【作者】 闫凤；

【导师】 孟庆雄；

【作者基本信息】 昆明理工大学 ， 生物化工， 2009， 硕士

【摘要】 我国的核桃资源非常丰富,其中所含的核桃蛋白是一种极优质的植物蛋白,除用于初级食品的制作外,还可作为食品基料或食品添加剂使用,应用前景广阔。目前,具有高附加值的核桃多肽已成为核桃蛋白的主要加工方向之一。生物活性肽是现今研究的热点活性物质之一,具有多种生理活性,而且与其它蛋白质类药物相比,它被机体吸收的效率更高,因此受到越来越多的关注。目前的研究主要是利用来源丰富的蛋白质通过蛋白酶水解来制备出大量具有活性的多肽。本论文选取核桃蛋白作为蛋白质来源,选用胰蛋白酶、胃蛋白酶和木瓜蛋白酶三种常用水解酶对核桃蛋白进行酶解,得到一系列酶解产物,并对酶解产物的活性进行了筛选。首先参考各种文献资料,确定三种蛋白酶的酶解条件,其中胰蛋白酶酶解条件为：加酶量E/S为1×104U/g,pH8.0,温度55℃,酶解时间8h；胃蛋白酶酶解条件为：pH 2,温度设定为37℃,加酶量E/S为1×104U/g,酶解时间8h；木瓜蛋白酶酶解条件为：pH8.0,温度55℃,加酶量E/S为1×104U/g,酶解时间8h。根据选定的酶解条件对核桃蛋白进行酶解,得到三种酶解产物：胰蛋白酶酶解产物(Y)、胃蛋白酶酶解产物(W)和木瓜蛋白酶酶解产物(M),并通过SDS-PAGE电泳对酶解产物的分子量进行了初步研究,发现三种酶解产物均为分子量在15KD以下的小肽,为后续的活性研究准备好了样品。接着对三种酶解产物(Y、W和M)进行了酶活性、血液活性、抑菌活性和抗肿瘤活性的筛选。结果表明：Y、W、M均具有一定的丝氨酸蛋白酶活性,无丝氨酸蛋白酶抑制活性；三种酶解产物中只能确定W具有一定的纤维蛋白原水解活性,并很有研究价值：W能水解纤维蛋白原的Aα、Bβ链；三种酶解产物均无磷脂酶A2活性及磷脂酶A2抑制活性；Y、W、M均具有一定的溶血活性,其中Y的溶血活性较高,溶血率高达199.33%,W和M的溶血率分别为47.49%和27.09%；三者均无红细胞凝集活性及血小板聚集活性；Y和M不具有血小板聚集抑制活性,而W的该活性较强,最大抑制率为70%左右；Y、W、M均对大肠杆菌、枯草芽孢杆菌、金黄色葡萄球菌、蜡样芽孢杆菌、巨大芽孢杆菌和白色念球菌六种细菌无抑制作用；Y、M对Hela细胞的抑制效果不明显,抑制率低于20%,W对Hela细胞的抑制率可达到75%,抑制效果明显；三者对PC-12细胞的抑制率都不超过15%；W对MCF-7细胞的抑制率为40%,其它两者不明显；Y、W、M均对MDA-435细胞具有一定的抑制作用,抑制率分别为56.18%、71.45%和26.56%；三者对Hep G2细胞几乎没有抑制作用。最后对酶解产物的丝氨酸蛋白酶活性进行了初步研究,排除了丝氨酸蛋白酶水解活性中的多种干扰因素,包括反应体系、酶解底物等,基本确证了丝氨酸蛋白酶活性的存在；初步确定了三种蛋白酶酶解核桃蛋白的最佳酶解时间；而且对M和W进行了初步的分离纯化,M分别选用Sephadex G-75、Sephadex G-25凝胶层析,W选用了Sephadex G-25凝胶层析和Sephadex C-25离子交换层析两种方法,结果得到了12个组分：MG-75Ⅰ、MG-75Ⅱ、MG-25Ⅰ、MG-25Ⅱ、MG-25Ⅲ、MG-25Ⅳ、WG-25Ⅰ、WG-25Ⅱ、WC-25Ⅰ、WC-25Ⅱ、WC-25Ⅲ、WC-25Ⅳ,但在对这12个组分进行丝氨酸蛋白酶活性的筛选过程中发现,各组分均不具有该活性。更多还原

