纳豆激酶的优化、提取分离纯化及基因克隆

发布时间：2018-06-26 12:35

本文选题：纳豆激酶 + 分离纯化　；参考：《河北联合大学》2011年硕士论文

【摘要】：心脑血管性疾病,具有“发病率高、致残率高、死亡率高、并发症多”等特性。2005年,世界卫生组织(WHO)调查表明,全世界每年约有1700万人死于心脑血管疾病。并且随着人类生活水平的提高以及生活压力的增加,而呈现年轻化的趋势,而血栓又是心脑血管疾病中致死率最高的疾病。所以,血栓的防治已是当务之急。虽然传统药物在心脑血管及血栓栓塞性疾病防治上疗效显著,但毒副作用严重;而传统大豆发酵食品不仅效果显著且安全无毒,可作为一种具有开发和利用价值的功能性预防保健食品。据文献记载,纳豆源于中国的豆豉,通过佛教由中国传入日本,日本人均寿命长的原因除了与较为优良的自然环境有关外,更重要的是其膳食结构中,发酵食品特别是消费量最大的纳豆,是日本人长寿的秘方,视为国宝级食品。寺院喜食大豆是因为其蛋白质含量高达35.3%,而由纳豆菌产出的酶,能使50%的蛋白质变为水溶性,消化率可达80%。还含氨基酸、维生素、植物性脂肪等。据科学研究表明,纳豆食品不仅调整胃肠功能明显,还具有治疗感冒、痢疾、胀气、胃肠炎、消化不良、残便、便秘等功效,还有防治糖尿病,抑制血栓的生成和溶解血栓的作用,是一种延年益寿、健康的美容食品。可以说纳豆是一种自然食品,是人类生活智慧的产物。 1987年日本的Sumi博士首次从日本传统食品纳豆中分离出一种具有强烈纤溶作用的碱性蛋白酶并命名纳豆激酶(nattokinase,NK)。它不但能直接作用交联纤维蛋白,而且还可激活体内的纤溶酶原,从而表现出很强的溶血栓作用。据报道其制品具有水解淀粉样蛋白,降低血浆纤维蛋白原的第七因子和第八因子,降低血液粘度、降血脂、降胆固醇,降血压,改善血液循环状态,维持血细胞的正常形态和功能,有利于神经损伤的再生,抑制骨质疏松症、抑制动脉粥样硬化等多种功能。由于NK具有安全性好、成本低、经口服后可迅速入血,作用时间长,胃肠道稳定性好,可由细菌发酵生产、也可由基因工程菌生产等优点,有望被开发为新一代的口服抗血栓药物用于血栓性疾病的预防和治疗。但是直到现在,纳豆激酶的来源主要是发酵的日本纳豆,因其独特的风味很多人不能接受。虽然含有纳豆激酶作为主要成分的胶囊制剂已在日本,韩国,朝鲜生产,但由于只能短期供应和高昂的代价,其应用很有限。为了使其既方便又广泛使用,人们从分子生物学角度寻找更好的表达载体和宿主菌,优化表达条件等方面做了进一步的研究,应用基因工程制品纯化出更为优质的溶栓药物,将具有更为广泛的应用前景,给临床带来新的曙光。目的本研究通过发酵液优化、盐析、层析、蛋白含量、纤溶活力测定、SDS-PAGE等技术和手段,通过传统方法提取分离纯化纳豆激酶,检测其活性。还通过分子生物学技术,提取纳豆枯草芽孢杆菌基因组DNA,设计引物,PCR克隆纳豆激酶及纳豆激酶原。选择合适的表达表达载体和宿主菌,以期应用基因工程制品纯化出更为安全优质溶栓药物,为临床应用提供理论依据。方法1.本实验通过比浊法[1]连续测定OD600值,绘制菌种生长曲线,确定纳豆菌培养条件,以便更好地应用于实际生产中。为了进一步提高酶的产量,降低发酵成本,我们采用单因素和正交实验法对发酵工艺进行优化。 2.将粗酶液经离心,40~45%盐析,CM-Cellulose离子交换层析和Sephadex G-100凝胶柱可以提取出单一酶,并用SDS-PAGE测定分子量,比较粗酶液与提取出的单一酶液的酶活力。 3.纳豆枯草芽孢杆菌基因组DNA提取后,经PCR扩增纳豆激酶及纳豆激酶原序列,并经1%琼脂糖凝胶电泳检测。为进一步构建重组质粒,并转化大肠杆菌做铺垫,后续工作有待进一步研究。结果 1.培养基成分的最优组合为MgSO4 0.02%、K2HPO4 0.02%、KH2PO4 0.01%、CaCl2 0.05%、麦芽糖3%、大豆蛋白胨3%、最适培养温度37℃、最适pH为8.0、最佳接种量为每5g黄豆接种500μL。优化发酵条件后生产的纳豆激酶,酶活由优化前的31.25IU/mL发酵液提高到158.74IU/mL发酵液,单位产量提高了约5.08倍。 2.经CM-Cellulose离子交换层析分离纳豆激酶酶,得到0.10 mol/LNaCl洗脱峰中含有活性组份,Folin-酚法测酶活为168.56 IU/m。将0.10mol/L NaCl洗脱峰中的纳豆激酶酶用Sephadex G-100凝胶过滤层析进行分离,Folin-酚法测酶活为137.16 IU/mL,并用SDS-PAGE测定它们的分子质量为28 000 Da。 3.纳豆枯草芽孢杆菌基因组DNA提取后,经PCR扩增纳豆激酶(275个氨基酸,碱基825bp)及纳豆激酶原序列(381个氨基酸,碱基1143bp),并经1%琼脂糖凝胶电泳检测得到确认。结论经液体培养基的优化后提取粗酶液,离心,40~45%盐析,CM-Cellulose离子交换层析和Sephadex G-100凝胶柱可以提取出单一酶,为减少操作过程中酶的损失,整个过程力求在4℃、无菌条件下操作。克隆了纳豆激酶及纳豆激酶原基因并回收。选择合适的表达载体和宿主菌,经多次重复表达未成功,本实验提供的方法可供以后科研工作者参考,以待进一步的研究。
