实验海洋生物学重点实验室知识库

近年来，螺旋藻、小球藻、莱茵衣藻、拟微球藻等多种微藻相继被批准成为新食品原料。微藻富含蛋白质、脂质、多糖、维生素等多种营养物质，因此在养殖、食品、医药、能源等多个领域都具有很大的应用潜力。当前我国年产微藻干粉超过了一万吨，产值超过20亿美元，占世界总产量的一半以上。但是这些微藻多以藻粉、藻片的形式直接消费，缺少精深加工工艺，产品附加值低，因而限制了微藻产业的发展。此前已有研究表明直接服用微藻和微藻制品具有降血压、降血糖、抗氧化和免疫调节等多种生物活性，生物活性肽是这些生物活性的物质基础之一。生物活性肽具有安全无毒、生物活性好、易于吸收等特点。目前从多种来源中鉴定到了具有抗氧化、抗菌、抗肿瘤、降血糖、降低体重、免疫调节和降血压的生物活性肽。其中，天然来源的ACE抑制肽被认为具有解决ACE抑制剂类药物的副作用的潜力，因而受到广泛研究。目前仅从螺旋藻和小球藻中鉴定到了25种ACE抑制肽，微藻ACE抑制肽还有很大的开发潜力。另一方面，目前我国高血压发病率逐渐上升，患病人数超过2.7亿人，还有众多前高血压人群缺乏有效的干预手段以保持正常血压。因此，开发微藻ACE抑制肽有助于进一步明确微藻功能，提高精深加工程度，实现精准应用，扩大市场需求，推动微藻产业发展。

本论文以螺旋藻（Spirulina platensis, Spirulina maxima.）、小球藻（Chlorella pyrenoidosa.）、拟微球藻（Nannochloropsis sp.）和莱茵衣藻（Chlamydomonas reinhardtii）等重要经济微藻为研究对象，对其蛋白提取方法及蛋白酶解物制备方法进行探究，并采用活性导向的分离纯化、肽组学方法对微藻蛋白酶解物所含降压肽进行研究。更进一步地，对鉴定得到的ACE抑制肽进行基于氨基酸替代的序列优化以提升其活性和稳定性。最后，利用分子动力学模拟对ACE-肽复合物的相互作用模式进行分析，以期从分子层面阐释微藻ACE抑制肽的抑制机理和构效关系。本论文取得的主要研究结果如下：

（1）对比不同的蛋白提取方法和微藻蛋白酶解条件发现，对于小球藻和拟微球藻，采用蛋白酶直接酶解可以得到更高的蛋白/蛋白酶解物提取率。最适pH为碱性的蛋白酶如胰蛋白酶、α-糜蛋白酶和碱性蛋白酶等，更适合用于制备微藻蛋白酶解物。针对四种微藻分别选取了最适蛋白酶，制备得到的相应微藻蛋白酶解物在100 mg/kg/d的剂量下可以将原发性高血压大鼠（SHR）的收缩压降低14.08~41.25 mmHg，具有显著的体内降压作用。

（2）为了探究不同蛋白酶制备得到的酶解物的差异，分别采用蛋白酶K、α-糜蛋白酶、以及加热变性联合α-糜蛋白酶对拟微球藻进行酶解，并进行肽组学分析。结果表明，酶解物ACE抑制活性显著提高，表明酶解产生了更多的ACE抑制肽。三种处理方式制备的寡肽的序列多样性升高，等电点更低，寡肽中必需氨基酸含量也有所提升。三种处理方式还通过改变寡肽的疏水性和C/N末端氨基酸产生了更多的ACE抑制肽。三种处理方式中，加热变性联合α-糜蛋白酶酶解处理得到的寡肽多样性最高，同时预测降压肽的数量也最多。因此选用合适的处理方式是高效制备微藻降压肽的关键，这些发现为深入理解微藻蛋白的营养价值和开发具有生物活性的拟微球藻寡肽提供了参考。

