本文選題：納豆激酶 + 分離純化��； 參考：《河北聯(lián)合大學(xué)》2011年碩士論文

【摘要】：心腦血管性疾病,具有“發(fā)病率高、致殘率高、死亡率高、并發(fā)癥多”等特性。2005年,世界衛(wèi)生組織(WHO)調(diào)查表明,全世界每年約有1700萬人死于心腦血管疾病。并且隨著人類生活水平的提高以及生活壓力的增加,而呈現(xiàn)年輕化的趨勢(shì),而血栓又是心腦血管疾病中致死率最高的疾病。所以,血栓的防治已是當(dāng)務(wù)之急。雖然傳統(tǒng)藥物在心腦血管及血栓栓塞性疾病防治上療效顯著,但毒副作用嚴(yán)重;而傳統(tǒng)大豆發(fā)酵食品不僅效果顯著且安全無毒,可作為一種具有開發(fā)和利用價(jià)值的功能性預(yù)防保健食品。 據(jù)文獻(xiàn)記載,納豆源于中國(guó)的豆豉,通過佛教由中國(guó)傳入日本,日本人均壽命長(zhǎng)的原因除了與較為優(yōu)良的自然環(huán)境有關(guān)外,更重要的是其膳食結(jié)構(gòu)中,發(fā)酵食品特別是消費(fèi)量最大的納豆,是日本人長(zhǎng)壽的秘方,視為國(guó)寶級(jí)食品。寺院喜食大豆是因?yàn)槠涞鞍踪|(zhì)含量高達(dá)35.3%,而由納豆菌產(chǎn)出的酶,能使50%的蛋白質(zhì)變?yōu)樗苄?消化率可達(dá)80%。還含氨基酸、維生素、植物性脂肪等。據(jù)科學(xué)研究表明,納豆食品不僅調(diào)整胃腸功能明顯,還具有治療感冒、痢疾、脹氣、胃腸炎、消化不良、殘便、便秘等功效,還有防治糖尿病,抑制血栓的生成和溶解血栓的作用,是一種延年益壽、健康的美容食品�？梢哉f納豆是一種自然食品,是人類生活智慧的產(chǎn)物。 1987年日本的Sumi博士首次從日本傳統(tǒng)食品納豆中分離出一種具有強(qiáng)烈纖溶作用的堿性蛋白酶并命名納豆激酶(nattokinase,NK)。它不但能直接作用交聯(lián)纖維蛋白,而且還可激活體內(nèi)的纖溶酶原,從而表現(xiàn)出很強(qiáng)的溶血栓作用。據(jù)報(bào)道其制品具有水解淀粉樣蛋白,降低血漿纖維蛋白原的第七因子和第八因子,降低血液粘度、降血脂、降膽固醇,降血壓,改善血液循環(huán)狀態(tài),維持血細(xì)胞的正常形態(tài)和功能,有利于神經(jīng)損傷的再生,抑制骨質(zhì)疏松癥、抑制動(dòng)脈粥樣硬化等多種功能。 由于NK具有安全性好、成本低、經(jīng)口服后可迅速入血,作用時(shí)間長(zhǎng),胃腸道穩(wěn)定性好,可由細(xì)菌發(fā)酵生產(chǎn)、也可由基因工程菌生產(chǎn)等優(yōu)點(diǎn),有望被開發(fā)為新一代的口服抗血栓藥物用于血栓性疾病的預(yù)防和治療。但是直到現(xiàn)在,納豆激酶的來源主要是發(fā)酵的日本納豆,因其獨(dú)特的風(fēng)味很多人不能接受。雖然含有納豆激酶作為主要成分的膠囊制劑已在日本,韓國(guó),朝鮮生產(chǎn),但由于只能短期供應(yīng)和高昂的代價(jià),其應(yīng)用很有限。為了使其既方便又廣泛使用,人們從分子生物學(xué)角度尋找更好的表達(dá)載體和宿主菌,優(yōu)化表達(dá)條件等方面做了進(jìn)一步的研究,應(yīng)用基因工程制品純化出更為優(yōu)質(zhì)的溶栓藥物,將具有更為廣泛的應(yīng)用前景,給臨床帶來新的曙光。 目的本研究通過發(fā)酵液優(yōu)化、鹽析、層析、蛋白含量、纖溶活力測(cè)定、SDS-PAGE等技術(shù)和手段,通過傳統(tǒng)方法提取分離純化納豆激酶,檢測(cè)其活性。