[Abstract]:Cardiovascular and cerebrovascular diseases have the characteristics of "high incidence, high disability rate, high mortality, and more complications" in.2005 years. The WHO (WHO) survey shows that about 17 million people die from cardiovascular and cerebrovascular diseases every year in the world. And with the increase of human living standard and the increase of life pressure, the trend of youth is younger and thrombosis. It is also the most fatal disease in cardiovascular and cerebrovascular diseases. Therefore, the prevention and treatment of thrombus is the urgent matter. Although the traditional medicine has significant effect on the prevention and treatment of cardiovascular and thrombotic embolism, the toxic and side effects are serious, but the traditional soybean fermented food is not only effective and safe, but also a kind of valuable development and utilization value. Functional preventive health food.
According to the documents, Nadu originated from the Chinese fermented bean and introduced into Japan by Buddhism. The reason for the long life expectancy of Japan is that in addition to the better natural environment, it is more important that the fermented food, especially the largest consumption of the Nata, is the secret of the longevity of the Japanese people and the national treasure food. The bean is because its protein content is as high as 35.3%, and the enzyme produced by natto bacteria can make 50% of the protein into water soluble, the digestibility can reach 80%. also contains amino acids, vitamins, and vegetable fat. According to scientific research, natto food not only adjusts the gastrointestinal function obviously, but also has the treatment of colds, dysentery, flatulence, gastroenteritis, indigestion and disability. Constipation and other effects, as well as the prevention and treatment of diabetes, inhibition of thrombosis and thrombolytic effect, is a kind of longevity, healthy beauty food. It can be said that natto is a natural food, is the product of the wisdom of human life.
In 1987, Dr. Sumi of Japan separated a alkaline protease with strong fibrinolysis from the traditional Japanese food natto and named nattokinase (NK). It not only directly acts on the crosslinked fibrin, but also activates the plasminogen in the body. It shows a strong hemolytic thrombolytic effect. It is reported that the product has a strong thrombolytic effect. It can hydrolyze amyloid protein, reduce the seventh factor and eighth factor of plasma fibrinogen, reduce blood viscosity, reduce blood lipid, reduce cholesterol, reduce blood pressure, improve blood circulation state, maintain normal form and function of blood cells, be beneficial to the regeneration of nerve injury, inhibit osteoporosis, inhibit atherosclerosis and other functions.
