group. A total of thirty-four peptides with high hydrophobicity were identified by liquid chromatograph-mass spectrometer/mass spectrometer (LC–MS/MS). Furthermore, the peptides were isolated and purified using gel filtration chromatography (GFC) and reversed-phased high-performance liquid chromatography (RP-HPLC). Eleven novel peptides were identified by Nano-LC-ESI-MS/MS, in which VALSLSRP with X-Pro structure exhibited the highest ACE inhibition rate (75.36 ± 2.45%). These results indicated that mixed starters are conducive to protein degradation and the formation of peptides with high ACE inhibitory activity, especially peptides less than 1 kDa, and could be used as a functional material for antihypertensive drugs.