（3）采用多种色谱技术对螺旋藻、小球藻和莱茵衣藻酶解物进行分离纯化，经过虚拟筛选和实验评价从螺旋藻、小球藻和莱茵衣藻中鉴定到了48个新型ACE抑制肽，其中6个ACE抑制肽具有微摩尔级ACE抑制活性，分别为TVLYEH（SpH-6，IC₅₀=2.88 μmol/L）、IDYRY（ID-5，IC₅₀=18.54 μmol/L）、LVAKA（LV-5，IC₅₀=26.66 μmol/L）、LKKAP（LK-5，IC₅₀=36.19 μmol/L）、PGLRP（PG-5，IC₅₀=44.78 μmol/L）和LQAGGLF（SpH-7，IC₅₀=66.83 μmol/L）。螺旋藻降压肽SpH-6和SpH-7为竞争性抑制，而小球藻和莱茵衣藻来源的降压肽LK-5、LV-5、PG-5和ID-5均为非竞争性抑制。此外，计算机模拟消化和体外模拟消化表明这些肽对消化酶不稳定，但是经过消化酶酶解后产生的小肽同样具有ACE抑制活性。质谱鉴定发现PG-5经模拟消化后产生PGL和RP的ACE抑制活性甚至优于PG-5；同样，LV-5的模拟消化产物经质谱鉴定发现一个序列新颖的ACE抑制肽VAK其IC₅₀为85.42 μmol/L。以SHR为模型评价几种微藻ACE抑制肽的体内降压作用，发现SpH-6和ID-5均可显著降低SHR的收缩压，具有显著的体内降血压作用。为期28天的连续给药和血压监测发现，SpH-6可以起到长期的降压效果。

（4）小球藻五肽LV-5序列中含有两个丙氨酸残基（Ala3、Ala5），而且丙氨酸扫描确定Leu1、Lys4是其发挥ACE抑制作用的关键残基，因此，以LV-5为例进行构效关系研究。采用Val2、Ala3和Ala5饱和突变和基于经验原则的氨基酸序列优化两种策略设计了35个小球藻系列五肽，其中LRAKA （LR-5，IC₅₀ = 0.35 μM）和LRAKP（RP-5，IC₅₀ = 0.93 μM）的活性相较LV-5分别提升了约76倍和26倍，腹腔注射LR-5使SHR的收缩压和舒张压产生最大降幅分别为20.5 mmHg和17.7 mmHg。采用D型氨基酸取代的方式对优化后的降压肽进行修饰以增强其稳定性。其中RP-5的首位亮氨酸被取代为D型氨基酸后，ACE抑制活性降低了约10倍，但是对消化道蛋白酶的稳定性显著提升。体内实验表明，RP-5不具有降血压作用，但是D型氨基酸取代后RP-5的体内降压活性显著增强，其对SHR的收缩压的降低幅度可达21.00 mmHg。这可能是由于D型氨基酸取代增强了RP-5的代谢稳定性，进而提高了其体内降血压作用。以上结果为开发抗高血压寡肽药物提供了重要参考。

（5）通过分子动力学模拟对LR-5发挥ACE抑制活性的分子基础进行研究。采用主成分分析得到了LR-5、LV-5和ACE结合的代表性构象，构象分析结果表明在LR-5和LV-5与ACE的结合模式存在差异。而氢键分析和结合自由能分析显示在LR-5和ACE结合过程中Glu376、Asp377和Asp453具有重要作用。分析Glu376和Asp453之间的距离显示，在LR-5和ACE结合过程中，这两个残基相互靠近形成负电荷位点并与LR-5中带正电的精氨酸残基产生稳定的相互作用，这可能是LR-5发挥其强力ACE抑制活性的关键。