NK has the advantages of good safety, low cost, rapid entry into blood after oral administration, long time, good stability of the gastrointestinal tract, the fermentation of bacteria and the production of genetic engineering bacteria. It is expected to be developed as a new generation of oral antithrombotic drugs for the prevention and treatment of thrombotic diseases. But to now, the source of Nattokinase The mainly fermented Japanese Nata is unacceptable because of its unique flavor. Although the capsules containing nattokinase are produced in Japan, Korea, and Korea, its application is limited due to the short supply and high cost. In order to make it both convenient and widely used, people find it from the molecular biology angle. To find a better expression vector and host bacteria, optimize the expression conditions and other aspects of further research, the application of genetic engineering products to purify more high quality thrombolytic drugs, will have a more extensive application prospects, to bring new dawn to the clinical.
Objective the purpose of this study was to optimize the fermentation liquid, salting out, chromatography, protein content, determination of fibrinolysis activity, SDS-PAGE and other techniques and methods. The traditional methods were used to extract and purify natto kinase by traditional methods, and to detect the activity of natto kinase, and to extract DNA from Bacillus subtilis group of Bacillus subtilis, the design primers, PCR cloning of natto kinase and nattokinase by molecular biology technology. The appropriate expression vector and host bacteria are selected to use genetic engineering products to purify more safe and high quality thrombolytic drugs, and provide a theoretical basis for clinical application.
Methods in 1. experiments, the OD600 value was continuously measured by turbidimetric [1], and the growth curve of strain was plotted. The culture conditions of natto bacteria were determined so as to be better applied to actual production. In order to further improve the production of enzymes and reduce the cost of fermentation, the fermentation process was optimized by single factor and orthogonal experiment.
2. the crude enzyme solution was centrifuged, 40~45% salting out, CM-Cellulose ion exchange chromatography and Sephadex G-100 gel column could extract the single enzyme, and the molecular weight was measured with SDS-PAGE, and the enzyme activity of the crude enzyme solution and the extracted single enzyme solution was compared.
3. natto bacillus subtilis genomic DNA was extracted by PCR amplification of Nattokinase and nattokinase sequence and detected by 1% agarose gel electrophoresis. In order to further construct the recombinant plasmid and transform the Escherichia coli into the paving, the follow-up work remains to be further studied.
Result
1. the optimum composition of the medium is MgSO4 0.02%, K2HPO4 0.02%, KH2PO4 0.01%, CaCl2 0.05%, maltose 3%, and soybean peptone 3%, the optimum culture temperature is 37, the optimum pH is 8. The optimum inoculation amount is the nattokinase after the optimized fermentation condition of 500 u L. per 5g yellow bean, and the enzyme activity is raised to 158.74IU/mL before the optimization of the 31.25IU/mL fermentation liquid. Fermentation broth, the unit output increased by about 5.08 times.
2. the nattokinase was separated by CM-Cellulose ion exchange chromatography, and the active components were found in the 0.10 mol/LNaCl elution peak. The enzyme activity of the Folin- phenol method was 168.56 IU/m., and the natto kinase enzyme in the 0.10mol/L NaCl elution peak was separated by Sephadex G-100 gel filtration chromatography, and the Folin- phenol method was used to measure the enzyme activity to 137.16 IU/mL, and it was determined by SDS-PAGE. The mass of the molecules is 28000 Da.
3. natto bacillus subtilis genomic DNA was extracted by PCR amplification of nattokinase (275 amino acids, base 825bp) and nattokinase (381 amino acids, base 1143bp) and confirmed by agarose gel electrophoresis (1% agarose gel electrophoresis).
Conclusion the crude enzyme solution, centrifugation, 40~45% salting out, CM-Cellulose ion exchange chromatography and Sephadex G-100 gel column can be extracted from the single enzyme after the optimization of liquid culture medium, which can reduce the loss of enzymes in the process of operation. The whole process should be operated at 4 degrees centigrade and aseptic conditions. The suitable expression vectors and host bacteria have not been successfully expressed after repeated expression. The methods provided in this study can be used for reference by future research workers for further study.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

【参考文献】