【Abstract】 Walnut resources are very rich in China. Walnut protein is one of them, which is an extremely high-quality plant protein resource. In addtion to the primary food production, walnut protein can be used as food base material or food additives. Nowadays, high value-added walnut protein peptide is a main direction of processing. Bioactive peptide is one of the most popular active substances in current study, which has a variety of biological activities. Moreover, bioactive peptides, compared with other proteinic drugs, can be absorbed by the human body more efficiently and therefore gain more and more attention. The current main method of active peptides preparing is performed by hydrolysing a rich source of protein with protease.In this paper, walnut protein was selected as a protein source, three kinds of common enzymes:trypsin, pepsin and papain were selected for hydrolyzation of walnut protein, then a series of hydrolysates were obtained and activities of hydrolysates of enzymes digestion were screened.Firstly, three kinds of protease digestion conditions were identified refered to a variety of literature, trypsin digestion conditions were:the amount of enzyme E/S is 1×104U/g, pH8.0, temperature 55℃, hydrolysis time is 8h; pepsin hydrolysis conditions:pH2.0, the temperature set to 37℃, the amount of enzyme E/S is1×104U/g, hydrolysis time is 8h; papain hydrolysis conditions:pH8.0, temperature 55℃, the amount of enzyme E/S is 1×104U/g, hydrolysis time is 8h. According to the selected enzyme hydrolysis conditions, walnut proteins were hydrolysed and three kinds of hydrolysates were obtained:trypsin hydrolysates (Y), pepsin hydrolysates (W), and papain hydrolysates (M), and through SDS-PAGE, a preliminary study of the molecular weight of hydrolysates was conducted, which suggested that three kinds of hydrolysates were small peptides with the molecular weight of below 15KD, by doing this, the samples of the follow-up activity study were ready.Enzymatic activity, blood activity, antibacterial activity and anti-tumor activity of the three kinds of hydrolysates (Y, W and M) were screened. The results showed that:Y, W, M all have a certain degree of serine protease activity, no serine protease inhibitory activity; In these three kinds of hydrolysates, W has fibrinogen hydrolysis activity, while the other two are uncertain. The fibrinogen hydrolysis activity of W is of great research value:W can hydrolyze fibrinogen Aα, Bβchain; All three kinds of hydrolysates have no phospholipase A2 activity and also no phospholipase A2 inhibitory activity; Y, W, M all have certain hemolytic activity, in which the hemolytic activity is higher in Y, hemolysis rate is as high as 199.33%, while for W and M, hemolysis rates are respectively 47.49% and 27.09%; none of them has hemagglutination activity and platelet aggregation activity; Y, and M do not have platelet aggregation inhibitory activity, while the W, has strong activity with maximum inhibition rate of about 70%; Y, W, M all have no inhibitory effect on E. coli, Bacillus subtilis, Staphylococcus aureus, Bacillus cereus, Bacillus megaterium and Monilia albican bacteria; the inhibitory effect of Y, M on Hela cells is not obvious, whice has a inhibition rate of less than 20%, while the inhibitory rate of W on Hela cells could reach 75% and its effect is obvious; the inhibitory rates of all three on PC-12 cells are no more than 15%; the inhibition rate of W on MCF-7 cells is 40% while the other two is not obvious; Y, W, M all have the certain inhibition effect on MDA-435 cells with a inhibitory rates of 56.18%,71.45% and 26.56% respectively; all three almost have no inhibitory effect on Hep G2 cells.Finally, the serine protease hydrolysis activities of the hydrolysates were elementrily studied, numbers of interfering factors in the hydrolysates, including reactive systems, walnut protein and the enzymes, were excluded. At last, the existence of serine protease activity were confirmed; and a preliminary separation and purification for the M and W were carried out, Sephadex G-75, Sephadex G-25 gel chromatography were selected for the M, Sephadex G-25 gel chromatography and Sephadex C-25 ion-exchange chromatography were selected for the W, the 12 components obtained are MG-75Ⅰ, MG-75Ⅱ, MG-25Ⅰ, MG-25Ⅱ, MG-25Ⅲ, MG-25Ⅳ, WG-25Ⅰ, WG-25Ⅱ, WC-25Ⅰ, WC-25Ⅱ, WC-25Ⅲ, WC-25Ⅳ, however none of these 12 compositions has serine protease activity.更多还原