目前对于微藻寡肽的研究仍然不够充分，同时，开发安全有效的ACE抑制剂仍然十分必要。本研究表明，螺旋藻、小球藻、拟微球藻和莱茵衣藻具有较高的营养价值，同时是强效ACE抑制肽的优良来源。对微藻ACE抑制肽的序列和结构进行优化，提升了微藻ACE抑制肽活性和稳定性，并对ACE抑制肽的构效关系进行了系统探讨。更进一步的分子动力学模拟则揭示了新的肽-ACE相互作用位点。以上结果拓展了对于微藻降血压肽的认知，为微藻高值化利用提供了理论依据，同时为开发更加安全高效的ACE抑制剂提供了理论基础。

In recent years, a variety of microalgae such as Spirulina, Chlorella, Chlamydomonas reinhardtii and Nanocloropsis sp. have been approved as new food ingredients. Microalgae are rich in proteins, lipids, polysaccharides, vitamins and other nutrients, so they have great potential for application in many fields such as aquaculture, food, medicine and energy. At present, China's annual production of dried microalgae powder exceeds 10,000 tonnes, with an output value of more than 2 billion US dollars, accounting for more than half of the world's total output. However, these microalgae are mostly consumed directly in the form of algal powder and algal tablets, lack of deep processing technology and low value-added products, thus limiting the development of the microalgae industry. Previous studies have shown that direct consumption of microalgae and microalgae products have a variety of biological activities such as lowering blood pressure, lowering blood sugar, antioxidant and immune regulation, and bioactive peptides are one of the material bases of these biological activities. Bioactive peptides are safe, non-toxic, biologically active and easily absorbed. Bioactive peptides with antioxidant, antimicrobial, antitumour, hypoglycaemic, body weight reduction, immunomodulation and blood pressure lowering properties have been identified from a variety of sources. Among them, ACE-inhibiting peptides from natural sources have been widely studied as they are believed to have the potential to address the side effects of ACE-inhibitor drugs. Currently, only 25 ACE inhibitory peptides have been identified from Spirulina and Chlorella, and there is still a great potential for the development of microalgae ACE inhibitory peptides. On the other hand, the incidence of hypertension is gradually increasing in China, with more than 270 million people suffering from the disease, and there are still many pre-hypertensive people who lack effective interventions to maintain normal blood pressure. Therefore, the development of microalgae ACE inhibitory peptide can help to further clarify the function of microalgae, improve the degree of deep processing, realise the precise application, expand the market demand and promote the development of microalgae industry.

Microalgae are rich in nutrients, fast growing and safe, and a variety of microalgae have been used as common food or new food ingredients. In this work, economically important microalgae such as Spirulina sp., Chlorella sp., Nannochloropsis sp. and Chlamydomonas reinhardtii were used as research objects to investigate the protein extraction method and the preparation of protein hydrolysate, and the activity-oriented separation and purification method and peptidomics methods were used to incestigate the antihypertensive peptides contained in the protein hydrolysate of microalgae. Furthermore, the identified ACE inhibitory peptides were subjected to sequence optimization based on amino acid substitutions to enhance their activity and stability. Finally, molecular dynamics simulation was used to study the peptide ACE to elucidate the conformational relationship of ACE inhibitory peptides at the molecular level. The main results of this study are as follows:

(1) Comparison of different protein extraction methods and hydrolysis conditions for microalgae revealed that for Chlorella and Nannochloropsis sp., which are difficult to extract proteins, direct digestion by protease resulted in higher protein/hydrolysate extract rate. In addition, proteases with optimal alkaline pH such as trypsin, α-chymotrypsin and alkaline protease were found to be more suitable for the preparation of microalgal protein hydrolysate. The protein hydrolysate obtained from the microalgaes could reduce the systolic blood pressure of SHRs by 14.08 to 41.25 mmHg at an oral dose of 100 mg/kg per day.