還通過分子生物學(xué)技術(shù),提取納豆枯草芽孢桿菌基因組DNA,設(shè)計(jì)引物,PCR克隆納豆激酶及納豆激酶原。選擇合適的表達(dá)表達(dá)載體和宿主菌,以期應(yīng)用基因工程制品純化出更為安全優(yōu)質(zhì)溶栓藥物,為臨床應(yīng)用提供理論依據(jù)。 方法1.本實(shí)驗(yàn)通過比濁法[1]連續(xù)測(cè)定OD600值,繪制菌種生長(zhǎng)曲線,確定納豆菌培養(yǎng)條件,以便更好地應(yīng)用于實(shí)際生產(chǎn)中。為了進(jìn)一步提高酶的產(chǎn)量,降低發(fā)酵成本,我們采用單因素和正交實(shí)驗(yàn)法對(duì)發(fā)酵工藝進(jìn)行優(yōu)化。 2.將粗酶液經(jīng)離心,40~45%鹽析,CM-Cellulose離子交換層析和Sephadex G-100凝膠柱可以提取出單一酶,并用SDS-PAGE測(cè)定分子量,比較粗酶液與提取出的單一酶液的酶活力。 3.納豆枯草芽孢桿菌基因組DNA提取后,經(jīng)PCR擴(kuò)增納豆激酶及納豆激酶原序列,并經(jīng)1%瓊脂糖凝膠電泳檢測(cè)。為進(jìn)一步構(gòu)建重組質(zhì)粒,并轉(zhuǎn)化大腸桿菌做鋪墊,后續(xù)工作有待進(jìn)一步研究。 結(jié)果 1.培養(yǎng)基成分的最優(yōu)組合為MgSO4 0.02%、K2HPO4 0.02%、KH2PO4 0.01%、CaCl2 0.05%、麥芽糖3%、大豆蛋白胨3%、最適培養(yǎng)溫度37℃、最適pH為8.0、最佳接種量為每5g黃豆接種500μL。優(yōu)化發(fā)酵條件后生產(chǎn)的納豆激酶,酶活由優(yōu)化前的31.25IU/mL發(fā)酵液提高到158.74IU/mL發(fā)酵液,單位產(chǎn)量提高了約5.08倍。 2.經(jīng)CM-Cellulose離子交換層析分離納豆激酶酶,得到0.10 mol/LNaCl洗脫峰中含有活性組份,Folin-酚法測(cè)酶活為168.56 IU/m。將0.10mol/L NaCl洗脫峰中的納豆激酶酶用Sephadex G-100凝膠過濾層析進(jìn)行分離,Folin-酚法測(cè)酶活為137.16 IU/mL,并用SDS-PAGE測(cè)定它們的分子質(zhì)量為28 000 Da。 3.納豆枯草芽孢桿菌基因組DNA提取后,經(jīng)PCR擴(kuò)增納豆激酶(275個(gè)氨基酸,堿基825bp)及納豆激酶原序列(381個(gè)氨基酸,堿基1143bp),并經(jīng)1%瓊脂糖凝膠電泳檢測(cè)得到確認(rèn)。 結(jié)論經(jīng)液體培養(yǎng)基的優(yōu)化后提取粗酶液,離心,40~45%鹽析,CM-Cellulose離子交換層析和Sephadex G-100凝膠柱可以提取出單一酶,為減少操作過程中酶的損失,整個(gè)過程力求在4℃、無菌條件下操作�？寺×思{豆激酶及納豆激酶原基因并回收。選擇合適的表達(dá)載體和宿主菌,經(jīng)多次重復(fù)表達(dá)未成功,本實(shí)驗(yàn)提供的方法可供以后科研工作者參考,以待進(jìn)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.
【學(xué)位授予單位】：河北聯(lián)合大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 凌均建,羅立新,楊汝德;納豆激酶的分子生物學(xué)研究進(jìn)展[J];廣東藥學(xué)院學(xué)報(bào);1999年04期

2 陸瑾,趙s，

本文編號(hào)：2070404