【作者】 庞广昌；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2007， 博士

【摘要】 牛乳酪蛋白经过适当的蛋白酶和适当的水解条件水解后具有抗菌活性,经过乙醇沉淀——透析——Sephadex G-25层析分离等程序获得一种富含酪氨酸的多肽。一系列的实验结果表明,这种多肽在体外表现出免疫刺激作用,并具有抗革兰氏阴性和阳性菌等广谱的抗菌作用。因其含有异常多的酪氨酸,故命名为富含酪氨酸的免疫活性多肽,缩写为:Trpi。对Trpi的氨基酸组分进行了分析,结合蛋白酶切位点和富含酪氨酸的特点对其一级结构进行了推测,并进行了χ~2-检验,结果证明了这种推断的正确性。这种富含酪氨酸具抗菌作用的免疫活性肽对应于κ-酪蛋白的11-63氨基酸残基片段(κ-CNf11-63),由53个氨基酸组成。数据库查询结果证明属于一种新的免疫活性肽和抗微生物肽。对其理化特性进行了研究,发现其具有热稳定性、带有较多的阳离子、等电点和乳铁蛋白相似等特性。在此基础上,对其抗菌机理进行了较为系统的研究,为进一步开发为食品防腐剂、口服抗菌药物或免疫增强功能性食品打下了理论和实验基础。本项研究还对其水解和纯化工艺进行了优化,获得了适合于放大规模制备Trpi的工艺路线和参数。研究结果显示:用胰蛋白酶水解牛乳酪蛋白制备Trpi的水解条件是:牛乳酪蛋白先在80℃条件下溶解,进行10min的适度热变性,然后再在45℃,pH=8,底物浓度为9.0%,酶与底物比为1∶120(U/mg)的条件下,酶解90min,所得Trpi抗菌活性最好。树脂NKA-8对Trpi有较好的吸附和解吸附效果,在上柱浓度为0.6mg/mL,上柱液的pH值为7.2,4倍树脂床体积的50%乙醇以1.5BV/h的流速进行洗脱即可基本将Trpi从NKA-8树脂上解吸下来;经等电聚焦电泳测定,Trpi的等电点为8.61。Trpi抗菌机制的初步研究结果为:Trpi有可能取代了细胞壁表面的镁离子,然后与负电性的脂多糖(LPS)紧密结合或者中和细胞壁上某一区域的电荷,导致细胞壁结构变形,Trpi穿过细胞壁,然后在细胞膜上打孔,改变细菌细胞膜的通透性而导致靶细胞死亡。此外,本课题还通过软件TheSwiss-PdbViewer(v.3.7)根据Trpi序列各组成、氨基酸之间潜在的键合力、非键合力、静电引力、扭矩等参数,结合Trpi的抗菌机制,对Trpi空间结构进行了模拟。更多还原

【Abstract】 The proteolytic solution of bovine casein which has antimicrobial and immunostimulating activity was found and a novel immunopeptide named Tyr-rich polypeptide was separated from the solution by the process of ethanolprecipitation——dialysis——Sephadex G-25 chromatography, the results of A serialexperiments showed that Trpi has antibacterial action to Gram-positive and Gram-negative bacteria, such as E.coli, S.aureus, and enhance lymphocyte cell mediated immunity markedly in vitro.The amino acid composition of the peptide was also tested, and its position in the bovine milk casein was determined with the help of the site-specific character of trypsin. The result of x~2 -test proved the deduce to be correct; The result showed that this immunopeptide was a novel peptide that hadn’t been reported comparing with the known antibacterial and immunopeptide sequences. This novel immunopeptide was composed of 53 amino acids, and it was the fragment of the bovine milkκ-CNf11-63. Some of the biochemical properties of the Tyr-rich polypeptide were tested, such as its high thermostablility, high contents of Tyr, pI and aminal acid sequence. On the basis of these works, the elucidation of the mechanisms of the action of Trpi was able to provide a basis for research and development of functional sanitarian food and oral medicament, more efficient methods to prepare immunopeptides are also being tried, so that a scale up method had established.The results of the study showed that the optimum enzyme hydrolysis conditions of enzymolysis of casein catalysed by trypsin were that dissolving casein in water at 80℃to denaturalize the casein moderately first, then trypsinized at 45℃, substrate 9%(w/v), enzyme: substrate ratio [E/S] l:120(U/mg), hydrolyzed for 90 minutes. NKA-8 , which has better adsorption and desorption function, was the most appropriate resin for the purification of Trpi. The concentration of 0.6mg/ml, pH 7.2 and being eluted with 4BV 50% ethanol in 1BV/h speed were the optimal conditions. The result of IEF showed that pI of Trpi is 8.61. Trpi was likely to displace the magnesium ions on the surface of the cellular wall, then either bind tightly to the negatively charged membrane lipopolysaccharide (LPS) or neutralize the charges over an area of the cellular wall, subsequently distorting the cellular wall structure. Once this is accomplished, Trpi can drill through the cellular wall. In addition, the three-dimensional structure of Trpi was studied in this thesis. The three-dimensional structure of Trpi was modeled according as the parameter of amino acid residue, formed Trpi, computed by The Swiss-PdbViewer(v.3.7), such as bonds, nonbonded, electrostatic constraint, torsion and so on.更多还原