核酸酶P1的初步分离纯化

论文编号:ZY113    论文字数:13293,页数:25

摘要：5’—磷酸二酯酶是—类可将核酸定向水解为5’—核苷酸的酶，核酸是生命之源,核苷酸作为核酸的基本组成单元,是参与新陈代谢不可缺少的物质,而5’—核苷酸用途广泛, 它具有强烈的增鲜作用，是—类很有价值的食品增鲜剂，同时也是生产多种药物的原料。目前国外从核糖核酸生产5’—核苷酸的工艺大多应用桔青霉的核酸酶P1。本文主要是对纯酶制剂的制备纯化过程做了初步的研究。本人采用桔青霉发酵法生产5’—磷酸二酯酶，接着将发酵液进行多个的纯化步骤来得到较纯的酶制剂——核酸酶P1。经过85小时的发酵，滤去菌种，得到粗酶液，在4℃温度下纯化过程，它包括第—次硫酸铵沉淀、热处理、第二次硫酸铵部分沉淀、丙酮沉淀、葡聚糖凝胶G—75层析、冷冻干燥浓缩酶液等六个环节，将酶液进行初步的分离纯化，得到较纯的目标物，其中，每个环节通过测定酶活力并且进行SDS—PAGE胶分离的方式检验纯化效果。试验数据中可以得出：实验方案还是能利用桔青霉发酵生产得到含5′- 磷酸二酯酶的液体，并将5′- 磷酸二酯酶进行个初步纯化。试验中菌种的无菌培养，含目标酶液的保存，丙酮浓度的控制是试验的关键环节。
关键词：桔青霉； 5’—磷酸二酯酶；5’—核苷酸；酶活力；纯化
 
ABSTRAT：5’-Phosphodiesterase is a kind enzyme which can directional hydrolyze the nucleic acid to be 5’-Nucleotides, The nucleic acid is source of the life,.the nucleotide .as nucleic acid the basic composition unit, participates in the metabolism essential matter, 5’-Nucleotides which use widely can intensely increases the fresh ,is kind of very valuable material for food to increases the fresh, also is raw materal of many kinds of medicines. At present, the craft of produces 5 ' -nucleotide from the ribonucleic acid (RNA) is using  Penicillium citrinum to produce nuclease P1 .This article mainly about the preliminary research on the pure enzyme preparation process. I use the Penicillium citrinum to produce 5’-Phosphodiesterase enzyme, then the fluid was carried on many pure steps ,then gain comparatively pure enzyme - nuclease P1. After 85 hours fermentation, filtrate the thalli out, obtain the crude fluid of enzyme, purefy in 4 ℃, the process of  purification including the first  deposit by (NH4)2SO4, the heat treatment, the second  partial deposit by (NH4)2SO4, deposit by the acetone, Chromatography on sephadex G – 75, freeze-drying and so on. six links obtain a purer enzyme.  each process carries on SDS-PAGE rubber separation way to test the purification effect .According to the results, the methord  can use  Penicillium citrinum to produce  liquid which  contain  nuclease P1 .In the process of experimants, the step of asepsis , preservation of liquid which  contain  nuclease P1 and  control the contents of  acetone  are important .
 Key word:  Penicillium citrinum; 5’-Phosphodiesterase; 5 ' -nucleotide; Enzyme activity;  Purification
 
 目录
中文摘要 I
英文摘要 II
目录 III
1．绪论 1
1.1  研究 5′- 磷酸二酯酶的背景和意义 1
1.1.1  5′- 磷酸二酯酶的定义 1
1.1.2   5′- 磷酸二酯酶的作用方式 2
1.1.3   5′-磷酸二酯酶的研究意义和前景 2
1.2  5′- 磷酸二酯酶的制备方法 3
1.2.1  提取法 3
1.2.2  发酵法 4
1.3  本课题的基本思路 4
1.3.1  实验的目的 4
1.3.2  实验流程 5
2．材料和方法 5
2.1 材料： 5
2.2 实验方法 6
2.2.1  菌种的活化 7
2.2.2  发酵培养 7
2.2.3  纯酶制剂的制备 7
2.2.4  酶活力的测定 13
3．结果与讨论 15
3.1 核酸酶P1的发酵 15
3.2 葡聚糖凝胶G-75层析纯化蛋白 15
3.3 各步骤纯化蛋白聚丙烯酰胺凝胶电泳 16
3.4 各步酶纯化试验结果 17
4．结束语 19
致谢 21
参考文献 22