(2) Three methods, protease K, α-chymotrypsin, and heat denaturation combined with α-chymotrypsin digestion, were used to prepare the enzymatic digests of Nanochloropsis sp. And the peptide in the hydrolysates were analyzed by peptidomics. The results showed that the ACE inhibitory activity of the hydrolysates was significantly increased, indicating that more ACE inhibitory peptides were produced after enzymatic digestion. The three treatments improved the sequence diversity, lowered the isoelectric point, and increased the content of essential amino acids in the generated peptides. The three treatments also changed the hydrophobicity and C/N-terminal amino acids of the peptides, which in turn produced more ACE inhibitory peptides. Among the three treatments, heat denaturation combined with α-chymotrypsin digestion yielded the highest diversity of peptides as well as the highest number of predicted ACE inhibitory peptides. Therefore, the selection of the appropriate treatment is the key to the efficient preparation of microalgae antihypertensive products, and these results provide an important reference for a deeper understanding of the nutritional value of protein sources and the development of biologically active peptides.

(3) The protein hydrolysates of Spirulina sp., Chlorella sp., and Chlamydomonas reinhartii were separated and purified by various chromatographic methods, and 48 novel ACE inhibitory peptides were identified after virtual screening and experimental evaluation, among which six ACE inhibitory peptides possessed micromolar inhibitory activities, namely, TVLYEH （SpH-6, IC₅₀=2.88 μmol/L）, LQAGGLF （SpH-7, IC₅₀=66.83 μmol/L）, LKKAP （LK-5, IC₅₀=36.19 μmol/L）, LVAKA （LV-5, IC₅₀=26.66 μmol/L）, PGLRP （PG-5, IC₅₀=44.78 μmol/L）, and IDYRY （ID-5, IC₅₀=18.54 μmol/L）. The inhibition mode against ACE of SpH-6 and SpH-7 from Spirulina was competitive, whereas the inhibition mode of LK-5, LV-5, PG-5, and ID-5 from Chlorella and Chlamydomonas reinhardtii were non-competitive. In addition, in-silico enzyme digestion and in vitro simulated digestion showed that these peptides were unstable to gastrointestinal proteases, but the fragments produced after simulated gastrointestinal digestion still had ACE inhibitory activity. Among them, the PGL and RP fragments produced by PG-5 after simulated digestion had even better ACE inhibitory activity than PG-5; the VAK fragment produced by LV-5 after simulated digestion was also an novel ACE inhibitory peptide （IC₅₀=85.42 μmol/L）. In vivo evaluation of antihypertensive activity showed that SpH-6 and ID-5 could significantly reduce the systolic and diastolic blood pressure of SHRs.

(4) Structural optimization of the Chlorella pentapeptide LV-5 discovered a nanomolar ACE inhibitors. Leu1, Val2 and Lys4 in LV-5 were identified as essential for LV-5 to exert ACE inhibition by virtual alanine scanning. Subsequently, a series of peptides were designed by saturating mutations of Val2, Ala3 and Ala5 and by an empirical amino acid sequence optimization strategy, including LRAKA （LR-5, IC₅₀=0.35 μmol/L） and LRAKP （RP-5, IC₅₀=0.93 μmol/L） showed approximately 26-fold and 76-fold enhanced activity compared to that of LV-5, respectively. Intraperitoneal injection of LR-5 produced maximum reductions in systolic and diastolic blood pressure of 20.5 and 17.7 mmHg, respectively. The optimized antihypertensive peptides were further modified by D-type amino acid substitutions to increase their stability. Substitution of the first leucine of RP-5 with a D-type amino acid resulted in an approximately 10-fold decrease in activity but a significant increase in stability against gastrointestinal proteases. In vivo experiments showed that although no significant antihypertensive effect was observed for RP-5, the in vivo antihypertensive activity of RP-5 after D-type amino acid substitution was significant and the reduction in systolic blood pressure in SHR could be as much as -21.00 mm Hg. This might be attributed to the enhancement of the stability of RP-5 by the substitution of the D-type amino acid. The above results provide an important reference for the development of antihypertensive peptide drugs.

(5) The molecular basis of the ACE inhibitory activity exerted by LR-5 was investigated by molecular dynamics simulation. The representative conformations of the binding of LR-5 and LV-5 with ACE were obtained by principal component analysis （PCA）, and the results of the conformational analysis showed that during the binding of LR-5 and ACE, the conformation of the active site of ACE was changed. Hydrogen bonding analysis and binding free energy analysis showed that Glu376, Asp377 and Asp453 played important roles in the binding process of LR-5 and ACE. The distance analysis between Glu376 and Asp453 showed that these two residues got closer to each other and formed a negatively charged site during the binding process and stably interacted with the positively charged arginine residue of LR-5, which may be the key to the potent ACE inhibitory activity of LR-5.

Although microalgae have gradually come to the public's attention as a food ingredient, research on microalgal peptides is still relatively scarce. On the other hand, there is still an urgent need for safe and effective ACE inhibitor-based antihypertensive drugs. This study shows that microalgae have excellent nutritional value and are also an excellent source of potent antihypertensive peptides, which provides a theoretical basis for the application of microalgae in the development of food, health food and drugs. Sequence optimization and structural modification of the peptides enhanced the activity and stability of the identified ACE inhibitory peptides, and molecular dynamic simulation identified a novel binding sites in the ACE active centers, which provides a new direction for the design of ACE inhibitory peptides with excellent activity and novel structure.

第1章 绪论... 1

1.1 微藻及其活性肽研究进展... 1

1.1.1 微藻的研究现状... 1

1.1.2 微藻蛋白概况... 2

1.1.3 微藻活性肽研究进展... 3

1.1.4 ACE抑制肽的序列结构特征... 8

1.2 高血压... 9

1.2.1 高血压概况... 9

1.2.2 高血压的病因、血压调控机制与高血压的控制方法... 10

1.3 食源生物活性肽改善高血压的研究进展... 12

1.3.1 生物活性肽概述... 12

1.3.2 生物活性肽制备方法... 13

1.3.3 生物活性肽纯化方法... 14

1.3.4 生物活性肽对肾素-血管紧张素-醛固酮系统 （RAAS） 的影响... 14

1.4 生物活性肽的优化... 15

1.4.1 氨基酸序列优化... 15

1.4.2 D型氨基酸取代... 16

1.4.3 化学修饰... 16

1.5 分子对接和分子动力学模拟在活性肽发现中的研究进展... 20

1.5.1 分子对接在活性肽发现中的研究现状... 20

1.5.2 分子动力学模拟在活性肽发现中的研究现状... 21

1.6 研究内容和研究意义... 23

第2章 四种微藻蛋白酶解物的制备及其降血压作用评价... 27

2.1 材料和仪器... 27

2.1.1 材料和试剂... 27

2.1.2 主要仪器... 28

2.2 实验方法... 28

2.2.1 微藻蛋白提取... 28

2.2.2 微藻蛋白提取物的表征... 30

2.2.3 蛋白酶筛选... 33

2.2.4 单因素实验... 34

2.2.5 响应面优化... 34

2.2.6 ACE抑制活性测定... 36

2.2.7 动物实验... 37

2.2.8 统计分析... 37

2.3 结果和讨论... 37

2.3.1 微藻化学组成分析... 37

2.3.2 微藻蛋白的提取与表征... 38

2.3.3 微藻酶解物制备工艺与响应面优化... 42

2.3.4 四种微藻蛋白酶解物的降血压潜力评价... 47

2.4 结论... 50

第3章 肽组学鉴定拟微球藻ACE抑制肽... 53

3.1 材料和仪器... 53

3.1.1 材料和试剂... 53

3.1.2 主要仪器... 54

3.2 实验方法... 54

3.2.1 拟微球藻蛋白酶酶解物制备... 54

3.2.2 拟微球藻蛋白酶解物中肽序列鉴定及定量... 54

3.2.3 肽活性预测... 56

3.2.4 ACE抑制活性测定... 56

3.2.5 寡肽的分子对接... 56

3.2.6 统计分析... 56

3.3 结果和讨论... 56

3.3.1 四个样品的体外ACE抑制活性... 56

3.3.2 酶解物肽组学概况... 57

3.3.3 寡肽来源蛋白分析... 61

3.3.4 寡肽的等电点、电荷和疏水性... 62

3.3.5 寡肽的氨基酸组成分析... 64

3.3.6 分子对接评价拟微球藻寡肽降压潜力... 66

3.4 结论... 67

第4章 螺旋藻、小球藻和莱茵衣藻降压肽的分离纯化与鉴定... 69

4.1 材料和仪器... 69

4.1.1 材料和试剂... 69

4.1.2 主要仪器... 69

4.2 实验方法... 70

4.2.1 ACE抑制活性测定... 70

4.2.2 螺旋藻蛋白酶K酶解物（SK）分离纯化... 70

4.2.3 小球藻胰蛋白酶酶解物（CT）的分离纯化... 70

4.2.4 莱茵衣藻碱性蛋白酶酶解物（CRPA）的分离纯化... 71

4.2.5 LC-MS/MS鉴定寡肽序列... 71

4.2.6 寡肽的分子对接及虚拟筛选... 72

4.2.7 寡肽固相合成... 72

4.2.8 寡肽对ACE的抑制模式... 72

4.2.9 ACE抑制肽的稳定性研究... 72

4.2.10 微藻ACE抑制肽的体内降压活性... 73

4.2.11 统计分析... 73

4.3 结果和讨论... 73

4.3.1 螺旋藻降压肽的分离、鉴定和活性评价... 73

4.3.2 小球藻降压肽的分离、鉴定和活性评价... 79

4.3.3 莱茵衣藻降压肽的分离、鉴定和活性评价... 83

4.3.4 微藻ACE抑制肽的稳定性研究... 87

4.3.5 微藻ACE抑制肽的体内降压活性... 91

4.4 结论... 93

第5章 小球藻降压肽序列优化、构效关系以及活性评价... 95

5.1 材料和仪器... 95

5.1.1 材料和试剂... 95

5.1.2 主要仪器... 95

5.2 实验方法... 95

5.2.1 LV-5的序列优化... 95

5.2.2 优化后候选肽的虚拟筛选... 96

5.2.3 ACE抑制活性测定... 96

5.2.4 ACE抑制肽消化道稳定性评价... 96

5.2.5 体内降压活性评价... 96

5.2.6 LR-5和RP-5的结构优化... 96

5.2.7 统计分析... 97

5.3 结果与讨论... 97

5.3.1 丙氨酸扫描确定关键氨基酸残基... 97

5.3.2 LV-5饱和突变... 98

5.3.3 LV-5经验原则序列优化... 99

5.3.4 LV-5衍生肽虚拟筛选和ACE抑制活性评价... 99

5.3.5 LR-5的体内降压活性... 101

5.3.6 LR-5的D型氨基酸取代及稳定性评价... 102

5.3.7 D型氨基酸取代后肽的体内降压作用... 104

5.3.8 基于小球藻五肽LV-5衍生肽的构效关系讨论... 105

5.4 结论... 107

第6章 分子动力学模拟探究降压肽LR-5的抑制机理... 109

6.1 实验方法... 109

6.1.1 初始肽-ACE复合物的构建... 109

6.1.2 分子动力学模拟... 109

6.1.3 统计分析... 110

6.2 结果和讨论... 110

6.2.1 分子动力学模拟过程中蛋白稳定性和氨基酸残基侧链运动... 110

6.2.2 主成分分析（PCA）和协方差分析... 111

6.2.3 LV-5和LR-5与ACE复合物的构象分析... 115

6.2.4 氢键和结合能分析... 117

6.3 结论... 121

第7章 结论与展望... 123

7.1 结论... 123

7.2 创新点... 124

7.3 展望... 125

参考文献... 127

附录... 143

致谢... 149

作者简历及攻读学位期间发表的学术论文与其他相关学术成果... 151